A ratiometric reversible responsive hydrogen sulfide fluorescent probe and its synthesis method and application

Abstract

The present application discloses a ratiometric reversibly responsive hydrogen sulfide fluorescent probe, a synthesis method thereof, and an application thereof. The ratiometric reversibly responsive hydrogen sulfide fluorescent probe is a DC probe, and the DC probe structure is: The DC probe that can be used to track changes in hydrogen sulfide content has a rapid response to hydrogen sulfide, high selectivity and sensitivity, and in particular can respond to changes in hydrogen sulfide content in real time. The hydrogen sulfide fluorescent probe of the present invention can indicate changes in hydrogen sulfide content through both fluorescence and colorimetric modes, and is particularly suitable for sensing fields such as solution / cell / tissue / in vivo imaging, biomarkers, or environmental and food testing.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis and detection, and in particular relates to a ratiometric reversible response hydrogen sulfide fluorescent probe and a synthesis method and application thereof. Background Art

[0002] Hydrogen sulfide (H2S) is the third gaseous signaling molecule after carbon monoxide (CO) and nitric oxide (NO), and is associated with various physiological functions in the human body. Hydrogen sulfide plays a crucial role in many pathological and physiological processes, such as those in the cardiovascular, respiratory, digestive, and nervous systems. Abnormal intracellular hydrogen sulfide concentrations may cause a variety of human diseases, such as Down syndrome, Alzheimer's disease, cirrhosis, diabetes, and gastric mucosal damage. Compared with traditional detection methods, visual detection of hydrogen sulfide concentration in biological systems is of great significance. Currently, various methods for detecting hydrogen sulfide have been reported, such as electrochemical analysis, gas chromatography, and capillary electrophoresis. However, these methods cannot be directly applied to living cells due to their destructive nature, time-consuming nature, and complex sample preparation. Organic small molecule fluorescent probes can rapidly, noninvasively, and sensitively detect target analytes and have become a powerful tool for in vivo imaging.

[0003] At present, people have reported many cases of fluorescent probes for detecting hydrogen sulfide. Most of the reports are irreversible fluorescent probes, which cannot detect hydrogen sulfide in real time. There are fewer reports of ratiometric fluorescent probes that can reversibly monitor hydrogen sulfide content in real time. (Yuming Zhang, Yuuncong Chen, Yang Bai, et al. FRET-based fluorescent ratiometric probes for the rapid detection of endogenous hydrogen sulphide in living cells, Analyst, 2020,145; Yuting Du, Hongliang Wang, Lu Qin, et al. Rational development of an ESIPT-based fluorescent probe with large Stokes shift for imaging of hydrogen sulfide in live cells, Bioorganic Chemistry, 2022, 129, 106158; Sha Li, Fangjun Huo, Caixia Yin. NIR fluorescent probe for dual-responseviscosity and hydrogen sulfide and its application in Parkinson's disease model, Dyes and Pigments, 2022, 197, 109825; Jianlong Ma, Feifei Li, Qiang Li, etal. Naked-eye and ratiometric fluorescence probe for fast and sensitive detection of hydrogen sulfide and its application in bioimaging,New Journal ofChemistry,2018,4,19272-19278;Yuncong Chen,Chengcheng Zhu,Zhenghao Yang,etal.A ratiometric fluorescent probe for rapid detection of hydrogen sulfide inmitochondria, Angewandte Chemie International Edition, 2013,52,6; Bing-Yu Wei a, Xiao-Meng Yan, Yu-Chang Yuan, et al. A rhodol-based fluorescent probe for ratiometric sensing of hydrogen sulfide in living cells, Journal of Photochemistry and PhotobiologyA,2024,450,115450.). . Summary of the Invention

[0004] Technical issues solved:

[0005] This application addresses the deficiencies in the existing technology and solves the technical problems that current methods for detecting hydrogen sulfide cannot be directly applied to living cells due to their destructiveness, time consumption, and complex sample preparation; there are many reports on irreversible fluorescent probes for detecting hydrogen sulfide, which cannot detect hydrogen sulfide dynamically in real time, and there are few reports on ratiometric fluorescent probes that can reversibly monitor hydrogen sulfide content in real time. The application provides a ratiometric reversible response hydrogen sulfide fluorescent probe, its synthesis method, and application.

[0006] Technical solution:

[0007] To achieve the above objectives, this application is implemented through the following technical solutions:

[0008] A ratiometric reversibly responsive hydrogen sulfide fluorescent probe, the structural formula of the ratiometric reversibly responsive hydrogen sulfide fluorescent probe is as follows:

[0009]

[0010] A method for synthesizing a ratiometric reversibly responsive hydrogen sulfide fluorescent probe comprises placing a fluorophore and anhydrous potassium carbonate in an organic solvent under argon protection and light-proofing, heating and reflux, and causing a substitution reaction to obtain a target probe DC.

[0011] Furthermore, the fluorophore is selected from one or more of rhodamine, coumarin, fluoroborane, fluorescein, naphthalimide, acridine, and cyanine dye fluorescent groups.

[0012] Furthermore, the fluorophore is an acridine fluorophore and / or a coumarin fluorophore.

[0013] Furthermore, the acridinium fluorophore is 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone DDAO.

[0014] Furthermore, the organic solvent is anhydrous acetonitrile, and the amount used is 40 mL.

[0015] Furthermore, the heating reflux temperature is 82° C., and the reflux time is 20 h.

[0016] Furthermore, after the reaction substitution generates the target probe DC, the product is separated and purified. The separation and purification steps are as follows: after the reaction solution is cooled to room temperature, potassium carbonate is removed by filtration, the filtrate is evaporated under reduced pressure to remove the organic solvent, and the dried solid is purified by silica gel column chromatography (dichloromethane / methanol = 40:1) to obtain an orange solid. The eluent in the silica gel column chromatography purification is a mixture of dichloromethane and methanol in a volume ratio of 40:1.

[0017] Furthermore, the fluorophore is 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone DDAO and a coumarin piperazine derivative; the stoichiometric ratio of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone DDAO, coumarin piperazine derivative, and anhydrous potassium carbonate is 1:2:1.1.

[0018] The present application also discloses the application of a ratiometric reversible response hydrogen sulfide fluorescent probe in analysis and detection in the fields of solution, cell, tissue, living body imaging, biomarker, environment, and food.

[0019] Principle explanation: The DDAO fluorophore is connected to a coumarin piperazine derivative through an ether bond to construct a hydrogen sulfide fluorescent probe, which can detect changes in hydrogen sulfide content in cells and living bodies in real time. DDAO is an excellent near-infrared fluorescent dye with a low pKa value, high water solubility and high quantum yield. The fluorescent probe synthesized by modifying the DDAO fluorophore has excellent sensing properties such as rapid response, high selectivity and sensitivity, low detection limit and obvious colorimetric signal changes. In addition, the probe has low cytotoxicity and has significant application value for the detection of living cells and organisms.

[0020] Beneficial effects:

[0021] This application provides a ratiometric reversible response hydrogen sulfide fluorescent probe and its synthesis method and application, which have the following beneficial effects compared with the existing technology:

[0022] 1. The hydrogen sulfide fluorescent probe described in this application has absorption peaks near 416nm and 482nm, and emission peaks near 476nm and 595nm. After the addition of hydrogen sulfide, the absorption peak near 482nm gradually weakens, the fluorescence emission peak at 595nm gradually weakens, and the fluorescence emission peak at 476nm significantly enhances;

[0023] 2. The probe appears orange in solution. After adding hydrogen sulfide, the solution changes from orange to light yellow. The fluorescence intensity has a clear linear relationship with the change of hydrogen sulfide concentration.

[0024] 3. When the probe solution containing hydrogen sulfide is placed in air, the solution gradually changes from light yellow back to its original orange color. At the same time, the UV absorption and fluorescence emission spectra return to the initial state of the probe before hydrogen sulfide is added. This change can be repeated many times, indicating that the probe color and fluorescence emission can change reversibly with the hydrogen sulfide content.

[0025] 4. The hydrogen sulfide fluorescent probe described in this application has the characteristics of rapid response, high selectivity and sensitivity in hydrogen sulfide recognition, is not affected by common species of living organisms under physiological pH conditions, and achieves dynamic and reversible imaging of changes in hydrogen sulfide content in cells;

[0026] 5. The probe of this application has broad application prospects in the fields of biology, medicine, environment and food testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the UV-visible absorption spectrum of the hydrogen sulfide fluorescent probe DC described in this application in response to hydrogen sulfide;

[0028] Figure 2 The fluorescence emission spectrum of the hydrogen sulfide fluorescent probe DC described in this application in response to hydrogen sulfide, wherein (a) is the fluorescence emission spectrum of the probe DC when HS is added dropwise. - Fluorescence spectrum of HS - Concentration change graph;

[0029] Figure 3 This is a comparison chart of the solution colors before and after adding hydrogen sulfide to the hydrogen sulfide fluorescent probe DC described in this application;

[0030] Figure 4 This is the fluorescence histogram of the hydrogen sulfide fluorescent probe DC described in this application for common species at 476nm / 595nm;

[0031] Figure 5 This is a graph showing the fluorescence ratio change of the hydrogen sulfide fluorescent probe DC described in this application at 476nm / 595nm in the pH range of 4-9;

[0032] Figure 6The UV absorption (a) and fluorescence emission (b) spectra of the probe DC solution described in this application, after adding hydrogen sulfide and exposing it to air, are plotted over time.

[0033] Figure 7 The UV absorption (a) and fluorescence emission (b) spectra of the probe DC solution described in this application after adding hydrogen sulfide in an argon atmosphere change over time;

[0034] Figure 8 This is the fluorescence imaging image of the probe DC described in this application on exogenous hydrogen sulfide in HeLa cells;

[0035] Figure 9 This is a bar graph of the ratio of the mean fluorescence intensity of the probe DC described in this application to exogenous hydrogen sulfide in HeLa cells;

[0036] Figure 10 This is a fluorescence imaging image of the probe DC described in this application on endogenous hydrogen sulfide in HeLa cells;

[0037] Figure 11 The bar graph is a ratio of the mean fluorescence intensity of the probe DC to the endogenous hydrogen sulfide in HeLa cells;

[0038] Figure 12 This is a fluorescence imaging diagram of the reversible cyclic changes of hydrogen sulfide content in HeLa cells by the probe DC described in this application;

[0039] Figure 13 The figure is a bar graph showing the ratio of the mean fluorescence intensity of HeLa cells to the reversible cyclic changes of hydrogen sulfide content in the cells by the probe DC. DETAILED DESCRIPTION

[0040] In order to further illustrate the present invention, the present invention is described in further detail below with reference to the embodiments.

[0041] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process. However, the protection scope of the present invention is not limited to the following examples.

[0042] Example 1

[0043] Preparation of compounds DDAO-Br 1 and DDAO-Br 2:

[0044]

[0045] Step 1: Synthesize compound DDAO by a method known in the literature (Zhao Wang, Weiwei Wang, Pengzhan, et al. Highly

[0046] sensitive near-infrared imaging ofperoxynitrite fluxes ininflammationprogress,Analytical chemistry,

[0047] 2021,93,5,3035-3041);

[0048] Step 2: Under nitrogen protection, DDAO, silver oxide and 1,2-dibromoethane were dissolved in anhydrous ethyl acetate and anhydrous quinoline was added, and the mixture was stirred at room temperature;

[0049] Step 3: The reaction solution was washed with ethyl acetate, 1M HCl solution, saturated NaHCO3 solution, and saturated brine, dried over anhydrous NaSO4, and then desolvated;

[0050] Step 4: The obtained mixture was purified by column chromatography (ethyl acetate / petroleum ether gradient elution) to obtain DDAO-Br1 and DDAO-Br2.

[0051] DDAO-Br 1:

[0052] 1 H NMR(400MHz,Chloroform-d,δ,ppm)7.81–7.70(m,1H),7.41–7.32(m,1H),6.68( dtt,J=5.5,3.9,1.8Hz,2H),4.43–4.34(m,2H),3.80–3.71(m,2H),1.79(s,6H). 13 C NMR(101MHz,Chloroform-d,δ,ppm)28.81,28.95,38.05,72.74,128.13,128.82,1 29.90,132.26,132.59,133.36,140.23,140.96,148.18,152.74,152.85,187.32.

[0053] DDAO-Br 2:

[0054] 1 H NMR(400MHz,Chloroform-d,δ,ppm)7.62(d,J=9.2Hz,2H),7.04(d,J=2.6Hz,1H),6. 91(dd,J=8.7,2.7Hz,1H),4.40(t,J=6.1Hz,2H),3.70(t,J=6.1Hz,2H),1.88(s,6H). 13C NMR(101MHz,Chloroform-d,δ,ppm)26.80,28.59,39.16,68.14,77.25,113.27,113.8 4,134.09,134.68,136.15,136.78,139.43,140.55,140.71,147.86,161.38,173.23.

[0055] Example 2

[0056] Preparation of DC probe:

[0057]

[0058] Step 1: Synthesize coumarin piperazine derivative 2 by a method known in the literature (Lin Yuan, Weiying Lin, Yinan Xie, et al.

[0059] Development of a ratiometric fluorescent sensor for ratiometric imaging of endogenously produced nitric oxide in macrophage cells, Chemical communications, 2011, 47, 33, 9372-9374.).

[0060] Step 2: Dissolve DDAO-Br 2 prepared in Example 1, the coumarin piperazine derivative 2 prepared in Step 1, and anhydrous potassium carbonate in anhydrous acetonitrile, and reflux at 82° C. for 20 h in an argon atmosphere in the dark;

[0061] Step 3: After the reaction is completed, the mixture is cooled to room temperature, filtered to remove potassium carbonate, and the filtrate is evaporated under reduced pressure. The resulting mixture is purified by silica gel column chromatography (dichloromethane / methanol = 40:1) to obtain an orange solid with a yield of approximately 43%.

[0062] 1H NMR(400MHz,Chloroform-d,δ,ppm)7.86(s,1H),7.62(s,1H),7.60(d,J=8.6Hz,1H),7 .31(d,J=8.9Hz,1H),7.03(d,J=2.7Hz,1H),6.91(dd,J=8.6,2.6Hz,1H),6.60(dd,J=8 .8,2.5Hz,1H),6.47(d,J=2.5Hz,1H),4.23(t,J=5.5Hz,2H),3.82(s,2H),3.45(p,J=7 .9,7.1Hz,6H),2.92(t,J=5.4Hz,2H),2.69(s,4H),1.88(s,6H),1.23(t,J=7.1Hz,6H). 13 C NMR(101MHz,Chloroform-d,δ,ppm)12.44,14.19,22.69,26.80,31.61,39.17, 42.16,44.98,47.29,53.16,53.51,53.68,56.88,66.36,76.76,96.90,107.72, 109.35,113.39,113.75,116.17,129.86,134.12,134.45,135.92,136.61,139.44,140.66,145.15,147.52,151.67,157.26,159.21,162.13,164.91,173.22.

[0063] Example 3

[0064] Fluorescence and UV spectrum responses of probe DC to hydrogen sulfide:

[0065] Sodium bisulfide solution was added dropwise to a PBS solution (pH 7.4) containing 10 μM probe DC, and data were collected after the spectrum stabilized. Figure 1 It can be seen that probe DC has two strong absorption peaks near 416nm and 482nm. After the addition of hydrogen sulfide, the intensity of the absorption peak at 482nm decreases, while the absorption peak near 416nm remains almost unchanged. Figure 2 As shown in (a), the fluorescence emission peaks of probe DC are near 476 nm and 595 nm, and the solution color is orange. As the amount of hydrogen sulfide increases, the fluorescence emission intensity of the probe near 476 nm gradually increases, and the fluorescence emission intensity near 595 nm gradually decreases. Figure 2 As shown in (b), the hydrogen sulfide concentration reaches equilibrium at 50 μM, and the solution color turns light yellow. Figure 3 shown.

[0066] Example 4

[0067] Probe DC's response behavior to common species of living organisms:

[0068] Common species of various organisms were added to a PBS solution (pH 7.4) containing 10 μM probe DC, and data were collected after the spectrum stabilized. Concentration of each species: 2 mM Na + , K + Mg 2+ , Ca 2+ ,50μM Zn 2+ 、Co 2+ 、Cu 2+ 、Fe 3+ 、Fe 2+ 、Ni 2+ 、Mn 2+ ,100μM SNP, NO2 - 、OCl - 、H2O2、HS - , 200μM Hcy, 1mM Cys, GSH, Hypoxia: 20μM NADPH, CYP 450 reductase 0.72μg / μL. Figure 4 As shown, the fluorescence ratio of probe DC did not change significantly after the addition of other species, and only after the addition of hydrogen sulfide did the fluorescence ratio at 476nm / 595nm change significantly enhance.

[0069] Example 5

[0070] Effect of pH on probe DC fluorescence emission:

[0071] HCl or NaOH was added to the PBS solution containing 10 μM probe DC to adjust the pH to the target value, and data was collected after the spectrum stabilized. Figure 5 The fluorescence ratio of probe DC at 476 nm / 595 nm within the physiological pH range shown was almost unaffected by pH changes.

[0072] Example 6

[0073] Reversibility test of the reaction with hydrogen sulfide in probe DC:

[0074] Add 5 times the amount of hydrogen sulfide to a 10 μM probe DC solution in PBS (pH 7.4) and place it in a normoxic state. Track the changes in the solution's UV and fluorescence spectra. Figure 6 As shown in (a), compared with the control group (probe only), the absorption peak of the probe near 482nm decreased rapidly after the addition of hydrogen sulfide (5min), and the absorption peak gradually recovered over time. Figure 6As shown in (b), after adding hydrogen sulfide, the emission peak near 476 nm rapidly increases, while the emission peak near 595 nm decreases rapidly (5 minutes). Over time, these two emission peaks gradually return to their initial levels, indicating a reversible reaction between the probe and hydrogen sulfide.

[0075] After adding 5 times the amount of hydrogen sulfide to the probe DC solution and placing it in an oxygen-free argon atmosphere, the changes in the solution's UV and fluorescence spectra were tracked. Compared to the control group (probe only), the absorption and emission spectra of the probe changed rapidly after adding hydrogen sulfide (5 minutes), and remained essentially unchanged over time, as shown in Figure 2. Figure 7 This indicates that the probe can react with hydrogen sulfide and return to its initial state in the presence of oxygen. That is, the reaction between the probe and hydrogen sulfide is reversible, and the change of hydrogen sulfide content can be dynamically tracked.

[0076] Example 7

[0077] Probe DC imaging of hydrogen sulfide in cells:

[0078] The experiment was tested on HeLa cells. Excitation was performed at 400nm-450nm, emission 1 (green channel) was collected at 465nm-495nm, and emission 2 (red channel) was collected at 590nm-640nm.

[0079] The cells in the control group were incubated with probe DC and then imaged. The cells in the experimental group were incubated with probe DC and different concentrations of NaHS and then imaged. Figure 8 、 9 As shown, with the increase of exogenous hydrogen sulfide, the fluorescence intensity of the green channel increased, the fluorescence intensity of the red channel decreased, and the ratio of the fluorescence intensity of the green channel to the red channel gradually increased, indicating that the probe DC can respond to changes in the exogenous hydrogen sulfide content in HeLa cells.

[0080] Cells were incubated with the endogenous hydrogen sulfide precursor L-cysteine ​​(200 μM) or the hydrogen sulfide scavenger ZnCl2 (1 mM) and then incubated with the probe DC and imaged. Figure 10 、 11 As shown, compared with the control group, the fluorescence signal of the ratio channel was enhanced after incubation with L-cysteine, and the fluorescence signal of the ratio channel was weakened after incubation with ZnCl2, indicating that the probe DC can respond to changes in endogenous hydrogen sulfide content in HeLa cells.

[0081] Example 8

[0082] Probe DC tracks and images the cyclic changes of intracellular hydrogen sulfide content:

[0083] Add probe DC to the cells, incubate under normoxia and then image; add 5 times sodium bisulfide to the cells, incubate under hypoxia and image; place the cells in normoxia and collect the fluorescence signal after it stabilizes; add sodium bisulfide to the cells, incubate under hypoxia and image; repeat the cycle. Figure 12 、 13 As shown, the ratiometric channel signal of the probe is weak under normoxia. The addition of sodium bisulfide significantly enhances the ratiometric channel signal. Over time under normoxia, the ratiometric channel signal weakens, and the addition of sodium bisulfide further enhances the ratiometric channel signal. This indicates that the ratiometric channel fluorescence signal changes with the cyclical changes in hydrogen sulfide content in the cells. This phenomenon is consistent with the results of reversible experimental tests in solution, confirming that the probe DC can reversibly respond to hydrogen sulfide, making it an example of a ratiometric, reversibly responsive hydrogen sulfide fluorescent probe.

[0084] The above is an exemplary description of the present application. It should be noted that, without departing from the core of the present application, any simple deformation, modification or equivalent replacement by other technical personnel in this field without expending creative labor falls within the scope of protection of the present application.

Claims

1. A ratiometric reversible response hydrogen sulfide fluorescent probe, characterized by: The structural formula of the ratiometric reversible response hydrogen sulfide fluorescent probe is as follows: .

2. A method for synthesizing the ratiometric reversible response hydrogen sulfide fluorescent probe according to claim 1, characterized in that: The synthetic route is as follows: .

3. The method for synthesizing a ratiometric reversible hydrogen sulfide fluorescent probe according to claim 2, wherein: After the reaction generates a ratiometric reversibly responsive hydrogen sulfide fluorescent probe, the product is separated and purified. The separation and purification steps are as follows: after the reaction solution is cooled to room temperature, potassium carbonate is removed by filtration, the filtrate is evaporated under reduced pressure to remove the organic solvent, and the dried solid is purified by silica gel column chromatography to obtain an orange solid. The eluent in the silica gel column chromatography purification is a mixture of dichloromethane and methanol in a volume ratio of 40:

1.

4. Use of the ratiometric reversibly responsive hydrogen sulfide fluorescent probe according to claim 1 in the preparation of reagents for analyzing and detecting hydrogen sulfide in solutions, cells, tissues, in vivo imaging, biomarkers, environment, and food.

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