Construction method of cysteine fluorescent probe based on coumarin

Abstract

The invention relates to the technical field of biosensing and fluorescent probes, in particular to a construction method of a cysteine fluorescent probe based on coumarin, dicyanoisophorone is introduced into a 7-hydroxycoumarin parent structure to serve as a fluorophore, acrylate serves as a recognition site of Cys, and the cysteine fluorescent probe based on coumarin is obtained. A near-infrared fluorescent probe DCI-Cu-Cys capable of specifically recognizing Cys is constructed, the maximum emission wavelength of the probe is 735 nm, and the probe is in a near-infrared band; the Stokes shift is 210 nm, which is very beneficial to biological imaging; the detection limit is as low as 23 nmol / L, the probe has high sensitivity to Cys, and in addition, the probe has low cytotoxicity and can rapidly reflect the change of the content of Cys in cells in real time in situ; lipopolysaccharide (LPS) is used for inducing cells and zebra fish to generate oxidative stress, different antioxidant drugs are used for treatment, in the process, the probe can detect the change of the mercaptan content, the treatment effect of the drugs is evaluated, and the potential application value in the biological field is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors and fluorescent probes, and in particular to a method for constructing a coumarin-based cysteine ​​fluorescent probe. Background Art

[0002] Over the past few decades, researchers have developed a variety of analytical methods for the detection of biothiols, including high-performance liquid chromatography, capillary electrophoresis, spectrophotometry, and electrochemical methods. In addition to the above four methods, methods for detecting thiol content also include high-performance liquid chromatography-mass spectrometry and mass spectrometry. Currently, although these methods are widely used, most of them require expensive instruments and cumbersome sample preparation processes, and have significant limitations in the detection of biological samples such as cells and living animals. Fluorescent probe methods are recognized by many researchers for their advantages such as high sensitivity, rapid response, and good biocompatibility. Fluorescent probes can dynamically detect thiol levels in organisms in real time, providing researchers with a powerful and effective tool for exploring the mechanism of action of thiols in physiological processes.

[0003] Biothiols are a class of compounds with unique structures and functions. They play an important role in antioxidant defense, protein structure and function regulation, signal transduction, and anti-toxicity. Among them, cysteine ​​(Cys), as a multifunctional biothiol, participates in important physiological processes such as cellular metabolism, detoxification, and maintaining redox balance in the body. However, when the cysteine ​​content in an organism is abnormal, it may induce a variety of major and minor diseases. For example, edema, lethargy, liver damage, and brain degenerative diseases not only reduce people's quality of life but also seriously affect human life and health.

[0004] In order to overcome the above technical problems, the present invention designs a method for constructing a coumarin-based cysteine ​​fluorescent probe, which solves the above technical problems. Summary of the Invention

[0005] The technical purpose to be achieved by the present invention is to develop a fluorescent probe for real-time monitoring of Cys content changes in an organism, which is of great significance for the early diagnosis and later treatment of related diseases.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] A method for constructing a coumarin-based cysteine ​​fluorescent probe includes a compound having the following molecular formula:

[0008]

[0009] The steps of the method for constructing the compound are as follows:

[0010] S1: Compounds isophorone and malononitrile are dissolved in anhydrous ethanol, and piperidine is added to the system and heated to react to produce DCI;

[0011] S2: 7-Hydroxycoumarin and hexamethylenetetramine (HMTA) are heated in trifluoroacetic acid (TFA) to generate Cou;

[0012] S3: Dissolve the compounds DCI and Cou in anhydrous ethanol, add piperidine to the stirring reaction system, raise the reaction temperature to 80°C, add acetic acid, and then reflux with stirring to produce DCI-Cou-OH;

[0013] S4: Use an analytical balance to accurately weigh the compound DCI-Cou-OH and dissolve it in DCM. Add acryloyl chloride at 0°C, then add triethylamine, and react at room temperature for 12 hours to generate DCI-Cou-Cys.

[0014] The preparation reaction formula of the above DCI-Cou-Cys is as follows:

[0015]

[0016] Preferably, the synthesis method of compound DCI in S1 is as follows:

[0017] In a 50 mL flask, add isophorone (4.5 mL, 30 mmol) and malononitrile (5.7 mL, 90 mmol), then add 45 mL of anhydrous ethanol to dissolve, then add piperidine (300 μL) to the system, and stir the reaction at 80 ° C under reflux for 6 h.

[0018] Preferably, the synthesis method of compound Cou in S2 is as follows:

[0019] 7-Hydroxycoumarin (3.3 g, 20 mmol) and hexamethylenetetramine (HMTA) (5.6 g, 40 mmol) were weighed separately using an analytical balance and placed in a 100 mL eggplant-shaped flask. 20 mL of trifluoroacetic acid (TFA) was added to completely dissolve the mixture. The reaction system was heated to 80° C. and refluxed for 8 h. The mixture was allowed to cool naturally. 20 mL of dilute hydrochloric acid (2 mol / L) and 60 mL of water were then added to the flask to quench the mixture. The mixture was extracted with ethyl acetate three times. The organic phase was adjusted to neutral with a saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate three times, dried, and concentrated in vacuo to remove the solvent. The mixture was separated and purified by flash column chromatography (V PE :V EA =1:1), to obtain 2.5 g of white solid,

[0020] Preferably, the synthesis method of the compound DCI-Cou-OH in S3 is as follows:

[0021] In a 50 mL eggplant-shaped flask, compound DCI (1.14 g, 6 mmol) was added and dissolved in 20 mL of anhydrous ethanol. Cou (1.08 g, 5.7 mmol) was then accurately weighed and added to the reaction flask. 1.5 mL of piperidine was then added to the stirring reaction system. The reaction temperature was raised to 80°C, and 1.5 mL of acetic acid was added. The mixture was refluxed and stirred for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation and separated by flash column chromatography to obtain 0.96 g of an orange-yellow solid.

[0022] Preferably, the synthesis method of the S4 probe DCI-Cou-Cys is as follows:

[0023] Use an analytical balance to accurately weigh the compound DCI-Cou-OH (0.18 g, 0.5 mmol) and dissolve it in DCM (10 mL). Add acryloyl chloride (135 μL, 1.5 mmol) at 0 ° C, then add triethylamine (75 μL, 0.5 mmol), and react at room temperature for 12 hours. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, pour it into a separatory funnel, remove the lower organic phase. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, then pour it into a separatory funnel, remove the lower organic phase, and repeatedly wash the crude product with saturated brine. Then use anhydrous sodium sulfate to dry the sample, then place it in a vacuum drying oven overnight. After drying overnight, separate and purify it by column chromatography (pure DCM) to successfully obtain 0.1 g of a yellow solid.

[0024] Preferably, the CCK-8 method is used to evaluate cell viability at different drug concentrations, and the cell viability of HeLa cells, HepG2 cells and A549 cells under incubation with 0, 1, 5, 10, 15 and 20 μmol / L of the probe is tested by a microplate reader. At a probe concentration of 20 μmol / L, the survival rate of all cells is still over 85%.

[0025] The beneficial effects of the present invention are as follows:

[0026] (1) The present invention designed and synthesized a cysteine ​​near-infrared fluorescent probe DCI-Cou-Cys based on coumarin-dicyanoisophorone as a fluorophore. The probe DCI-Cou-Cys undergoes a Michael addition reaction with the added Cys, followed by intramolecular cyclization to remove the seven-membered ring, exposing the hydroxyl group, allowing the probe to recover fluorescence at 735 nm. The fluorescence intensity reaches stability within 290 s, with a Stokes shift of up to 210 nm and a minimum detection limit of 23 nmol / L.

[0027] (2) The probe of the present invention has low cytotoxicity and can quickly, in real time, and in situ reflect changes in intracellular Cys content. Subsequently, LPS was used to induce oxidative stress in cells and zebrafish and treated with different antioxidant drugs. During this process, the probe can detect changes in thiol content and evaluate the therapeutic effect of the drug, which has potential application value in the biological field. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] The above and other aspects of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0030] Figure 1 : Synthesis route of probe DCI-Cou-Cys;

[0031] Figure 2 : HRMS spectrum of the probe DCI-Cou-Cys after reaction with 0.5 equivalents of Cys;

[0032] Figure 3 :Diagram of the recognition mechanism of probe DCI-Cou-Cys and Cys;

[0033] Figure 4 :Compound DCI 1 HNMR (400 MHz) spectrum;

[0034] Figure 5 :Compound DCI 13 CNMR (400MHz) images;

[0035] Figure 6 : Compound Cou 1 HNMR (400 MHz) spectrum;

[0036] Figure 7 : Compound Cou 13 CNMR (400MHz) images;

[0037] Figure 8 :Compound DCI-Cou-OH 1 HNMR (400 MHz) spectrum;

[0038] Figure 9 :Compound DCI-Cou-OH 13CNMR (400MHz) images;

[0039] Figure 10 : Probe DCI-Cou-Cys 1 HNMR (400 MHz) spectrum;

[0040] Figure 11 : Probe DCI-Cou-Cys 13 CNMR (400MHz) images;

[0041] Figure 12 : Mass spectrum of compound DCI-Cou-OH;

[0042] Figure 13 : Mass spectrum of compound DCI-Cou-Cys;

[0043] Figure 14 :(A) Normalized images of UV absorption and fluorescence spectra of probe DCI-Cou-Cys after reaction with Cys;(B) Fluorescence changes of probe DCI-Cou-Cys before and after reaction with Cys under different pH test conditions;(C) UV titration spectra of probe DCI-Cou-Cys for determination of Cys concentration changes (0-26μmol / L);(D) Fluorescence titration spectra of probe DCI-Cou-Cys for determination of different Cys concentrations (0-22μmol / L);(E) Linear relationship between fluorescence intensity of probe DCI-Cou-Cys and Cys concentration changes (0-14μmol / L);(F) Kinetic spectrum of reaction between probe DCI-Cou-Cys and Cys;(G,H) Fluorescence response spectra of probe DCI-Cou-Cys and different analytes;

[0044] Figure 15 : 24h toxicity test of different concentrations of probe DCI-Cou-Cys on cells;

[0045] Figure 16 :(A) Fluorescence imaging of HepG2 cells with different concentrations of probe DCI-Cou-Cys;(B) Fluorescence intensity graph corresponding to the imaging of each group of cells in Figure A;

[0046] Figure 17 :(A) Real-time imaging of the probe DCI-Cou-Cys in HepG2 cells for 22 consecutive minutes;(B) Fluorescence intensity graph corresponding to the imaging of each group of cells in Figure A;

[0047] Figure 18 :(A) Imaging of the probe DCI-Cou-Cys in HepG2 cells after treatment with different drugs;(B) Fluorescence intensity graph corresponding to the imaging of each group of cells in Figure A;

[0048] Figure 19 :(A)(C) Imaging of the probe in HepG2 cells after treatment with different drugs;(B)(D) Corresponding fluorescence intensity images of cells in groups A and C;

[0049] Figure 20 :(A) Real-time imaging of the probe DCI-Cou-Cys in zebrafish for 33 consecutive minutes;(B) Fluorescence intensity graph corresponding to the imaging of each group of zebrafish in Figure A;

[0050] Figure 21 :(A) Imaging of the probe DCI-Cou-Cys in zebrafish after treatment with different drugs;(B) Fluorescence intensity graph corresponding to the imaging of each group in Figure A;

[0051] Figure 22 :(A)(C) Imaging of the probe DCI-Cou-Cys in zebrafish after treatment with different drugs;(B)(D) Corresponding fluorescence intensity images of each group in A and C; DETAILED DESCRIPTION

[0052] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0053] Example 1: Synthesis of fluorescent probe:

[0054] A method for constructing a coumarin-based cysteine ​​fluorescent probe includes a compound having the following molecular formula:

[0055]

[0056] The steps of the method for constructing the compound are as follows:

[0057] S1: Compounds isophorone and malononitrile are dissolved in anhydrous ethanol, and piperidine is added to the system and heated to react to produce DCI;

[0058] S2: 7-Hydroxycoumarin and hexamethylenetetramine (HMTA) are heated in trifluoroacetic acid (TFA) to generate Cou;

[0059] S3: Dissolve the compounds DCI and Cou in anhydrous ethanol, add piperidine to the stirring reaction system, raise the reaction temperature to 80°C, add acetic acid, and then reflux with stirring to produce DCI-Cou-OH;

[0060] S4: Use an analytical balance to accurately weigh the compound DCI-Cou-OH and dissolve it in DCM. Add acryloyl chloride at 0°C, then add triethylamine, and react at room temperature for 12 hours to generate DCI-Cou-Cys.

[0061] The preparation reaction formula of the above DCI-Cou-Cys is as follows:

[0062]

[0063] Synthesis of compound DCI: In a 50 mL flask, isophorone (4.5 mL, 30 mmol) and malononitrile (5.7 mL, 90 mmol) were added, and then 45 mL of anhydrous ethanol was added to dissolve them. Piperidine (300 μL) was then added to the system and stirred at 80°C for 6 h. After the reaction was completed, the system was left to cool at room temperature, the solvent was removed by vacuum concentration, and the product was purified by column chromatography (V PE :V EA =10:1), and finally 4.4 g of white solid was obtained successfully with a yield of 77%. 1 H NMR (400MHz, Chloroform-d) δ6.61 (q, J = 1.5Hz, 1H), 2.51 (s, 2H), 2.17 (s, 2H), 2.04–2.00 (m, 3H), 1.00 (s, 6H). 13 C NMR (101MHz, Chloroform-d) δ170.55,159.94,120.69,113.33,112.54,45.78,42.74,32.50,27.94,25.46.

[0064] Synthesis of compound Cou: 7-hydroxycoumarin (3.3 g, 20 mmol) and hexamethylenetetramine (HMTA) (5.6 g, 40 mmol) were weighed separately using an analytical balance and placed in a 100 mL eggplant-shaped flask. 20 mL of trifluoroacetic acid (TFA) was added to completely dissolve the mixture. The reaction system was heated to 80° C. and refluxed for 8 h. The mixture was allowed to cool naturally. 20 mL of dilute hydrochloric acid (2 mol / L) and 60 mL of water were then added to the flask to quench the mixture. The mixture was extracted three times with ethyl acetate. The organic phase was adjusted to neutral with a saturated aqueous sodium bicarbonate solution, extracted three times with ethyl acetate, dried, and concentrated in vacuo to remove the solvent. The mixture was separated and purified by flash column chromatography (V PE :V EA =1:1), and finally 2.5 g of white solid was obtained successfully with a yield of 66%. 1 H NMR (400MHz, Chloroform-d) δ12.21(s,1H),10.59(s,1H),7.66(d,J=9.6Hz,1H),7.60(d,J=8.8Hz,1H),6.89(s,1H),6.34(s,1H). 13C NMR (101MHz, Chloroform-d) δ193.10,165.63,159.30,156.85,143.56,136.18,114.85,113.54,110.99,108.78.

[0065] Synthesis of compound DCI-Cou-OH: In a 50 mL eggplant-shaped flask, compound DCI (1.14 g, 6 mmol) was added and dissolved in 20 mL of anhydrous ethanol. Cou (1.08 g, 5.7 mmol) was then accurately weighed and added to the reaction flask. 1.5 mL of piperidine was then added to the stirring reaction system. The reaction temperature was raised to 80°C, 1.5 mL of acetic acid was added, and the mixture was refluxed and stirred for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation and separated by flash column chromatography to obtain 0.96 g of an orange-yellow solid with a yield of 47%. 1 H NMR (400MHz, DMSO-d6) δ11.48(s,1H),7.97(d,J=9.5Hz,1H),7.68(d,J=16.4Hz,1H),7.54(d,J=8.6Hz,1H),7.45(d ,J=16.4Hz,1H),6.96(d,J=8.5Hz,1H),6.71(s,1H),6.30(d,J=9.4Hz,1H),2.62(s,2H),2.56(s,2H),1.06(s,6H). 13 C NMR (101MHz, DMSO-d6) δ160.90,160.38,156.61,153.91,145.53,133.97,130.70,127.01,113.52,110.61,77.15,42.85,38.30,32.22,27.92.

[0066] Synthesis of the probe DCI-Cou-Cys: Use an analytical balance to accurately weigh the compound DCI-Cou-OH (0.18 g, 0.5 mmol) and dissolve it in DCM (10 mL). Add acryloyl chloride (135 μL, 1.5 mmol) at 0°C, then add triethylamine (75 μL, 0.5 mmol), and react at room temperature for 12 hours. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, pour it into a separatory funnel, and remove the lower organic phase. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, then pour it into a separatory funnel, remove the lower organic phase, and repeatedly wash the crude product with saturated brine. Then, dry the sample with anhydrous sodium sulfate and place it in a vacuum drying oven overnight. After drying overnight, separate and purify it by column chromatography (pure DCM), and finally successfully obtain 0.1 g of a yellow solid with a yield of 56%. 1H NMR(400MHz,Chloroform-d)δ7.76(d,J=9.6Hz,1H),7.51(d,J=8.5Hz,1H),7.38–7.23(m,2H),7.16(d,J=7. 5Hz,1H),6.87–6.67(m,2H),6.55–6.34(m,2H),6.22(d,J=12.0Hz,1H),2.64(d,J=5.3Hz,4H),1.11(s,6H). 13 CNMR(101MHz,Chloroform-d)δ169.14,163.56,159.60,153.58,152.76,151.11,143.35,136.26,134.58,12 8.33,127.00,124.98,124.37,119.80,118.11,117.19,116.28,113.22,112.40,43.13,38.79,32.08,28.04.

[0067] Example 2: Study on Stokes shift before and after reaction of probe DCI-Cou-Cys with Cys:

[0068] The Stokes shift of the probe DCI-Cou-Cys before and after the addition of Cys was tested by UV and fluorescence spectrophotometer. Figure 14 As shown in (A), it can be seen from the figure that after the reaction, the Stokes shift between the probes DCI-Cou-Cys and DCI-Cou-OH is 210 nm, which is an ideal bioimaging material.

[0069] Example 3: Study on the effect of probe DCI-Cou-Cys on Cys detection under different pH conditions:

[0070] Under different conditions, we studied whether the change of pH affects the fluorescence of the probe itself, and observed the pH range in which the probe DCI-Cou-Cys can recognize Cys. Figure 14 As shown in (B), the probe DCI-Cou-Cys exhibits low fluorescence intensity and a stable baseline profile in a PBS buffer system with a pH range of 3.0-8.0, demonstrating good chemical stability within this pH range. After the addition of Cys, fluorescence enhancement was observed at all pH levels. The fluorescence intensity increased with increasing pH, reaching a near-maximum value at pH 7.4. Based on the fluorescence intensity curves and the requirements for in vivo imaging applications, pH 7.4 was ultimately selected as the optimal physiological mimic.

[0071] Example 4: UV titration spectrum study of Cys using probe DCI-Cou-Cys:

[0072] In a 2 mL test system, the changes in the UV absorption spectrum of the probe DCI-Cou-Cys before and after the addition of Cys were measured by UV spectrophotometer. Figure 14 As shown in (C), when Cys (0-26 μmol / L) was gradually added to the test system, the original UV absorption peak of the probe at 385 nm gradually attenuated, while the absorption peak at 525 nm gradually increased, indicating that new products were generated during the reaction.

[0073] Example 5: Fluorescence titration spectrum study of probe DCI-Cou-Cys on Cys:

[0074] The change in fluorescence intensity of the system caused by the addition of Cys to the probe DCI-Cou-Cys (15 μmol / L) was tested by fluorescence spectrophotometry. Figure 14 As shown in (D), it can be seen that as the Cys concentration (0-22μmol / L) gradually increases, the fluorescence intensity also continues to rise. When the Cys concentration reaches 22μmol / L, the fluorescence intensity no longer changes. According to the calculation formula of the detection limit, the minimum detection limit LOD of the probe DCI-Cou-Cys is calculated to be 23nmol / L. Then, a linear fit is performed with the Cys concentration as the horizontal axis and the fluorescence intensity as the vertical axis, as shown in the figure below. Figure 14 As shown in (E), within the range of 0-14 μmol / L, there is a strong linear relationship y=18639x+86411, and a good linear correlation (R 2 =0.9942), DCI-Cou-Cys can sensitively recognize Cys and detect trace amounts of Cys.

[0075] Example 6: Kinetic analysis of probe DCI-Cou-Cys:

[0076] The response speed of the probe to Cys was evaluated by fluorescence spectrometer. Figure 14 As shown in Figure 5 (F), before the addition of Cys, the fluorescence intensity of the DCI-Cou-Cys probe was weak and exhibited strong stability and minimal fluctuation within 1800 s. After the addition of an excess of Cys, the fluorescence signal of the test system increased rapidly, reaching a maximum within 290 s, and remained essentially unchanged for 30 minutes. This indicates that the DCI-Cou-Cys probe can rapidly and sensitively recognize Cys, and both the probe itself and after recognition have good photostability.

[0077] Example 7: Selective evaluation of the probe DCI-Cou-Cys for Cys recognition:

[0078] In order to evaluate the ability of probe DCI-Cou-Cys to selectively recognize Cys, the fluorescence changes of probe DCI-Cou-Cys after adding different analytes were tested by fluorescence spectrophotometer. Figure 14 As shown in (G) and (H), when different analytes are added to the test system, the fluorescence intensity of the test system changes slightly. However, when Cys is added, the fluorescence intensity rises rapidly, and the emission wavelength is 735 nm. These experimental results demonstrate that the probe can specifically recognize Cys.

[0079] Example 8: Cytotoxicity test of probe DCI-Cou-Cys:

[0080] In order to study the toxicity of the probe in vivo, a series of cytotoxicity tests on different cell lines were conducted. The CCK-8 method was used to evaluate the cell viability at different drug concentrations. The following microplate reader was used to test the cell viability of HeLa cells, HepG2 cells and A549 cells under 0, 1, 5, 10, 15 and 20 μmol / L probe incubation. The experimental results showed that under the condition of 20 μmol / L probe concentration, the survival rate of all cells was still more than 85% (such as Figure 15 Therefore, the subsequent experiments selected HepG2 cells with a higher survival rate for testing.

[0081] Example 9: Imaging analysis of different concentrations of probe DCI-Cou-Cys in cells:

[0082] In order to explore the sensitivity of the probe DCI-Cou-Cys in detecting thiols in cells, four different concentrations of the probe were set up to incubate HepG2 cells. The experimental results are shown in Figure 2. Figure 16 As shown, it can be seen that when no probe is added, almost no fluorescence is detected. When different concentrations of the probe are added, the fluorescence intensity gradually increases, and when the probe is 1 μmol / L, fluorescence can be observed, which proves that the probe can sensitively detect the thiol content in the cell.

[0083] Example 10: Evaluation of the photostability of probe DCI-Cou-Cys in cells:

[0084] The long-term imaging of the probe in HepG2 cells was investigated. Figure 17As shown in the figure, the fluorescence intensity within the cell gradually increases from 0 to 10 minutes and reaches equilibrium at 12 minutes. The fluorescence of the probe remains nearly constant over the next 22 minutes of imaging. Therefore, this probe can rapidly detect changes in intracellular Cys content, and its fluorescence remains stable within the cell.

[0085] Example 11: Imaging of endogenous and exogenous Cys using the probe DCI-Cou-Cys:

[0086] We investigated whether the probe DCI-Cou-Cys can detect both endogenous thiols and exogenous thiols. Figure 18 As shown, the control group showed a significant increase in fluorescence after the probe was added, while the fluorescence decreased when a thiol scavenger (NEM) was added. In the third group, fluorescence increased significantly when Cys was exogenously added. In the fourth group, fluorescence also increased significantly when DTT was added to stimulate cells to produce endogenous Cys. This demonstrates that the DCI-Cou-Cys probe can detect both endogenously produced and exogenous thiols.

[0087] Example 12: Detection and analysis of Cys in oxidative stress cells using probe DCI-Cou-Cys:

[0088] Lipopolysaccharide (LPS) can cause oxidative stress in cells. To determine the concentration of LPS that induces oxidative stress, HepG2 cells were incubated with different concentrations of LPS (1.0, 1.5, and 2.0 μg / mL) and then imaged after incubation with the same concentration of probe. The results are shown in Figure 2. Figure 19 As shown in Figure A, it can be seen that when the concentration of lipopolysaccharide increases, the fluorescence intensity of the cells gradually decreases, and at a concentration of 2.0μg / mL, almost no fluorescence is seen. Therefore, a concentration of 2.0μg / mL is used as the concentration to induce oxidative stress in cells. Afterwards, the effects of different antioxidant drugs on the treatment of oxidative stress were explored, and the results are shown in Figure 4. Figure 19 As shown in Figure 2B, it can be seen that they all have certain therapeutic effects, and NAC has the most obvious effect.

[0089] Example 13: Evaluation of photostability of probe DCI-Cou-Cys in zebrafish:

[0090] In order to explore whether the probe DCI-Cou-Cys can be used for tracking imaging of living organisms, a set of zebrafish photostability imaging experiments were designed. Figure 20(A) In the initial 0-12 minutes, the fluorescence intensity of the zebrafish body can respond quickly to the Cys molecules in the zebrafish body, and gradually increases. After a 33-minute long-term scan, the fluorescence intensity in the zebrafish body remains almost unchanged. Figure 20 , B). This experiment shows that the probe can detect Cys in zebrafish and has good stability.

[0091] Example 14: Imaging of endogenous and exogenous Cys in zebrafish using the probe DCI-Cou-Cys:

[0092] We investigated whether the probe DCI-Cou-Cys could detect both endogenous Cys and exogenous Cys in zebrafish. Figure 21 As shown, it can be seen that the control group had a significant fluorescence enhancement after the probe was added. When a thiol scavenger (NEM) was added, the fluorescence weakened. After adding different concentrations of Cys (20μmol / L) and Cys (50μmol / L), the fluorescence increased to varying degrees, which proves that the probe DCI-Cou-Cys can detect both endogenous Cys produced in zebrafish and exogenous Cys.

[0093] Example 15: Detection and analysis of Cys in oxidative stress zebrafish using probe DCI-Cou-Cys:

[0094] Lipopolysaccharide (LPS) can induce oxidative stress in zebrafish. In order to determine the modeling time of 2.0μg / mL LPS, zebrafish were incubated with 2.0μg / mL LPS and imaged at 1, 3, 6, and 12 hours, respectively. The control group was incubated with only the probe. The experimental results are shown in Figure 22, A. It can be seen that as the LPS incubation time increases, the fluorescence intensity gradually decreases. After incubation for 12 hours, almost no obvious fluorescence can be seen, proving that incubation with 2.0μg / mL LPS for 12 hours can construct an oxidative stress model in zebrafish. Afterwards, the effects of different antioxidant drugs on the treatment of oxidative stress were explored, and the results are shown in Figure 22, A. Figure 22 As shown in Figure 2B, it can be seen that they all have certain therapeutic effects, and NAC has the most obvious effect.

[0095] Various modifications to the present disclosure will be apparent to those skilled in the art, and the general principles defined herein may be applied to other variations without departing from the scope of the present disclosure. Therefore, the present disclosure is not limited to the examples and designs described herein, but should be given the widest scope consistent with the principles and novel features disclosed herein. Although one or more exemplary embodiments of the present disclosure have been described with reference to the accompanying drawings, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as defined in the appended claims.

Claims

1. A method for constructing a coumarin-based cysteine ​​fluorescent probe, characterized in that: The final fluorescent probe includes a compound having the following molecular formula: The steps of the method for constructing the compound are as follows: S1: Compounds isophorone and malononitrile are dissolved in anhydrous ethanol, and piperidine is added to the system and heated to react to produce DCI; S2: 7-Hydroxycoumarin and hexamethylenetetramine (HMTA) are heated in trifluoroacetic acid (TFA) to react to form compound Cou; S3: Dissolve the compounds DCI and Cou in anhydrous ethanol with stirring, then add piperidine to the stirring reaction system, raise the reaction temperature to 80°C, add acetic acid, and then reflux with stirring to generate DCI-Cou-OH; S4: Use an analytical balance to accurately weigh the compound DCI-Cou-OH and dissolve it in DCM. Add acryloyl chloride at 0°C, then add triethylamine, and react at room temperature for 12 hours to generate DCI-Cou-Cys.

2. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The preparation reaction formula of the above DCI-Cou-Cys is as follows:

3. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The synthesis method of compound DCI in S1 is as follows: In a 50 mL flask, add isophorone (4.5 mL, 30 mmol) and malononitrile (5.7 mL, 90 mmol), then add 45 mL of anhydrous ethanol to dissolve, then add piperidine (300 μL) to the system, and stir the reaction at 80 ° C under reflux for 6 h.

4. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The synthesis method of compound Cou in S2 is as follows: 7-Hydroxycoumarin (3.3 g, 20 mmol) and hexamethylenetetramine (HMTA) (5.6 g, 40 mmol) were weighed separately using an analytical balance and placed in a 100 mL eggplant-shaped flask. 20 mL of trifluoroacetic acid (TFA) was added to completely dissolve the mixture. The reaction system was heated to 80° C. and refluxed for 8 h. The mixture was allowed to cool naturally. 20 mL of dilute hydrochloric acid (2 mol / L) and 60 mL of water were then added to the flask to quench the mixture. The mixture was extracted with ethyl acetate three times. The organic phase was adjusted to neutral with a saturated aqueous sodium bicarbonate solution, extracted with ethyl acetate three times, dried, and concentrated in vacuo to remove the solvent. The mixture was separated and purified by flash column chromatography (V PE :V EA =1:1), to obtain 2.5 g of a white solid.

5. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The synthesis method of the compound DCI-Cou-OH in S3 is as follows: In a 50 mL eggplant-shaped flask, compound DCI (1.14 g, 6 mmol) was added and dissolved in 20 mL of anhydrous ethanol. Cou (1.08 g, 5.7 mmol) was then accurately weighed and added to the reaction flask. 1.5 mL of piperidine was added to the stirring reaction system. The reaction temperature was raised to 80°C, and 1.5 mL of acetic acid was added. The mixture was refluxed and stirred for 6 h. After the reaction was completed, the solvent was removed by rotary evaporation and separated by flash column chromatography to obtain 0.96 g of an orange-yellow solid.

6. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The synthesis method of the S4 probe DCI-Cou-Cys is as follows: Use an analytical balance to accurately weigh the compound DCI-Cou-OH (0.18 g, 0.5 mmol) and dissolve it in DCM (10 mL). Add acryloyl chloride (135 μL, 1.5 mmol) at 0 ° C, then add triethylamine (75 μL, 0.5 mmol), and react at room temperature for 12 hours. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, pour it into a separatory funnel, remove the lower organic phase. After the reaction is completed, add an appropriate amount of water to remove excess acryloyl chloride, then pour it into a separatory funnel, remove the lower organic phase, and repeatedly wash the crude product with saturated brine. Then use anhydrous sodium sulfate to dry the sample, then place it in a vacuum drying oven overnight. After drying overnight, separate and purify it by column chromatography (pure DCM) to successfully obtain 0.1 g of a yellow solid.

7. The method for constructing a coumarin-based cysteine ​​fluorescent probe according to claim 1, wherein: The CCK-8 method was used to evaluate cell viability at different drug concentrations. The cell viability of HeLa cells, HepG2 cells, and A549 cells incubated with 0, 1, 5, 10, 15, and 20 μmol / L probes was tested using a microplate reader. At a probe concentration of 20 μmol / L, the survival rate of all cells was still over 85%.

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