A coumarin-based lysosome-targeting probe for hypochlorous acid detection, preparation method and application thereof

Abstract

The application provides a coumarin lysosome-targeting probe for hypochlorous acid detection, a preparation method and application thereof. The probe is Cou-CN-Mor, the probe is prepared from 7-diethylamino-4-methylcoumarin and N-bromosuccinimide reaction to obtain compound 2, then reacts with cuprous cyanide to obtain compound 3, then reacts with 4-(4-morpholinyl)benzaldehyde to obtain compound 5, and then reacts with Lawesson's reagent to obtain the probe; the probe is a coumarin fluorescent probe with a large Stokes shift, can realize sensitive detection of HClO, has specific targeting of lysosomes, has low cytotoxicity, and through fluorescence imaging research on biological samples, the probe is successfully applied to detection of HClO in living cells, zebra fish and plant rhizome tissues, and exhibits good bioimaging performance.

Description

Technical Field

[0001] This invention belongs to the field of coumarin fluorescent probes, and in particular relates to a coumarin lysosomal targeting probe for the detection of hypochlorous acid, its preparation method and its application. Background Technology

[0002] Reactive oxygen species (ROS) play a crucial role in various physiological processes, including maintaining cell signal transduction, antimicrobial activity, and immune defense. Hypochlorous acid (HClO) is one such endogenously produced ROS. In biological systems, hypochlorous acid is catalyzed by myeloperoxidase (MPO) to react with hydrogen peroxide (H₂O₂) and chloride ions (Cl₂O₃). - Hypochlorous acid (HClO) is produced through a reaction. Normally, HClO exerts its antimicrobial protective effect due to its strong oxidizing properties; however, excessive production of HClO leads to oxidative stress, causing alterations in the structure and function of biomolecules, resulting in various negative consequences. These changes can lead to disease and tissue damage.

[0003] Currently, various techniques exist for detecting hypochlorous acid, including chemiluminescence, colorimetry, electrochemical methods, and spectrophotometry, all of which exhibit excellent sensitivity and selectivity. However, these methods cannot meet the need for non-invasive real-time imaging of hypochlorous acid in live samples. Over the past few decades, fluorescence analysis has been widely used in bioimaging due to its high sensitivity, high spatiotemporal resolution, ease of operation, and non-invasiveness. Furthermore, numerous fluorescent probes for hypochlorous acid detection have been developed. Using these probes, the production of hypochlorous acid in various biological processes has been visualized, such as the production of hypochlorous acid in the ankle joints of rheumatoid arthritis mice. However, these probes still have some drawbacks, such as interference from other reactive oxygen species (ROS) and poor stability, high detection limits, poor sensitivity, and long response times to hypochlorous acid. CN121873023A discloses a single-atom switching fluorescent probe, its preparation method, and its application, specifically disclosing a fluorescent probe with the following structure: ; The fluorescent probe with the above structure has a detection limit of 0.8224 μM for hypochlorous acid, which is relatively high. It has poor sensitivity and poor fluorescence stability. Furthermore, it has no targeting effect on lysosomes and cannot be used for the detection of exogenous HClO in organisms or environmental HClO. Summary of the Invention

[0004] To address the technical problems existing in the prior art, this invention provides a coumarin-based lysosomal targeting probe for hypochlorous acid detection, its preparation method, and its application, achieving the following objectives: low detection limit for hypochlorous acid, short response time, good fluorescence stability after response, achieving targeting effect on intracellular lysosomes, and achieving effective detection of organelles, exogenous HClO, and environmental HClO.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A coumarin-based fluorescent probe for sensitive detection of HClO has the following molecular structure:

[0006] A method for preparing a coumarin-based lysosomal targeting probe for sensitive HClO detection, comprising the following steps: a first reaction, a second reaction, a third reaction, and a fourth reaction; The method of the first reaction is as follows: 7-diethylamino-4-methylcoumarin (compound 1) and N-bromosuccinimide (NBS) are reacted at room temperature in a solvent environment to obtain compound 2; Compound 2 has the following molecular structural formula: ; The secondary reaction method is as follows: in a solvent environment, cuprous cyanide is contacted with compound 2, and the reaction is carried out by heating to obtain compound 3. Compound 3 has the following molecular structural formula: ; The method for the three reactions is as follows: in a solvent environment, under the protection of an inert gas, in the presence of piperidine, compound 3 and compound 4 are contacted and reacted by heating to obtain compound 5; Compound 4 has the following molecular structural formula: ; Compound 5 has the following molecular structural formula: ; The four-reaction method is as follows: in a solvent environment, compound 5 is contacted with Lawson's reagent and the reaction is carried out at a higher temperature to obtain a coumarin-based fluorescent probe (Cou-CN-Mor) for sensitive hypochlorous acid detection. Preferably, in the primary reaction, the molar ratio of 7-diethylamino-4-methylcoumarin to N-bromosuccinimide (NBS) is 1:1.3-1.7.

[0007] Preferably, in the primary reaction, the reaction is carried out at room temperature for 46-50 hours.

[0008] Preferably, in the secondary reaction, the molar ratio of compound 2 to cuprous cyanide is 1:1.1-1.3.

[0009] Preferably, in the secondary reaction, the reaction temperature is 183-187℃ and the time is 3.5-4.5h.

[0010] Preferably, in the three reactions, the molar ratio of compound 3 to compound 4 is 1:1.1-1.3.

[0011] Preferably, in the three reactions, the reaction temperature is 88-92℃ and the reaction time is 11-13h.

[0012] Preferably, in the four reactions, the molar ratio of compound 5 to Lawesson reagent is 1:2.8-3.

[0013] Preferably, in the four reactions, the reaction temperature is 108-112℃ and the holding time is 5.5-6.5h.

[0014] The aforementioned probe is used for lysosomal targeting in living cells and for detecting exogenous HClO at intracellular and zebrafish levels, as well as for detecting hypochlorous acid levels at plant root tips.

[0015] The synthetic route for the above probe is shown below:

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The coumarin fluorescent probe of the present invention has a large Stokes shift (>140nm) and achieves sensitive detection of HClO. The fluorescent probe utilizes the sulfur atom effect after modification of the coumarin parent ring to weaken the fluorescence. Through the strong oxidizing property of HClO, the synthesis of the “OFF-ON” type probe is achieved, which restores the fluorescence. In addition, a morpholine group is introduced at position 4 to achieve specific targeting of lysosomes.

[0017] (2) The probe of the present invention has a detection limit of 0.1178 μM for hypochlorous acid, a response time of <2 min, and a stable fluorescence intensity within 30 minutes. It also maintains high selectivity in the presence of pH 4-10 and various physiological ions / ROS.

[0018] (3) The fluorescent probe of the present invention has low cytotoxicity. Through fluorescence imaging studies of biological samples, the probe has been successfully applied to the detection of HClO in living cells, zebrafish and plant root and stem tissues, showing good bioimaging performance. Attached Figure Description

[0019] Figure 1 The fluorescent probe Cou-CN-Mor prepared in Example 1 1 H NMR spectrum; Figure 2 The fluorescent probe Cou-CN-Mor prepared in Example 1 13 C NMR spectrum; Figure 3 The HR-MS spectrum of the fluorescent probe Cou-CN-Mor prepared in Example 1; Figure 4 The image shows the HR-MS spectrum of the fluorescent probe Cou-CN-Mor prepared in Example 1 after its response. Figure 5 The UV absorption and fluorescence emission spectra of Cou-CN-Mor in different solvents, and the UV and fluorescence emission spectra of the probe before and after its response in PBS / ethanol solution; Where a represents the UV absorption spectrum in different solvents; b represents the fluorescence emission spectrum in different solvents; c represents the UV absorption spectrum of the probe before and after responding to HClO in PBS / ethanol solution; and d represents the fluorescence emission spectrum of the probe before and after responding to HClO in PBS / ethanol solution. Figure 6 The fluorescence titration spectra and linearity graphs of the fluorescent probe Cou-CN-Mor in response to HClO are shown; where a is the fluorescence titration spectrum at different HClO concentration gradients; and b is the fluorescence titration linearity graph at 637 nm wavelength. Figure 7 Figures show the interference resistance and pH stability tests of the fluorescent probe Cou-CN-Mor for HClO detection; Where a is the anti-interference test diagram (1-21 correspond to Cu respectively). 2+ SO4 2- Na + NO2 - HS - S 2- Fe 3+ Cys, Hcy, CO3 2- K + Ca 2+ Mg 2+ Hg 2+ Zn 2+ (N2H4·H2O, Arg, Trp, Ala, H2O2, Ser); b is the pH stability test graph; Figure 8 The kinetic stability fluorescence spectrum and fluorescence stability dot plot of the fluorescent probe Cou-CN-Mor in the HClO system are shown. Where a is the kinetic stability fluorescence spectrum; b is the fluorescence stability dot plot at 637 nm; Figure 9 This is a graph showing the HepG2 cytotoxicity assay of the fluorescent probe Cou-CN-Mor. Figure 10 This is an image showing the lysosomal colocalization of the fluorescent probe Cou-CN-Mor in HepG2 cells. Where a is the green channel imaging of the commercial lysosomal probe Lyso Tracker Green; b is the red channel imaging of Cou-CN-Mor; c is the bright field portion; d is the overlapping channel imaging; and e is the Pearson coefficient. Figure 11 The fluorescent probe Cou-CN-Mor is used to visualize the fluorescence of exogenous HClO in HepG2 cells, and the fluorescence intensity bar graph is shown. Where a is a fluorescence imaging diagram of the fluorescent probe Cou-CN-Mor for different concentrations of exogenous HClO in HepG2 cells; b is a bar chart corresponding to the fluorescence intensity shown in figure a. Figure 12 The fluorescent probe Cou-CN-Mor is used to image the exogenous HClO in zebrafish. Figure 13 This is a fluorescence image of the fluorescent probe Cou-CN-Mor on the root tip of tobacco containing HClO from the external environment. Detailed Implementation

[0020] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific embodiments are now described. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below in conjunction with some specific embodiments.

[0022] Example 1 A method for preparing a coumarin-based fluorescent probe Cou-CN-Mor with a large Stokes shift, the specific steps of which are as follows: 1. 7-Diethylamino-4-methylcoumarin (462.0 mg, 2.0 mmol) and N-bromosuccinimide (NBS) (534 mg, 3.0 mmol) were dissolved in anhydrous acetonitrile (5.0 mL). The mixture was stirred at room temperature for 48 h. The solvent was removed directly by rotary evaporation. The residual solid was purified by column chromatography to give compound 2 as a green solid powder (449.4 mg, yield 72.5%).

[0023] 2. Compound 2 (309.0 mg, 1.0 mmol) and cuprous cyanide (108.0 mg, 1.2 mmol) were dissolved in N-methylpyrrolidone (10 mL) and refluxed at 185 °C for 4 h. The mixture was then cooled to room temperature and diluted with ethyl acetate (100 mL). The pre-packing procedure was the same as in step 1. The residual solid was purified by column chromatography to obtain compound 3, which was a yellow-green solid powder (214.3 mg, yield 83.7%). 1 H NMR (400 MHz, DMSO- D 6) δ 7.63 (d, J = 9.2 Hz, 1H), 6.77(dd, J = 9.3, 2.6 Hz, 1H), 6.53 (d, J = 2.5 Hz, 1H), 3.45 (q, J = 7.1 Hz, 4H), 2.53(s, 3H), 1.09 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, DMSO- D 6) δ 162.83, 158.84,156.54, 153.45, 128.98, 116.21, 110.57, 107.57, 96.97, 92.14, 44.90, 18.01,12.85.

[0024] 3. Compound 3 (128.1 mg, 0.50 mmol) and 4-(4-morpholine)benzaldehyde (114.7 mg, 0.6 mmol) were added to a pressure-resistant tube, with ethanol (5 mL) as the solvent. Piperidine (100 μL) was then added dropwise. The tube was sealed, and the mixture was evacuated and purged with nitrogen three times. The mixture was then stirred at 90 °C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, diluted with ethyl acetate (100 mL), and washed with deionized water (3 × 100 mL). The pre-packing procedures were the same as in step 1. The mixture was purified by column chromatography to obtain compound 5 (144.6 mg, yield 67.4%). 1 H NMR (400 MHz, DMSO- D 6) δ 7.84 (d, J = 9.3 Hz, 1H),7.67 – 7.60 (m, 2H), 7.54 (d, J= 16.1 Hz, 1H), 7.38 – 7.30 (m, 1H), 7.01 –6.95 (m, 2H), 6.80 – 6.74 (m, 1H), 6.59 (dd, J = 12.9, 2.5 Hz, 1H), 3.71 (t, J =4.9 Hz, 4H), 3.46 (q, J = 7.1 Hz, 4H), 3.22 (dd, J = 5.8, 3.9 Hz, 4H), 1.14 –1.05 (m, 6H). 13 C NMR (101 MHz, DMSO- D 6) δ 159.83, 158.56, 156.67, 153.21,152.92, 143.13, 130.61, 130.21, 129.27,125.81, 117.42, 115.83, 114.83,114.71, 110.49, 106.65, 97.44, 86.55, 66.43, 47.82, 44.89, 12.90.

[0025] 4. Compound 5 (144.6 mg, 0.34 mmol) and Lawson's reagent (404.2 mg, 1.0 mmol) were dissolved in anhydrous toluene (5.0 mL). The mixture was stirred at 110 °C for 6 hours, then cooled to room temperature and diluted with ethyl acetate (100 mL). The organic phase was washed with deionized water (3 × 100 mL). The pre-column loading procedure was the same as in step 1. The residual solid was purified by column chromatography to obtain Cou-CN-Mor, a dark red solid powder (114.7 mg, yield 75.8%). 1 H NMR (400 MHz, DMSO- D 6) δ 7.90 (d, J = 9.4 Hz, 1H), 7.68 – 7.61 (m, 2H), 7.57 (d, J = 4.7 Hz, 1H), 7.30 (d, J = 16.2 Hz, 1H), 7.02 – 6.96 (m, 2H), 6.89 (dd, J = 9.4, 2.6 Hz,1H), 6.79 – 6.72 (m, 1H), 3.71 (dd, J = 6.2, 3.6 Hz, 4H), 3.50 (q,J = 7.1 Hz, 4H), 3.23 (q, J = 5.7 Hz, 4H), 1.13 (td, J = 7.1, 3.9 Hz, 6H). 13 C NMR (101 MHz, DMSO- D 6) δ 192.86, 158.93, 153.68, 153.07, 151.89, 144.47, 130.46, 129.55, 125.88, 117.91, 115.43, 114.77, 112.30, 108.50, 101.55, 97.02, 66.41, 60.28, 47.72, 45.12, 14.62, 12.92. Pre-response HRMS (ESI) Found: 446.1901 [M+H] + Molecular formula C 32 H 30 N4OS requires [M+H] + 446.19. Post-response HRMS (ESI) Found: 428.1659, consistent with the theoretical high-resolution mass spectrometry value of the Cou-CN-Mor compound. Figure 1-4 ).

[0026] Example 2 Measurement of UV absorption and fluorescence emission spectra of probe Cou-CN-Mor in different solvents, and UV and fluorescence emission spectra of probe before and after response in ethanol solution. First, dimethyl sulfoxide (DMSO) was selected as the solvent for the probe stock solution. The Cou-CN-Mor solid prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a probe stock solution with a concentration of 1 mM.

[0027] Take the probe stock solution of Cou-CN-Mor and add acetonitrile, dichloromethane, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, ethanol, methanol, phosphate buffered saline (PBS, pH 7.4, concentration 10 mM), and tetrahydrofuran to the cuvette, controlling the final detection solution volume to 2 mL, so that the final probe concentration is 10 μM.

[0028] For the probe Cou-CN-Mor, its absorption and emission spectra in different solvents were first analyzed. The absorption wavelength of Cou-CN-Mor fluctuated between 480 nm and 510 nm in different polar solvents. Figure 5a). Furthermore, the probe also exhibited significant changes in its fluorescence spectrum; with increasing solvent polarity, the emission of the probe Cou-CN-Mor showed a significant red shift ( Figure 5 (b) The emission wavelength redshifts by nearly 90 nm from the less polar Dio to the more polar DMSO, demonstrating that the probe has good polar sensitivity, which provides conditions for subsequent lysosomal targeting.

[0029] To verify the probe's response to HClO, solvents were screened. Through a series of experiments, the PBS / ethanol (1 / 1, volume ratio) system was finally selected as the solvent for subsequent spectral performance testing.

[0030] Take an appropriate amount of 10wt% sodium hypochlorite solution and prepare a 1mM sodium hypochlorite working solution with 0.1mol / L sodium hydroxide aqueous solution.

[0031] Add 1 mM probe stock solution and 1 mM sodium hypochlorite working solution to PBS / ethanol (1 / 1, v / v), resulting in a final detection solution volume of 2 mL. This prepares detection solutions with a final probe concentration of 10 μM and final sodium hypochlorite concentrations of 10 μM and 20 μM, respectively. After reacting for 2 min, perform UV absorption spectroscopy measurements. The results are shown below. Figure 5 c); The detection solution with a probe final concentration of 10 μM and a sodium hypochlorite final concentration of 40 μM was prepared according to the above method. After reacting for 2 min, the fluorescence spectrum was tested, and the results are shown on day 5.

[0032] like Figure 5 As shown in Figure c, after the probe responds to HClO, the UV absorption peak at 500 nm decreases, while the UV absorption peak at 400 nm increases, demonstrating that the UV absorption peak changes with the increase of the concentration of the detected substance. Similarly, as... Figure 5 As shown in d, the fluorescence spectra before and after the response show that the fluorescence intensity increases significantly with the addition of HClO, indicating that the probe can detect HClO in ethanol solvent.

[0033] Example 3 Determination of the fluorescence titration spectrum of the fluorescent probe Cou-CN-Mor in response to HClO Take an appropriate amount of 10wt% sodium hypochlorite solution and prepare a 1mM sodium hypochlorite working solution with 0.1mol / L sodium hydroxide aqueous solution; Add 20 μL of Cou-CN-Mor probe stock solution to a cuvette, then add a mixture of ethanol and PBS buffer (V:V=1:1) to the cuvette, followed by sodium hypochlorite working solution to prepare the detection solution. The total volume of the detection solution is 2 mL. The final concentration of the probe is 10 μM, and the final concentrations of sodium hypochlorite are 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM, respectively. Incubate for 10 min before testing. Figure 6 As shown, with the continuous increase of HClO concentration, the fluorescence of the probe Cou-CN-Mor at 637 nm gradually increases. The fluorescence intensity of Cou-CN-Mor at 637 nm shows a linear correlation with the sodium hypochlorite (NaClO) concentration (0-50 μM), and the linear regression equation is y = 85.5174 + 5.2234[ClO] - The correlation coefficient was 0.9939. Under these conditions, the detection limit of the probe was calculated to be 0.1178 μM based on 3δ / k. This demonstrates that the probe exhibits excellent sensitivity to HClO and can be used as a sensitive tool for detecting HClO.

[0034] Example 4 Interference resistance and pH stability tests of the fluorescent probe Cou-CN-Mor for HClO detection.

[0035] To investigate the anti-interference ability of Cou-CN-Mor against HClO, the effects of common interfering substances were tested.

[0036] Take 20 μL of the 1 mM probe stock solution prepared in Example 2 and add it to a mixed solution of ethanol (EtOH) and PBS buffer (V:V = 1:1). Then add 20 μL of the interfering standard solution containing different interfering ions (final concentration 10 μM) to prepare a final volume of 2 mL detection solution. After shaking well, perform fluorescence detection (λ) after 5 min. ex =470nm, λ em =630nm); then add an equivalent amount of NaClO as the interfering substance, incubate for 2 minutes, and then perform fluorescence detection (λ). ex =470nm, λ em (630nm) Establish a bar chart of fluorescence intensity versus each interfering ion.

[0037] Specific test results are as follows: Figure 7 As shown in Figure a, Cu was added to a PBS / ethanol (1:1) solution containing Cou-CN-Mor. 2+ SO4 2- Na + NO2 - HS - S2- Fe 3+ Cys, Hcy, CO3 2- K + Ca 2+ Mg 2+ Hg 2+ Zn 2+ After being treated with N2H4·H2O, Arg, Trp, Ala, H2O2, and Ser, the probe Cou-CN-Mor exhibits a slight fluorescence enhancement due to the influence of mercury ion desulfurization; otherwise, the change in fluorescence intensity is negligible. When sodium hypochlorite is added to a PBS / ethanol (1:1, volume ratio) solution containing Cou-CN-Mor and the interfering agent, a strong fluorescence response is observed. Therefore, the probe Cou-CN-Mor exhibits high selectivity for hypochlorous acid.

[0038] The fluorescence response of Cou-CN-Mor to hypochlorous acid under different pH conditions was further investigated. 20 μL of the 1 mM probe stock solution prepared in Example 2 was diluted to 2 mL with PBS / ethanol solutions (volume ratio 1:1) at different pH values ​​(4-10) to achieve a final probe concentration of 10 μM. Fluorescence intensity was then measured. Sodium hypochlorite working solution was then added to achieve a final sodium hypochlorite concentration of 50 μM. After incubation for 2 minutes, fluorescence intensity was measured again.

[0039] like Figure 7 As shown in b, when hypochlorous acid was not added to the PBS / ethanol (1:1, volume ratio) solution containing Cou-CN-Mor, the fluorescence intensity of the solution remained essentially unchanged under different pH conditions, indicating that the probe molecule can maintain good stability within the pH range of 4-10. After the addition of sodium hypochlorite, the fluorescence intensity significantly increased, and the change in fluorescence intensity within the pH range of 4-10 was small, indicating good stability. Therefore, within cells, Cou-CN-Mor detects hypochlorous acid with almost no interference.

[0040] Example 5 Kinetic stability test of fluorescent probe Cou-CN-Mor in HClO system: The 1 mM probe stock solution prepared in Example 2 was diluted with a mixed solution of ethanol (EtOH) and PBS buffer (V:V=1:1), and then the prepared 1 mM sodium hypochlorite working solution was added to obtain the detection solution. The final concentration of the probe in the detection solution was 10 μM, the final concentration of sodium hypochlorite was 40 μM, and the total volume of the detection solution was 2 mL.

[0041] After preparing the detection solution, the test was performed directly, with continuous fluorescence detection over 30 minutes. The PBS buffer concentration in the detection solution was 0.01 M, pH = 7.4. like Figure 8 As shown, the fluorescence intensity of the probe Cou-CN-Mor rapidly reached its maximum within 2 minutes after the addition of 40 μM sodium hypochlorite; and after reaching its maximum value, the fluorescence intensity of Cou-CN-Mor remained relatively stable for the next 30 minutes. These experimental phenomena indicate that Cou-CN-Mor is more sensitive to low concentrations of hypochlorous acid, exhibiting a larger fluorescence gradient and a faster response speed, making it suitable for the detection of HClO in cells, organisms, and the environment.

[0042] Example 6 HepG2 cytotoxicity assay of the fluorescent probe Cou-CN-Mor.

[0043] HepG2 cells were placed in a CO2 incubator and maintained at a constant temperature of 37°C with a constant CO2 concentration. The MTT assay was used to test the toxicity of the probe: cells were seeded in 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 Cell viability was determined by incubating the cells at 0, 5, 10, 15, and 20 μM probe concentrations per well for 24 hours, followed by adding 10 μL of MTT (5 mg / mL) to each well and incubating for another 4 hours. After incubation, the culture medium was removed, and 100 μL of DMSO was added. The absorbance at 570 nm was measured using a microplate reader. The absorbance of probe Cou-CN-Mor was compared with that of the control group to determine the cell viability.

[0044] Different concentrations of probes were cultured with HepG2 cells for 24 hours, and cell viability was observed. The experimental results are as follows: Figure 9 As shown, the probe Cou-CN-Mor exhibits low biotoxicity and a cell survival rate exceeding 85%.

[0045] Example 7 Lysosomal colocalization imaging assay of fluorescent probe Cou-CN-Mor in HepG2 cells HepG2 cells were cultured in Dulbecco modified Eagle medium (DMEM, Hyclone) containing 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin, Hyclone) for 3 days at a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL, 1mL of density 1×10 5 / mL of cell culture medium was seeded into a glass-bottomed culture dish and placed in an incubator to allow the cells to adhere to the wall. When the adhered cells exceeded 70% of the bottom of the culture dish, the following steps were taken: Probe set: Wash cells three times with PBS, then incubate at 37°C, 5% CO2 and 95% air for 30 min with Cou-CN-Mor (final concentration 10 μM) and Lyso Tracker Green (final concentration 10 μM).

[0046] In the fluorescence imaging experiment of this embodiment, the green channel has the following parameters: excitation wavelength = 405 nm, acquisition wavelength = 580-660 nm, and scale bar = 25 μm.

[0047] like Figure 10 As shown, the red channel containing the probe Cou-CN-Mor exhibits red sheet-like fluorescence that overlaps well with the green sheet-like fluorescence emitted by the commercial green lysosomal probe Lyso Tracker Green. The Pearson correlation coefficient between the fluorescence signals of the two probes is 0.93. This demonstrates that the probe Cou-CN-Mor has excellent lysosomal targeting efficacy.

[0048] Example 8 Fluorescence imaging of exogenous HClO in HepG2 cells by fluorescent probe Cou-CN-Mor. The cell culture method before washing with PBS was the same as in Example 7.

[0049] Probe control group: Wash cells three times with PBS, and then incubate with Cou-CN-Mor (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min.

[0050] Exogenous HClO stimulation group: cells were washed three times with PBS, then incubated with Cou-CN-Mor (10 μM) at 37°C, 5% carbon dioxide and 95% air for 30 min, and then incubated with NaClO (20, 40 or 60 μM) under the same conditions for 30 min.

[0051] In the fluorescence imaging experiment of this embodiment, the red channel has an excitation wavelength of 405 nm and an acquisition wavelength of 580-660 nm. The scale bar is 25 μm.

[0052] Cou-CN-Mor was incubated with HepG2 cells and different concentrations of hypochlorous acid for 30 minutes, respectively, followed by fluorescence imaging. Figure 11 As shown, the red channel exhibits a weak signal without the addition of sodium hypochlorite. With increasing HClO concentration, the red channel shows a significant gradient fluorescence enhancement. This demonstrates that Cou-CN-Mor possesses excellent detection capabilities for exogenous hypochlorous acid in living cells.

[0053] Example 9 Fluorescence imaging of exogenous HClO in zebrafish using the fluorescent probe Cou-CN-Mor.

[0054] Probe control group: Zebrafish and probe Cou-CN-Mor (10 μM) were added together in PBS buffer and incubated for 30 minutes. The images were then captured using a laser confocal microscope at an excitation wavelength of 405 nm and an emission wavelength of 580-660 nm.

[0055] Exogenous HClO stimulation group: Zebrafish and probe Cou-CN-Mor (10 μM) were added together in PBS buffer and incubated for 30 minutes, and then incubated with NaClO (20, 40 or 60 μM) under the same conditions for 30 minutes.

[0056] In the fluorescence imaging experiment of this embodiment, the red channel has an excitation wavelength of 405 nm and an acquisition wavelength of 580-660 nm. The scale bar is 25 μm.

[0057] like Figure 12 As shown, after co-culturing the probe Cou-CN-Mor with zebrafish for 30 minutes, confocal imaging revealed that the probe exhibited weak red fluorescence. However, with the gradual increase of the added NaClO concentration (20 µM, 40 µM, and 60 µM), the fluorescence intensity within the zebrafish during imaging continuously increased with the increase of HClO concentration. The results indicate that the probe Cou-CN-Mor can sensitively detect exogenous HClO in zebrafish.

[0058] Example 10 Fluorescence imaging of HClO from the external environment at the root tip of tobacco by the fluorescent probe Cou-CN-Mor.

[0059] Probe control group: Tobacco seedlings were first incubated in culture medium for three days, the root tips were removed and washed with distilled water, and then incubated with Cou-CN-Mor (10 μM) for 30 min.

[0060] Exogenous HClO stimulation group: Tobacco seedlings were first incubated in culture medium for three days, the root tips were removed and washed with distilled water, and then incubated with Cou-CN-Mor (10 μM) for 30 min. Then, different concentration gradients of NaClO solution (20, 40 or 60 μM) were added and incubated for 30 min.

[0061] Confocal imaging revealed that the fluorescence intensity in the red channel gradually increased with increasing HClO concentration. Figure 13 The imaging results show that Cou-CN-Mor can successfully detect HClO at the root tip of tobacco, enabling the detection of HClO in the environment.

[0062] Unless otherwise stated, all percentages used in this invention are mass percentages.

[0063] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A coumarin-based lysosomal targeting probe for the detection of hypochlorous acid, characterized in that, The structural formula of the probe is as follows: 。 2. The method for preparing the probe according to claim 1, characterized in that: The probe is prepared by reacting compound 3 with 4-(4-morpholine)benzaldehyde to obtain compound 5, and then reacting compound 5 with Lawson's reagent to obtain the probe. The structural formula of compound 3 is as follows: ； The structural formula of compound 5 is as follows: 。 3. The preparation method according to claim 2, characterized in that: Compound 3 was reacted with 4-(4-morpholine)benzaldehyde at a molar ratio of 1:1.1-1.3 and a reaction temperature of 88-92℃ for 11-13 h to obtain compound 5.

4. The preparation method according to claim 2, characterized in that: The probe was prepared by reacting compound 5 and Lawson's reagent at a molar ratio of 1:2.8-3 and a reaction temperature of 108-112℃ for 5.5-6.5 h.

5. The preparation method according to claim 2, characterized in that: Compound 3 was prepared by reacting compound 2 with cuprous cyanide. The structural formula of compound 2 is as follows: 。 6. The preparation method according to claim 5, characterized in that: Compound 3 was prepared by reacting compound 2 with cuprous cyanide at a molar ratio of 1:1.1-1.3 and a reaction temperature of 183-187℃ for 3.5-4.5 h.

7. The preparation method according to claim 6, characterized in that: Compound 2 was prepared by reacting 7-diethylamino-4-methylcoumarin with N-bromosuccinimide.

8. The preparation method according to claim 7, characterized in that: Compound 2 was prepared by reacting 7-diethylamino-4-methylcoumarin and N-bromosuccinimide at a molar ratio of 1:1.3-1.7 at room temperature for 46-50 h.

9. The application of the probe according to claim 1 in the detection of exogenous hypochlorous acid in cells, in vivo exogenous hypochlorous acid, and in the environment.

Images