Fluorescent probe for detecting mitochondrial autophagy as well as preparation method and application of fluorescent probe

Abstract

The invention relates to a preparation method and application of a micromolecular fluorescent probe Mcp5 for detecting mitochondrial autophagy. According to the mitochondrial autophagy fluorescent probe Mcp5, the limitation of dye single-band emission is broken through through a hybridization design thought, and the mitochondrial autophagy fluorescent probe Mcp5 has excellent responsiveness to pH, namely, the mitochondrial autophagy fluorescent probe Mcp5 shows green fluorescence under an alkaline condition (such as mitochondria) and shows red fluorescence under an acidic condition (such as lysosome). Meanwhile, the probe has excellent mitochondrial targeting ability and is suitable for visual detection of the whole mitochondrial autophagy process. The probe Mcp5 can specifically track the mitochondrial autophagy process through the change of the ratio of dual-band emitted (F500 / F600) fluorescence, and can detect mitochondrial autophagy in a Parkinson's disease cell model and brain glioma cells. In addition, the probe can realize two-photon excitation, so that the penetration depth of the probe is improved, and mitochondrial autophagy in brain tissues of Parkinson's disease drosophila is successfully detected.

Description

TECHNICAL FIELD

[0001] The present application relates to a small-molecule fluorescent probe for detecting mitochondrial autophagy, a preparation method and application thereof, and belongs to the field of molecular probes. BACKGROUND

[0002] Mitochondria are known as "energy factories" and play a crucial role in cell metabolism and physiological functions. To maintain the balance of healthy mitochondria and eliminate damaged or aged mitochondria, cells have evolved a mitochondrial quality control system, of which the most important system is "mitochondrial autophagy". Functionally abnormal mitochondria first form autophagosomes and then fuse with lysosomes for degradation, which is a selective autophagy process. Mitochondrial autophagy plays a key role in regulating and maintaining cell functions, including immune signaling, aging and survival. It is related to cancer and central nervous system diseases, such as Parkinson's disease (PD) and many other diseases.

[0003] At present, more and more evidence shows that mitochondrial dysfunction is a prominent phenomenon in the pathogenesis of PD, and the impairment of mitochondrial autophagy function may further promote the occurrence and development of PD. Due to the important role of mitochondrial autophagy in physiological and pathological processes, people have developed various detection methods for analyzing the process of mitochondrial autophagy. Visualization of the process of mitochondrial autophagy can be achieved by electron microscopy (EM) or fluorescence microscopy (FM), and quantification of mitochondrial degradation can be achieved by detecting the level of mitochondrial marker proteins. However, the shortcomings of these detections limit their application to some extent. For example, the method of using FM to check the colocalization of mitochondrial and autophagosome-related proteins requires gene fusion of fluorescent proteins or cell fixation for immunofluorescence imaging. Therefore, complex transfection steps are needed to establish the fusion protein; and the fixed cells have died and cannot monitor the process of mitochondrial autophagy in living cells.

[0004] Small molecule fluorescent probes have been attracting much attention from researchers due to their easy use and real-time tracking. Lee et al. reported a pH-sensitive fluorescent probe based on a fluorescent switch signal unit to detect mitochondrial autophagy; Iwashita et al. synthesized a dye named Mtphagy dye using 3,4-dimethyl imine and triphenylphosphine to visualize mitochondrial autophagy; Li et al. used cyano-3-linked cucurbituril and cyano 5-linked adamantine amine (AdA-Cy5) to visualize the fusion of mitochondria and lysosomes. However, these probes can only show the late stage of autophagy (autophagosome has been formed), and cannot detect early mitochondria, so they cannot track the entire autophagy process; the current probes also have some unavoidable shortcomings such as poor light stability and anti-interference, and long-term real-time monitoring of mitochondrial autophagy has not been achieved, which limits the application of the probes in the study of mitochondrial autophagy-related disease models. SUMMARY

[0005] In order to solve the problems existing in the current detection of mitochondrial autophagy, the purpose of the present application is to provide a fluorescent probe capable of long-term real-time monitoring of the process of mitochondrial autophagy, which can be used to detect the process of mitochondrial autophagy in PD cells, Drosophila models and brain glioma cells.

[0006] The probe can specifically target mitochondria and accurately locate on mitochondria, thereby helping to real-time monitor the entire process of mitochondrial autophagy. The probe has good stability under different pH conditions and is not interfered by other molecules in the cell; and has good emission performance under long-time (up to 36h) laser excitation, which helps to achieve long-time monitoring of mitochondrial autophagy. The probe can be used for in vivo monitoring and real-time monitoring. Since the probe has small molecular weight, it is easy to enter cells and tissues in vivo. In addition, we also conducted a cytotoxicity experiment of the fluorescent probe molecule, and the results showed that the fluorescent probe molecule has small toxicity to cells.

[0007] A small molecule fluorescent probe Mcp5 for specifically monitoring mitochondrial autophagy has a general formula (I): The compound Mcp5 shown in the formula.

[0008] Advantages of the present application

[0009] The two-photon fluorescent probe designed and synthesized in the present application has the characteristics of strong biological safety, good selectivity and rapid response, and compared with the existing fluorescent probes for detecting the process of mitochondrial autophagy, the advantages of the present application include the following points:

[0010] 1. High specificity: the probe can specifically target mitochondria and accurately locate on mitochondria, thereby helping to real-time monitor the entire process of mitochondrial autophagy.

[0011] 2. High stability: The probe has good stability under different pH conditions, is not interfered by other molecules in the cell, and has good emission performance under long-time (up to 36h) laser excitation, which helps to realize long-time monitoring of mitochondrial autophagy.

[0012] 3. Real-time monitoring: The probe can be used for in vivo monitoring and real-time monitoring.

[0013] 4. High biocompatibility: Since the probe has small molecular weight, it is easy to enter cells and tissues in vivo. In addition, we also conducted a cytotoxicity experiment of the fluorescent probe molecule, and the results show that the fluorescent probe molecule has small toxicity to cells.

[0014] 5, The mitochondrial autophagy fluorescent probe Mcp5 of the application, the probe breaks through the limitation of single waveband emission of dye through hybrid design idea, has excellent response ability to pH: presents green fluorescence under alkaline condition (such as mitochondria), and presents red fluorescence under acidic condition (such as lysosome). Meanwhile, the probe has excellent mitochondrial targeting ability, and is suitable for visual detection of whole process of mitochondrial autophagy. The probe Mcp5 can specifically track the process of mitochondrial autophagy through the change of the ratio of double waveband emission (F500 / F600) fluorescence, can detect mitochondrial autophagy in a Parkinson's disease cell model and a brain glioma cell, and can realize two-photon excitation, thereby improving the penetration depth, and successfully detects mitochondrial autophagy in brain tissues of a Parkinson's disease fruit fly. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a synthesis route map of the two-photon fluorescent probe Mcp5;

[0016] Figure 2 It is a nuclear magnetic hydrogen spectrum (a), carbon spectrum (b) and high-resolution mass spectrum (c) of Mcp5;

[0017] Figure 3 It is a detection mechanism diagram of the fluorescent probe Mcp5;

[0018] Figure 4 It is an absorption (a) and emission spectrum analysis (b) of Mcp5 under different pH conditions;

[0019] Figure 5 It is a fluorescence ratio change diagram of Mcp5 under alternating acid-base conditions of pH 3 and pH 8;

[0020] Figure 6 It is an anti-interference test of Mcp5: (a) 500nm fluorescence emission diagram; (b) 600nm fluorescence emission diagram;

[0021] Figure 7 It is a two-photon fluorescence spectrum of Mcp5

[0022] Figure 8 Cytotoxicity test for Mcp5;

[0023] Figure 9 Test chart for intracellular mitochondrial targeting of Mcp5;

[0024] Figure 10 Chart for real-time monitoring of mitophagy by Mcp5 in HeLa-GFP cells (a) and quantification of the fluorescence ratio of F600 nm and F500 nm of Mcp5 (b);

[0025] Figure 11 Chart for real-time monitoring of mitophagy by Mcp5 in wild-type and Parkinson's disease cell models (a) and quantification of the fluorescence ratio of F600 nm and F500 nm of Mcp5 (b);

[0026] Figure 12 Chart for real-time monitoring of mitophagy by Mcp5 in glioma cell models (a) and quantification of the fluorescence ratio of F600 nm and F500 nm of Mcp5 (b);

[0027] Figure 13 Chart for monitoring of mitophagy by Mcp5 in brain tissue of Parkinson's disease fruit fly models; DETAILED DESCRIPTION

[0028] The following detailed description of the application, specifically with reference to the drawings attached to the specification of the application, is intended to provide a more clear and complete description of the embodiments of the application, and does not limit the content thereof. The embodiments described in the application are only a part, not all of the embodiments, which are within the scope of protection of the application.

[0029] Example 1 Method for chemical synthesis of Mcp5

[0030]

[0031] Step 1: 6-nitronaphthalene-2,3-dicarboxylic acid (compound 2)

[0032] Compound 1 was dissolved in 100 mL of N,N-dimethylformamide and stirred. Maleic anhydride (7.6 g, 77.5 mmol) and potassium iodide (27.72 g, 167 mmol) were added and the reaction was stirred and heated to 65 °C overnight. 350 mL of pure water was added and sodium bisulfite (5 g) was stirred in. The solution was cooled and a yellow solid appeared. The solid was filtered and dissolved in 1 N sodium hydroxide solution. The pH was adjusted to precipitate a solid using concentrated sulfuric acid and the solid was filtered. Column chromatography using ethyl acetate yielded Compound 2 as a brown solid in 78% yield. 1 H NMR (300 MHz, DMSO-d6): δ 9.04 (s, 1H), 8.61 (s, 1H), 8.40 (s, 1H), 8.25 (s, 2H).

[0033] Step 2: 6-Aminonaphthalene-2,3-dicarboxylic acid dimethyl ester (Compound 3)

[0034] Compound 2 was dissolved in 35 mL of methanol and stirred. A 3 M tin chloride solution in hydrochloric acid was added dropwise using a syringe. The reaction was refluxed overnight, cooled and 120 mL of a 10% aqueous sodium carbonate solution was added. A yellow-brown solid precipitated and column chromatography yielded Compound 3 as a brown solid in 65% yield. 1 H NMR (300 MHz, CDCl3): δ 8.15 (s, 1H), 7.81 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H), 6.91 (S, 1H), 3.90 (dd, J1= 3.15 Hz, J2= 1.5 Hz, 6H).

[0035] Step 3: 6-(Dimethylamino)naphthalene-2,3-dicarboxylic acid dimethyl ester (Compound 4)

[0036] Compound 3 (0.55 g, 2.1 mmol) was dissolved in 10 mL of methanol and 0.6 mL of a 37% formaldehyde solution and 2 mL of acetic acid were added. Sodium cyanoborohydride (0.6 g, 9.5 mmol) was dissolved in methanol and added dropwise to the flask in an ice bath while stirring. The reaction was allowed to continue at room temperature for 4.5 h. The reaction was stopped by adding water and a precipitate appeared. The solid was filtered and column chromatography yielded Compound 4 as a brown solid in 82% yield. 1H NMR (300 MHz, CDC13): δ 8.18 (s, 1H), 7.89 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 6.6 Hz, 1H), 6.86 (s, 1H), 3.93 (d, J = 6.6 Hz, 6H), 3.08 (s, 6H).

[0037] Step 4: 6-(dimethylamino)naphthalene-2,3-dicarboxylic acid (Compound 5)

[0038] Compound 4 (373 mg, 1.3 mmol) was dissolved in a mixed solution of methanol and sodium hydroxide solution, and the reaction was heated to 130 °C for 3 h. The reaction was stopped by adding water, and the pH was adjusted to 6 with hydrochloric acid. The product was extracted with 30 mL of ethyl acetate three times, and the organic phase was dried with anhydrous sodium sulfate and purified by column chromatography to obtain Compound 5 as a red-brown solid in a yield of 73%. 1 H NMR (300 MHz, DMSO-d6): δ 8.22 (s, 1H), 7.96 (s, 1H), 7.89 (d, J = 6.9 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.05 (s, 1H), 3.05 (s, 6H).

[0039] Step 5: 6-(dimethylamino)naphtho[2,3-c]furan-1,3-dione (Compound 6)

[0040] Compound 5 (100 mg, 0.38 mmol) was stirred and dissolved in 2 mL of acetic anhydride, and the reaction was heated to 140 °C for 4 h. Then, it was cooled and filtered, washed with methanol, and purified by column chromatography to obtain Compound 6 as a yellow solid in a yield of 90%. 1 H NMR (300 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.29 (s, 1H), 8.08 (d, J = 9 Hz, 1H), 7.48 (d, J = 9 Hz, 1H), 7.24 (s, 1H), 3.12 (s, 6H).

[0041] Step 6: N-(9-(3-carboxy-6-(dimethylamino)naphthalen-2-yl)-6-(dimethylamino)-3H-anthracen-3-ylidene)-N-methylmethanaminium (Compound Mcp5)

[0042] Compound 6 (200 mg, 0.83 mmol) and 3-dimethylamino-phenol (228 mg, 1.66 mmol) were dissolved in a mixed solution of toluene (5 mL) and nitrobenzene (5 mL). The reaction was reacted at 180 °C for 36 h. Purification by column chromatography resulted in white solids of Mcp4 and Mcp5 in a yield of 8%. Finally, Mcp5 was obtained after column purification with DCM:MeOH = 10:1.

[0043] 1 H NMR (400 MHz, CDC13): δ 8.527 (s, 1H), 7.913 (d, J = 9.2 Hz, 1H), 7.355 (s, 1H), 7.224 (s, 1H), 6.924 (d, 8 Hz, 1H), 6.726 (s, 1H), 6.573 (s, 2H), 6.320 (d, 9.2 Hz, 1H), 3.073 (s, 6H), 3.068 (s, 12H). 13 C NMR (100 MHz, CDC13): δ 169.14, 154.79, 154.04, 150.51, 137.85, 131.68, 130.85, 130.40, 128.88, 127.82, 126.08, 125.07, 123.25, 122.70, 119.07, 117.23, 110.95, 110.81, 107.19, 105.31, 97.75, 40.67. HRMS: Calculated: [M+H] + = 480.2282, found: 480.22789. (Mcp4)

[0044] 1 H NMR (400 MHz, CDC13): δ 8.433 (s, 1H), 7.647 (d, J = 9.2 Hz, 1H), 7.458 (s, 1H), 7.284 (s, 1H), 7.100 (s, 1H), 6.860 (d, J = 9.2 Hz, 2H), 6.553 (s, 2H), 6.484 (d, J = 8.8 Hz, 2H), 3.113 (s, 6H), 3.050 (s, 12H). 13 C NMR (100 MHz, CDC13): δ 169.01, 155.40, 154.57, 149.56, 135.16, 130.71, 129.05, 128.46, 128.27, 127.51, 126.20, 119.08, 111.69, 111.33, 106.96, 97.50, 40.68. HRMS: Calculated: [M+H] + = 480.2282, found: 480.22789. (Mcp5)

[0045] Example 2 Characterization of Mcp5 and detection mechanism

[0046] The final structure of the Mcp5 fluorescent probe can be completely confirmed by the nuclear magnetic resonance hydrogen spectrum (a), nuclear magnetic resonance carbon spectrum (b) and high resolution mass spectrum (c) shown in the following figure. Figure 2 The final structure of the Mcp5 fluorescent probe can be completely confirmed by the nuclear magnetic resonance hydrogen spectrum (a), nuclear magnetic resonance carbon spectrum (b) and high resolution mass spectrum (c) shown in the following figure.

[0047] As Figure 3 shown, the fluorescent probe molecule of Mcp5 in the present application presents green fluorescence in mitochondria (basic condition); when the mitochondria is wrapped by autophagosome under damaged or other stress conditions and fused with lysosome, Mcp5 presents red fluorescence in lysosome (acidic condition), indicating the occurrence of mitochondrial autophagy.

[0048] Example 3 Absorption and emission fluorescence spectrum of Mcp5

[0049] UV-visible absorption spectrum was tested on UV-3600 UV-Vis-NIR and Synergy HTX microplate reader, the sample cell was a two-side light transmission quartz sample cell with a light transmission length of 1.0 cm and a 384-hole bottom transparent full-black hole plate, and the final concentration of the probe solution tested was 5 x 10 -6 M, fluorescence spectrum was determined on a HITACHI F4600 fluorescence instrument, a 150W Xe lamp was used as the light source, the sample cell was a four-side light transmission quartz cuvette with a light transmission length of 1.0 cm, and the solution concentration was 5 x 10 -6 M, the test solutions used were all 25 mM Hepes buffer solution, the quantum yield of the two compounds in different solvents was determined by reference method, and fluorescein was used as the reference. The two-photon fluorescence spectrum used a laser with a wavelength of 800 nm, a pulse width of 200 fs, a repetition frequency of 76 MHz and an energy of 1.26 W provided by a Tisapphire femtosecond laser (Coherent Mira 900F) as the light source. The specific experimental results are as follows:

[0050] (1) As Figure 4 (a) shown, the absorption spectrum properties of Mcp5 in different acid-base environments, Mcp5 has a weak absorption peak at 360 nm and a strong absorption peak at 550 nm; the absorption peaks of the probe under acidic and basic conditions do not change significantly. Based on the results of the absorption spectrum test, we tested the fluorescence spectrum of Mcp5 under different acid-base conditions using 360 nm as the excitation wavelength. The test results are as Figure 4 (b) shown, when 360 nm is used as the excitation light, two emission peaks of the probe at 500 nm and 600 nm are measured; the fluorescence intensity of the probe at 500 nm gradually weakens with the increase of the environmental acidity, and the fluorescence intensity at 600 nm gradually increases.

[0051] (2) The effect of acid-base alternation on the fluorescence intensity of the probe: We tested the effect of acid-base on the fluorescence shift and intensity of the probe at 500 nm and 600 nm by alternately changing the acid-base environment of the same sample probe. The results are shown in the figure. The results prove that the probe can well adapt to the changes of acid-base alternation and can stably respond to the changes of pH. Moreover, the fluorescence can recover to the initial fluorescence intensity each time. Figure 5 The effect of acid-base alternation on the fluorescence intensity of the probe: We tested the effect of acid-base on the fluorescence shift and intensity of the probe at 500 nm and 600 nm by alternately changing the acid-base environment of the same sample probe. The results are shown in the figure. The results prove that the probe can well adapt to the changes of acid-base alternation and can stably respond to the changes of pH. Moreover, the fluorescence can recover to the initial fluorescence intensity each time.

[0052] (3) Anti-interference ability test: As shown in Figure 6 , we selected two small molecules of oxidation and eight amino acids essential for the human body as interference factors for fluorescence intensity interference experiments. The concentration of Mcp5 was 5 μM, and the final concentration of the interference substance was 50 μM (1. Control; 2. Ba 2+ ; 3. Ca 2+ ; 4. Ni 2+ ; 5. Cu 2+ ; 6. Mg 2+ ; 7. Pd 2 + ; 8. Cr 3+ ; 9. Zn 2+ ; 10. Co 2+ ; 11. Fe 2+ ; 12. Fe 3+ ; 13. Na + ; 14. K + ; 15. F - ; 16. Cl - ; 17. SO4 2- ; 18. HPO4 2- ; 19. SO3 2 -; 20. CO3 2- ; 21. NO3 -; 22. Threonine; 23. Asparagine; 24. Cysteine; 25. Arginine; 26. Histidine; 27. Methionine; 28. Proline; 29. Phenylalanine; 30. Tryptophan; 31. Isoleucine; 32. Ornithine; 33. Serine; 34. Tyrosine; 35. Cystine; 36. Glycine; 37. Glutamic acid; 38. Leucine), the fluorescence intensity at 600 nm and 500 nm was taken respectively under the excitation of 360 nm, pH 4 and 8 buffer. The experimental results are shown in Figure 6 As shown in the figure, the probes have strong anti-interference ability and stability under acidic and alkaline conditions.

[0053] (4) Test and analysis of two-photon fluorescence spectrum: the excitation wavelength of two-photon fluorescence spectrum test is 800 nm, and the test results of the two probes are shown in Figure 7 As shown in the figure, there is only one fluorescence peak near 580 nm, and the two-photon fluorescence intensity is also stronger and stronger with the decrease of pH, which is the same as the single-photon fluorescence spectrum.

[0054] (5) Comparison of fluorescence properties of probes Mcp5 and Mcp4: we also tested Mcp4 by the same method, as shown in Table 1, the absorption and emission of the two probes are similar, but the fluorescence brightness of Mcp5 is much larger than that of Mcp4, and the performance is better, so we selected Mcp5 for subsequent test.

[0055] Table 1 Comparison of optical properties of Mcp4 and Mcp5

[0056]

[0057] Example 4 Test of intracellular mitochondrial targeting property of Mcp5

[0058] The HeLa cell line was incubated with high-sugar medium added with 10% FBS and 1% P / S. 5 μM mitochondrial autophagy probe Mcp5 was first added and incubated for 0.5 h, and then 100 nM mitochondrial tracer MitoTracker Red (MTR) was added and incubated for 0.5 h. The fluorescence of the cells was recorded by confocal, and the fluorescence map of the probe was overlapped with the map of MitoTracker Red. As shown in Figure 8 Mcp5 and MTR have good co-localization, and the image fitting coefficient (Pearsen coefficient) reaches 0.87, which indicates that Mcp5 can specifically target mitochondria.

[0059] Example 5 Test of cytotoxicity of Mcp5

[0060] The cytotoxicity of the probe Mcp5 was detected. Specifically, a standard MTT assay was used, HeLa cells were digested with 0.25% trypsin, then a cell suspension was prepared with cell culture medium and inoculated in a 96-well plate, which was incubated at 37°C, 5% carbon dioxide for 24 h, different concentrations (0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 25 μM) of the probe were added to the well plate and incubated for 24 h, MTT was added and incubated for 4 h, and finally triplex dissolving solution was added for 2 h, the absorbance value of each well was tested by an enzyme-labeled instrument, and control wells and zero wells were set. The results are shown in Figure 9 It can be seen that the survival rate of cells can reach 80% when the intracellular concentration of the fluorescent probe material reaches 25 μM, indicating that the cytotoxicity of the probe is very small.

[0061] Example 6 Mcp5 detects mitochondrial autophagy in cells

[0062] Parkin-overexpressing HeLa cells (Parkin-GFP) were induced for mitochondrial autophagy (a classical model of mitochondrial autophagy) with 10 μM CCCP, and the cells were monitored in real time by confocal microscopy. Incubation with 5 μM Mcp5 was performed for 0.5 h, followed by the addition of lysosome-specific near-infrared dye LysoTacker DeepRed (LTDR, 1 μM) for another 0.5 h. Then, the basic (F500 nm) and acidic (F600 nm) fluorescence channels and the LTDR fluorescence channel (F668 nm, 640 nm excitation) were recorded, respectively. Because the fluorescence of LTDR is also red, in order to distinguish the red fluorescence of the acidic channel of Mcp5, we set the fluorescence of the F500 nm channel to blue and the fluorescence of the F600 nm channel to green. As shown in Figure 10 a, with the extension of CCCP treatment time, Parkin first aggregated (Parkin-GFP showed a dotted pattern), and then gradually degraded (Parkin-GFP fluorescence disappeared); the fluorescence intensity of the F500 nm channel gradually weakened, and the fluorescence intensity of the F600 nm channel gradually increased, indicating that after Mcp5 targeted mitochondria, it gradually entered lysosomes and was eventually degraded. Finally, taking the ratio of the basic (F500 nm) and acidic (F600 nm) fluorescence intensities as the ordinate and the CCCP treatment time as the abscissa Figure 10 b), it can be clearly seen that with the increase of time, the F600 / F500 (Ratio channel) significantly increases, which indicates that when exposed to CCCP, the mitochondrial membrane potential is unbalanced, the damaged mitochondria are wrapped by the autophagic membrane, and then fuse with lysosomes for degradation.

[0063] Example 7 Mitophagy detection in Parkinson's disease cell model using Mcp5

[0064] SH-5YSY cell line was incubated with high glucose medium added with 10% FBS and 1% P / S. The SH-5YSY cell line was treated with 0.5 μΜ rotenone for 24 h to obtain a Parkinson's disease cell model (PD model). The untreated SH-5YSY cells (wild type) and the PD model SH-5YSY cells were incubated at 37 °C in a constant temperature incubator containing 5% carbon dioxide for 24 h, then 5 μΜ probe Mcp5 was added to the cells for 0.5 h, the cells were washed with PBS for three times, then 10 μΜ CCCP was added to induce mitophagy, the cells were monitored in real time by confocal microscope, and the fluorescence of F600 and F500 was recorded by confocal microscope and analyzed. As shown in Figure 11 a-11b, F600 / F500 increased with the increase of CCCP treatment time, and the wild type cells reached the maximum value of F600 / F500 at 12 h of CCCP treatment, indicating that the mitophagy in the cells was completely occurred; while the PD model cells reached the maximum value of F600 / F500 at 20 h of CCCP treatment, 8 h later than the wild type cells, indicating that the process of mitophagy in the PD model cells was blocked, thus the efficiency of mitophagy decreased.

[0065] Example 8 Mitophagy detection in brain glioma cell model using Mcp5

[0066] The brain glioma U87 cell line was incubated with high glucose medium added with 10% FBS, 1% P / S, 1% non-essential amino acids and sodium pyruvate. 5 μΜ probe Mcp5 was added to the cells for 0.5 h, the cells were washed with PBS for three times, then the cells were placed in a live cell workstation (37 °C, 5% CO2), 10 μΜ CCCP was added to induce autophagy, and the cells were monitored in real time by confocal microscope. As shown in Figure 12 a-12b, F600 / F500 increased with the increase of CCCP treatment time (CCCP treatment time is the horizontal coordinate), and reached the highest value at 28 h, indicating that the mitophagy in U87 cells was completely occurred. Cancer cells obtain energy from glycolysis with low efficiency, therefore more mitochondria need to be accumulated to maintain the energy supply of the cells, and this process actively inhibits the level of mitophagy, thus the efficiency of mitophagy is lower than that of SH-5YSY.

[0067] Example 9 Mitophagy detection in Drosophila brain tissue using Mcp5

[0068] Brain tissue from a Parkinson's disease model of fruit flies was utilized. Specifically, the brains of wild-type and Pakin-null fruit flies were incubated with 5 μM Mcp5 for 2 hours and imaged using two-photon fluorescence microscopy (using a Tisapphire femtosecond laser (Coherent Mira 900F) with a wavelength of 800 nm, a pulse width of 200 fs, a repetition rate of 76 MHz, and an energy of 1.26 W). Figure 13 As shown, no fluorescent signal was observed in the brains of fruit flies without Mcp5 incubation. Compared to the wild type, the brain tissue of fruit flies with Parkin knockout showed weak fluorescence, indicating a defect in mitophagy in the brain tissue of the Parkinson's disease model, consistent with reports of impaired mitophagy in Parkinson's disease models in the literature. In contrast, the fluorescent signal of fruit flies with Parkin knockout fed with nicotine was similar in intensity to that of the wild type, indicating that nicotine treatment induced mitophagy in the brain of the Parkinson's disease model.

[0069] The materials, methods, and embodiments described herein are exemplary and not restrictive. Those skilled in the art can appropriately substitute and / or modify process parameters based on the content of this document; however, it should be particularly noted that all such substitutions and / or modifications are obvious to those skilled in the art and do not depart from the spirit of the invention or exceed the scope defined by the appended claims. Therefore, they are all considered to be included in the present invention.

Claims

1. A fluorescent probe for detecting mitophagy, characterized by, The fluorescent probe compound Mcp5 has a structural formula as shown in formula (I):

2. The method of claim 1, wherein the method is characterized by, The preparation route is as follows:

3. The method of claim 2, wherein the method is characterized by, The method comprises the following steps: Step 1: 6-nitro naphthalene-2,3-dicarboxylic acid (compound 2) Dissolve p-nitro-o-xylene (5 g, 33.1 mmol) in dichloroethane, and then add N-bromosuccinimide (24 g, 135.7 mmol) and azobisisobutyronitrile (0.6 g, 3.65 mmol) under stirring; heat the reaction to 70 DEG C overnight; cool the reaction to room temperature, remove the solvent to obtain an oily liquid compound 1, dissolve the compound 1 in 100 mL of N,N-dimethylformamide, and then add maleic anhydride (7.6 g, 77.5 mmol) and potassium iodide (27.72 g, 167 mmol) under stirring; heat the reaction to 65 DEG C overnight, add 350 mL of pure water, and then add sodium bisulfite (5 g) under stirring; cool the solution to obtain a yellow solid, and then perform suction filtration; dissolve the solid in 1N sodium hydroxide solution, adjust the pH to a solid by using concentrated sulfuric acid, and then perform suction filtration; and purify the solid by column chromatography with ethyl acetate to obtain brown solid compound 2, and the yield is 78%. Step 2: 6-amino naphthalene-2,3-dicarboxylic acid dimethyl ester (compound 3) Dissolve the obtained compound 2 in 35 mL of methanol under stirring, and then add 3M tin chloride hydrochloride solution drop by drop through a syringe; reflux the reaction overnight, cool, and then add 120 mL of 10% sodium carbonate aqueous solution, and a yellow-brown solid is precipitated; purify the solid by column chromatography to obtain brown solid compound 3, and the yield is 65%. Step 3: 6-(dimethylamino) naphthalene-2,3-dicarboxylic acid dimethyl ester (compound 4) Dissolve compound 3 (0.55 g, 2.1 mmol) in 10 mL of methanol, and then add 0.6 mL of 37% formaldehyde solution and 2 mL of acetic acid; dissolve sodium cyanoborohydride (0.6 g, 9.5 mmol) in methanol, and then add the solution drop by drop into the flask in an ice bath under stirring; wait for the solution to return to room temperature, and then continue to react for 4.5 hours (4.5 h); stop the reaction by adding water, and then perform suction filtration; and purify the solid by column chromatography to obtain brown solid compound 4, and the yield is 82%. Step 4: 6-(dimethylamino) naphthalene-2,3-dicarboxylic acid (compound 5) Dissolve compound 4 (373 mg, 1.3 mmol) in a mixed solution of methanol and sodium hydroxide solution, heat the reaction to 130 DEG C for 3 h; stop the reaction by adding water, adjust the pH to 6 by using hydrochloric acid, extract the reaction solution with 30 mL of ethyl acetate for three times, dry the organic phase with anhydrous sodium sulfate, and then purify the organic phase by column chromatography to obtain red-brown solid compound 5, and the yield is 73%. Step 5: 6-(dimethylamino) naphtho[2,3-c]furan-1,3-dione (compound 6) Dissolve compound 5 (100 mg, 0.38 mmol) in 2 mL of acetic anhydride under stirring, heat the reaction to 140 DEG C for 4 h, then cool, perform suction filtration, wash the solid with methanol, and then purify the solid by column chromatography to obtain yellow solid compound 6, and the yield is 90%. Step 6: N-(9-(3-carboxy-6-(dimethylamino)naphthalen-2-yl)-6- (dimethylamino)-3H-anthracen-3-ylidene)-N-methylmethanaminium (Compound Mcp5) Compound 6 (200 mg, 0.83 mmol) and 3-dimethylamino-phenol (228 mg, 1.66 mmol) were dissolved in a mixture solution of toluene (5 mL) and nitrobenzene (5 ml). The reaction was reacted at 180 °C for 36 h; purified by column chromatography to give white solid Mcp4 and Mcp5, yield 8%.

4. The use of the mitochondrial autophagy fluorescent probe according to claim 1, characterized in that The fluorescent probe compound Mcp5 is used for preparing a fluorescent probe for detecting mitochondrial autophagy.

5. The use of the mitochondrial autophagy fluorescent probe according to claim 4, characterized in that The fluorescent probe compound Mcp5 is used for preparing a fluorescent probe for detecting mitochondrial autophagy process in cells.

6. The use of the mitochondrial autophagy fluorescent probe according to claim 4, characterized in that The fluorescent probe compound Mcp5 is used for preparing a fluorescent probe for detecting mitochondrial autophagy in brain diseases.

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