Ratio-type pH fluorescent probe for fixing mitochondria as well as preparation method and application thereof

Abstract

The invention discloses a mitochondrial fixed ratio type pH fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The mitochondria-fixed ratio-type pH fluorescent probe shows ratio-type fluorescent response to pH, can well eliminate interference of various factors such as probe concentration, test environment and optical path length, and is suitable for accurate quantitative determination of pH of complex biological samples. The polymer can be stably fixed in mitochondria and is not influenced by mitochondrial membrane potential. The mitochondrial pH tracer can be used for preparing a mitochondrial pH tracer reagent, can realize quantitative determination of mitochondrial pH change in cell models such as Parkinson's disease and the like, has good biocompatibility, and provides a powerful tool for mitochondrial function and disease related mechanism research. The preparation method disclosed by the invention has the advantages of easily available raw materials, simplicity and convenience in operation, low cost, rapidness and sensitivity, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a mitochondrial-fixed ratiometric pH fluorescent probe and its preparation method and application. Background Art

[0002] Mitochondria are the primary site of cellular aerobic respiration and are involved in processes such as cell differentiation, information transmission, apoptosis, and autophagy. The mitochondrial microenvironment, including membrane potential, polarity, pH, and viscosity, is crucial for its function. pH, in particular, plays a crucial role in the dynamic regulation of cellular behaviors such as migration, proliferation, differentiation, and apoptosis. Even small changes in pH can lead to mitochondrial dysfunction. Therefore, abnormal mitochondrial pH is closely associated with the development and progression of many diseases, such as neurodegenerative diseases, cardiovascular diseases, and tumors. Therefore, developing methods to monitor changes in mitochondrial pH is crucial for understanding mitochondrial function and elucidating the mechanisms underlying the development and progression of related diseases.

[0003] Fluorescent labeling techniques based on fluorescent probes are widely used to study changes in the mitochondrial microenvironment due to their high sensitivity, dynamics, and real-time properties. Because the mitochondrial matrix is ​​alkaline, most mitochondrial-targeting pH probes are based on cationic dyes. These probes accumulate in mitochondria due to the negative potential of the mitochondrial inner membrane. However, the mitochondrial membrane potential is dynamic. In many pathological processes, the inner membrane potential difference decreases or even disappears, causing these cationic dyes to escape from the mitochondrial matrix, a result that is extremely detrimental to studying changes in mitochondrial pH. Mitochondrial-immobilized probes, which are unaffected by membrane potential, circumvent this problem and better track mitochondrial pH changes. Furthermore, ratiometric fluorescent probes, which detect based on the ratio of fluorescence intensity at two wavelengths, can effectively eliminate interference from various factors such as probe concentration, test environment, and optical path length, making them more suitable for accurate pH measurement in biological samples. Therefore, there is an urgent need to develop ratiometric pH fluorescent probes that can be immobilized on mitochondria and are unaffected by membrane potential to quantitatively monitor changes in mitochondrial pH during pathological processes. This approach is expected to provide a new and convenient method for studying mitochondrial function and disease-related mechanisms. Summary of the Invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a mitochondrially fixed ratiometric pH fluorescent probe, its preparation method, and its application. This mitochondrially fixed ratiometric pH fluorescent probe can be stably fixed in mitochondria without being affected by mitochondrial membrane potential. It can be used to prepare a mitochondrial pH tracer reagent, enabling quantitative measurement of mitochondrial pH changes in cell models such as Parkinson's disease. It has good biocompatibility and provides a powerful tool for studying mitochondrial function and disease-related mechanisms.

[0006] In order to achieve these objects and other advantages according to the present invention, a mitochondrial-immobilized ratiometric pH fluorescent probe is provided, which has the structure of the following formula (I): (I) Wherein, R is a straight chain or branched chain alkyl group.

[0007] Preferably, R is a linear dodecyl group.

[0008] The object of the present invention can be further achieved by a method for preparing a mitochondrial-immobilized ratiometric pH fluorescent probe, which comprises: using a compound of formula (II) and an alkylamine as raw materials, dissolving them in an organic solvent, carrying out an amidation reaction under the action of an organic base and a condensing agent, and purifying the mixture by silica gel column chromatography to obtain a compound of formula (I); (II).

[0009] Preferably, the molar ratio of the compound of formula (II) to the alkylamine is 1:0.5-3.

[0010] Preferably, the organic base is N,N-diisopropylethylamine, and the molar ratio of the compound of formula (II) to the organic base is 1:1-5.

[0011] Preferably, the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the molar ratio of the compound of formula (II) to the condensing agent is 1:1-5.

[0012] Preferably, the organic solvent is dichloromethane, and the molar volume ratio of the compound of formula (II) to dichloromethane is 1:60-100 mmol / mL.

[0013] Preferably, the amidation reaction temperature is 20-50° C., and the reaction time is 2-24 h.

[0014] Preferably, the eluent used for silica gel column chromatography is dichloromethane and methanol in a volume ratio of 30 to 100:1.

[0015] The purpose of the present invention can be further achieved by using a mitochondrial-fixed ratiometric pH fluorescent probe in the preparation of a mitochondrial pH tracer for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0016] The present invention has at least the following beneficial effects: 1. The mitochondrial-immobilized ratiometric pH fluorescent probe of the present invention exhibits a ratiometric fluorescence response to pH, which can effectively eliminate interference from various factors such as probe concentration, test environment, and optical path length, and is suitable for accurate quantitative determination of pH in complex biological samples.

[0017] 2. The mitochondria-fixed ratiometric pH fluorescent probe of the present invention can be stably fixed in mitochondria without being affected by mitochondrial membrane potential.

[0018] 3. Compared with existing commercial mitochondrial dyes, the mitochondrial-fixed ratiometric pH fluorescent probe of the present invention has better photostability, providing a viable means for continuous observation and research of mitochondrial function; it has a near-infrared emission wavelength and has broad application prospects in mitochondrial tracing at the tissue and in vivo levels.

[0019] 4. Compared with existing commercial mitochondrial fluorescent dyes, the mitochondrial-fixed ratiometric pH fluorescent probe of the present invention can also monitor small changes in mitochondrial pH and can be used to prepare mitochondrial pH tracing reagents.

[0020] 5. The mitochondrial pH tracer reagent prepared by the present invention can realize the quantitative measurement of mitochondrial pH changes in cell models such as Parkinson's disease and Alzheimer's disease, providing a powerful tool for the study of mitochondrial function and disease-related mechanisms.

[0021] 6. The composition of the mitochondria-fixed ratiometric pH fluorescent probe of the present invention is determined, and it has good biocompatibility and meets the requirements of medication.

[0022] 7. The preparation method of the mitochondrial-fixed ratiometric pH fluorescent probe of the present invention has the advantages of readily available raw materials, simple operation, low cost, rapidity and sensitivity, and is easy to promote and apply.

[0023] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The mitochondrial fixed ratiometric pH fluorescent probe MIpH synthesized in Example 1 1 H NMR spectrum.

[0025] Figure 2The mitochondrial fixed ratiometric pH fluorescent probe MIpH synthesized in Example 1 13 C NMR spectrum.

[0026] Figure 3 This is a high-resolution mass spectrum of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH synthesized in Example 1.

[0027] Figure 4 Figure 2 shows the fluorescence spectra of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH in PBS at different pH values ​​(4, 5, 6, 6.5, 7, 7.4, 8, 9, and 10) in Example 2.

[0028] Figure 5 The fluorescence intensity ratio of the mitochondrial-fixed ratiometric pH fluorescent probe MIpH at 704 and 670 nm changes with pH in Example 2. The inset shows the relationship between pH and log(I max -I) / (II min ) linear relationship graph.

[0029] Figure 6 This is a confocal image of PC12 cells co-stained with the mitochondria-immobilized ratiometric pH fluorescent probe MIpH and the commercial dye Mitotracker green in Example 3 (scale bar: 10 μm).

[0030] Figure 7 Confocal imaging of PC12 cells treated with CCCP in Example 3 co-incubated with the mitochondria-immobilized ratiometric pH fluorescent probe MIpH and Mito tracker green (scale bar: 10 μm).

[0031] Figure 8 These are confocal images of PC12 cells co-stained with the mitochondria-immobilized ratiometric pH fluorescent probe MIpH and Mito tracker in Example 3 after laser irradiation for different times (scale: 20 μm).

[0032] Figure 9 Confocal images (A) and corresponding mitochondrial pH calibration curves (B) of PC12 cells stained with the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH in Example 4 after incubation in buffers with different pH values. Scale bar: 20 μm. The ratio in Figure B refers to the ratio of fluorescence intensity between the green and red channels in Figure A.

[0033] Figure 10 Confocal images of PC12 cells stained with the mitochondria-immobilized ratiometric pH fluorescent probe MIpH after rotenone induction for different times (A), changes in the ratio of fluorescence intensity between the green and red channels (B), and changes in mitochondrial pH (C) in Example 4. Scale bar: 20 μm. DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0035] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0036] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0037] Main equipment: UV-visible spectrophotometer (UH-5300, Hitachi), fluorescence spectrophotometer (F-7100, Hitachi), 400 MHz narrow-cavity liquid nuclear magnetic resonance spectrometer (AVANCE III, Bruker), laser confocal microscopy imager (FV3100, Olympus).

[0038] Main chemical reagents: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (AR, Shanghai Aladdin Biochemical Technology Co., Ltd.), N,N-diisopropylethylamine (DIPEA) (AR, Shanghai Aladdin Biochemical Technology Co., Ltd.), dodecylamine (AR, Anhui Zesheng Technology Co., Ltd.), dichloromethane (AR, Tianjin Damao Chemical Reagent Factory), methanol (AR, Tianjin Damao Chemical Reagent Factory), dimethyl sulfoxide (AR, Thermo Fisher Scientific), DMEM medium (Wuhan Pronose Life Science Technology Co., Ltd.), fetal bovine serum (Wuhan Pronose Life Science Technology Co., Ltd.), Mito tracker green (Shanghai Beyotime Biotechnology Co., Ltd.), carbonyl cyanide chlorophenylhydrazone (CCCP) (Beijing Solebao Technology Co., Ltd.), and rotenone (Beijing Solebao Technology Co., Ltd.).

[0039] Example 1 A mitochondrial-immobilized ratiometric pH fluorescent probe MIpH has the following structure: The specific synthetic route is as follows: The specific synthesis steps are as follows: HATU (69 mg, 0.18 mmol), DIPEA (32 μL, 0.18 mmol), dodecylamine (40 mg, 0.22 mmol), and near-infrared fluorescent dye 1 (80 mg, 0.18 mmol) were dissolved in 15 mL of dichloromethane and stirred at room temperature for 4 hours. The organic solvent was evaporated under reduced pressure, and the resulting residue was separated by silica gel column chromatography using dichloromethane / methanol (40:1, v / v) as the eluent to obtain the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH (51 mg, 46% yield) as a blue powder. 1 H NMR (400 MHz, 298 K, CDCl3) δ 8.55 (d, J = 13.2 Hz, 1H), 7.40 (s, 3H), 7.28 (s, 1H), 7.16 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 6.92 (s, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.64 (s,1H), 6.31 (s, 1H), 4.47 (s, 2H), 3.17 (s, 2H), 2.86 (s, 2H), 2.65 (s, 4H),1.88 (s, 2H), 1.73 (s, 6H), 1.43 (s, 2H), 1.25-1.19 (m,18H), 0.86 (t, J = 6.4Hz, 3H). 13 C NMR (101 MHz, 298 K, DMSO-d6) δ 177.0, 169.2, 161.8, 161.1, 154.2,144.4, 141.8, 141.4, 134.6, 129.3, 128.8, 126.6, 125.7, 122.6, 114.7, 114.4,113.9, 112.9, 104.2, 101.9,50.1, 41.8, 38.8, 38.2, 33.8, 31.3, 29.1, 29.0,28.9, 28.7, 28.4, 27.6, 26.4, 23.5, 22.1, 20.1, 14.0. High resolution mass spectrometry: C 40 H 53 N2O3 + , M + ; Calculated value: 609.4051; Measured value: 609.4059.

[0040] Among them, the synthesis reference of near-infrared fluorescent dye 1 is: Adv. Healthc. Mater., 2024, 13,2400593; Biosens. Bioelectron., 2016, 81, 395-400. Among them, MIpH 1 H NMR spectrum Figure 1 , 13 CNMR spectrum Figure 2 , high resolution mass spectrometry Figure 3 .

[0041] Example 2 Fluorescence response of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH to pH.

[0042] 6.1 mg of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH synthesized in Example 1 was weighed and dissolved in 10 mL of dimethyl sulfoxide to prepare a probe stock solution (concentration of 1 mM). 20 µL of the probe stock solution was diluted to 2 mL with phosphate buffer solutions (PBS) of different pH values, and the fluorescence emission spectrum was measured at room temperature with an excitation wavelength of 640 nm.

[0043] The results of the test are as follows Figure 4 and Figure 5 As shown, Figure 4 Shown are the fluorescence spectra of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH (10 μM) in PBS at different pH values ​​(4, 5, 6, 6.5, 7, 7.4, 8, 9, and 10). As can be seen, the fluorescence intensity of the mitochondria-immobilized ratiometric pH fluorescent probe MIpH at 670 nm gradually decreases with increasing pH, while the fluorescence intensity at 704 nm significantly increases. Figure 5 The ratio of the fluorescence intensity at 704 nm and 670 nm changes with pH. It can be seen that the change is most significant when the pH is between 6 and 8. The p value of the mitochondrial fixed ratiometric pH fluorescent probe MIpH is calculated. K a The pH value is 6.93, which is close to the pH of the normal mitochondrial microenvironment (7.8), indicating that the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH is suitable for monitoring small pH changes in mitochondria.

[0044] Example 3 Mitochondrial targeting and fixation ability of the mitochondrial-fixed ratiometric pH fluorescent probe MIpH.

[0045] 1) Rat adrenal medullary pheochromocytoma (PC12) cells were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 µg / mL streptomycin at 37°C and 5% CO2.

[0046] 2) PC12 cells in the logarithmic growth phase were seeded in a 24-well plate. After 24 hours of culture, the culture medium was discarded and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). The cells were then incubated with the mitochondria-immobilized ratiometric pH fluorescent probe MIpH (5 µM) synthesized in Example 1 for 30 minutes. The probe was discarded and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). The cells were then incubated with the commercial mitochondria-immobilized fluorescent dye Mitotracker Green (250 nM) for 30 minutes before confocal imaging. Mitotracker Green was imaged at an excitation wavelength of 488 nm and an emission channel of 500-540 nm. The mitochondria-immobilized ratiometric pH fluorescent probe MIpH was imaged at an excitation wavelength of 635 nm and an emission channel of 655-725 nm. To assess photostability, the cells were illuminated with laser light for 15 seconds at 1-minute intervals, followed by imaging, and repeated five times.

[0047] Figure 6 Shown are confocal images of PC12 cells using the mitochondria-immobilized ratiometric pH fluorescent probe MIpH synthesized in Example 1. The results show a high colocalization coefficient between the mitochondria-immobilized ratiometric pH fluorescent probe MIpH and the commercial dye MitotrackerGreen, demonstrating that the mitochondria-immobilized ratiometric pH fluorescent probe MIpH has good mitochondrial targeting capability.

[0048] Carbonyl cyanide chlorophenylhydrazone (CCCP) is an inhibitor of oxidative phosphorylation that increases the permeability of the mitochondrial membrane to protons, thereby disrupting the mitochondrial membrane potential and further affecting the normal physiological function of the mitochondria. To further verify that the mitochondrial-fixed ratiometric pH fluorescent probe MIpH can be fixed to mitochondria without being affected by the membrane potential, PC12 cells were first incubated with CCCP (20 μM) for 30 minutes (specific reference: Anal. Chem. , 2021, 93, 3241-3249) to damage mitochondrial function and induce a decrease in its membrane potential, and then incubated with the mitochondrial fixed ratiometric pH fluorescent probe MIpH for imaging. The results are shown in Figure 7 As shown. Figure 7It can be seen that in cells treated with CCCP, the mitochondrial-fixed ratiometric pH fluorescent probe MIpH still has a good colocalization coefficient with the commercial dye, indicating that the mitochondrial-fixed ratiometric pH fluorescent probe MIpH has good mitochondrial fixation ability and is not affected by mitochondrial membrane potential.

[0049] However, compared with the commercial mitochondrial fixed dye Mito tracker green (emission wavelength 516 nm), the emission wavelength of the mitochondrial fixed ratiometric pH fluorescent probe MIpH is greater than 650 nm, which is located in the near-infrared light region. The light in this wavelength band can effectively avoid the interference of biological background fluorescence and has stronger tissue penetration, making it suitable for tissue and in vivo imaging. In addition, Figure 8 The figure shows the photostability comparison of the mitochondria-fixed ratiometric pH fluorescent probe MIpH and Mito tracker green. It can be seen that after continuous laser irradiation, the fluorescence intensity of Mito tracker green decays sharply, while the mitochondria-fixed ratiometric pH fluorescent probe MIpH has better resistance to photobleaching. This indicates that the mitochondria-fixed ratiometric pH fluorescent probe MIpH has better photostability and is expected to provide a viable means for the continuous observation of mitochondrial function.

[0050] Example 4 The mitochondrial-immobilized ratiometric pH fluorescent probe MIpH was used as a mitochondrial pH tracer to monitor mitochondrial pH changes.

[0051] (1) Plotting a mitochondrial pH calibration curve: PC12 cells in the logarithmic growth phase were seeded in a 24-well plate. After culturing for 24 h, the culture medium was discarded and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). The cells were then incubated with the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH (5 µM) synthesized in Example 1 for 30 min. The probe was discarded and washed three times with PBS (pH = 7.4) (1 mL each time). The cells were then incubated with high potassium buffer (containing 10 µM nigericin) at pH values ​​of 4.0, 5.0, 6.0, 7.0, and 8.0 for 30 min, respectively. The imaging was performed with an excitation wavelength of 635 nm, a green channel emission wavelength collection range of 655-685 nm, and a red channel emission wavelength collection range of 695-725 nm. The ratio of the fluorescence intensity of the green channel to the red channel in the fluorescence images at each pH was calculated and plotted to obtain the mitochondrial pH calibration curve.

[0052] (2) Monitoring the changes in mitochondrial pH in the rotenone-induced PD model: PC12 cells in the logarithmic growth phase were seeded in a 24-well plate. After culturing for 24 h, the culture medium was discarded and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). They were then incubated with the mitochondrial-fixed ratiometric pH fluorescent probe MIpH (5 μM) synthesized in Example 1 for 30 min. The probe was discarded and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). The cells were cultured in a medium containing 1 μM rotenone to induce a Parkinson's disease cell model. The culture medium was removed after 0, 1, 2, and 3 h, and the cells were washed three times with PBS (pH = 7.4) (1 mL each time). The cells were then imaged under the same conditions as above. The ratio of the green channel fluorescence intensity to the red channel fluorescence intensity in the fluorescence images under different conditions was calculated, and the changes in the mitochondrial pH of PC12 cells during rotenone induction were quantitatively calculated based on the above intracellular pH calibration curve.

[0053] Figure 9 Figure A shows confocal images of PC12 cells stained with the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH after incubation in high potassium buffers with different pH values ​​containing nigericin. Nigericin can promote K + / H + The results showed that as the intracellular pH increased from 4 to 8, the fluorescence of the green channel of the cell gradually weakened while the fluorescence of the red channel gradually increased. Figure 9 Figure B shows that the ratio of the green to red channel fluorescence intensity exhibits a good linear relationship with pH. These results indicate that the mitochondrial-immobilized ratiometric pH fluorescent probe, MIpH, can be used as a ratiometric fluorescent probe to track changes in mitochondrial pH and is promising for the quantitative determination of mitochondrial pH in physiological and pathological processes.

[0054] Rotenone, a widely used insecticide and water reservoir cleaner, is a mitochondrial complex I inhibitor that can impair energy synthesis in substantia nigra dopaminergic neurons, leading to Parkinson's disease. Studies have shown that rotenone induces the production of large amounts of reactive oxygen species and disrupts mitochondrial membrane potential, but changes in mitochondrial pH during this process remain largely unexplored. To demonstrate the potential of the mitochondrial-immobilized ratiometric pH fluorescent probe, MIpH, for monitoring mitochondrial pH changes in Parkinson's disease, PC12 cells were co-incubated with rotenone (1 μM) to induce a Parkinson's disease cell model. Figure 10 A is a confocal image of PC12 cells stained with mitochondria-fixed ratiometric pH fluorescent probe MIpH after rotenone treatment for different time periods. It can be seen that with the extension of rotenone induction time, the green fluorescence of the cells increased and the red fluorescence decreased, indicating that the mitochondria of PC12 cells were acidified during rotenone treatment. The ratio of the fluorescence intensity of the green and red channels was calculated ( Figure 10Combined with the mitochondrial pH calibration curve established above, it can be calculated that the mitochondrial pH decreased from 7.87 to 7.41, 6.87, and 6.40 after 1, 2, and 3 h of rotenone induction, respectively ( Figure 10 These experimental results demonstrate that the mitochondrial-immobilized ratiometric pH fluorescent probe MIpH can quantitatively measure mitochondrial pH changes in Parkinson's disease, providing a powerful tool for monitoring mitochondrial pH during pathological processes and for studying mitochondrial function and disease mechanisms.

[0055] Although the technical solution of the present invention has been disclosed as above, it is not limited to the applications listed in the description and implementation methods. It can be fully applied to various fields suitable for the present invention. For those familiar with this field, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and implementation methods shown and described herein.

Claims

1. A mitochondrial-fixed ratiometric pH fluorescent probe, characterized in that: It has the structure of formula (I): （I） Wherein, R is a straight chain or branched chain alkyl group.

2. The mitochondria-immobilized ratiometric pH fluorescent probe according to claim 1, wherein R is a straight-chain dodecyl group.

3. A method for preparing the mitochondria-immobilized ratiometric pH fluorescent probe according to claim 1 or 2, characterized in that: include: The compound of formula (II) and alkylamine are used as raw materials, dissolved in an organic solvent, and subjected to amidation reaction in the presence of an organic base and a condensing agent. After the reaction, the compound of formula (I) is purified by silica gel column chromatography; (II)。 4. The method according to claim 3, wherein The molar ratio of the compound of formula (II) to the alkylamine is 1:0.5-3.

5. The method according to claim 3, wherein The organic base is N,N-diisopropylethylamine, and the molar ratio of the compound of formula (II) to the organic base is 1:1-5.

6. The method according to claim 3, wherein The condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the molar ratio of the compound of formula (II) to the condensing agent is 1:1-5.

7. The method according to claim 3, wherein The organic solvent is dichloromethane, and the molar volume ratio of the compound of formula (II) to dichloromethane is 1:60-100 mmol / mL.

8. The method according to claim 3, wherein The temperature of the amidation reaction is 20-50°C, and the reaction time is 2-24 h.

9. The method according to claim 3, wherein The eluent used for silica gel column chromatography is dichloromethane and methanol in a volume ratio of 30 to 100:

1.

10. Use of the mitochondrial-immobilized ratiometric pH fluorescent probe according to claim 1 or 2 in the preparation of a mitochondrial pH tracer for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

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