Covalent mitochondrial fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to a covalent mitochondrial fluorescent probe as well as a preparation method and application thereof, the covalent fluorescent probe takes Cy3 or Cy5 with low phototoxicity as a fluorophore and chloroacetyl chloride as a covalent group, the Cy3 or Cy5 is also used as a mitochondrial targeting group, and the chloroacetyl chloride is used as a covalent group. The covalent group chloroacetyl chloride on the fluorescent probe molecule is covalently bound with the mitochondrial inner membrane protein to realize targeted covalent labeling, and compared with other fluorescent probes synthesized by covalent groups, the covalent fluorescent probe has more excellent labeling stability, has good labeling stability, strong light stability and low phototoxicity, and can be used for detecting the mitochondrial inner membrane protein of the mitochondrial inner membrane protein of the mitochondrial inner membrane protein of the mitochondrial inner membrane protein. And the requirements of multicolor imaging and long-term dynamic imaging of mitochondria can be met.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and more particularly to a covalent mitochondrial fluorescent probe and a preparation method and application thereof. Background Art

[0002] As important bioenergetic centers and key signal integrators, mitochondria exhibit highly dynamic properties characterized by fission, fusion, and complex intercellular interactions. These dynamic processes are the basis of cellular homeostasis, and their dysregulation is increasingly recognized as a key factor in a range of human diseases, including cancer, Alzheimer's disease, and cardiovascular disease (References 1 and 2). Therefore, long-term monitoring of mitochondrial dynamics has become a key strategy to decipher its multifaceted role in disease pathogenesis. As an important tool for studying mitochondrial function, mitochondrial fluorescent probes have made significant progress in design and application in recent years. Due to the ease of synthesis, customizable performance adjustment, and minimal impact on cell physiology of organic small molecules, a variety of mitochondrial fluorescent probes have been developed for mitochondrial imaging. They mainly include mitochondrial localization probes and probes for detecting active components within mitochondria.

[0003] Among them, the probe used to detect the active components in mitochondria is used to detect peroxynitrite (ONOO - ) and the ATP fluorescent probe for monitoring changes in mitochondrial ATP concentration in patent CN 109456349B. Mitochondrial localization probes such as the water-soluble red fluorescent probe for targeting mitochondria in patent CN 112079771 B and the commercially available Mito Tracker Red. The mainstream strategy of these mitochondrial localization probes is to achieve targeted positioning by electrostatic adsorption of the mitochondrial inner membrane. However, the fluctuating mitochondrial membrane potential (MMP) greatly challenges probes that rely solely on electrostatic interactions for positioning. Because this mechanism essentially relies on the charge distribution on the mitochondrial membrane, this limitation can easily lead to significant off-target effects, which ultimately manifests as a decrease in signal-to-noise ratio and a decrease in fluorescence intensity, especially during long-term imaging. In addition, probes with high phototoxicity can induce structural and functional abnormalities in mitochondria, leading to rapid and large fluctuations in membrane potential, thereby further exacerbating off-target effects.

[0004] Furthermore, while the advancement of live-cell super-resolution microscopy has enabled long-term observation of mitochondrial dynamics, this advanced imaging approach places higher demands on mitochondrial fluorescent probes. First, the probes should exhibit excellent labeling stability to ensure accurate targeting; second, strong photostability, which is crucial for maintaining image quality during long-term imaging; and finally, low phototoxicity to prevent artifacts and functional alterations during long-term dynamic imaging. However, existing fluorescent probes targeting mitochondria struggle to simultaneously meet all three requirements. In particular, probes that rely solely on electrostatic adsorption to the mitochondrial inner membrane for targeting suffer from poor labeling stability. For example, the commercially available MitoTracker Red exhibits both poor labeling stability and high phototoxicity. While the covalent labeling strategy explored by Yamaguchi and colleagues offers a potential avenue for enhancing labeling stability, the inherent high phototoxicity of these probes remains a key limitation, hindering their effective use in long-term dynamic studies. Furthermore, it remains unclear whether different fluorophores and covalent groups can be combined to successfully synthesize fluorescent probes and how this combination affects mitochondrial localization and labeling. Therefore, it is necessary to develop an ideal mitochondrial fluorescent probe that can simultaneously meet the requirements of good labeling stability, strong photostability and low phototoxicity, which is conducive to the long-term observation of mitochondrial dynamics and aims to reveal the potential role of mitochondrial changes in the pathogenesis of diseases.

[0005] Source:

[0006] Document 1: A. Suomalainen, J. Nunnari, Cell 2024, 187, 2601~2627.

[0007] Document 2: J.Wang, W.-J.Liu, H.-Z.Shi, H.-R.Zhai, J.-J.Qian, W.-N.Zhang, Cells2022,11,2849. Summary of the Invention

[0008] In response to the above-mentioned defects or improvement needs of the prior art, the present invention provides a mitochondrial-targeted covalent fluorescent probe, a preparation method and application thereof, the purpose of which is to discover that using chloroacetyl chloride as a covalent group, the fluorescent group Cy3 or Cy5, the covalent group and the COT group through a coupling reaction can not only synthesize a fluorescent probe, but also the fluorescent probe can achieve targeted positioning of mitochondria through electrostatic adsorption and covalent binding, and has the characteristics of good labeling stability, strong light stability and low phototoxicity. It is an ideal mitochondrial fluorescent probe, thereby solving the technical problems that the existing commercially available mitochondrial fluorescent probes only rely on electrostatic adsorption of the mitochondrial inner membrane for positioning, the probe is prone to off-target and has high phototoxicity.

[0009] To achieve the above objectives, according to one aspect of the present invention, a covalent mitochondrial fluorescent probe is provided, which can achieve targeted localization of mitochondria in cells through electrostatic adsorption and covalent binding, and its structural formula is shown below:

[0010]

[0011] According to another aspect of the present invention, a method for preparing the mitochondrial covalent fluorescent probe according to the present invention is also provided, which comprises the steps of synthesizing the fluorescent probe by coupling a fluorescent group, a covalent group and a COT group; the fluorescent group is Cy3 or Cy5, the covalent group is chloroacetyl chloride, and the COT group is cyclooctatetraene.

[0012] Preferably, in the preparation method, the equivalent ratio of the fluorescent group, the covalent group and the COT group is 1:2:1.

[0013] According to another aspect of the present invention, there is also provided a use of the mitochondrial covalent fluorescent probe according to the present invention in targeted cell mitochondrial fluorescence imaging.

[0014] Preferably, in the application, cells are incubated with a cell incubation solution containing the mitochondrial covalent fluorescent probe, so that the fluorescent probe penetrates the cells and covalently binds to the mitochondrial inner membrane protein for targeted positioning and fluorescence imaging.

[0015] Preferably, in the application, the cells include fibroblasts, cardiomyocytes and / or cancer cells.

[0016] Preferably, in the application, the cells are COS-7, BHK21, Cardiomyocytes or A2780.

[0017] Preferably, in the application, the concentration of the mitochondrial covalent fluorescent probe in the cell incubation medium is 10 μM.

[0018] Preferably, the application is applied to mitochondrial dual-color super-resolution imaging and long-term dynamic super-resolution imaging.

[0019] In general, the above technical solution conceived by the present invention is compared with the prior art, because chloroacetyl chloride is used as a covalent group, the following can be achieved:

[0020] The mitochondrial covalent fluorescent probe provided by the present invention has a low phototoxic fluorophore that can be used as a mitochondrial targeting group, and the covalent group chloroacetyl chloride on the fluorescent probe molecule is covalently bound to the mitochondrial inner membrane protein to achieve targeted covalent labeling. Compared with other covalent groups, the probe synthesized with chloroacetyl chloride as the covalent group has better labeling stability. The covalent fluorescent probe provided by the present invention can still stably label mitochondria after losing the mitochondrial membrane potential, and the colocalization coefficient can reach more than 0.9, which can avoid the off-target problem caused by changes in the mitochondrial inner membrane potential, and the introduced COT group can further reduce the phototoxicity of Cy3 and Cy5 on the one hand, and on the other hand, it can also enhance the anti-photobleaching performance of the fluorescent probe and improve its photostability. Compared with existing mitochondrial fluorescent probes, the covalent fluorescent probe synthesized by the present invention has good labeling stability, strong photostability and low phototoxicity, and is an ideal mitochondrial-targeted covalent fluorescent probe that can achieve long-term high signal-to-noise ratio super-resolution dynamic imaging of mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the NMR and mass spectrometry results of the HZ Mito Red probe;

[0022] Figure 2 These are the NMR and mass spectrometry results of the HZ Mito Deep Red probe;

[0023] Figure 3 These are the spectral test results of the covalent mitochondrial probes HZ Mito Red and HZ Mito Deep Red;

[0024] Figure 4 This is the fluorescence imaging image of mitochondria in cells labeled with the fluorescent probes HZ Mito Red and HZ Mito Deep Red;

[0025] Figure 5 This is a fluorescence imaging image of mitochondria labeled with probes HZ Mito Red and HZ Mito Deep Red after the mitochondrial membrane potential is lost;

[0026] Figure 6 It is the result of labeling stability of mitochondria by different fluorescent probes;

[0027] Figure 7 This is a comparison of the mitochondrial localization labeling effects of the fluorescent probes HZ Mito Red and HZ Mito Deep Red with the commercially available Mito Tracker Red. Figure 7 (a) is the long-term super-resolution imaging of different probes, (b) is the photobleaching curve of different probes, (c) is the number of frames taken when the fluorescence loss was 20%, and (d) is the number of frames taken when the mitochondrial width increased by 25%. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0029] Existing targeting mitochondrial fluorescent probe mainly relies on the electrostatic adsorption of mitochondrial inner membrane to realize targeting, and its labeling stability is poor.In addition the fluorescent group in these probe molecules mainly comprises rhodamine group, cyanine group and naphthalene phosphine pore group, and wherein the phototoxicity of naphthalene phosphine pore group fluorescent group is higher. The present invention has selected two kinds of fluorescent groups Cy3 and Cy5 that phototoxicity itself is lower when designing synthetic fluorescent probe, corresponding respectively to redness and crimson. For further reducing the phototoxicity of fluorescent probe, we also introduce COT group (cyclooctatetraene) and fluorescent group is modified, described COT group can quench the reactive oxygen species that mitochondria produces and then reduce the generation amount of reactive oxygen species on the one hand, can further reduce the phototoxicity of this fluorescent probe, can also strengthen the anti-photobleaching performance of fluorescent probe on the other hand, improve its photostability.

[0030] Next, we synthesized fluorescent probes by screening different covalent groups for coupling with the above-mentioned fluorescent groups (Cy3 or Cy5) and COT groups. Among them, the chloroacetyl chloride group is used in proteomics, but its application in mitochondrial fluorescent probes has not yet been seen. We first attempted to introduce the chloroacetyl chloride group. The results showed that using chloroacetyl chloride as a covalent group, coupling the fluorescent group, covalent group and COT group not only allowed the synthesis of fluorescent probes (named HZ Mito Red and HZ MitoDeep Red, respectively), but also that these two fluorescent probes could be targeted to mitochondria through electrostatic adsorption and covalent binding. Compared with probes synthesized with other covalent groups, this probe has better labeling stability. It can still stably label mitochondria after losing the mitochondrial membrane potential, and the colocalization coefficient can reach above 0.9, which can avoid the off-target problem caused by changes in the mitochondrial inner membrane potential. It has good labeling stability, strong photostability and low phototoxicity, making it an ideal mitochondrial-targeted covalent fluorescent probe.

[0031] Based on this, the present invention provides a mitochondrial-targeted covalent fluorescent probe, which can achieve targeted positioning of mitochondria in cells through electrostatic adsorption and covalent binding. Its structural formula is shown below:

[0032]

[0033] The probe with red fluorophore (Cy3) is named HZ Mito Red, and its structural formula is as follows:

[0034]

[0035] The probe with a deep red fluorophore (Cy5) is named HZ Mito Deep Red, and its structural formula is as follows:

[0036]

[0037] The positive charge contained in the fluorophore in the covalent fluorescent probe described in the present invention can also be used as a mitochondrial targeting group for targeting mitochondria. After the fluorescent probe penetrates the cell membrane and enters the interior of the cell, it first reaches the mitochondria through electrostatic adsorption with the negatively charged mitochondria. The covalent group chloroacetyl chloride on the fluorescent probe molecule covalently binds to the mitochondrial inner membrane protein to achieve covalent labeling. Therefore, the present fluorescent probe can achieve mitochondrial fluorescent labeling imaging based on a method combining electrostatic adsorption and covalent coupling. Compared with probes synthesized by other covalent groups, the present probe has better labeling stability and can meet the needs of multi-color imaging and long-term dynamic imaging of mitochondria.

[0038] In addition, the present invention also provides a method for preparing the mitochondrial-targeted covalent fluorescent probe as described in the present invention, which includes the steps of coupling a fluorescent group, a covalent group and a COT group to synthesize a fluorescent probe, wherein the fluorescent group is Cy3 or Cy5, and the covalent group is chloroacetyl chloride, wherein the probe synthesized with the fluorescent group being Cy3 is HZ Mito Red, and the probe synthesized with the fluorescent group being Cy5 is HZ Mito Deep Red.

[0039] In some embodiments, the fluorescent group and the covalent group are added at an equivalent ratio of 1:2; the COT group is cyclooctatetraene, and the reaction synthesis is carried out at an equivalent ratio of 1:2:1 among the fluorescent group, the covalent group and the COT group.

[0040] In addition, the present invention also provides an application of the covalent fluorescent probe described in the present invention in targeted cell mitochondrial fluorescence imaging, specifically: cells are incubated with a cell incubation solution containing the mitochondrial covalent fluorescent probe described in the present invention, so that the covalent fluorescent probe penetrates the cells and covalently binds to the mitochondrial inner membrane protein for targeted positioning and fluorescence imaging.

[0041] The cells include fibroblast cell lines, cardiomyocytes and / or cancer cells; in some embodiments, they are used to prepare mitochondria in COS-7, BHK21, Cardiomyocytes and / or A2780 cells for targeted positioning.

[0042] In some embodiments, the concentration of the mitochondrial covalent fluorescent probe in the cell incubation medium is 10 μM.

[0043] The following are examples

[0044] Example 1 Preparation of mitochondrial-targeted covalent fluorescent probes

[0045] The structural formulas of the mitochondria-targeted covalent fluorescent probes (HZ Mito Red and HZ Mito Deep Red) are shown below:

[0046]

[0047] The synthesis route of the covalent fluorescent probe HZ Mito Red is as follows:

[0048]

[0049] The synthesis route of the covalent fluorescent probe HZ Mito Deep Red is as follows:

[0050]

[0051] The specific synthesis process of the covalent fluorescent probe HZ Mito Red is as follows:

[0052] (1) 4-Hydrazinylbenzoic acid (2 g, 13.15 mmol) and 3-methyl-2-butanone (1.7 g, 19.72 mmol) were refluxed in glacial acetic acid (10 mL) for about 16 h, cooled to room temperature, and quenched with water. The crude oil was extracted with dichloromethane, washed with water, and the organic phase was dried over anhydrous sodium sulfate. The dichloromethane was evaporated in vacuo and purified by silica gel column chromatography to give compound 1 (2.22 g, 10.92 mmol, 83%) as a light yellow solid.

[0053] (2) Compound 1 (2 g, 9.84 mmol) and methyl iodide (8.38 g, 59.08 mmol) were mixed in a 50 mL round-bottom flask and refluxed overnight. After the reaction was completed, the mixture was cooled to room temperature, the residue was filtered, and washed with acetonitrile and hexane to obtain compound 2 (1.8 g, 8.25 mmol, 84%) as a white solid.

[0054] (3) A solution of Boc2O (1.2 g, 5.5 mmol) in DCM (30 mL) was added dropwise to a solution of diamine (2.03 mL, 27.5 mmol) in DCM (6 mL) at 0°C for 30 minutes. The solution was stirred at room temperature for 2 hours. The reaction mixture was filtered and concentrated under reduced pressure. Some azeotropes were removed with toluene. The crude residue was dissolved in ethyl acetate (200 mL) and washed with saline (50 mL). It was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 3 (957.75 mg, 5.5 mmol, 100%).

[0055] (4) Compound 3 (800 mg, 4.59 mmol) was dissolved in dry pyridine. The mixture was stirred at 0°C for 10 minutes. Chloroacetyl chloride (617.17 mg, 5.51 mmol) was added. The reaction mixture was warmed to room temperature. Stirring was continued for 12 hours. The reaction mixture was poured into crushed ice. The mixture was carefully extracted with ethyl acetate. The residue in the extract was purified by DCM-MeOH gradient column chromatography to obtain compound 4 (917.9 mg, 3.67 mmol, 80%) as a dark brown solid.

[0056] (5) A mixture of 1.5 mL of TFA and 2 mL of DCM was added dropwise to a round-bottom flask containing compound 4 (70 mg, 0.4 mmol) at 0°C. The reaction solution was stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was concentrated in vacuo, the solvent was removed, and the product was azeotroped with toluene three times. The product was then washed with DCM to obtain compound 5 (87.17 mg, 0.35 mmol, 0.875%) as a light yellow oil.

[0057] (6) 2,3,3-Trimethylindene (1.03 g, 6.22 mmol) and 2-bromoethylamine hydrobromide (1.91 g, 9.41 mmol) were added to a pressure tube. The mixture was stirred at 120°C in argon for 14 hours. After the reaction, the solid was washed with CHCl3, MeOH and acetone. It was then dissolved in DCM, washed with saturated NaHCO3 aqueous solution and saline, and dried over anhydrous Na2SO4. The crude product was further purified by silica gel column (eluent: n-hexane / AcOEt = 60 / 40 to 20 / 80, DCM / MeOH = 100 / 0 to 90 / 10) to obtain compound 6 (0.73 g, 3.51 mmol, 56%).

[0058] (7) Compound 6 (300 mg, 1.48 mmol), Boc2O (288 mg, 1.78 mmol), and DIEA (0.39 mL, 2.22 mmol) were added to a 10 mL round-bottom flask containing CHCl3 (5 mL) and stirred overnight at room temperature under nitrogen. After the reaction, the mixture was diluted with DCM, washed with saturated aqueous NaHCO3 and saline, and dried over Na2SO4. The solvent was evaporated and purified by silica gel column chromatography (eluent: DCM / MeOH = 100 / 0 to 90 / 10) to afford compound 7 (362 mg, 1.19 mmol, 80%) as a pink oil.

[0059] (8) Magnesium particles (1.0 g, 42 mmol) were added to a dry 50 mL two-necked round-bottom flask and purged with argon. COTBr (1.0 g, 5.8 mmol) was dissolved in ultra-dry THF and then slowly added dropwise to the two-necked round-bottom flask containing the magnesium particles in an ice bath under argon and continued stirring for 1 h. The system was then raised to room temperature and stirred for 4 hours until a blue-green solution was formed. The resulting solution was cooled to -78°C and an excess of solid dry ice was added. The reaction was quenched with 20 mL of water and acidified to pH = 2 with 1 M hydrochloric acid. The resulting carboxylic acid was extracted from the aqueous solution with n-hexane and then purified by flash chromatography on a silica gel column (EA / PE = 5 / 1) to give compound 8 (430 mg, 2.9 mmol, 50%).

[0060] (9) Compound 8 (180 mg, 1.22 mmol), DIEA (236.5 mg, 1.83 mmol), and N,N-disuccinyl carbonate (625.05 mg, 2.44 mmol) were dissolved in a round-bottom flask containing 5 mL of dry DMF. The reaction solution was stirred at room temperature overnight. After the reaction, the resulting mixture was purified using silica gel (eluent: ethyl acetate: hexane = 1:3) to obtain compound 9 (187 mg, 0.73 mmol) as a light yellow oil.

[0061] (10) Compound 7 (120 mg, 0.56 mmol) and N,N'-diphenylformamidine (129.2 mg, 0.68 mmol) were dissolved in a 50 mL round-bottom flask containing 5 mL of acetic anhydride, heated to 120°C for 30 min, and then cooled to room temperature. A solution of compound 2 (206 mg, 0.68 mmol) in dry pyridine (10 mL) was added to the mixture. The mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residual oil was dissolved in chloroform (50 mL). The mixture was washed with water and 0.1 M hydrochloric acid in sequence. The organic layer was dried over Na2SO4. The crude product was purified by column chromatography (eluent: DCM / MeOH = 50 / 1 to 20 / 1) to give compound 10 (106 mg, 0.2 mmol, 36%).

[0062] (11) Compound 10 (100 mg, 0.19 mmol) was added to a round-bottom flask containing 3 ml of DCM. TFA (2 mL) was added dropwise in an ice bath. Subsequently, the reaction mixture was transferred to room temperature and stirred for 4 hours. The solvent was removed in vacuo to obtain the product. No further purification was required and it was used directly in the next step. The product was dissolved in 3 ml of DMF, and then compound 9 (46.6 mg, 0.19 mmol) and DIEA (49 mg, 0.38 mmol) were added. The reaction mixture was stirred overnight in an Ar atmosphere. After the reaction was completed, the solvent was removed in vacuo and the crude product was purified by silica gel column chromatography (eluent: DCM / MeOH = 50 / 1 to 10 / 1) to obtain compound 11 (89.6 mg, 0.16 mmol, 84%) as a red solid.

[0063] (12) Compound 11 (997 mg, 2.49 mmol) and N-hydroxysuccinimide (285.99 mg, 2.49 mmol) were placed in a 100 mL round-bottom flask, and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) (285.84 mg, 2.49 mmol) was added, followed by 16 mL of DMF. The system was allowed to react overnight at room temperature. After the reaction, the solvent was removed in vacuo, and the product Cy3-COT-NHS (1001.46 mg, 2.01 mmol) was obtained by extraction with DCM.

[0064] (13) Cy3-COT-NHS (166 mg, 0.33 mmol) and compound 5 (82.19 mg, 0.33 mmol) were placed separately in a 100 mL round-necked flask, and then Et3N (33.72 mg, 0.66 mmol) and 2 mL of DMF were added. The reaction was stirred at room temperature overnight. The solution was then concentrated in vacuo. To obtain high-purity HZ Mito Red, silica gel column chromatography was performed using methanol / dichloromethane 5% to 8% (100.30 mg, 0.19 mmol, 57%).

[0065] The synthesis of HZ Mito Deep Red was similar to that of HZ Mito Red, except that N-(3-(phenylamino)allyl)aniline was used.

[0066] The nuclear magnetic resonance and mass spectrometry results of the HZ Mito Red and HZ Mito Deep Red probes synthesized in this example are shown in Figure 2. Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 The middle green is the integral of the nuclear magnetic resonance signal, which comes with the original image and is a normal phenomenon.

[0067] The structure of HZ Mito Red is as follows:

[0068]

[0069] The structure of HZ Mito Deep Red is as follows:

[0070]

[0071] The synthetic routes of the comparative covalent probes are as follows:

[0072]

[0073]

[0074] Example 2 Spectral testing of mitochondria-targeted covalent fluorescent probes HZ Mito Red and HZ Mito Deep Red

[0075] The probes HZ Mito Red and HZ Mito Deep Red were prepared into 1mM stock solutions using DMSO. 20μL of the stock solution was diluted with PBS buffer to prepare a 10M test solution. The UV absorption spectra of the probes were measured using an Agilent Cary 60 UV-visible spectrophotometer, and the fluorescence emission spectra were measured using an Agilent Cary Eclipse fluorescence spectrophotometer. The results are shown in Figure 2. Figure 3 shown.

[0076] Depend on Figure 3 The results show that the absorption and emission of the probes HZ Mito Red and HZ Mito Deep Red meet the requirements of dual-channel fluorescence imaging.

[0077] Example 3 Labeling Effects of Different Fluorescent Probes on Mitochondria

[0078] Preparation of cells before imaging:

[0079] 1. Prepare cells: COS-7, BHK21, Cardiomyocyte, and A2780;

[0080] 2. Dilute 2 μL of 1 mM HZ Mito Red, HZ MitoDeep Red, Cy3 (commercially available), Cy3-NHS, Cy3-COT-NHS, Cy3-Py, Cy3-COT-Py, Cy3-EP, Cy3-COT-EP, or commercially available Mito Tracker Red to 200 μL in DMEM containing fetal bovine serum (FBS) to prepare the cell incubation medium.

[0081] 3. Take an appropriate amount of PBS to wash the cells grown in the well plate or confocal culture dish, and then take an appropriate amount of serum-free medium to rinse the cell surface;

[0082] 4. Add incubation solution and incubate the cells with the incubation solution for 30 minutes;

[0083] 5. Wash with an appropriate amount of PBS two to three times and then perform SIM super-resolution imaging. The labeling results of HZ Mito Red and HZ MitoDeep Red probes are as follows: Figure 4 shown.

[0084] from Figure 4 It can be seen that both HZ Mito Red and HZ Mito Deep Red can well label mitochondria and are suitable for mitochondrial labeling in different cell lines. In addition, after fixing the cells transfected with COX4-EGFP and labeled with HZ Mito Red or HZ Mito Deep Red, it was found that after losing the mitochondrial membrane potential, HZ Mito Red and HZ MitoDeep Red can still stably label mitochondria, with colocalization coefficients as high as 0.91 and 0.89 ( Figure 5 ) while the other covalent probes maintained at around 0.7, and the probe relying solely on electrostatic adsorption could only maintain at 0.18 ( Figure 6 ).

[0085] In addition, the results of HiS-SIM long-term dynamic super-resolution imaging with the same shooting parameters (laser power 10%, exposure time 10ms) are as follows: Figure 7 Figure a, Compared with the commercially available Mito Tracker Red, HZ Mito Red and HZ Mito Deep Red have significantly improved signal-to-noise ratios during long-term dynamic imaging of mitochondria, and still show negligible background signals after continuously capturing 500 frames of super-resolution images; while Mito Tracker Red-labeled mitochondria show significantly increased fluorescence background after only 50 consecutive frames.

[0086] In addition, HZ Mito Red and HZ Mito Deep Red showed significantly improved resistance to photobleaching ( Figure 7 After continuously capturing nearly 300 frames of super-resolution images, HZ Mito Red and HZ Mito Deep Red still retained more than 80% of their fluorescence intensity ( Figure 7Meanwhile, HZ Mito Red and HZ Mito Deep Red exhibit low phototoxicity and can maintain normal mitochondrial morphology. Nearly 400 frames of super-resolution images were continuously taken. Compared with Mito Tracker Red, the phototoxicity was reduced by nearly 10 times ( Figure 7 (d)

[0087] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A covalent mitochondrial fluorescent probe, characterized in that It can achieve targeted positioning of mitochondria in cells through electrostatic adsorption and covalent binding. Its structural formula is shown below:

2. A method for preparing a mitochondrial covalent fluorescent probe according to claim 1, characterized in that: The method comprises the steps of subjecting a fluorescent group, a covalent group and a COT group to a coupling reaction to synthesize a fluorescent probe; the fluorescent group is Cy3 or Cy5, and the covalent group is chloroacetyl chloride.

3. The preparation method according to claim 2, wherein The equivalent ratio of the fluorescent group to the covalent group is 1:

2.

4. The preparation method according to claim 3, wherein The COT group is cyclooctatetraene, and the equivalent ratio of the fluorescent group, the covalent group and the COT group is 1:2:

1.

5. Use of the mitochondrial covalent fluorescent probe according to claim 1 in targeted cell mitochondrial fluorescence imaging.

6. The use according to claim 5, characterized in that Cells are incubated with a cell incubation solution containing the mitochondrial covalent fluorescent probe according to claim 1, so that the fluorescent probe penetrates the cells and covalently binds to the mitochondrial inner membrane protein for targeted positioning and fluorescence imaging.

7. The use according to claim 6, characterized in that The cells include fibroblasts, cardiomyocytes and / or cancer cells.

8. The use according to claim 7, characterized in that The cells are COS-7, BHK21, Cardiomyocyt or A2780.

9. The use according to claim 8, characterized in that The concentration of the mitochondrial covalent fluorescent probe in the cell incubation solution is 10 μM.

10. The use according to claim 9, characterized in that Applied to mitochondrial multi-color super-resolution imaging and long-term dynamic super-resolution imaging.

Images