CN111848509A

CN111848509A - Molecular rotor type red light mitochondrial probe and preparation method and application thereof

Info

Publication number: CN111848509A
Application number: CN202010815453.5A
Authority: CN
Inventors: 徐景坤; 张革; 刘芳; 李慧
Original assignee: Jiangxi Science and Technology Normal University
Current assignee: Jiangxi Science and Technology Normal University
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-10-30

Abstract

The invention discloses a molecular rotor type red light mitochondrial probe, a preparation method thereof and application thereof in living cells and tissues. The molecular rotor type red mitochondrial probe has the following structural formula:

is named SQ. The invention also provides a preparation method of SQ, which comprises the following steps: using triphenylamine, DMF and POCl₃Reacting to obtain 4- (diphenylamine) benzaldehyde; 4-methyl-quinoline reacts with 1-iodoethanol to obtain 1- (2-hydroxyethyl) -2-methyl quinoline-1-indole iodonium salt; 4- (diphenylamine) benzaldehyde and 1- (2-hydroxyethyl) -2-methylquinoline-1-indole iodonium salt are reacted through Knoevenagel to obtain SQ. The molecular rotor type red light mitochondrial probe has high signal-to-noise ratio and high selectivitySelectivity, good biocompatibility and lower cytotoxicity, and has good application prospect in the field of biomarkers.

Description

Molecular rotor type red light mitochondrial probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of mitochondrial fluorescent probes, in particular to a molecular rotor type red-light mitochondrial probe and a preparation method and application thereof.

Background

Mitochondria are the main site of aerobic respiration and play an important role in the life activities of cells. Research shows that mitochondria have special interface structure, can participate in processes such as cell differentiation, cell information transmission, apoptosis and the like, and also have the capacity of regulating cell growth and cell cycle. Changes in mitochondrial function, as reflected by changes in morphology and number, are associated with various human diseases, including Alzheimer's disease and Parkinson's disease. Therefore, high-fidelity visualization and long-term tracking of mitochondrial dynamics are critical to the fields of physiology, pathology, and pharmacology.

Currently, there are several methods available for mitochondrial visualization, including mitochondrial western blotting, citrate synthase activity assay, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and the like. However, these methods are costly, time consuming and not useful for visualization of the entire process of mitochondria in living cells or tissues. To solve this problem, a fluorescence imaging method is proposed, which has many advantages in terms of sensitivity, visualization and non-invasive detection of living cells and tissues. Traditional mitochondrial probes such as rhodamine 123 can clearly image mitochondria in living cells and tissues, but these probes are highly dependent on Mitochondrial Membrane Potential (MMP). When MMPs in living cells decrease, they will shed from mitochondria, let alone track and visualize mitochondria in tissues; and rhodamine 123 has the disadvantages of high cytotoxicity, unsuitability for long-time mitochondrial imaging and the like. Therefore, it is necessary to design a molecular rotor type mitochondrial probe which is not affected by MMP, so that the molecular rotor type mitochondrial probe is tightly embedded into a mitochondrial inner membrane phospholipid bilayer membrane, the binding affinity with mitochondria is enhanced, and the cytotoxicity is low, which is of great significance for promoting the practical application of the fluorescent probe.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a molecular rotor type red mitochondrial probe and a preparation method and application thereof. The molecular rotor type red light mitochondrial probe prepared by the invention has high signal-to-noise ratio, high selectivity, good biocompatibility and lower cytotoxicity, and has good application prospect in the field of biomarkers.

The invention is realized by the following technical scheme:

in a first aspect of the present invention, a molecular rotor type red mitochondrial probe is provided, which has a chemical structural formula as shown in formula (I):

the chemical name is (E) -2- (4- (diphenylamine) styryl) -1- (2-hydroxyethyl) quinoline-1-indole iodonium salt, and the name is SQ.

In a second aspect of the present invention, a method for preparing the molecular rotor type red mitochondrial probe comprises the following steps:

(1) in an ice bath, POCl was added dropwise to DMF₃Stirring to obtain DMF and POCl₃Mixing the solution; dissolving triphenylamine in CHCl₃Obtaining triphenylamine solution, adding DMF and POCl₃Mixing the mixed solution with a triphenylamine solution to obtain a reaction solution, heating and refluxing the reaction solution in an oil bath at 60 ℃ for 12 hours, cooling to room temperature, pouring the reaction solution into ice water, neutralizing with a NaOH solution, extracting with dichloromethane, collecting an organic phase, washing the organic phase with saturated saline water, and drying with anhydrous magnesium sulfate; drying and separating by column chromatography to obtain 4- (diphenylamine) benzaldehyde;

(2) mixing 4-methylpyridine and 1-iodoethanol, and heating and refluxing in an oil bath; after the reaction is finished, adding the obtained product into dichloromethane until the product is completely dissolved, and then pouring the product into petroleum ether to generate brown solid, namely 1- (2-hydroxyethyl) -2-methylquinoline-1-indole iodonium salt;

(3) and (2) mixing 1mmol of 1- (2-hydroxyethyl) -2-methylquinoline-1-indole iodonium salt obtained in the step (1) and 1mmol of 4- (diphenylamine) benzaldehyde obtained in the step (2), adding absolute ethyl alcohol to completely dissolve the mixture, then dropwise adding piperidine, carrying out reflux reaction, and separating by using a column chromatography after the reaction is finished to obtain SQ.

The reaction formula for the above preparation is as follows:

preferably, in step (1), DMF is reacted with POCl₃The volume ratio of (A) to (B) is 30: 5.7; triphenylamine and CHCl₃The adding amount ratio of (1) is 5g to 30 mL; DMF and CHCl₃The volume ratio of (A) to (B) is 1: 1.

Preferably, in the step (1), the temperature of the oil bath is 60 ℃ and the time is 12 h.

Preferably, in the step (1), the mass fraction of the NaOH solution is 20%.

Preferably, in the step (1), the column chromatography is silica gel column chromatography; the eluent is ethyl acetate/petroleum ether (1: 8, v/v).

Preferably, in step (2), the ratio of the amount of 4-methylpyridine to the amount of 1-iodoethanol added is 1.43 g/0.936 mL.

Preferably, in the step (2), the temperature of the oil bath is 105 ℃ and the time is 12 h.

Preferably, in the step (3), the ratio of the addition volume of the absolute ethyl alcohol to the addition volume of the piperidine is 50: 1.

Preferably, in the step (3), the column chromatography is silica gel column chromatography; the eluent was dichloromethane/methanol (15: 1, v/v).

In a third aspect of the present invention, there is provided a use of the above molecular rotor type red light mitochondrial probe for marking or displaying mitochondria.

Preferably, the mitochondria are marked or indicated for distribution in living cells and tissues.

Preferably, the living cells are immortalized cells.

Preferably, the immortalized cells are HeLa cells.

Preferably, the tissue is skeletal muscle tissue.

Preferably, the molecular rotor type red light mitochondrial probe targets the mitochondrial inner membrane of a high viscosity environment in a manner of fluorescence enhancement in glycerol, a high viscosity solvent, such that SQ is highly selective.

The invention has the beneficial effects that:

1. due to the restriction of intramolecular movement, the rotator type indole salt compound SQ of the invention is in H₂The physical property that the fluorescence intensity in O is low and the fluorescence intensity in the high-viscosity solvent glycerol (Gly) is obviously enhanced enables SQ to target the mitochondrial inner membrane in a high-viscosity environment with high selectivity. SQ stains mitochondria independently of MMP, it can track mitochondria and mitochondrial autophagy processes in living cells in real time and for a long period of time, and can image four mitochondria in tissues.

2. Compared with the mitochondrial fluorescent probe with similar functions, the molecular rotor type red-light mitochondrial probe has high signal-to-noise ratio, high selectivity, good biocompatibility and lower cytotoxicity, and has good application prospect in the field of biomarkers.

3. The molecular rotor type red light probe can target mitochondria in living cells and tissues with high selectivity and is not influenced by MMP. Provides a quick, convenient and visual biological detection reagent for the pathological research related to mitochondria. Therefore, the molecular rotor type red light mitochondrial probe has a very good application prospect.

Drawings

FIG. 1: nuclear magnetic hydrogen spectrum of SQ.

FIG. 2: nuclear magnetic carbon spectrum of SQ.

FIG. 3: pseudo-color multicolor images of live, CCCP treated, fixed HeLa cells after 10min incubation with 1. mu.M SQ followed by 1. mu.M MTDR (commercial mitochondrial tracking Red Probe). The number in the merged image is the co-localization coefficient of SQ and MTDR. SQ: ex 473nm, Em 540-; MTDR: ex 635nm, Em 655 and 755 nm. Scale bar 10 μm.

FIG. 4: (a) deconvolution high-resolution three-dimensional reconstruction images obtained by aerial scanning after 10min HeLa cells were stained with 1 μ M SQ. (b, c) enlarged image of green frame in (a) with ridge at arrow. Ex 473nm, Em 540-.

FIG. 5: confocal images of mitochondria in skeletal muscle tissue. Four mitochondria in skeletal muscle tissue after 20min staining with 5 μ M SQ and 5 μ M Hoechst 33342. Confocal fluorescence images of skeletal muscle tissue at (a)20 ×, (b)40 ×, (c)60 × magnification; (d) FIG. (c) is an enlarged view of a selected part. SQ: ex 473nm, Em 540-; hoechst 33342: ex 405nm, Em 420-460 nm. Scale bar 20 μm.

FIG. 6: SQ stained optical section fluorescence images of skeletal muscle (5. mu.M, 20 min). SQ: ex 473nm, Em 540-.

FIG. 7: (a) costain images of HeLa cells at different time points were stained with SQ and LTDR (commercial lysosome tracking red probe) after treatment with 10. mu.M CCCP and 7.5. mu.M pepstatin. Scale bar 10 μm; (b) co-localization coefficient plots of SQ and LTDR at different time points in active HeLa cells. SQ: ex 473nm, Em 540-; LTDR: ex 635nm, Em 655 and 755 nm.

FIG. 8: (a) survival rate of HeLa cells after 12 hours of SQ and MTDR culture at different concentrations; (b) viability of HeLa cells after incubation with 1. mu.M SQ and 0.2. mu.M MTDR for various periods.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background, existing mitochondrial probes are highly dependent on mitochondrial membrane potential and are toxic and unsuitable for long-line mitochondrial imaging. Based on the molecular rotator type red ray mitochondrial probe, the rotator type indole salt compound SQ of the invention is H due to the restriction of intramolecular movement₂The physical property that the fluorescence intensity in O is low and the fluorescence intensity in the high-viscosity solvent glycerol (Gly) is obviously enhanced enables SQ to target the mitochondrial inner membrane in a high-viscosity environment with high selectivity. The mitochondrial inner membrane can provide a high-viscosity environment, and experiments prove that the fluorescence intensity of SQ in the high-viscosity environment can be obviously enhanced, so that SQ has high-intensity fluorescence in the mitochondrial inner membrane and has the capability of high-resolution imaging.

The invention also provides a preparation method of SQ, which comprises the following steps: (1) using triphenylamine, DMF and POCl₃Reacting to obtain 4- (diphenylamine) benzaldehyde; (2) 4-methyl-quinoline reacts with 1-iodoethanol to obtain 1- (2-hydroxyethyl) -2-methyl quinoline-1-indole iodonium salt; (3) 4- (diphenylamine) benzaldehyde and 1- (2-hydroxyethyl) -2-methylquinoline-1-indole iodonium salt are reacted through Knoevenagel to obtain SQ.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified.

Example 1

A method for preparing a molecular rotor type red light mitochondrial probe comprises the following steps:

(1) synthesis of 4- (diphenylamine) benzaldehyde: the three-necked flask was placed in an ice bath at 0 ℃ and 30mL of DMF was added, followed by dropwise addition of 5.7mL of POCl₃Stirring for 30 min; 5g of triphenylamine was dissolved in 30mL of CHCl₃Then transferring the solution into a flask, and placing the flask in an oil bath for refluxing at 60 ℃ for 12 h; cooling to room temperature, pouring the reaction solution into ice water, neutralizing with a 20% NaOH solution, extracting with dichloromethane, collecting an organic phase, washing the collected organic phase with saturated saline water for three times, and drying over anhydrous magnesium sulfate overnight; the final product was isolated by column chromatography on silica gel using ethyl acetate/petroleum ether (1: 8, v/v) as eluent in 55% yield.

(2) Synthesis of 1- (2-hydroxyethyl) -2-methylquinoline-1-indolium iodide salt: 1.43g of 4-methylpyridine and 0.936mL of 1-iodoethanol are added into a 100mL flask, and heated and refluxed for 12 hours in an oil bath at 105 ℃; after the reaction was complete, the product was added to dichloromethane until the product was completely dissolved, and then poured into 500mL of petroleum ether to yield a brown solid in 85% yield.

(3) Synthesis of SQ: 1mmol of 1- (2-hydroxyethyl) -2-methylquinoline-1-indole iodide (0.310g) and 1mmol of 4- (diphenylamine) benzaldehyde (0.273g) were charged in a 100mL three-necked flask, and 20mL of anhydrous ethanol was added thereto to completely dissolve it, followed by dropwise addition of 0.4mL of piperidine, reflux reaction for 12 hours, and separation by silica gel column chromatography using dichloromethane/methanol (15: 1, v/v) as an eluent to give a red solid in a yield of 70%.

The prepared red solid is subjected to nuclear magnetic detection, and the result is as follows:¹H NMR(400MHz，DMSO-d₆)(ppm)：8.99(d，J＝8.0Hz，1H)，8.59(d，J＝12Hz，1H)，8.54(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.20(d，J＝16.0Hz，1H)，8.13(t，J＝10.0Hz，1H)，7.92(t，J＝8.0Hz，1H)，7.845(d，J＝4.0Hz，2H)，7.81(s，1H)，7.42(t，J＝8.0Hz，4H)，6.97(d，2H)，5.24(t，J＝12.0Hz，2H)，5.20(d，J＝8.0Hz，1H)，4.01-4.05(m，2H)；

¹³C NMR(400MHz，DMSO-d₆)，(ppm)：157.23，150.92，147.46，146.35，144.05，139.33，134.98，131.41，130.63，130.40，129.07，128.25，128.10，126.19，125.40，121.34，120.46，119.91，116.85，59.88，52.90.HRMS(m/z)：calculated443.12；found：443.46.C₃₁H₂₇INO₂ ⁺and the successful preparation of SQ (the nuclear magnetic hydrogen spectrogram and the nuclear magnetic carbon spectrogram of SQ are shown in figures 1-2) is proved.

Example 2 cell (HeLa) culture and staining

Placing human cervical cancer cell line (HeLa) in 5% CO at 37 deg.C₂The saturated humidity incubator. Human cervical cancer cell lines were cultured in cell culture medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin (penicillin concentration 100U/mL, streptomycin concentration 100. mu.g/mL). SQ was dissolved in DMSO solution to prepare a stock solution with a concentration of 1 mM. MTDR (commercial mitochondrial tracking Red Probe) and LTDR (commercial lysosomal tracking Red Probe) (both MTDR and LTDR available from molecular probes) were prepared as 1mM aqueous solutions. Live cell staining experiment, cultured cells grown in glass coverslips were stained with 1 μ M SQ for 10 minutes in culture medium at 37 ℃ and then imaged with a fluorescence microscope. Study of dead cells: HeLa cells were pre-incubated with 1. mu.M SQ and 200nM MTDR for 10min, respectively. PBS Wash 2 times in PBS solutionPre-incubating with 4% paraformaldehyde for 30min, and directly imaging under FV-1200 microscope.

Example 3 counterstaining experiments of live/CCCP treated/fixed HeLa cells with SQ and MTDR

The bioimaging properties of SQ in living cells were studied with confocal laser scanning microscopy. Live HeLa cells were stained for 10min with SQ and observed for filamentous structures in the cytoplasm. This is typical of mitochondrial morphology according to our previous work and other literature. To verify this hypothesis, we performed co-staining experiments and selected a co-staining reagent (MTDR), a commercial mitochondrial probe, to test the selectivity of SQ. As can be seen from fig. 3, the mean co-localization coefficient for SQ and MTDR was 0.86, indicating targeting of the trochanter to mitochondria.

MMP as a major parameter of mitochondrial function is associated with many physiological processes. Its reduction or disappearance can cause cell dysfunction and even death. But according to our previous work and other literature, it was found that probes with cationic salts are highly dependent on MMPs. Thus in the present invention, we evaluated the ability of SQ to target mitochondria when MMP decreases. Live HeLa cells were first pretreated with MTDR (MMP remains fixed in the mitochondria when altered) and SQ, and then with the proton carrier CCCP, which allows MMP to decline, resulting in rapid mitochondrial acidification. As shown in FIG. 3, the probes showed good overlap with MTDR and co-localization coefficient of 0.89 after CCCP treatment. This phenomenon suggests that SQ can be firmly localized in mitochondria, regardless of a reduction in MMP.

When cells are fixed, MMPs disappear. Therefore, we investigated the staining ability of SQ in fixed cells to verify if the probe could be immobilized in mitochondria after MMP disappearance. Viable HeLa cells were first stained with SQ and MTDR and then fixed with 4% paraformaldehyde (30 min). As shown in fig. 3, the staining pattern of SQ overlaps well with MTDR, and the co-localization coefficient of SQ and MTDR is 0.89, which indicates that SQ can still be fixed in mitochondria alone when MMP disappears.

Example 4SQ high resolution visualization of mitochondrial inner Membrane experiments

Usually, cationic mitochondrial probes are enriched in the inner mitochondrial membrane, so we investigated whether SQ could localize to the inner mitochondrial membrane. After staining HeLa cells with SQ, magnified mitochondrial images were obtained (fig. 4). In FIG. 4a, the individual mitochondrial morphology is elliptical and the hollow structure is clearly visible. The hollow spheres in the magnified image show SQ can target the inner mitochondrial membrane. From the SEM image of the fixed muscle fibers we can see the ridges of the inner mitochondrial membrane. Also, this characteristic structure of the inner mitochondrial membrane was also obscured (marked by arrows in fig. 4b and 4 c). The above results further demonstrate that SQ does target the inner mitochondrial membrane.

Example 5SQ experiment for high fidelity imaging in skeletal muscle tissue

Since SQ has trochanteric properties, the probe can also visualize mitochondria in tissue with high fidelity. Therefore, we attempted to image the morphology of mitochondria in tissues. Skeletal muscle tissue of Wistar rats (purchased from the centre of laboratory animals, university of Shandong) was stained with SQ and Hoechst33342, and mitochondrial morphology was observed with a confocal microscope without washing. As shown by the enlarged muscle tissue in fig. 5a and 5b, we can clearly identify a fine and regular mitochondrial network. The actual myofibrillar (IMF) mitochondrial morphology is tubular, consistent with that observed by SEM. In addition, cross-fibro-connective mitochondria (CFCM), I-band mitochondria (IBM), perivascular mitochondria (PVM) and fibro-parallel mitochondria (FPM) can also be easily observed (fig. 5c and 5d), which affect the energy distribution in skeletal muscle through conduction pathways. By optically sectioning the tissue, a fluorescence image in the z-axis direction can be obtained (fig. 6), clearly showing the intracellular mitochondrial network and the different mitochondria, further showing that mitochondria can be observed at a depth of 66 μm at SQ according to fig. 6(a), 6 (h).

Example 6 SQ staining HeLa cell experiment to follow mitochondrial autophagy Process

Autophagy is a regulated lysosomal pathway that is used for the degradation and circulation of organelles and long-lived proteins. During autophagy, a portion of the mitochondria is sequestered in a double membrane autophagy vesicle. Therefore, the mitochondrial autophagy process is closely related to mitochondria and lysosomes. The present invention performs SQ bioimaging experiments to test its ability to track mitochondrial autophagy. The autophagy process was initiated by first staining HeLa cells with SQ and LTDR, respectively, and then incubating the cells with CCCP and a pepsin inhibitor. Fluorescence images of the cells at different time points (0h, 0.25h, 0.5h, 0.75h, 1.0h, 1.5h and 2.0h) are obtained, and corresponding co-localization coefficients are obtained. And detecting the overlapping condition of SQ and LTDR by using the co-localization coefficient, and observing the autophagy process of mitochondria in real time. With increasing time, the fluorescent signal for SQ gradually overlaps the fluorescent signal for LTDR (FIG. 7 a). The co-localization coefficient for SQ and LTDR increased from 22% to 82% (fig. 7 b). The high degree of overlap data successfully demonstrated the occurrence of the phenomenon of mitochondrial autophagy. Notably, this probe monitors mitophagy against MMP changes as compared to other mitophagy probes. Therefore, according to the above results, SQ can track mitophagy in real time in living cells for a long period of time in situ.

Example 7 cytotoxicity test of SQ-stained HeLa cells

Cytotoxicity is essential for acceptable biological probes, particularly probes for long-range tracking and imaging. The present invention uses MTT reagents to detect cytotoxicity of SQ and MTDR. As shown in FIG. 8a, the viability of HeLa cells was higher than 80% after 12h of incubation with 1.5. mu.M SQ. However, after 12h of culture with 1.5. mu.M MTDR, the viability of HeLa cells decreased to 40%. Meanwhile, the invention researches the viability of HeLa cells stained with commonly used MTDR and SQ concentrations. As the incubation time increased, the viability of HeLa cells stained with SQ was not less than 80%, much higher than that of the culture with MTDR (fig. 8 b). The results indicate that SQ is less cytotoxic and more biocompatible than MTDR. Therefore, SQ is almost non-toxic and suitable for imaging and tracking of live cell mitochondria.

In addition, the Stokes shift of SQ is 84nm, interference of light scattering on tissue and cell imaging is avoided, and cells or mitochondria in the tissue can be imaged with high resolution. The change of the fluorescence intensity of SQ in the physiological pH range (pH is 4.0-9.0) is not obvious, which shows that the SQ is not sensitive to the pH change and is beneficial to the application of the SQ in high-fidelity visualization mitochondria.

In summary, compared with MTDR, the molecular rotor type red mitochondrial probe SQ has high signal-to-noise ratio, high selectivity, good biocompatibility and lower cytotoxicity, and is suitable for imaging and tracking mitochondria in living cells.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The molecular rotor type red mitochondrial probe is characterized in that the chemical structural formula is shown as the formula (I):

2. The method for preparing the molecular rotor type red mitochondrial probe as claimed in claim 1, wherein the method comprises the following steps:

(1) in ice bath, POCl was added dropwise to DMF₃Stirring to obtain DMF and POCl₃Mixing the solution; dissolving triphenylamine in CHCl₃Obtaining triphenylamine solution, adding DMF and POCl₃Mixing the mixed solution with a triphenylamine solution to obtain a reaction solution, heating and refluxing the reaction solution in an oil bath at 60 ℃ for 12 hours, cooling to room temperature, pouring the reaction solution into ice water, neutralizing with a NaOH solution, extracting with dichloromethane, collecting an organic phase, washing the organic phase with saturated saline water, and drying with anhydrous magnesium sulfate; drying and separating by column chromatography to obtain 4- (diphenylamine) benzaldehyde;

3. The method according to claim 2, wherein in the step (1), DMF and POCl are used₃The volume ratio of (A) to (B) is 30: 5.7; triphenylamine and CHCl₃The adding amount ratio of (1) is 5g to 30 mL; DMF and CHCl₃The volume ratio of (A) to (B) is 1: 1.

4. The production method according to claim 2, wherein in the step (1), the temperature of the oil bath is 60 ℃ and the time is 12 hours.

5. The process according to claim 2, wherein in the step (2), the amount of 4-methylpyridine to 1-iodoethanol is 1.43 g/0.936 mL.

6. The production method according to claim 2, wherein in the step (2), the temperature of the oil bath is 105 ℃ and the time is 12 hours.

7. The method according to claim 2, wherein in the step (3), the volume ratio of the absolute ethanol to the piperidine is 50: 1.

8. The use of the molecular rotor type red light mitochondrial probe of claim 1 for marking or displaying mitochondria.

9. Use according to claim 8, characterized in that mitochondria are marked or indicated for distribution in living cells and tissues.

10. The use of claim 9, wherein the molecular rotator-type red light mitochondrial probe of claim 1 is used in a manner of fluorescence enhancement in glycerol, a high viscosity solvent, such that SQ is targeted to the inner mitochondrial membrane in a high viscosity environment with high selectivity.