阅读说明：本技术 分子转子型红光线粒体探针及其制备方法和应用 (Molecular rotor type red light mitochondrial probe and preparation method and application thereof ) 是由 徐景坤 张革 刘芳 李慧 于 2020-08-13 设计创作，主要内容包括：本发明公开了分子转子型红光线粒体探针及其制备方法和在活细胞和组织中的应用。所述分子转子型红光线粒体探针结构式如下：<Image he="409" wi="700" file="DDA0002631129040000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>命名为SQ。本发明还给出了SQ的制备方法：利用三苯胺,DMF和POCl<Sub>3</Sub>反应得到4-(二苯胺)苯甲醛；利用4-甲基-喹啉与1-碘乙醇反应得到1-(2-羟乙基)-2-甲基喹啉-1-吲哚碘盐；将4-(二苯胺)苯甲醛与1-(2-羟乙基)-2-甲基喹啉-1-吲哚碘盐通过Knoevenagel反应得到SQ。本发明的分子转子型红光线粒体探针具有高信噪比、高选择性、良好的生物兼容性和较低的细胞毒性,在生物标记领域具有良好的应用前景。(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 POCl 3 Reacting 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.)

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

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

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;

(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.

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.

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.