A mitochondrial fluorescent probe independent of mitochondrial membrane potential and preparation method and application thereof

Abstract

The application discloses a kind of mitochondrial fluorescent probe independent of mitochondrial membrane potential and its preparation method and application, the mitochondrial fluorescent probe independent of mitochondrial membrane potential provided by the application can target and image mitochondria when mitochondrial membrane potential decreases and disappears.

Description

Technical Field

[0001] This invention relates to the field of probes, and more particularly to a mitochondrial fluorescent probe that is independent of mitochondrial membrane potential, its preparation method, and its application. Background Technology

[0002] Mitochondria are organelles in cells that produce energy and are the primary site of cellular aerobic respiration. Besides providing energy, mitochondria participate in vital processes such as cell differentiation, cell signaling, and apoptosis, and possess the ability to regulate cell growth and the cell cycle. Many diseases are closely related to the morphology and number of mitochondria. Mitochondrial fluorescence imaging is an important tool in mitochondrial research; therefore, mitochondrial fluorescent probes are widely used in medical, life science research, and clinical diagnostics.

[0003] Current mitochondrial fluorescent probes are driven by the high mitochondrial membrane potential of living cells to accumulate in mitochondria. They can only be used for mitochondrial staining and imaging in living cells and cannot specifically stain biological samples with reduced or even lost mitochondrial membrane potential, including mitochondria in fixed cells. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a mitochondrial fluorescent probe, its preparation method, and its application, to solve the problem that mitochondrial fluorescent probes in the prior art rely on high mitochondrial membrane potential.

[0005] A mitochondrial fluorescent probe independent of mitochondrial membrane potential according to a first aspect embodiment of the present invention, wherein the line

[0006] The particulate fluorescent probe is a compound having the structure shown in formula (I), or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Wherein, the R 1 Including any one of hydrogen or C1-C4 alkyl groups; the R 2 It includes any one of hydrogen, C1-C4 alkyl groups, and C1-C4 alkoxy groups; X includes any one of halogen atoms, BF4, and ClO4.

[0009] According to some embodiments of the present invention, the halogen atom is selected from any one of iodine, bromine, and chlorine.

[0010] According to some embodiments of the present invention, the R 1 In this context, the C1 to C4 alkyl groups include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0011] According to some embodiments of the present invention, the R 2 In this context, the C1 to C4 alkyl groups include any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0012] According to some embodiments of the present invention, the R 2 In this context, the C1 to C4 alkoxy groups include any one of methoxy, ethoxy, propoxy, and butoxy groups.

[0013] According to some preferred embodiments of the present invention, the mitochondrial fluorescent probe comprises (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexylquinoline iodide.

[0014] In this invention, R 1 Selected from hydrogen; R 2 When X is selected from methoxy; and X is selected from iodine, the resulting mitochondrial fluorescent probe independent of mitochondrial membrane potential is (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide.

[0015] A method for preparing a mitochondrial fluorescent probe according to a second aspect embodiment of the present invention, the method comprising the following steps:

[0016] S1. 4-methylquinoline (Formula II) and halohexane (Formula III) are placed in a solvent and refluxed to generate 1-n-hexyl-4-methylquinoline salt (Formula IV);

[0017] S2. Mix 1-n-hexyl-4-methylquinoline salt, indole-3-carboxaldehyde (Formula V) and catalyst, reflux and then remove impurities;

[0018] The catalyst includes piperidine.

[0019] According to some embodiments of the present invention, the solvent includes ethanol.

[0020] According to some embodiments of the present invention, the molar ratio of indole-3-carboxaldehyde to 4-methylquinoline is 1:(1.0 to 2.0).

[0021] According to some embodiments of the present invention, in step S2, the reflux reaction time is 6h to 24h.

[0022]

[0023] According to some preferred embodiments of the present invention, the indole-3-carboxaldehyde is selected from 5-methoxy-3-formylindole; the halohexane is selected from iodohexane. The method for preparing the mitochondrial fluorescent probe using 5-methoxy-3-formylindole and iodohexane as reactants is as follows:

[0024] S01. Prepare an ethanolic mixed solution of 4-methylquinoline and iodohexane;

[0025] S02. Heat and stir under reflux for three days;

[0026] S03. Add an ethanolic solution of 5-methoxy-3-formylindole;

[0027] S04. Add the catalyst piperidine to the ethanol mixture, heat the ethanol mixture with piperidine to reflux at 85°C for one day, evaporate the excess solvent, and slowly cool to room temperature to obtain the organic solid product to be purified.

[0028] S05. The organic solid product to be purified is subjected to column chromatography for purification. The eluent is dichloromethane / methanol. After drying, a purple-red powder is obtained. The purple-red powder is a mitochondrial fluorescent probe (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide that is independent of mitochondrial membrane potential.

[0029] Application of the mitochondrial fluorescent probe according to a third aspect of the present invention in any one of A1) to A4):

[0030] A1) Markers and / or localization of mitochondria for non-disease treatment diagnosis;

[0031] A2) Monitoring mitochondrial-related life activities for non-disease treatment diagnosis;

[0032] A3) Prepare products for labeling and / or locating mitochondria;

[0033] A4) Prepare products for monitoring relevant life activities of mitochondria.

[0034] According to some embodiments of the present invention, the mitochondria include mitochondria in cells with reduced mitochondrial membrane potential, such as fixed cells.

[0035] The application of the mitochondrial fluorescent probe according to a fourth aspect of the present invention in the preparation of products targeting mitochondria.

[0036] The application of the mitochondrial fluorescent probe according to a fifth aspect of the present invention in the preparation of products for mitochondrial imaging.

[0037] The mitochondrial fluorescent probe provided by this invention is independent of mitochondrial membrane potential, enabling its application in imaging mitochondria in cells, particularly in imaging biological samples with reduced or even lost mitochondrial membrane potential, including the morphology, number, and distribution of mitochondria in fixed cells.

[0038] The indoquinoline hexyl salt fluorescent probe described in this invention is a novel type of mitochondrial-specific fluorescent probe molecule in cells. Compared with existing mitochondrial fluorescent probes, the probe described in this invention is unique in that it does not rely on a high mitochondrial membrane potential. It can image biological samples with reduced or even lost mitochondrial membrane potential, including the morphology, number, and distribution of mitochondria in fixed cells. It does not fluoresce in water / PBS / culture medium (without serum and antibiotics) solution, but emits red fluorescence after binding to mitochondria in cells. At the same time, it has good membrane permeability and good counterstain compatibility.

[0039] The mitochondrial fluorescent probe provided by this invention, which is independent of mitochondrial membrane potential, can be used as a fluorescent probe to label the morphology, number and distribution of mitochondria in cells. It can provide a simple and intuitive biological detection reagent for mitochondrial-related physiological and pathological research and clinical diagnosis, with wide applications and good effects. Attached Figure Description

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0041] Figure 1 This is a confocal fluorescence micrograph of HeLa cancer cells after co-staining (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide with mitochondrial deep red fluorescent probe (MitoTracker Deep Red) and mitochondrial green fluorescent probe (MitoTracker Green).

[0042] Figure 2 This is a confocal fluorescence micrograph of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide, along with mitochondrial deep red and mitochondrial green fluorescent probes, after co-staining HeLa cells treated with CCCP.

[0043] Figure 3 This is a confocal fluorescence micrograph of normal HEK293 fixed cells after co-staining with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide and the mitochondrial deep red fluorescent probe (MitoTracker Deep Red).

[0044] Figure 4This is a confocal fluorescence micrograph of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide, a molecule with a structure similar to (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide, used to stain HeLa fixed cells and CCCP-treated cells. Detailed Implementation

[0045] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0046] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0047] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0048] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0049] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0050] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0051] Example 1

[0052] This embodiment discloses the synthesis of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide, the specific steps of which are as follows:

[0053] First, 200 μL of 4-methylquinoline and 236 μL of iodohexane were dissolved in ethanol and refluxed at 85 °C with stirring for three days. Then, an ethanol solution containing 0.263 g of 5-methoxy-3-formylindole was added, and after stirring evenly, 4-5 drops of piperidine were added, and the solution gradually turned red. After refluxing for one day, excess solvent was evaporated, and the product was purified by column chromatography using dichloromethane / methanol as eluent to obtain a purple-red powder with a yield of about 26%.

[0054] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 12.09 (s, 1H), 9.12 (d, J=4.0Hz, 1H), 8.96 (d, J=8.0Hz, 1H), 8.62 (d, J=16.0Hz, 1H), 8.44 (m, 3H), 8.18 (t, J=8.0Hz, 1H), 7.98 (m, 2H), 7.69 (s, 1H), 7.42 (d, J=8.0Hz, 1H), 6.91 (dd, J=4.0, 8.0Hz, 1H), 4.85 ( t, J=8.0Hz, 2H), 3.90 (s, 3H), 1.92 (m, 2H), 1.32 (m, 6H), 0.86 (t, J=8.0Hz, 3H). 13 C NMR (400MHz, DMSO-d6), δ (ppm): 155.72, 154.17, 146.12, 139.13, 138.29, 135.13, 132.63, 132.44, 128.80, 127.24, 126. 96, 126.14, 119.25, 115.07, 113.92, 113.79, 112.96, 112.82, 102.75, 56.17, 56.11, 31.17, 29.67, 25.97, 22.45, 14.32. HRMS: calculated 385.23, found 385.23.

[0055] To develop more compounds with similar functions, the molecular core skeleton remains unchanged in this invention, R 1 It can also be selected from any C1-C4 alkyl group, R 2 It can also be selected from any one of hydrogen, C1-C4 alkyl, and C1-C4 alkoxy groups, and X is selected from any one of halogen atoms, BF4, and ClO4. According to the inventors' research, the function of mitochondrial localization independent of mitochondrial membrane potential in this invention is determined by the conjugated organic cation group, and the interaction with the anion X... - Irrelevant; R 1 Selected from hydrogen or any C1-C4 alkyl group; R 2 The molecules can be selected from any of hydrogen, C1-C4 alkyl groups, and C1-C4 alkoxy groups. Changes within this range do not affect the function of the molecule. However, the length of the carbon chain attached to the N-chain of the quinoline has a significant impact on the molecule's targeting, protein binding, and molecular aggregation state. For example, indolemethylquinoline salt binds to RNA in fixed cells, while indolehexylquinoline salt of this invention is located in mitochondria.

[0056] Test Example 1

[0057] HeLa and HEK293 cell cultures:

[0058] Cancer cells HeLa and normal cells HEK293 were cultured in a saturated humidity incubator at 37°C and 5% CO2 using medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and passaged every 2-3 days.

[0059] Once the cells reach the logarithmic growth phase, transfer them to confocal dishes for culture: Wash the confluent T25 cells in the culture flask with PBS, then digest with 1 mL of trypsin for 1–2 minutes (0.25% trypsin for HeLa, 0.025% trypsin for HEK293). Remove the trypsin, add fresh culture medium, mix thoroughly, and count the cells. Control the cell density by adjusting the amount of culture medium added, aiming for a final cell concentration of 1 x 10⁻⁶ cells / mL. 5 Then, the cells were seeded into confocal glass-bottom culture dishes and placed in a 5% CO2 incubator for culture until the cells grew to a coverage of about 70% for cell experiments.

[0060] Test Example 2

[0061] Staining observation of HeLa fixed cells with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide: After washing the HeLa cells in the confocal dish prepared in Test Example 1 twice with PBS, the following staining steps were performed: (1) Incubation with 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min, followed by washing with PBS; (2) Fixation of cells with 4% paraformaldehyde solution for 20 min, followed by washing with PBS; (3) Incubation with 1 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide fluorescent probe solution for 30 min, followed by washing with PBS; (4) Incubation with 0.5 μM commercial mitochondrial green fluorescent probe solution for 30 min, followed by washing with PBS. The stained cell samples were observed using a confocal fluorescence microscope for multi-channel fluorescence co-localization.

[0062] The results are as follows Figure 1 As shown, Figure 1 (A) is the red fluorescence image of the probe molecule of this invention. Figure 1 (B) is a fluorescence image of a commercial mitochondrial deep red fluorescent probe. Figure 1 (C) is a fluorescence image of a commercial mitochondrial green fluorescent probe. Figure 1 (D) is Figure 1 (A) and Figure 1 (B) overlay image, Figure 1 (E) is Figure 1 (C) and Figure 1 (B) overlay diagram. Figure 1 The high overlap between (A) and 1(B) indicates that the fluorescence of the probe molecule of this invention is completely distributed in the mitochondria; and Figure 1 The fluorescence of (C) and 1(B) does not overlap well, indicating that commercial mitochondrial fluorescent probes cannot be used for fixed cells. This result demonstrates that (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide, independent of mitochondrial membrane potential, can be used for mitochondrial staining and fluorescence imaging of fixed cancer cells.

[0063] Test Example 3

[0064] Staining observation of (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide on CCCP-treated HeLa cells:

[0065] After washing the HeLa cells in the confocal dish prepared in Test Example 1 twice with PBS, the following treatments and staining were performed: (1) incubation with 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min, followed by washing with PBS; (2) incubation with 20 μM mitochondrial proton-binding agent CCCP solution for 3 h, followed by washing with PBS; (3) incubation with 1 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide fluorescent probe solution for 30 min, followed by washing with PBS; (4) incubation with 0.5 μM commercial mitochondrial green fluorescent probe solution for 30 min, followed by washing with PBS. The stained cell samples were then observed using a confocal fluorescence microscope for multi-channel fluorescence co-localization.

[0066] The results are as follows Figure 2 As shown, Figure 2 (A) is the red fluorescence image of the probe molecule of this invention. Figure 2 (B) is a fluorescence image of a commercial mitochondrial deep red fluorescent probe. Figure 2 (C) is a fluorescence image of a commercial mitochondrial green fluorescent probe. Figure 2 (D) is Figure 2 (A) and Figure 2 (B) overlay image, Figure 2 (E) is Figure 2 (C) and Figure 2 (B) overlay diagram. Figure 2 The high overlap between (A) and 2(B) indicates that the fluorescence of the probe molecule of this invention is completely distributed in the mitochondria; and Figure 2 The fluorescence in (C) and 2(B) does not overlap well, indicating that commercial mitochondrial fluorescent probes cannot be used for mitochondrial staining in cells after loss of mitochondrial membrane potential. This result demonstrates that (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide can be used for mitochondrial staining and fluorescence imaging in live cells after loss of mitochondrial membrane potential.

[0067] Test Example 4

[0068] Observation of staining of HEK293 cells with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide

[0069] HEK293 cells in the confocal dish prepared in Test Example 1 were washed twice with PBS, and then subjected to the following treatments and staining: (1) incubated with 0.5 μM commercial mitochondrial deep red fluorescent probe solution for 30 min, followed by washing with PBS; (2) fixed with 4% paraformaldehyde solution for 20 min, followed by washing with PBS; (3) incubated with 1 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-hexylquinoline iodide fluorescent probe solution for 30 min, followed by washing with PBS. The stained cell samples were then observed using a confocal fluorescence microscope for multichannel fluorescence co-localization.

[0070] The results are as follows Figure 3 As shown, Figure 3 (A) is the red fluorescence image of the probe molecule of this invention. Figure 3 (B) is a fluorescence image of a commercial mitochondrial deep red fluorescent probe. Figure 3 (C) is Figure 3 (A) and Figure 3 (B) overlay diagram. Figure 1 The good overlap between (A) and 1(B) indicates that the fluorescence of the probe molecule of the present invention is completely distributed in the mitochondria, proving that (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide can be used for mitochondrial staining and fluorescence imaging of normal cells that have lost mitochondrial membrane potential and have been fixed.

[0071] Test Example 5

[0072] Staining observation of HeLa cells with mitochondrial membrane potential loss using (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide, a structurally similar control molecule.

[0073] Two groups of HeLa cells in the confocal dish prepared in Test Example 1 were washed twice with PBS. One group was treated and stained as follows: (1) cells were fixed with 4% paraformaldehyde solution for 20 min and washed with PBS; (2) cells were incubated with 10 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide solution for 30 min and washed with PBS. The other group was treated and stained as follows: (1) cells were incubated with 10 μM (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide solution for 30 min and washed with PBS; (2) cells were incubated and preserved with 20 μM mitochondrial proton-binding agent CCCP solution. The two groups of cell samples were observed using a confocal fluorescence microscope.

[0074] The results are as follows Figure 4 As shown, Figure 4(A) is a fluorescent photograph of fixed cells stained with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide. Figure 4 (B) is a fluorescence image of CCCP-treated cells stained with (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide. As can be seen from the image, the fluorescence distribution in both groups of cells is concentrated in the cytoplasm and nucleolus, significantly different from the distribution in the mitochondria. This indicates that (E)-4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-methylquinoline iodide, which has a similar molecular structure to the probe of this invention, cannot be used for mitochondrial fluorescence imaging in cells with lost mitochondrial membrane potential.

[0075] In summary, the mitochondrial fluorescent probe provided in this application, which is independent of mitochondrial membrane potential and has applications in mitochondrial imaging observation for non-diagnostic and therapeutic methods, is unique in that it does not rely on a high mitochondrial membrane potential compared to other mitochondrial fluorescent probes. It can target and image biological samples with reduced or even lost mitochondrial membrane potential, including the morphology, number, and distribution of mitochondria in fixed cells. It does not fluoresce in water / PBS / culture medium (without serum and antibiotics) solution, but emits red fluorescence after binding with mitochondria in cells. At the same time, it has good membrane permeability and good counterstain compatibility.

[0076] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mitochondrial fluorescent probe independent of mitochondrial membrane potential, characterized in that, The mitochondrial fluorescent probe, which is independent of mitochondrial membrane potential, has the following characteristics: Formula I; Wherein, the R 1 It is hydrogen; the R 2 It is a methoxy group; X includes any one of halogen atoms, BF4, and ClO4.

2. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1, characterized in that, The halogen atom is selected from any one of iodine, bromine, and chlorine.

3. The mitochondrial fluorescent probe independent of mitochondrial membrane potential according to claim 1 or 2, characterized in that, The mitochondrial fluorescent probes that are independent of mitochondrial membrane potential include (E) -4-(2-(5-methoxy-1H-indole-3-)vinyl)-1-n-hexylquinoline iodide.

4. A method for preparing a mitochondrial fluorescent probe independent of mitochondrial membrane potential as described in any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: S1. 4-methylquinoline and halohexane are placed in a solvent and refluxed to generate 1-n-hexyl-4-methylquinoline salt; S2. Mix 1-n-hexyl-4-methylquinoline salt, indole-3-carboxaldehyde and catalyst, reflux and then remove impurities; The catalyst includes piperidine.

5. The application of a mitochondrial fluorescent probe, independent of mitochondrial membrane potential, as described in any one of claims 1 to 3, in any one of A1) to A4): A1) Markers and / or localization of mitochondria for non-disease treatment diagnosis; A2) Monitoring mitochondrial-related life activities for non-disease treatment diagnosis; A3) Prepare products for labeling and / or locating mitochondria; A4) Prepare products for monitoring relevant life activities of mitochondria.

6. The use of a mitochondrial fluorescent probe, independent of mitochondrial membrane potential, as described in any one of claims 1 to 3, in the preparation of products targeting mitochondria.

7. The use of a mitochondrial fluorescent probe, independent of mitochondrial membrane potential, as described in any one of claims 1 to 3, in the preparation of products for mitochondrial imaging.

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