A cell mitochondrial organic fluorescent probe and a preparation method thereof

Abstract

The application relates to the technical field of biological materials, and mainly relates to a cell mitochondrial organic fluorescent probe and a preparation method thereof. The cell mitochondrial organic fluorescent probe comprises the following structural formula: The cell mitochondrial organic fluorescent probe provided by the application has the effect of targeting cell mitochondria, accurate positioning, good biocompatibility, AIE characteristics and low cytotoxicity, does not need washing operation, can avoid water washing, makes the dyeing process more convenient and economical, is an excellent mitochondrial imaging material, and can effectively realize rapid targeted positioning and imaging of mitochondria.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and mainly to a mitochondrial organic fluorescent probe and its preparation method. Background Technology

[0002] Mitochondria, as the power source of eukaryotic cells, play a crucial role in converting oxygen and nutrients into adenosine triphosphate (ATP), which powers cellular metabolic activities. Many types of human diseases are currently attributed to mitochondrial dysfunction, such as myopathy, diabetes, and cardiovascular disease. Therefore, mitochondrial targeting and imaging are of great significance to biological and medical sciences. The design and synthesis of mitochondrial fluorescent probes with diverse fluorescence colors and signal-to-noise ratios has become a research area of ​​great interest to biologists and chemists.

[0003] Researchers have developed various fluorescent probes based on organic nanostructures in recent years. With the advent of aggregation-induced emission (AIE) molecules, scientists have developed numerous AIE probes for imaging cells and organs due to their excellent photostability and high signal-to-noise ratio. AIE probe molecules emit fluorescence under aggregation conditions, making them a better choice for biosensing and bioimaging. Some AIE probes can target certain subcellular organelles, such as mitochondria and lysosomes. Researchers have already reported AIE molecules targeting mitochondria, applying tetraphenylethylene-indole (TPE-indo) to mitochondrial targeting in living cells. TPE-indo is a red fluorescent mitochondrial probe that can monitor changes in mitochondrial membrane potential and exhibits superior bioimaging capabilities compared to other reported AIE probes. However, some existing probes still suffer from complex synthesis steps, poor biocompatibility, poor photostability, and insufficient targeting precision, failing to meet the requirements for high-fidelity, real-time, and dynamic imaging of mitochondria. Therefore, the research and development of novel mitochondrial probes with high sensitivity, high selectivity, good photostability, and strong targeting is of great significance. Thus, existing technologies still need improvement and development. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide a cell mitochondrial organic fluorescent probe and its preparation method, which aims to solve the problems of complex synthesis steps, poor biocompatibility, poor photostability and insufficient targeting of existing mitochondrial probes.

[0005] The technical solution of this application is as follows: In a first aspect, this application provides a cellular mitochondrial organic fluorescent probe, comprising the following structural formula: .

[0006] The organic fluorescent probe for mitochondria provided in this application has the effect of targeting mitochondria, with precise positioning, good biocompatibility, and no need for washing, making it an excellent mitochondrial imaging material.

[0007] Secondly, this application also provides a method for preparing a cellular mitochondrial organic fluorescent probe as described in the first aspect, comprising the following steps: 4-Methylquinoline iodide and 4-(1,2,2-triphenylvinyl)benzaldehyde were dissolved in a solvent, pyrrolidine was added, and the mixture was reacted at 20-40°C for 12-48 hours. The solvent was removed to obtain the residue, which was then purified by silica gel column chromatography using dichloromethane / anhydrous methanol as the elution buffer to obtain the mitochondrial organic fluorescent probe.

[0008] Furthermore, the molar ratio of 4-methylquinoline iodide to 4-(1,2,2-triphenylvinyl)benzaldehyde is 40-70:58; The concentration of the 4-methylquinoline iodide is 0.01~0.10 mol / L; the concentration of the 4-(1,2,2-triphenylvinyl)benzaldehyde is 0.01~0.10 mol / L; and the volume ratio of the solvent to pyrrolidine is 50-100:1.

[0009] Furthermore, the molar ratio of 4-methylquinoline iodide to 4-(1,2,2-triphenylvinyl)benzaldehyde is 53:58; The concentration of the 4-methylquinoline iodide is 0.0353 mol / L; the concentration of the 4-(1,2,2-triphenylvinyl)benzaldehyde is 0.0387 mol / L; and the volume ratio of the solvent to pyrrolidine is 75:1.

[0010] Furthermore, the solvent is anhydrous ethanol; The volume ratio (v / v) of the dichloromethane / anhydrous methanol is 12:1-3:1.

[0011] Further, the steps for preparing the 4-(1,2,2-triphenylvinyl)benzaldehyde include: Bromotriphenylethylene and 4-formylphenylboronic acid were dissolved in toluene, and tetrabutylammonium bromide was added to obtain a mixed solution. Then, an aqueous solution of potassium carbonate was added to obtain a reaction solution. The reaction was carried out at room temperature under argon atmosphere for 0.5-2 hours. Then, tetra(triphenylphosphine)palladium was added and the mixture was heated to 70-100℃ and reacted for 12-48 hours. After cooling the reaction solution to room temperature, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate to obtain an organic phase. The organic phase was dried with Na2SO4 and then evaporated to dryness to obtain a crude product. The crude product was purified by silica gel column chromatography using n-hexane / dichloromethane as eluent to obtain 4-(1,2,2-triphenylvinyl)benzaldehyde.

[0012] Further, the concentration of the bromotriphenylethylene dissolved in toluene is 0.1~1 mol / L; the concentration of the 4-formylphenylboronic acid dissolved in toluene is 0.1~0.4 mol / L; and the concentration of the tetrabutylammonium bromide dissolved in toluene is 0.01~0.1 mol / L.

[0013] Further, the concentration of the potassium carbonate aqueous solution is 1-3 mol / L, and its volume percentage in the reaction solution is 15-35%; the concentration of the tetra(triphenylphosphine)palladium in the reaction solution is 2 × 10⁻⁶. -3 -4×10 -3 mol / L.

[0014] Further, the step of preparing 4-methylquinoline iodide includes: 4-Methylquinoline was dissolved in benzene, and excess iodomethane was added at room temperature to obtain a mixed solution. The mixed solution was heated to reflux, then cooled to room temperature, the precipitate was filtered out, washed with benzene, and dried to obtain 4-methylquinoline iodide.

[0015] Further, the concentration of 4-methylquinoline is 0.02~0.1 mol / L; the concentration of iodomethane is 0.1~0.4 mol / L; The temperature at which the heating reaches reflux is 85-100℃; The reflux time is 0.5-3 hours.

[0016] Beneficial effects: The organic fluorescent probe for mitochondria provided in this application has the effect of targeting mitochondria, with precise positioning and good biocompatibility. Its AIE properties and low cytotoxicity eliminate the need for washing operations, making the staining process more convenient and economical. It is an excellent mitochondrial imaging material that can effectively achieve rapid targeting and imaging of mitochondria. Attached Figure Description

[0017] Figure 1 This is a chemical synthesis route diagram for the organic fluorescent probe for mitochondria in this application.

[0018] Figure 2 This is the fluorescence spectrum of the cellular mitochondrial organic fluorescent probe of this application.

[0019] Figure 3 This is an image of mitochondria by the organic fluorescent probe of mitochondria in Example 1 of this application.

[0020] Figure 4 Images showing the cellular mitochondrial organic fluorescent probe of this application before and after washing with commercial probes. Detailed Implementation

[0021] This application provides a mitochondrial organic fluorescent probe and its preparation method. To make the purpose, technical solution, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0022] This application provides a cellular mitochondrial organic fluorescent probe, comprising the following structural formula: .

[0023] The organic fluorescent probe for mitochondria provided in this application has the effect of targeting mitochondria, with precise positioning, good biocompatibility, and no need for washing, making it an excellent mitochondrial imaging material.

[0024] This application also provides a method for preparing the above-mentioned organic fluorescent probe for mitochondria, comprising the following steps: Preparation of 4-methylquinoline iodide; Preparation of 4-(1,2,2-triphenylvinyl)benzaldehyde; Organic fluorescent probes for cell mitochondria were prepared using 4-methylquinoline iodide and 4-(1,2,2-triphenylvinyl)benzaldehyde.

[0025] More specifically, such as Figure 1 As shown ( Figure 1 In this text, Mel represents iodomethane, benzene represents benzene, reflux represents reflux, pyrrolidine represents pyrrolidine, and EtOH represents anhydrous ethanol. The preparation method of the organic fluorescent probe for mitochondria includes the following steps: S1. Dissolve 4-methylquinoline in benzene, add excess iodomethane at room temperature to obtain a mixed solution, heat the mixed solution to reflux, cool to room temperature, filter out the precipitate, wash with benzene, and dry to obtain 4-methylquinoline iodide.

[0026] Further, in the mixed solution of step S1, the concentration of 4-methylquinoline is 0.02~0.1 mol / L, preferably 0.02~0.08 mol / L, and most preferably 0.07 mol / L; the concentration of iodomethane is 0.1~0.4 mol / L, preferably 0.1~0.3 mol / L, and most preferably 0.24 mol / L; the temperature for heating to reflux is 85-100℃, preferably 90-100℃, and most preferably 95℃; the reflux time is 0.5-3 hours, preferably 0.5-2 hours, and most preferably 1 hour.

[0027] With the preparation method provided in this application and appropriate concentration, the yield of 4-methylquinoline iodide can reach 95%.

[0028] S2. Dissolve triphenylethylene bromide and 4-formylphenylboronic acid in toluene, then add tetrabutylammonium bromide (TBAB) to obtain a mixed solution, then add potassium carbonate aqueous solution to obtain a reaction solution, and react at room temperature under argon atmosphere for 0.5-2 hours. Then add tetra(triphenylphosphine)palladium (Pd(PPh3)4) and heat to 70-100℃ to react for 12-48 hours. After cooling the reaction solution to room temperature, pour water into the reaction solution, and extract three times with ethyl acetate to obtain an organic phase. Add Na2SO4 to dry the organic phase and then evaporate to dryness to obtain 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO).

[0029] The rotary evaporation can be performed using a rotary evaporator. After preparation, the product can be used as a crude product, eluted with hexane / dichloromethane, and purified by silica gel column chromatography to obtain 4-(1,2,2-triphenylvinyl)benzaldehyde with higher purity. Further, the volume ratio (v / v) of hexane / dichloromethane is 10:1-2:1, preferably 8:1-3:1, and most preferably 4:1.

[0030] Further, in step S2, the concentration of triphenyl bromoethylene dissolved in toluene is 0.1~1 mol / L, preferably 0.1~0.7 mol / L, and most preferably 0.17 mol / L; the concentration of 4-formylphenylboronic acid dissolved in toluene is 0.1~0.4 mol / L, preferably 0.1~0.3 mol / L, and most preferably 0.25 mol / L; and the concentration of tetrabutylammonium bromide dissolved in toluene is 0.01~0.1 mol / L, preferably 0.01~0.08 mol / L, and most preferably 0.05 mol / L. The addition of tetrabutylammonium bromide as a phase transfer catalyst promotes the chemical reaction in the toluene / water mixture.

[0031] Further, in step S2, the concentration of the potassium carbonate aqueous solution is 1-3 mol / L, preferably 1-2.5 mol / L, and most preferably 2 mol / L. The volume percentage of the potassium carbonate aqueous solution in the reaction solution is 15-35%, preferably 20-30%, and most preferably 25%.

[0032] Furthermore, in the reaction solution of step S2, the concentration of tetra(triphenylphosphine)palladium is 2 × 10⁻⁶. -3 -4×10 -3 mol / L, preferably 2×10 -3 -3×10 -3 mol / L, the optimal value is 2.5 × 10⁻⁶ mol / L. -3 mol / L. Among them, tetra(triphenylphosphine)palladium, as a catalyst for the Suzuki coupling reaction, can promote the coupling of triphenylethylene bromide and 4-formylphenylboronic acid.

[0033] Further, in step S2, the reaction time at room temperature and under argon atmosphere is 0.5-2 hours, preferably 0.5-1 hour, and most preferably 0.5 hours; the heating temperature after adding tetra(triphenylphosphine)palladium is 70-100°C, preferably 80-95°C, and most preferably 90°C; the reaction time is 12-48 hours, preferably 12-36 hours, and most preferably 24 hours.

[0034] With the preparation method provided in this application and appropriate concentration, the yield of 4-(1,2,2-triphenylvinyl)benzaldehyde can reach 95%.

[0035] S3. Dissolve the 4-methylquinoline iodide obtained in step S1 and the 4-(1,2,2-triphenylvinyl)benzaldehyde obtained in step S2 in a solvent, add pyrrolidine, and react at 20-40℃ for 12-48 hours. Remove the solvent to obtain the residue, purify the residue by silica gel column chromatography, elute with dichloromethane / anhydrous methanol, collect the eluent and concentrate to obtain the purified mitochondrial organic fluorescent probe.

[0036] The solvent is anhydrous ethanol.

[0037] Further, in step S3, the molar ratio of 4-methylquinoline iodide to 4-(1,2,2-triphenylvinyl)benzaldehyde is 40-70:58, preferably 50-55:58, and most preferably 53:58; the concentration of 4-methylquinoline iodide is 0.01-0.1 mol / L, preferably 0.01-0.08 mol / L, and most preferably 0.0353 mol / L; the concentration of 4-(1,2,2-triphenylvinyl)benzaldehyde is 0.01-0.1 mol / L, preferably 0.01-0.07 mol / L, and most preferably 0.0387 mol / L; the volume ratio (v / v) of solvent to pyrrolidine is 50-100:1, preferably 70-80:1, and most preferably 75:1.

[0038] Further, in step S3, the reaction temperature is 20-40℃, preferably 20-35℃, and most preferably 30℃; the reaction time is 12-48 hours, preferably 12-36 hours, and most preferably 24 hours; the volume ratio (v / v) of dichloromethane / anhydrous methanol is 12:1-3:1, preferably 10:1-4:1, and most preferably 5:1.

[0039] The preparation method described in this application enables the fabrication of an organic fluorescent probe that targets and binds to mitochondria. This probe generates a strong signal upon excitation with 405 nm light and can be effectively used as a fluorescent probe for mitochondria. Specifically, this mitochondrial organic fluorescent probe comprises a structure where 4-methylquinoline is coupled to tetraphenylethylene via a double bond (TPE-Quino). Due to its aggregation-induced emission effect, this mitochondrial organic fluorescent probe eliminates the need for washing during cell imaging, making it more convenient than typical quinoline-coupled fluorescent probes.

[0040] The following specific examples provide further details.

[0041] The sources of some of the reagents and instruments used in the embodiments of this application are as follows: 4-Methylquinoline, iodomethane, triphenyl bromoethylene, 4-formylphenylboronic acid, tetrabutylammonium bromide, potassium carbonate, tetra(triphenylphosphine)palladium, pyrrolidine: purchased from Beijing Bailingwei Company; Ethyl acetate, dichloromethane, toluene, methanol, and n-hexane: purchased from Sinopharm Group; Deionized water (18.2 M.Wcm): Provided via a Milli-Q purification system; HeLa cells: purchased from the Shanghai Cell Bank, Chinese Academy of Sciences; Eagle's medium: purchased from Dulbecco's modified Eagle's medium, DMEM; MitoTracker FM: Purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0042] Rotary evaporator: purchased from IKA, Germany, model IKA RV 10; Continuous spectrum multi-functional microplate reader: Tecan Infinite M200; CO2 incubator: Sanyo Corporation; Confocal microscope: Zeiss.

[0043] Example 1 The preparation method of the cellular mitochondrial organic fluorescent probe in Example 1 includes the following steps: (1) Dissolve 4-methylquinoline (250 mg, 1.75 mmol) in benzene (25 mL), add excess iodomethane (853 mg, 6 mmol) at room temperature to obtain a mixed solution, then heat the mixed solution to 95 °C and reflux for 1 hour, then cool to room temperature, filter out the precipitate, wash with benzene, and dry to obtain a yellow solid as 4-methylquinoline iodide (474 ​​mg, yield: 95%).

[0044] (2) Synthesis of 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO): Triphenyl bromoethylene (1.68 g, 5 mmol) and 4-formylphenylboronic acid (1.13 g, 7.5 mmol) were dissolved in toluene (30 mL), and tetrabutylammonium bromide (0.48 g, 1.5 mmol) was added to obtain a mixture. Then, a 2 mol / L potassium carbonate aqueous solution (10 mL) was added, and the reaction was carried out at room temperature under argon protection for 0.5 hours. Then, tetra(triphenylphosphine)palladium (0.067 g, 100 × 10⁻⁶ mmol) was added to the reaction solution. -3 After heating to 90°C for 24 hours, the reaction solution was cooled to room temperature, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate to obtain the organic phase. Na2SO4 was added to dry the organic phase, and the mixture was evaporated to dryness using a rotary evaporator. The product was eluted with n-hexane / dichloromethane (v / v=4:1) and purified by silica gel column chromatography. The resulting yellow solid was 4-(1,2,2-triphenylvinyl)benzaldehyde (1.71 g, yield: 95%).

[0045] (3) Synthesis of compound TPE-Quino: 4-methylquinoline iodide (151 mg, 0.53 mmol) obtained in step (1) and 4-(1,2,2-triphenylvinyl)benzaldehyde (210 mg, 0.58 mmol) obtained in step S2 were dissolved in anhydrous ethanol (15 mL), pyrrolidine (0.2 mL) was added, and the mixture was reacted at 30 °C for 24 hours. The solvent was removed by vacuum evaporation to obtain the residue. The residue was purified by silica gel column chromatography using dichloromethane / anhydrous methanol (v / v = 5:1) as the elution buffer. The orange solid obtained was the mitochondrial organic fluorescent probe (TPE-Quino) (110 mg, yield: 53%).

[0046] The fluorescence spectrum of the mitochondrial organic fluorescent probe (TPE-Quino) prepared in Example 1 is shown below. Figure 2 As shown.

[0047] HeLa cells were grown overnight on confocal culture dishes. After adding 5 μmol / L of the mitochondrial organic fluorescent probe (TPE-Quino) prepared in Example 1, and incubating for 20 minutes in a cell culture incubator at 37°C with 5% CO2, the cells were directly imaged using a confocal microscope (Zesis LSM710) without washing. The mitochondrial organic fluorescent probe (TPE-Quino) localized and imaged almost all HeLa cells. When excited with 405 nm excitation light, we observed a very strong signal from the mitochondrial regions of live cells. To verify this finding, we co-localized HeLa cells using the far-red fluorescent mitochondrial staining dye MitoTrackerFM and the mitochondrial organic fluorescent probe (TPE-Quino) prepared in Example 1. The localization map is shown below. Figure 3 As shown in the colocalization images, TPE-Quino can specifically image mitochondria in living cells. The Pearson colocalization coefficient also confirms that the mitochondrial organic fluorescent probe (TPE-Quino) can precisely target mitochondria. The same imaging procedure was repeated using a commercially available mitochondrial dye (MitoTracker) for comparison. A comparison of the mitochondrial organic fluorescent probe prepared in this application embodiment with the commercially available mitochondrial dye before and after washing is shown below. Figure 4 As shown, compared with commercially available mitochondrial dyes, this application maintains good clarity both before and after washing. The prepared mitochondrial organic fluorescent probe (TPE-Quino) does not require washing due to its AIE properties and low cytotoxicity, making the staining process more convenient and economical.

[0048] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A cell mitochondrial organic fluorescent probe, characterized in that, Includes the following structural formulas: 。 2. A method for preparing the cell mitochondrial organic fluorescent probe according to claim 1, characterized in that, Includes the following steps: 4-Methylquinoline iodide and 4-(1,2,2-triphenylvinyl)benzaldehyde were dissolved in a solvent, pyrrolidine was added, and the mixture was reacted at 20-40°C for 12-48 hours. The solvent was removed to obtain the residue, which was then purified by silica gel column chromatography using dichloromethane / anhydrous methanol as the elution buffer to obtain the mitochondrial organic fluorescent probe.

3. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 2, characterized in that, The molar ratio of 4-methylquinoline iodide to 4-(1,2,2-triphenylvinyl)benzaldehyde is 40-70:58; The concentration of the 4-methylquinoline iodide is 0.01~0.10 mol / L; the concentration of the 4-(1,2,2-triphenylvinyl)benzaldehyde is 0.01~0.10 mol / L; and the volume ratio of the solvent to pyrrolidine is 50-100:

1.

4. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 3, characterized in that, The molar ratio of 4-methylquinoline iodide to 4-(1,2,2-triphenylvinyl)benzaldehyde is 53:58; The concentration of the 4-methylquinoline iodide is 0.0353 mol / L; the concentration of the 4-(1,2,2-triphenylvinyl)benzaldehyde is 0.0387 mol / L; and the volume ratio of the solvent to pyrrolidine is 75:

1.

5. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 2, characterized in that, The solvent is anhydrous ethanol; The volume ratio (v / v) of the dichloromethane / anhydrous methanol is 12:1-3:

1.

6. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 2, characterized in that, The steps for preparing the 4-(1,2,2-triphenylvinyl)benzaldehyde include: Bromotriphenylethylene and 4-formylphenylboronic acid were dissolved in toluene, and tetrabutylammonium bromide was added to obtain a mixed solution. Then, an aqueous solution of potassium carbonate was added to obtain a reaction solution. The reaction was carried out at room temperature under argon atmosphere for 0.5-2 hours. Then, tetra(triphenylphosphine)palladium was added and the mixture was heated to 70-100℃ and reacted for 12-48 hours. After cooling the reaction solution to room temperature, water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate to obtain an organic phase. The organic phase was dried with Na2SO4 and then evaporated to dryness to obtain a crude product. The crude product was purified by silica gel column chromatography using n-hexane / dichloromethane as eluent to obtain 4-(1,2,2-triphenylvinyl)benzaldehyde.

7. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 6, characterized in that, The concentration of the bromotriphenylethylene dissolved in toluene is 0.1~1 mol / L; the concentration of the 4-formylphenylboronic acid dissolved in toluene is 0.1~0.4 mol / L; and the concentration of the tetrabutylammonium bromide dissolved in toluene is 0.01~0.1 mol / L.

8. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 6, characterized in that, The concentration of the potassium carbonate aqueous solution is 1-3 mol / L, the volume ratio in the reaction solution is 15-35%, and the concentration of the tetrakis(triphenylphosphine)palladium in the reaction solution is 2x10 -3 -4x10 -3 mol / L.

9. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 2, characterized in that, The steps for preparing the 4-methylquinoline iodide salt include: 4-Methylquinoline was dissolved in benzene, and excess iodomethane was added at room temperature to obtain a mixed solution. The mixed solution was heated to reflux, then cooled to room temperature, the precipitate was filtered out, washed with benzene, and dried to obtain 4-methylquinoline iodide.

10. The method for preparing the cellular mitochondrial organic fluorescent probe according to claim 9, characterized in that, The concentration of 4-methylquinoline is 0.02~0.1 mol / L; the concentration of iodomethane is 0.1~0.4 mol / L; The temperature at which the heating reaches reflux is 85-100℃; The reflux time is 0.5-3 hours.

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