Pyridinium cationic mitochondrial fluorescent probe and preparation and application thereof

Abstract

The application discloses a pyridine cation mitochondrial fluorescent probe, the chemical name of the probe is 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodine salt, and the chemical structural formula is shown as formula (I). The application further discloses application of the fluorescent probe in labeling or imaging display of mitochondrial distribution or morphology in living cells and in long-range tracking display of mitochondria in living cells and imaging display of nucleoli in cells. Experiments prove that the labeled mitochondrial fluorescent probe has high light bleaching resistance and a high fluorescence quantum yield, can realize long-range tracking of mitochondria in living cells, and can label fixed nucleoli in cells. It is indicated that the fluorescent probe has potential application and development value in the fields of fluorescent probes and tissue imaging, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a mitochondrial fluorescent probe, its preparation and application, and more particularly to a pyridine cation mitochondrial-targeting fluorescent probe, its preparation method and application. It belongs to the field of organic small molecule fluorescent probes and bioimaging technology. Background Technology

[0002] Mitochondria are among the most important organelles in cells, distributed throughout the cytoplasm, and vary in size and shape, ranging from small particles to highly filamentous structures. Abnormal changes in mitochondrial substances and unusual mitochondrial behavior are closely related to various important diseases, such as Alzheimer's disease, diabetes, and cardiovascular disease.

[0003] To date, mitochondrial fluorescent probes have been widely developed and applied. It is well known that the transmembrane potential of the inner membrane is as high as -180 mV, which distinguishes mitochondria from other organelles. Therefore, designing positively charged probes to target mitochondria is an effective strategy. Pyridinium salt organic fluorescent probes have been widely designed for mitochondrial imaging; however, their quantum yield is typically low. Furthermore, some molecules remain in the culture medium after staining, generating a strong background signal and resulting in excessively high observational background. Washing, on the other hand, can stimulate cells, leading to changes in mitochondrial morphology.

[0004] Photobleaching is a common problem faced by fluorescent probes. In bioimaging, prolonged laser irradiation is unavoidable, especially when observing dynamic processes within cells. Therefore, probes with strong resistance to photobleaching have higher application value, enabling long-term imaging under continuous laser scanning. Simultaneously, fluorescent probes with long-range tracking capabilities are also crucial for long-term studies of mitochondrial morphology. Therefore, developing wash-free fluorescent probes with high quantum yield and long-range tracking capabilities is both urgent and of practical value.

[0005] A search revealed few reports on pyridine cationic mitochondrial-targeting fluorescent probes that can accurately label mitochondria in living cells without background signal and have strong resistance to photobleaching and long-range tracking capabilities, as well as their preparation and applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a pyridine cation mitochondrial-targeting fluorescent probe, its preparation method, and its application.

[0007] The pyridine cationic mitochondrial fluorescent probe of the present invention is characterized in that: the chemical name of the fluorescent probe is: 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide; its chemical structural formula is shown in formula (I):

[0008] .

[0009] The preparation methods of the above-mentioned pyridine cationic mitochondrial fluorescent probe compounds are summarized as follows:

[0010] To increase the rigidity of the molecule, the inventors first added a methyl group to the pyridine nitrogen to obtain a nitrogen cation structure, thereby improving the mitochondrial targeting function of the molecule. At the same time, based on compound 3, the synthetic route was optimized through an intramolecular esterification strategy to obtain an intramolecularly cyclized pyridine salt compound.

[0011] The preparation reaction formulas for compound 3 and the pyridine salt compound are as follows:

[0012]

[0013] The above-mentioned pyridine cationic mitochondrial fluorescent probes are used in labeling or imaging to show the distribution or morphology of mitochondria in living cells.

[0014] The above-mentioned pyridine cationic mitochondrial fluorescent probe demonstrates its application in mitochondria in living cells during long-range tracking.

[0015] In the aforementioned application, the live cells are preferably HeLa cells.

[0016] The above-mentioned pyridine cationic mitochondrial fluorescent probes are used in imaging to display the nucleolus in cells.

[0017] Experimental results show that the fluorescent probe provided by this invention can accurately label mitochondria in living cells without washing and without background signal, while also exhibiting high resistance to photobleaching and long-range tracking ability, providing an intuitive and convenient biomonitoring dye for mitochondrial-related research. Fixed cell experiments show that the fluorescent probe provided by this invention can stain the nucleolus, demonstrating its potential for staining tissue cell nucleoli.

[0018] Further experiments confirmed that the pyridine cationic mitochondrial fluorescent probe provided by this invention is a probe molecule with high fluorescence quantum yield and selective labeling of mitochondria. Furthermore, the intramolecular esterification strategy involved in the technical solution is effective in improving intramolecular rigidity and photostability. In fixed cells, the nucleoli stained by the fluorescent probe provided by this invention are clear and bright. Moreover, this fluorescent probe exhibits good biocompatibility with Hoechst 33342 and mitochondrial green, suggesting that this fluorescent probe has potential application and development value in the fields of fluorescent probes and tissue imaging, and has broad application prospects. Attached Figure Description

[0019] Figure 1 Absorption and emission spectra of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide in different polar solvents.

[0020] Figure a shows the absorption spectrum, and figure b shows the emission spectrum.

[0021] Figure 2 Comparison of fluorescence quantum yields of compounds 3 and 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide in different solvents.

[0022] Figure 3 Image showing the colocalization imaging results of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide and mitochondrial green in HeLa cell mitochondria.

[0023] Both dyes are 488 excitation. Figure a is a staining image of the probe described in this invention, Figure b is a staining image of mitochondrial green, Figure c is a bright-field cell image, and Figure d is a combined image of the three images on the left.

[0024] Figure 4 Figure 1: Photobleaching results of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide in HeLa.

[0025] Figures a, b, c, d, and e show the images obtained under continuous laser scanning conditions at times of 0, 10, 20, 30, and 40 minutes, respectively.

[0026] Figure 5 Image of long-range tracking experiment of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide in HeLa.

[0027] Figures a, b, c, d, and e show images obtained by confocal microscopy at incubation times of 1, 15, 30, 45, and 60 hours, respectively.

[0028] Figure 6 : Staining results of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide and Hoechst 33342 in fixed HeLa cells.

[0029] The two dyes are 488 and 405 for excitation, respectively. Figure a is the staining image of the probe described in this invention, Figure b is the staining image of Hoechst 33342, Figure c is the bright field cell image, and Figure d is a composite image of the three images on the left. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0031] Unless otherwise specified, the materials, reagents, cells, etc. used in the following examples are all commercially available.

[0032] Example 1: Synthesis of Compound 2

[0033] Compound 1 (1.08 g, 5 mmol), K₂CO₃ (1.04 g, 7.5 mmol), and CH₃I (1.06 g, 7.5 mmol) were added to 10 mL of DMF, and the mixture was stirred at room temperature for 4 hours. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was separated by column chromatography using n-hexane / ethyl acetate (8:1) as the eluent to give a bright yellow solid in 73% yield.

[0034] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 10.32 (d, J = 6.4Hz, 1H), 8.04 (d, J = 9.3Hz, 1H), 7.58 (d, J = 8.7Hz, 1H), 7.47(d,J=8.8Hz,1H),7.29(dd,J=9.4,2.6Hz,,1H),6.98(d,J=2.6Hz,1H),4.06(s,3H),3.09(s,6H).

[0035] The reaction formula is as follows:

[0036]

[0037] Example 2: Synthesis of Compound 3

[0038] Compound 2 (0.1 g, 0.44 mmol) and 1-methyl-4-pyridine iodide (0.123 mg, 0.52 mmol) were added to 8 mL of acetonitrile, followed by 5 drops of piperidine as a catalyst. The mixture was heated at 80 °C for 24 hours. The solvent was removed by evaporation under reduced pressure, and the residue was separated by column chromatography using dichloromethane / methanol (50:1) as eluent to give a brown solid in 28% yield.

[0039] 1H NMR (400MHz, DMSO-d6), δ (ppm): 8.79 (d, J = 6.8Hz, 2H), 8.25 (d, J = 6.8Hz, 2H), 8.15 (d, J = 16.4Hz, 1H), 7.97 (d, J = 9.3Hz, 1H), 7.8 0(d,J=8.9Hz,1H),7.54–7.47(m,2H),7.28(dd,J=9.3,2.5Hz,1H),7.00(d,J=2.4Hz,1H),4.25(s,3H),3.97(s,3H),3.08(s,6H).

[0040] The reaction formula is as follows:

[0041]

[0042] Example 3: Synthesis of Compound 4

[0043] 4-Pyridineacetonitrile (0.1546 g, 1 mmol) and t-BuOK (0.1346 g, 1.2 mmol) were added to 20 mL of anhydrous ethanol and stirred for 10 minutes. Then, compound 1 (0.1935 g, 0.9 mmol) was added and the mixture was stirred at 90 °C for 24 hours. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, and the reaction mixture was extracted with ethyl acetate in an acidic (HCl, 2N) medium. The organic layer was then dried over anhydrous MgSO4 for 2 hours, and the solvent was removed by evaporation under reduced pressure.

[0044] The residue was separated by column chromatography using dichloromethane / methanol (90:1-70:1) as the eluent to give a reddish-brown solid, yield: 35%.

[0045] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 8.67 (dd, J=4.6, 1.5Hz, 2H), 8.57 (s, 1H), 8.23 ​​(d, J=9.3Hz, 1H), 7.86 (dd,J=4.6,1.6Hz,2H),7.61(s,2H),7.38(dd,J=9.4,2.5Hz,1H),7.05(d,J=2.5Hz,1H),3.12(s,6H).

[0046] Example 4: Synthesis of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide

[0047] Compound 4 (165 mg, 0.52 mmol) and iodomethane (0.6 mg, 4.23 mmol) were added to 16 mL of 1,4-dioxane and stirred at 95 °C for 15 hours. The mixture was cooled to room temperature and filtered to obtain a dark red precipitate. Recrystallization from ethyl acetate gave a deep red solid in 31% yield.

[0048] The reaction formula is as follows:

[0049]

[0050] 1 H NMR (400MHz, Methanol-d4), δ (ppm): 8.83 (s, 1H), 8.80 (d, J = 6.9Hz, 2H), 8.64 (d, J = 7.0Hz, 2H), 8.33 (d, J = 9 .4Hz,1H),7.57–7.51(m,2H),7.33(dd,J=9.4,2.5Hz,1H),7.00(d,J=2.5Hz,1H),4.34(s,3H),3.15(s,6H).

[0051] Example 5: HeLa cell culture

[0052] HeLa cell lines were cultured adherently in medium containing 10% fetal bovine serum in a humidified incubator at 37°C and 5% CO2, with medium changes and passages every 48 hours. Once cells reached the logarithmic growth phase, they were seeded into imaging dishes: cells were washed three times with PBS, digested with 1 mL of 0.25% trypsin for 3 minutes, then 2 mL of fresh medium was added and resuspended. 3 mL of the mixture was added to a centrifuge tube and centrifuged for 4 minutes. The liquid was aspirated, and immediately 3 mL of fresh medium was added and resuspended. 40 μL of this mixture was added to an imaging dish containing 1 mL of fresh medium and mixed well. The dish was then incubated in a CO2 incubator. The cells were ready for use.

[0053] Example 6: 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide and mitochondrial green counterstaining experiment

[0054] 0.5 μL of the prepared stock solution (1 mM) of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide and 1 μL of mitochondrial green (200 μM) were added to an imaging dish containing HeLa cells cultured for 36 hours. The cells were stained in a CO2 incubator for 30 minutes. After staining, no washing was required; the cells were directly imaged under a confocal microscope. The focus was adjusted to obtain images of the corresponding fluorescence channels. Results are shown below. Figure 3 .

[0055] Figure 3The image shows fluorescence micrographs obtained by staining viable HeLa cells with 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide and mitochondrial green, collected in two channels under 488 nm laser irradiation. The results showed that the staining regions of mitochondrial green and the probe described in this invention are similar within the cells, and the co-localization coefficient is 0.89. Therefore, the probe described in this invention can specifically target mitochondria.

[0056] Example 7: Photobleaching experiment of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide

[0057] After culturing HeLa cells for 36 hours, the cells were washed three times with PBS, then 1 mL of 4% paraformaldehyde was added, shaken well, and the cells were fixed for 2 hours. The paraformaldehyde was then removed, the cells were washed three times with PBS, 0.5% Triton-100 was added, shaken well, and allowed to stand for 5 minutes. The cells were then washed three times with PBS, and incubated with 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide for 30 minutes before microscopic imaging. Results are shown below. Figure 4 .

[0058] Figure 4 The results showed that after staining cells with 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide, the laser intensity was adjusted to obtain a clear and observable staining effect. The confocal microscope was then set to automatic continuous scanning, taking one image every 10 minutes. The results showed that obvious fluorescent staining effects were observed within 40 minutes, therefore, the probe described in this invention has good anti-bleaching ability.

[0059] Example 8: Long-range tracking experiment of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide

[0060] Starting 30 minutes after staining viable HeLa cells with 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide, the cells were washed three times with PBS, followed by the addition of fresh culture medium. The culture medium was changed every 24 hours to ensure normal cell physiological processes. An image was taken at 1, 15, 30, 45, and 60 hours. Results are shown below. Figure 5 .

[0061] The results showed that the probe described in this invention could still effectively stain the mitochondria in the cells after 60 hours of incubation with the cells and was not excluded from the cells by HeLa cells.

[0062] Example 9: Long-range tracking experiment of 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide

[0063] The process for fixing cells is the same as in Example 7.

[0064] See results Figure 6 .

[0065] Figure 6 The results showed that after staining cells with 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide for 20 minutes, Hoechst 33342 was added for staining for 10 minutes, and the staining effect was observed using a confocal microscope. The results showed that only the nucleolus in the cell nucleus was stained red, indicating that the probe described in this invention has a certain affinity for RNA. Since a small amount of RNA is also present in the cytoplasm, the probe also stained some areas of the cytoplasm.

Claims

1. 4-(8-dimethylamino-5-benzocoumarin)-methylpyridine iodide is used in the preparation of a long-term tracking fluorescent probe for displaying mitochondria in live cells; wherein the chemical structure of the methylpyridine iodide is shown in Formula (I): Formula (I).

2. The application according to claim 1, characterized in that: The living cells were HeLa cells.

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