Pyridinium fluorescent probe compound, preparation method thereof and application of pyridinium fluorescent probe compound in cell mitochondrial staining

Abstract

The invention discloses a pyridinium fluorescent probe compound as well as a preparation method and application thereof in cell mitochondrial staining, and belongs to the technical field of biomedical detection. The compound is formed by combining a 5-cyano indanone derivative and pyridinium salt, and the synthesis method is simple. The compound Y1OPy has large Stokes shift and low cytotoxicity, can specifically target living cell mitochondria, presents clear mitochondrial network structure fluorescence imaging, and does not have specific targeting to fixed cells, and the probe is suitable for real-time, dynamic and high-resolution fluorescence imaging of living cell mitochondria, and has wide application prospects. The method has a wide application prospect in the field of biomedical research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically a pyridine salt fluorescent probe compound, its preparation method, and its application in cell mitochondrial staining. Background Technology

[0002] Mitochondria originated from alpha-proteobacteria that were engulfed by primitive eukaryotic cells billions of years ago and are among the most important and oldest organelles in eukaryotic cells. Mitochondria are considered the cell's power source, converting oxygen and nutrients into adenosine triphosphate (ATP), providing the energy for various biochemical and physiological processes. They are also an important biosynthetic platform for fatty acids, cholesterol, amino acids, glucose, heme, and other substances, maintaining the organism's growth, movement, and homeostasis.

[0003] Furthermore, mitochondria are inextricably linked to the occurrence of diseases and cellular metabolic disorders, playing a crucial role in human evolution and disease. Studies have confirmed that mitochondrial dysfunction is highly correlated with various metabolic and neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD). Studying mitochondrial morphology can reveal mitochondrial stress and dysfunction, thereby providing a better understanding of the relationship between mitochondrial defects and diseases. Mitochondrial morphology is highly dynamic, exhibiting characteristics ranging from giant filamentous networks to small round entities. Their morphological changes are mainly determined by interactions such as fusion and division events, and can be affected by hypoxia, exposure to mitochondrial toxins, and metabolic disorders. For example, when cells experience hypoxia-reoxygenation stress, fragmented, ring-shaped mitochondria are observed. Studies have shown that mitochondria in living cells have a negative internal membrane potential and a positive external membrane potential, thus allowing them to enrich membrane-permeable organic cations through electrostatic interactions. The higher the membrane potential, the higher the concentration of cation enrichment. Therefore, membrane potential (MMP) is a key factor to consider when designing mitochondrial-targeting probes.

[0004] Biological and medical research often requires observation and monitoring without causing physical damage to samples. Labeling mitochondria within cells with specific fluorescent compounds and observing different depth planes of biological samples such as living cells using laser scanning confocal microscopy (LSCM) enables clear fluorescence imaging while maintaining the integrity of the biological sample, meeting the needs of basic research and clinical applications in the biomedical field. A crucial aspect of this is the development of water-soluble fluorescent probes that can target and label mitochondria. Organic small-molecule ionic salts are simple to synthesize and structurally controllable, allowing for a balance between water and lipid solubility, facilitating successful crossing of cell membranes. The introduction of specific functional groups enables the modulation of emission wavelengths and targeted binding to specific cellular structures. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the present invention aims to design and provide a pyridine salt fluorescent probe compound, its preparation method, and its application in cell mitochondrial staining. The compound Y1OPY of the present invention has low toxicity to live cells and can specifically label the mitochondria of live cells at low concentrations in a short time with clear laser confocal imaging, indicating that this small organic molecule ionic salt is an effective mitochondrial fluorescent probe and has promising applications in the field of live cell mitochondrial imaging.

[0006] A pyridine salt fluorescent probe compound represented by general formula (I):

[0007] General Formula (I) Its chemical name is: ( E )-1-(2-(2-((5-cyano-1-oxo-1,3-dihydro-2H-inden-2-ylidene))methyl)-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as Y1OPy, has the chemical formula C 26 H 24 BrN3O2.

[0008] A method for preparing a pyridine salt fluorescent probe compound, characterized by comprising the following steps: 1) 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde (QBr) is condensed with 5-cyanoindanone in an alcohol solvent to generate the intermediate Y1OBr, i.e. ( E )-2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethyl)-1-oxo-2,3-dihydro-1H-indene-5-nitrile; 2) The intermediate Y1OBr is subjected to a quaternization reaction with pyridine under heating conditions to generate the target pyridine salt fluorescent probe compound Y1OPy.

[0009] The method for preparing a pyridine salt fluorescent probe compound is characterized by the following synthetic route: .

[0010] The method for preparing a pyridine salt fluorescent probe compound is characterized in that, in step 1), the molar ratio of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde to 5-cyanoindanone is 1:1; the alcohol solvent is ethanol; the reaction is carried out at room temperature for 1.5-2.5 hours, preferably 2 hours.

[0011] The method for preparing a pyridine salt fluorescent probe compound is characterized in that, in step 2), the reaction temperature is 75-85℃, preferably 80℃; the reaction time is 16-20 hours, preferably 18 hours; after the reaction, the target compound is obtained by cooling, filtration, and washing.

[0012] The application of the pyridine salt fluorescent probe compound in mitochondrial staining.

[0013] The application of the pyridine salt fluorescent probe compound described in 6 is characterized by the following steps: co-incubating the cells to be tested with a culture medium or buffer containing an effective concentration of the pyridine salt fluorescent probe compound, allowing the probe to enter the cells and label the mitochondria, and then performing fluorescence imaging observation.

[0014] The application of the pyridine salt fluorescent probe compound is characterized in that: the concentration of the pyridine salt fluorescent probe compound in the culture medium or buffer is 10nM-50μM, preferably 500nM-10μM; the co-incubation temperature is 36-38℃, preferably 37℃; and the co-incubation time is 10-60 minutes, preferably 30-40 minutes.

[0015] The application of the pyridine salt fluorescent probe compound is characterized in that: the fluorescence imaging observation is performed using a laser scanning confocal microscope with an excitation wavelength of 450nm-490nm and an emission light collection wavelength of 560nm-660nm. The present invention has the following technical effects: 1) The compound Y1OPy of this invention has a large Stokes shift in aqueous solution, with a Stokes shift as high as 128 nm, which can effectively reduce the mutual interference between excitation light and emission light in fluorescence imaging, and improve imaging resolution and signal-to-noise ratio; its maximum absorption wavelength is located at 480 nm, which causes little light damage to cells and is beneficial for long-term observation of live cells. 2) Low cytotoxicity, suitable for long-term live cell studies; cell viability remains >80% at a concentration of 50 μM. 3) The probe of this invention can rapidly enter living cells and specifically accumulate in mitochondria, with a clear filamentous network structure of fluorescence signal that matches the morphology of mitochondria; however, it does not have this specific targeting effect on fixed cells, indicating that its targeting depends on the physiological characteristics of living cells, such as mitochondrial membrane potential. 4) Low concentration, short incubation time, and simple operation help maintain cell state and obtain high-quality fluorescence images. Attached Figure Description

[0016] Figure 1 The UV-Vis absorption spectrum and fluorescence emission spectrum of the compound Y1OPy in aqueous solution are shown below. Figure 2This is a graph showing the effect of the compound Y1OPy of the present invention on cell activity at different concentrations; Figure 3 These are confocal fluorescence micrographs of live and fixed cells after staining with the compound Y1OPy of this invention; Figure 4 These are confocal fluorescence micrographs of live cells stained with the compound Y1OPy of this invention at different incubation times. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0018] The pyridine salt fluorescent probe compound of the present invention is shown in general formula (I):

[0019] General Formula (I) Its chemical name is: ( E )-1-(2-(2-((5-cyano-1-oxo-1,3-dihydro-2H-inden-2-ylidene))methyl)-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as Y1OPy, has the chemical formula C 26 H 24 BrN3O2.

[0020] The preparation method of the compound Y1OPy of the present invention includes the following steps: 1) Synthesis of intermediate Y1OBr: Equimolar amounts of QBr (2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde, C 11 H 14 BrNO2 (refer to published patent CN119661424A) was mixed with 5-cyanoindanone in ethanol, and the mixture was stirred at room temperature for 2 hours. The mixture was then filtered and the filter cake was washed with ethyl acetate to obtain the intermediate. E Y1OBr, abbreviated as Y1OBr, has the chemical formula C1-2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethyl)-1-oxo-2,3-dihydro-1H-indene-5-onitrile. 21 H 19 BrN2O2.

[0021] The molecular structure of Y1OBr, labeled with atomic numbers, and its precise NMR data are as follows:

[0022] The structure of the product was confirmed by 1H NMR spectroscopy, and the results were consistent with the target structure. 1H NMR(400 MHz, Chloroform-d) δ 8.19 (d, J = 2.1 Hz, 1H, 19), 7.97 (d, J = 7.8 Hz,1H, 2), 7.85–7.81 (m, 1H, 20), 7.72–7.63 (m,2H, 16, 17), 6.65 (d, J = 8.5 Hz,1H, 5), 6.57 (s, 1H, 7), 4.40 (t, J = 6.4 Hz, 2H, 22), 4.01 (d, J = 1.9 Hz,2H, 1), 3.73 (t, J = 6.4 Hz, 2H, 23), 3.11 (s, 6H,10, 11). 2) Synthesis of the target compound Y1OPy: An appropriate amount of Y1OBr was added to pyridine and stirred uniformly. The reaction was carried out at 80℃ for 18 hours. The system was cooled to room temperature, and an orange-red solid precipitated. The solid was filtered, and the filter cake was washed with dichloromethane to obtain an orange solid, which was the target product (E)-1-(2-(2-((5-cyano-1-oxo-1,3-dihydro-2H-inden-2-ylidene))methyl)-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as Y1OPy, with the chemical formula C 26 H 24 BrN3O2.

[0023] The molecular structure of Y1OPy with atomic numbers marked and its precise NMR data are as follows:

[0024] The structure of the product was confirmed by 1H NMR spectroscopy, and the results were consistent with the target structure: 1H NMR (400 MHz, Methanol-d4) δ 9.26–9.22 (m, 2H, 25, 29), 8.65 (tt, J = 7.8, 1.4Hz, 1H, 27), 8.28–8.23 (m, 2H, 26, 28), 7.99 (t, J = 1.1 Hz, 1H, 20), 7.96–7.90 (m, 2H, 2, 19), 7.80 (dd, J = 7.9, 1.4 Hz, 1H, 17), 7.67 (dd, J = 9.0, 3.7 Hz, 1H, 16), 6.49 (dd, J = 8.9, 2.4 Hz, 1H , 5), 6.27 (d, J =2.4 Hz, 1H ,7), 5.24–5.17 (m, 2H , 22), 4.67–4.61 (m, 2H , 23), 4.02–3.95 (m, 2H , 1),3.09 (d, J = 3.1 Hz, 6H, 10, 11).

[0025] In the above preparation method, in step 1), the reaction time is 1.5 or 2.5 hours, and in step 2), the reaction temperature is 75 or 85°C, and the reaction time is 16 or 20 hours, which can also produce the target product.

[0026] The following experimental data and application examples further illustrate the beneficial effects of the present invention.

[0027] Experiment 1: Determination of the spectroscopic properties of the compound Y1OPy of this invention The concentration to be prepared is 10 μM (i.e., 10 × 10⁻⁶). -5 An aqueous solution of Y1OPy (M) was prepared. Its UV-Vis absorption spectrum was obtained using a UV-Vis spectrophotometer with water as a reference, scanning in the wavelength range of 300-600 nm. Subsequently, its fluorescence emission spectrum was scanned using a fluorescence spectrometer with the maximum absorption wavelength as the excitation wavelength. The test results are shown in [Figure number missing]. Figure 1 .

[0028] Figure 1 This indicates that the maximum UV-Vis absorption peak is located at 480 nm, and the only emission peak in the emission spectrum measured using this value as the excitation wavelength is located at 608 nm. Therefore, the emission peak of compound Y1OPy at 10 nm can be calculated. -5The Stokes shift in the aqueous solution of M is as high as 128 nm. This large Stokes shift value can largely avoid the overlap of light excitation and reception signals during subsequent laser scanning confocal microscopy, reducing incident light interference and improving fluorescence imaging resolution. Furthermore, the excitation source, compatible with the 480 nm absorption peak, causes minimal light damage to cells or biological tissues, ensuring the survival time of the test samples and increasing the probability of acquiring high-resolution images.

[0029] Experiment 2: Cytotoxicity test of compound Y1OPy of the present invention (CCK-8 assay), see [link to experiment]. Figure 2 .

[0030] Figure 2 The results showed that healthy cells were digested, centrifuged, and seeded into 96-well plates with 100 μL of cell suspension (approximately 3000-5000 cells / well) added to each well. Different concentration gradients of Y1OPy solution (2 μM, 5 μM, 10 μM, 20 μM, and 50 μM) were prepared for cell treatment, with 4-6 replicates. Cell controls and culture medium controls were also included. Cells were cultured overnight at 37℃, 5% CO2, and 90% humidity. After treatment, cells were washed 1-2 times with PBS, and 100 μL of culture medium containing 10% CCK-8 solution was added to each well. After incubation in the dark for 2 h, the absorbance at 450 nm was measured using a microplate reader. Cell viability in all experimental groups was above 80%, indicating that Y1OPY has low cytotoxicity and is suitable for live cell labeling.

[0031] Application Example 1: Staining and imaging of mitochondria in live cells using the compound Y1OPy of this invention. 1) Cell Culture The HeLa cells used in the experiment were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture, under the conditions of 37°C and 5% carbon dioxide.

[0032] 2) Cell imaging Before the confocal imaging experiment, all cells were seeded in a confocal culture dish and cultured for 48 hours.

[0033] Confocal imaging parameters: Y1OPY concentration 500 nM, excitation wavelength (Ex) 473 nm, emission wavelength (Em) 560-660 nm.

[0034] Probe and live cell experiments: Cells were incubated with Y1OPY at 37°C and 5% CO2 for 30 minutes, and then imaged using an FV1200 confocal microscope; for experiments with different staining times, cells were incubated with Y1OPY under the same conditions for 10 minutes, 20 minutes and 30 minutes, and then imaged using an FV1200 confocal microscope.

[0035] Probe and fixed cell experiments: Live cells were fixed in 4% paraformaldehyde at room temperature for 2 hours, washed twice with 1 mL PBS, and then incubated with Y1OPY for 30 minutes. Finally, the cells were imaged using an FV1200 confocal microscope.

[0036] 3) Experimental Results a. such as Figure 3 As shown, imaging of live cells 30 minutes after staining with the probe revealed a filamentous fluorescence signal, consistent with the typical morphological characteristics of mitochondria. However, imaging of fixed cells after staining showed no obvious organelle targeting, with the fluorescence signal diffusely distributed in the cytoplasm. The scale bar for this set of images is 10 μm.

[0037] b. To determine the optimal staining time for the probe, confocal imaging was performed after incubating the probe with live cells for different durations: such as... Figure 4 After 10 minutes of incubation, the fluorescence signal was mainly concentrated in the cell membrane and extracellular culture medium, and the intensity was relatively weak. After 20 minutes of incubation, a fluorescence signal appeared inside the cell, but there were still residues on the cell membrane. After 30 minutes of incubation, the probe completely entered the cell and specifically stained the mitochondria. The scale bar of this set of images is 10 μm.

[0038] Based on the above experimental results, the Y1OPY emission wavelength can avoid biological autofluorescence, has low toxicity to live cells, and can specifically label the mitochondria of live cells at low concentrations in a short time with clear laser confocal imaging. However, it loses its specific labeling function for fixed cells (where the membrane potential is almost gone), confirming that the probe molecule relies on the mitochondrial membrane potential for specific binding. This indicates that the organic small molecule ionic salt is an effective mitochondrial fluorescent probe and has application prospects in the field of live cell mitochondrial imaging.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pyridine salt fluorescent probe compound of general formula (I):

2. General Formula (I) Its chemical name is: ( E )-1-(2-(2-(((5-cyano-1-oxo-1,3-dihydro-2H-inden-2-ylidene))methyl)-5-(dimethylaminophenoxy)ethyl)pyridine-1-ammonium bromide, abbreviated as Y1OPy, has the chemical formula C 26 H 24 BrN3O2.

3. The method for preparing a pyridine salt fluorescent probe compound as described in claim 1, characterized in that... Includes the following steps: 1) 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde (QBr) is condensed with 5-cyanoindanone in an alcohol solvent to generate the intermediate Y1OBr, i.e. ( E )-2-(2-(2-bromoethoxy)-4-(dimethylamino)benzylmethyl)-1-oxo-2,3-dihydro-1H-indene-5-nitrile; 2) The intermediate Y1OBr is subjected to a quaternization reaction with pyridine under heating conditions to generate the target pyridine salt fluorescent probe compound Y1OPy.

4. The method for preparing a pyridine salt fluorescent probe compound as described in claim 2, characterized in that... Its synthesis route is as follows: 。 5. The method for preparing a pyridine salt fluorescent probe compound as described in claim 2, characterized in that... In step 1), the molar ratio of 2-(2-bromoethoxy)-4-(dimethylamino)benzaldehyde to 5-cyanoindanone is 1:1; the alcohol solvent is ethanol; the reaction is carried out at room temperature for 1.5-2.5 hours, preferably 2 hours.

6. The method for preparing a pyridine salt fluorescent probe compound as described in claim 2, characterized in that... In step 2): the reaction temperature is 75-85℃, preferably 80℃; the reaction time is 16-20 hours, preferably 18 hours; after the reaction is completed, the target compound is obtained by cooling, filtration and washing.

7. The application of the pyridine salt fluorescent probe compound as described in claim 1 or 2 in mitochondrial staining.

8. The application of the pyridine salt fluorescent probe compound as described in claim 6, characterized in that... The specific application method includes the following steps: co-incubate the cells to be tested with a culture medium or buffer containing an effective concentration of pyridine salt fluorescent probe compound, so that the probe enters the cells and labels the mitochondria, and then perform fluorescence imaging observation.

9. The application of the pyridine salt fluorescent probe compound as described in claim 7, characterized in that: The concentration of the pyridine salt fluorescent probe compound in the culture medium or buffer is 10 nM-50 μM, preferably 500 nM-10 μM; the co-incubation temperature is 36-38℃, preferably 37℃; and the co-incubation time is 10-60 minutes, preferably 30-40 minutes.

10. The application of the pyridine salt fluorescent probe compound as described in claim 7, characterized in that: The fluorescence imaging observation was performed using a laser scanning confocal microscope with an excitation wavelength of 450nm-490nm and an emission light collection wavelength of 560nm-660nm.

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