Tri-emission dual-ratio organic fluorescent compounds, their preparation methods and applications

Abstract

A three-emission dual-ratio organic fluorescent compound, its preparation method, and its applications are disclosed. The compound has the structure shown in Formula I. The compound provided by this invention exhibits vibration-induced three-emission fluorescence, and its fluorescence changes are influenced by viscosity. Based on the characteristic that the maximum emission peaks of its VIE molecules in bent, planar, and twisted conformations are different and do not interfere with each other, it can be used as a fluorescent viscosity probe. By grouping the three emission peaks into pairs, the ratio of the emission peak intensities of the bent and twisted conformations to the ratio of the emission peak intensities of the planar and twisted conformations is used to monitor changes in mitochondrial viscosity, which can greatly improve its accuracy, thereby enabling precise quantitative analysis of mitochondrial viscosity changes during ferroptosis.

Description

Technical Field

[0001] This invention relates to an organic fluorescent compound, its preparation method and application, and more particularly to a three-emission dual-ratio organic fluorescent compound, its preparation method and application, belonging to the technical field of organic fluorescent compound preparation and application. Background Technology

[0002] As one of the most important organelles in eukaryotic cells, mitochondria are the main energy production sites in cells and are known as the cell's energy factories. Many important cellular processes are inseparable from mitochondria, such as the production of adenosine triphosphate (ATP), central metabolism, calcium regulation and redox signal transduction, and the apoptosis process of cell death.

[0003] Mitochondrial dysfunction is associated with many diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and atherosclerosis.

[0004] In addition, some studies have shown that mitochondrial morphology is also related to the physiological state of cells, that is, the size, shape and number of mitochondria change with the real-time state of the cells.

[0005] Intracellular viscosity is an important physical parameter describing cell state and a key factor in a series of physiological processes such as material transport and signal transduction, which are controlled by diffusion. The consequences of changes in mitochondrial network organization caused by the viscosity matrix in mitochondria through mechanical or osmotic induction are closely related to the respiratory state of mitochondria. This suggests that changes in mitochondrial matrix viscosity may regulate metabolite diffusion and thus regulate mitochondrial metabolism. Therefore, monitoring changes in mitochondrial viscosity and thus mitochondrial metabolism can be a powerful means of monitoring cellular physiological state and related activities.

[0006] Ferroptosis is an iron-dependent, novel form of programmed cell death, distinct from apoptosis, necrosis, and autophagy. Its main mechanism is that under the action of ferrous iron or ester oxygenase, highly expressed unsaturated fatty acids on the cell membrane undergo lipid peroxidation, thereby inducing cell death. In addition, it is also characterized by a decrease in the expression of antioxidant systems (glutathione (GSH) and glutathione peroxidase-4 (GPX4)). It is a novel form of cell death proposed by Stockwell et al. in 2012.

[0007] Unlike other forms of cell death, cells undergoing ferroptosis do not exhibit characteristics such as nuclear condensation, cytoplasmic or organelle swelling. Their only morphological feature is that mitochondria appear smaller than normal cells and have increased membrane density.

[0008] Although the role of mitochondria in ferroptosis remains controversial, mounting evidence suggests that mitochondria do indeed participate in the process. On the one hand, ferroptosis is induced by mitochondrial voltage anion channels; on the other hand, mitochondria play an important role in promoting ferroptosis through environmentally dependent metabolic effects. Therefore, it is essential to study mitochondrial changes during ferroptosis in order to gain a better understanding of the process.

[0009] Currently, there are quantitative studies on changes in mitochondrial viscosity during ferroptosis. Among them, fluorescent probes have attracted the attention of many researchers due to their advantages such as simple staining process, real-time detection, in situ detection, and high spatial resolution.

[0010] However, there is still a lack of research on the precise quantitative analysis of mitochondrial viscosity during ferroptosis. Therefore, it is necessary to develop a highly sensitive fluorescent probe that is mitochondrial-targeting and can be used to quantitatively analyze changes in mitochondrial viscosity. Summary of the Invention

[0011] To quantitatively analyze changes in mitochondrial viscosity, this invention first provides a highly accurate three-emission, dual-ratio organic fluorescent compound, along with its preparation method, and proposes its application as a highly sensitive fluorescent probe for precise quantitative analysis of mitochondrial viscosity changes during ferroptosis.

[0012] The aforementioned three-emission dual-ratio organic fluorescent compound is a compound having the structure shown in Formula I, abbreviated as PPAC-C4:

[0013]

[0014] A method for preparing the above-mentioned three-emission dual-ratio organic fluorescent compound (i.e., PPAC-C4) includes steps a to h, or steps b to h, or steps c to h, or steps e to h, or steps f to h, or steps g to h in the following synthetic route:

[0015]

[0016]

[0017] in:

[0018] Step a includes:

[0019] Pyrene and ruthenium trichloride hydrate were dissolved in a mixed solution of dichloromethane, acetonitrile and water. Sodium periodate was then added in batches to the mixed solution to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected and evaporated to dryness. The filtrate was then purified by silica gel column chromatography to obtain an orange-red solid, namely compound 1.

[0020] Step b includes:

[0021] Compound 1 was added to a reactor, and ultra-dry dichloromethane was added to the reactor under nitrogen protection. Aniline, pyridine and titanium tetrachloride solution were added sequentially under ice bath to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, and then the filtrate was evaporated to dryness and slurried with ethanol to obtain a dark red solid, namely compound 2.

[0022] Step c includes:

[0023] Compound 2 was added to the reactor, and tetrahydrofuran was added to the reactor under nitrogen protection at 0°C to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, and after the filtrate was evaporated to dryness, it was purified by silica gel column chromatography to obtain a bright yellow-green solid, namely compound 3.

[0024] Step d includes:

[0025] Compound 3, copper sulfate, potassium carbonate and m-trichlorobenzene were added to a reactor, heated to 170°C for reaction, then heated to 210°C, and iodobenzene was added for reaction. After the reaction was completed, the mixture was cooled to room temperature, and m-trichlorobenzene was removed. The mixture was purified by silica gel column chromatography to obtain a light yellow-green solid, namely compound 4.

[0026] Step e includes:

[0027] Compound 4 was dissolved in N,N-dimethylformamide and added to a reactor. Phosphorus oxychloride was added dropwise under nitrogen protection at 0°C and the reaction was stirred. The reaction mixture was then heated to 70°C and stirred continuously. The resulting solution was then cooled to room temperature and poured into an ice-water mixture. The precipitate was then filtered out and purified by silica gel column chromatography to obtain a light yellow solid, namely compound 5.

[0028] Step f includes:

[0029] Compound 5 was dissolved in tetrahydrofuran, sodium borohydride was added at 0°C to carry out the reaction, and then the reaction was stirred at room temperature. The reaction mixture was then poured into ice water, and the crude product was filtered out and recrystallized from toluene to obtain a light yellow solid, namely compound 6.

[0030] Step g includes:

[0031] Compound 6 and 4-dimethylaminopyridine were dissolved together in N,N-dimethylformamide and then added to a reactor. The mixture was stirred at 0°C, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The mixture was stirred at 0°C, and then 4-bromobutyric acid was added to the reaction mixture. The reaction mixture was then poured into a separatory funnel, extracted with water and dichloromethane, and the organic phase was collected. The solvent was then removed to obtain a pale yellow crude product. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid, namely compound 7.

[0032] Step h includes:

[0033] Compound 7 was dissolved in tetrahydrofuran and added to a reactor. 4-methylpyridine was then added, and the temperature was raised to 70°C. The mixture was refluxed and stirred. The reaction mixture was then cooled to room temperature, the precipitate was filtered off, and purified by silica gel column chromatography to obtain a light yellow solid, namely compound I.

[0034] An application of the above-mentioned three-emission dual-ratio organic fluorescent compound, namely:

[0035] The aforementioned three-emission, dual-ratio organic fluorescent compound was used as a fluorescent probe in mitochondrial imaging in live cells, thereby enabling precise quantitative analysis of changes in mitochondrial viscosity within the cells.

[0036] The living cells are either immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells, while the normal cells are HUVEC cells.

[0037] Compared with the prior art, the beneficial effects and significant progress of the present invention are as follows:

[0038] 1) First, the compound provided by this invention has been experimentally proven to be a three-emission fluorescent compound molecule with vibration-induced emission (VIE) effect, and its fluorescence change is affected by viscosity. Based on the unique three-emission phenomenon of the compound with Formula I structure and the characteristic that its fluorescence change is affected by viscosity, it can be used as a fluorescent viscosity probe. By utilizing the characteristic that the maximum emission peaks of the VIE molecule are different and do not interfere with each other in the three different conformations of bending, planar and twisted, the three emission peaks are grouped into two groups. The ratio of the emission peak intensity of the bending conformation and the twisted conformation and the ratio of the emission peak intensity of the planar conformation and the twisted conformation are used to monitor the change of mitochondrial viscosity, which can greatly improve the accuracy of the probe.

[0039] 2) Secondly, the compound with the structure shown in Formula I (i.e., PPAC-C4, the same below) provided by the present invention is connected with a commonly used mitochondrial targeting group - pyridine salt, which can not only improve its water solubility, but also utilize its positive ion structure to interact with the electrostatic potential of the mitochondrial negative membrane, so that the probe can better specifically target the mitochondria.

[0040] 3) Experiments have shown that the PPAC-C4 provided by this invention has dual-ratio fluorescence emission characteristics. With the help of this characteristic, it can not only accurately characterize the increase in mitochondrial viscosity caused by nystatin, but also perform relatively accurate quantitative analysis of mitochondrial viscosity changes during ferroptosis. For example, as can be seen from the results of the following effect examples, after ferroptosis, the average mitochondrial viscosity of A549 cells can be detected to increase from the original 43 cP to 152 cP. Moreover, thanks to the good mitochondrial targeting of PPAC-C4, the shrunken mitochondrial morphology of cells undergoing ferroptosis can also be observed. Therefore, the PPAC-C4 provided by this invention can perform relatively accurate quantitative analysis of mitochondrial viscosity changes, thus providing a promising strategy for the diagnosis of certain mitochondrial-related diseases.

[0041] 4) Since the PPAC-C4 provided by this invention has ultra-high precision and good mitochondrial targeting, it can be used as a viscosity fluorescent probe for specific labeling or displaying mitochondrial morphology in living cells or tissues, and can quantitatively analyze mitochondrial viscosity during ferroptosis with relatively high accuracy. The living cells are preferably immortalized cells or normal cells. The immortalized cells are preferably HeLa cells or A549 cells, and the normal cells are preferably HUVEC cells.

[0042] 5) Results of the implementation examples confirm that the PPAC-C4 provided by this invention, thanks to its unique three-emission characteristic that is only sensitive to viscosity, can serve as an excellent dual-ratio probe for accurately monitoring changes in mitochondrial viscosity under different conditions. It is also the first known probe that can accurately measure mitochondrial viscosity during ferroptosis, enabling real-time and accurate monitoring of changes in mitochondrial viscosity. This not only helps to more effectively understand mitochondrial function during ferroptosis but also provides a new strategy for the diagnosis of other mitochondrial-related diseases. Furthermore, as the first fluorescent probe to use dual-ratio imaging, it also provides a novel approach for the design of fluorescent sensors for precise quantitative analysis.

[0043] 6) Compared with other mitochondrial viscosity probes with similar functions, the viscosity detection principle of PPAC-C4 provided by this invention is based on the structural characteristics of the VIE molecule itself, rather than the traditional intramolecular charge transfer effect, i.e., the TICT effect. Furthermore, the viscosity of PPAC-C4 in an ethanol-glycerol mixture is similar to that of I... 480nm / I 628nm and I 533nm / I 628nm Both ratios exhibit a good linear relationship, and R0 2 All values ​​are above 0.9, giving it the characteristics of ultra-high precision, high sensitivity, and high accuracy in detecting changes in mitochondrial viscosity. It also exhibits high selectivity in response to viscosity changes and is not affected by environmental pH, polarity, or other bioanalytes.

[0044] In summary, the PPAC-C4 provided by this invention is a novel fluorescent probe compound with high selectivity, high precision, simple detection, and low cytotoxicity. It can specifically image mitochondria in active cells and accurately monitor their viscosity changes in real time. Compared with existing technologies, it has substantial features and significant progress, with broad application prospects and great promotion and application value. Attached Figure Description

[0045] To more clearly illustrate the technical solution of the present invention and the technical effects of implementing the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly introduced below.

[0046] Obviously, the accompanying drawings described below are only some of the drawings of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of the drawings required for the embodiments of the present invention.

[0047] Figure 1 The absorption spectra of PPAC-C4 provided by this invention in dichloromethane, DMF (i.e., N,N-dimethylformamide), acetonitrile, ethanol, glycerol ethanol solution with a volume percentage concentration of 85% and glycerol.

[0048] Figure 2 Emission spectra of PPAC-C4 provided by this invention in dichloromethane, DMF (i.e., N,N-dimethylformamide), acetonitrile, ethanol, glycerol ethanol solution with a volume percentage concentration of 85% and glycerol.

[0049] Figure 3 The fluorescence spectra of PPAC-C4 in solutions of different viscosities obtained by mixing ethanol and glycerol at different volume ratios provided by the present invention are shown, wherein the excitation wavelength is 365 nm and the concentration of PPAC-C4 is 10 μM.

[0050] Figure 4 The PPAC-C4 provided by this invention has I 480nm / I 628nm with I 533nm / I 628nmThe graph shows the relationship between the emission spectrum of the fluorescence emission group and the ratio of the emission spectrum to the low viscosity range solutions composed of ethanol-glycerol mixtures with different volume ratios.

[0051] Figure 5 The PPAC-C4 provided by this invention has I 480nm / I 628nm and I 533nm / I 628nm The graph shows the relationship between the emission spectrum of the fluorescence emission group and the ratio of the emission spectrum to the high viscosity range of solutions composed of ethanol-glycerol mixtures with different volume ratios.

[0052] Figure 6 To adopt the PPAC-C4 provided by this invention, its I 480nm / I 628nm and I 533nm / I 628nm The graph shows the relative ratios of the selectivity of the three-emission dual-ratio emission spectra to the viscosity of ions or molecules, where:

[0053] The excitation wavelength was 365 nm, the concentration of PPAC-C4 was 10 μM, and the concentration of each selective ion or molecule was 0.2 mM.

[0054] Figure 7 This collection of images, provided by the present invention, shows images obtained from co-localization experiments of HeLa cells seeded on confocal microplates using PPAC-C4 and the commercially available mitochondrial red fluorescent dye Mito-Tracker Deep Red.

[0055] Photo A1 is a fluorescence image of PPAC-C4 provided by the present invention under 405nm excitation irradiation, with λem = 520-620nm and scale bar = 20μm;

[0056] Photo B1 is a fluorescence image of the red fluorescent dye Mito-Tracker Deep Red under 633nm laser irradiation, with λem = 650-650nm and scale bar = 20μm;

[0057] Photo C1 is a superimposed image of Photo A1 and Photo B1, with a scale bar of 20 μm.

[0058] Figure 8 This invention provides a photo gallery of images obtained after staining different cells with PPAC-C4, wherein:

[0059] Column A2 contains images of A549 cells, column B2 contains images of HeLa cells, and column C2 contains images of HUVEC cells.

[0060] Photo a 21 -a 23The image is an image captured using the blue channel, with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0061] Photo b 21 -b 23 The image is an image captured using the green channel, with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0062] Photo c 21 -c3 is an image captured using the red channel, with imaging conditions of λex = 405nm, λem = 620-700nm, and scale bar = 20μm.

[0063] Figure 9 This is a collection of confocal fluorescence and ratio images of HeLa cells after nystatin treatment at different time points, including:

[0064] Column A3 shows photos of the control group, column B3 shows photos of the group treated with nystatin for 1 hour, and column C3 shows photos of the group treated with nystatin for 2 hours.

[0065] Photo a 31 -a 33 The image is a Blue channel image with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0066] Photo b 31 -b 33 The image is a Green channel image with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0067] Photo c 31 -c 33 The image is a Red channel image with imaging conditions of λex = 405 nm, λem = 620-700 nm, and scale bar = 20 μm.

[0068] Photo d 31 -d 33 In Matlab software, use (a 31 -a 33 ) divided by (c 31 -c 33 The resulting overlay image has a scale bar of 20 μm.

[0069] Photo e 31-e 33 In Matlab software, use (b 31 -b 33 ) divided by (c 31 -c 33 The resulting overlay image has a scale bar of 20 μm.

[0070] Figure 10 The image shows the biproportional ratios of HeLa cells after nystatin treatment at different time points, calculated using Matlab software.

[0071] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0072] Figure 11 A bar chart showing the mitochondrial viscosity values ​​of HeLa cells after nystatin treatment at different time points, calculated using Matlab software in a dual-ratio manner.

[0073] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0074] Figure 12 This is a collection of confocal fluorescence and ratio images of A549 cells at different time points after treatment with the ferroptosis inducer FIN56.

[0075] Column A4 contains photos taken at 0 hours, column B4 contains photos taken at 0.5 hours, and column C4 contains photos taken at 4.5 hours.

[0076] Photo a 41 -a 43 The image is a Blue channel image with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0077] Photo b 41 -b 43 The image is a Green channel image with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0078] Photo c 41 -c 43The image is a Red channel image with imaging conditions of λex = 405 nm, λem = 620-700 nm, and scale bar = 20 μm.

[0079] Photo d 41 -d 43 In Matlab software, use (a 41 -a 43 ) divided by (c 41 -c 43 The resulting overlay image has a scale bar of 20 μm.

[0080] Photo e 41 -e 43 In Matlab software, use (b 31 -b 33 ) divided by (c 41 -c 43 The resulting overlay image has a scale bar of 20 μm.

[0081] Figure 13 The image shows a dual proportion plot calculated using Matlab software for A549 cells treated with the ferroptosis inducer FIN56 at different time points and the control group.

[0082] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0083] Figure 14 The bar chart shows the mitochondrial viscosity values ​​calculated by Matlab software for A549 cells after treatment with the ferroptosis inducer FIN56 at different time points and for the control group.

[0084] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel. Detailed Implementation

[0085] To make the objectives, technical solutions, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings provided in the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0086] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection claimed by the present invention.

[0087] It should be noted that:

[0088] The terms "firstly," "secondly," etc., used in the specification, claims, and accompanying drawings of the embodiments of this invention are merely for distinguishing different objects and are not for describing a specific order.

[0089] Furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion, such as a process, method, system, product, or apparatus that includes a series of steps or units that is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0090] What needs to be understood is:

[0091] In the description of the embodiments of the present invention, some basic operational terms commonly used in the art are used, such as "heating", "stirring", "mixing", "dissolving", "purifying", "filtering" and "drying". These terms should be interpreted broadly, and can refer to routine operations performed using various conventional equipment and instruments in the art, as well as operations performed using the latest equipment, such as programmed operations and unmanned automatic operations. Unless otherwise explicitly limited, those skilled in the art can understand the specific meaning of the above terms in the present invention according to the specific circumstances and adopt specific operating methods to achieve their operational objectives.

[0092] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.

[0093] Furthermore, the raw materials, auxiliary materials, and reaction equipment and facilities involved in the following specific embodiments are all commercially available.

[0094] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0095] Example 1

[0096] This embodiment provides a three-emission dual-ratio organic fluorescent compound and its preparation method.

[0097] A three-emission, dual-ratio organic fluorescent compound, having the structure shown in Formula I, abbreviated as PPAC-C4:

[0098]

[0099] A method for preparing PPAC-C4 includes steps a to h, or steps b to h, or steps c to h, or steps e to h, or steps f to h, or steps g to h in the following synthetic route:

[0100]

[0101] in:

[0102] Step a includes:

[0103] Pyrene and ruthenium trichloride hydrate were dissolved in a mixed solution of dichloromethane, acetonitrile and water. Sodium periodate was then added in batches to the mixed solution to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected and evaporated to dryness. The filtrate was then purified by silica gel column chromatography to obtain an orange-red solid, namely compound 1.

[0104] Step b includes:

[0105] Compound 1 was added to a reactor, and ultra-dry dichloromethane was added to the reactor under nitrogen protection. Aniline, pyridine and titanium tetrachloride solution were added sequentially under ice bath to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, and then the filtrate was evaporated to dryness and slurried with ethanol to obtain a dark red solid, namely compound 2.

[0106] Step c includes:

[0107] Compound 2 was added to the reactor, and tetrahydrofuran was added to the reactor under nitrogen protection at 0°C to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, evaporated to dryness, and purified by silica gel column chromatography to obtain a bright yellow-green solid, namely compound 3.

[0108] Step d includes:

[0109] Compound 3, copper sulfate, potassium carbonate and m-trichlorobenzene were added to a reactor and heated to 170°C for reaction. Then, the temperature was raised to 210°C and iodobenzene was added for reaction. After the reaction was completed, the mixture was cooled to room temperature and m-trichlorobenzene was removed. The mixture was purified by silica gel column chromatography to obtain a light yellow-green solid, namely compound 4.

[0110] Step e includes:

[0111] Compound 4 was dissolved in N,N-dimethylformamide and added to a reactor. Phosphorus oxychloride was added dropwise under nitrogen protection at 0°C and the reaction was stirred. The reaction mixture was then heated to 70°C and stirred continuously. The resulting solution was then cooled to room temperature and poured into an ice-water mixture. The precipitate was then filtered out and purified by silica gel column chromatography to obtain a light yellow solid, namely compound 5.

[0112] Step f includes:

[0113] Compound 5 was dissolved in tetrahydrofuran, sodium borohydride was added at 0°C to carry out the reaction, and then the reaction was stirred at room temperature. The reaction mixture was then poured into ice water, and the crude product was filtered out and recrystallized from toluene to obtain a light yellow solid, namely compound 6.

[0114] Step g includes:

[0115] Compound 6 and 4-dimethylaminopyridine were dissolved together in N,N-dimethylformamide and then added to a reactor. The mixture was stirred at 0°C, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The mixture was stirred at 0°C, and then 4-bromobutyric acid was added to the reaction mixture. The reaction mixture was then poured into a separatory funnel, extracted with water and dichloromethane, and the organic phase was collected. The solvent was then removed to obtain a pale yellow crude product. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid, namely compound 7.

[0116] Step h includes:

[0117] Compound 7 was dissolved in tetrahydrofuran and added to a reactor. After adding 4-methylpyridine, the temperature was raised to 70°C, refluxed, and stirred. The reaction mixture was then cooled to room temperature, the precipitate was filtered off, and purified by silica gel column chromatography to obtain a light yellow solid, namely compound I.

[0118] To further aid in understanding the technical solution provided in this embodiment, as well as the specific operation process and the effects that can be obtained, the preparation method will be further explained below through specific examples.

[0119] Case 1, Synthesis of Chemical 1

[0120] 10 g (50 mmol) of pyrene and 418 mg (2 mmol) of ruthenium trichloride hydrate were dissolved in a mixed solution of 230 mL of dichloromethane, 230 mL of acetonitrile, and 150 mL of water. The solution was then added to a round-bottom flask. 50 g (235 mmol) of sodium periodate was added in portions to the mixed solution. The reaction was allowed to proceed overnight. The reactants were then washed with water and extracted with dichloromethane. The organic phase was dried over anhydrous NaSO4. After removing the solvent under reduced pressure, the residue was purified by column chromatography. The mobile phase used for purification was a mixed solvent of dichloromethane and petroleum ether in a volume ratio of 1:1. 5.3 g (46% molar yield) of orange solid compound 1 was obtained.

[0121] Test results:

[0122] 1H NMR (400MHz, DMSO-d6) δ 8.36-8.32 (m, 4H), 8.03 (s, 2H), 7.85 (t, J = 7.6Hz, 2H).

[0123] Case 2, Synthesis of Chemical 2

[0124] 5.0 g (21.5 mmol) of compound 1 was added to a three-necked flask. Under nitrogen protection, more than 500 mL of dry dichloromethane was added to the flask. Under ice bath conditions, 5.90 mL of aniline, 64.5 mmol of pyridine, and 7.1 mL of pure titanium tetrachloride were added sequentially, and the reaction was allowed to proceed overnight. After the reaction was completed, the mixture was first filtered and the filter cake was washed three times with dichloromethane. The filtrate was collected, and then the filtrate was evaporated to dryness using a rotary evaporator and slurried with ethanol to obtain 5.9 g (72% molar yield) of a dark red solid, namely compound 2.

[0125] Test results:

[0126] 1 H NMR (400MHz, DMSOd6) δ8.32 (d, J=7.2Hz, 2H), 8.25 (d, J=6.8Hz, 2H), 8.03 (s, 2H), 7.87 (t, J=7.6Hz, 2H), 7.10 (t, J=7.6Hz, 4H), 7.01 (t, J=7.4Hz, 2H), 6.30 (d, J=7.2Hz, 4H).

[0127] Case 3, Synthesis of Chemical 3

[0128] 5.5 g (14.4 mmol) of compound 2 and catalyst Pd / C were added to a three-necked flask containing 80 mL of tetrahydrofuran. Under nitrogen protection, 2.68 mL (86.3 mmol) of hydrazine hydrate was added, and the mixture was stirred overnight at room temperature. The mixture was filtered and washed with dichloromethane. The organic solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography, with a mobile phase of dichloromethane:petroleum ether mixed solvent in a volume ratio of 1:1, to obtain 3.85 g (69% molar yield) of yellow-green chemical 3.

[0129] Test results:

[0130] 1 HNMR (400MHz, DMSO-d6) δ8.26 (dd, J=7.6, 1.2Hz, 2H), 8.23-8.20 (m, 4H), 7.99 (t, J=7.6Hz, 2H), 7.89 (s, 2H), 7.01 (dd, J=8.4, 7.2Hz, 4H), 6.67-6.55 (m, 6H).

[0131] Case 4, Synthesis of Chemical 4

[0132] 1.15 g (3 mmol) of compound 3, 0.65 g (4.05 mmol) of copper sulfate, 0.54 g (3.9 mmol) of potassium carbonate, and 15 g of m-trichlorobenzene were added to a round-bottom flask. The mixture was heated to 170 °C for 2 hours, then heated to 210 °C. 3.36 mL (30 mmol) of iodobenzene was added in portions, and the reaction was carried out for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, and the m-trichlorobenzene was removed with a hot gun. The mixture was purified by silica gel column chromatography to obtain a pale yellow-green solid, compound 4, with a molar yield of approximately 40%.

[0133] Test results:

[0134] 1 HNMR (600MHz, DMSO-d6) δ8.33(d, J=7.6Hz, 2H), 8.27–8.22(m, 4H), 8.04(t, J=7.7Hz, 2H), 7.97(d d, J=6.0, 3.5Hz, 2H), 7.44 (dd, J=6.0, 3.4Hz, 2H), 7.13–7.08 (m, 8H), 6.87 (tt, J=6.5, 2.0Hz, 2H).

[0135] Case 5, Synthesis of Chemical 5

[0136] 1.00 g (2.1 mmol) of compound 4 was dissolved in 20 mL of ultradry N,N-dimethylformamide and then added to a three-necked round-bottom flask. 2.00 mL (21.0 mmol) of phosphorus oxychloride was added dropwise under nitrogen protection at 0 °C. After stirring at 0 °C for 1 hour, the reaction mixture was heated to 70 °C and stirred overnight. The resulting solution was then cooled to room temperature and poured into an ice-water mixture, during which a precipitate formed. The precipitate was filtered off and purified by column chromatography using silica gel, with petroleum ether / dichloromethane (DCM) as the eluent, to give a pale yellow solid, compound 5, with a molar yield of approximately 30%.

[0137] Test results:

[0138] 1HNMR (600MHz, DMSO-d6) δ9.70 (s, 1H), 8.39 (ddd, J=15.0, 7.6, 1.1Hz, 2H), 8.29 (d, J=1.3Hz, 2H), 8.24 (ddd, J=8.9, 7.8, 1.1Hz, 2H), 8.12 (t, J=7.7Hz, 1H), 8.1 0–8.08(m, 1H), 8.06–8.02(m, 2H), 7.63–7.60(m, 2H), 7.53–7.50(m, 2H), 7.17( dt, J=7.2, 1.2Hz, 2H), 7.11 (dd, J=8.9, 7.1Hz, 4H), 6.88 (td, J=7.2, 1.1Hz, 1H).

[0139] Case 6, Synthesis of Chemical 6

[0140] 977 mg (2.0 mmol) of compound 5 was dissolved in 30 mL of tetrahydrofuran. The resulting mixture was added to a round-bottom flask and cooled to 0 °C using an ice-water bath. Then, 20 mmol of sodium borohydride was added in batches. After stirring overnight at room temperature, the reaction mixture was poured into an ice-water mixture to obtain a beige precipitate. The precipitate was filtered out and recrystallized from toluene to give a pale yellow solid, compound 6, with a molar yield of approximately 70%.

[0141] Test results:

[0142] 1 HNMR (600MHz, DMSO-d6) δ8.32 (t, J=6.6Hz, 2H), 8.26–8.20 (m, 4H), 8.06–8.00 (m, 2H), 7.98–7.93 (m, 2H), 7.44 –7.41 (m, 2H), 7.15–7.09 (m, 6H), 7.05 (d, J=8.5Hz, 2H), 6.88–6.85 (m, 1H), 4.99 (s, 1H), 4.32 (d, J=5.7Hz, 2H).

[0143] Case 7, Synthesis of Chemical 7

[0144] 489 mg (1.0 mmol) of compound 6 and 519 mg (3.0 mmol) of 4-dimethylaminopyridine were dissolved in 30 mL of N,N-dimethylformamide and then added to a round-bottom flask. After stirring at 0 °C for 0.5 hours, 244 mg (2.0 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and stirring was continued at 0 °C for another 0.5 hours. Then, 648 mg (4.0 mmol) of 4-bromobutyric acid was added to the reaction mixture. After stirring overnight at room temperature, the reaction mixture was poured into a separatory funnel, extracted with water and dichloromethane, and the organic phase was collected. The organic phase was dried by rotary evaporation to obtain a pale yellow crude product. The crude product was purified by column chromatography with silica gel, using a petroleum ether / dichloromethane (DCM) mixed solution as the eluent, to obtain a pale yellow solid, compound 7, with a molar yield of 50%.

[0145] Test results:

[0146] 1 HNMR (600MHz, DMSO-d6) δ8.34 (dt, J=7.6, 1.3Hz, 2H), 8.26 (s, 2H), 8.22 (ddd, J=7.5, 6.3, 1.1Hz, 2H), 8.05 (td, J=7.7, 6.5Hz, 2H), 7.98 (ddd, J=6.0, 3.5, 2.5 Hz, 2H), 7.45 (dd, J=6.0, 3.4Hz, 2H), 7.14–7.08 (m, 8H), 6.89–6.86 (m, 1H), 4. 91 (s, 2H), 3.49 (t, J=6.6Hz, 2H), 2.42 (t, J=7.3Hz, 2H), 2.00 (p, J=6.9Hz, 2H).

[0147] Case 8: Synthesis of Chemical I, PPAC-C4

[0148] 319 mg (0.5 mmol) of compound 7 was dissolved in 20 mL of tetrahydrofuran and added to a round-bottom flask. 0.5 mL (5 mmol) of 4-methylpyridine was added to the resulting mixture, and the mixture was heated to 70 °C and refluxed. After stirring overnight, the reaction mixture was cooled to room temperature, during which a precipitate formed. The precipitate was filtered off and purified by column chromatography using silica gel. The eluent used for purification was a methanol / dichloromethane mixture, yielding a pale yellow solid of chemical I, PPAC-C4, in a molar yield of 60%.

[0149] Test results:

[0150] 1HNMR (600MHz, DMSO-d6) δ8.83 (d, J=6.3Hz, 2H), 8.35 (d, J=7.6Hz, 2H), 8.27 (s, 2H), 8.21 (dd, J=11.1, 7.8Hz, 2H), 8.05 (t, J=7.7Hz, 2H), 8.00–7.97 (m, 2H), 7.89 (d, J=6.2Hz, 2H), 7.46 (dd, J=6.0, 3.4Hz, 2H), 7.11 (td, J=9.8, 9.3, 6.3Hz, 8H), 6.86 (t, J=7.1Hz, 1H), 4.87 (s, 2H), 4.48 (t, J=7.1Hz, 2H), 2.55 (s, 3H), 2.37 (t, J=7.5Hz, 2H), 2.11 (t, J=7.3Hz, 2H);

[0151] 13 C NMR (151MHz, DMSO-d6) δ158.24, 146.57, 143.28, 143.17, 142.98, 137.23, 136.83, 130.55, 128.83, 128.49, 128.21, 127.7 2, 127.70, 127.01, 126.96, 126.53, 126.47, 126.37, 126.28, 125.92, 125.29, 125.04, 64.79, 58.54, 29.38, 25.14, 20.73;

[0152] HRMS(m / z): [M] + Calculated for C 45 H 37 N3O2, 651.2880; found, 650.2817.

[0153] Example 2

[0154] This embodiment provides an application of a three-emission, dual-ratio organic fluorescent compound.

[0155] The application described in this embodiment involves using the tri-emission dual-ratio organic fluorescent compound obtained in Example 1 as a fluorescent probe in mitochondrial imaging of live cells, thereby accurately and quantitatively analyzing changes in mitochondrial viscosity in cells, wherein:

[0156] The living cells are either immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells, while the normal cells are HUVEC cells.

[0157] Effect Example

[0158] Example 1: Response of PPAC-C4 to viscosity in different solutions

[0159] The PPAC-C4 obtained in Example 1 above was dissolved in a dimethyl sulfoxide solution to prepare a PPAC-C4 / dimethyl sulfoxide solution containing 1 mM of PPAC-C4;

[0160] Take 30 μL of the above PPAC-C4 / dimethyl sulfoxide solution into multiple 5 mL centrifuge tubes, and then dilute the PPAC-C4 / dimethyl sulfoxide solution in each centrifuge tube to 3 mL with dichloromethane, DMF (N,N-dimethylformamide), acetonitrile, ethanol, 85% (v / v) glycerol aqueous solution, and glycerol, respectively. Then, perform absorption and emission spectra measurements on the solutions in each centrifuge tube, as shown below:

[0161] Figure 1 The absorption spectra of PPAC-C4 provided by the present invention in dichloromethane, DMF (N,N-dimethylformamide), acetonitrile, ethanol, a glycerol ethanol solution with a volume percentage concentration of 85% (Glycerol), and glycerol are shown below;

[0162] Figure 2 The emission spectra of PPAC-C4 provided by the present invention in dichloromethane, DMF (i.e., N,N-dimethylformamide), acetonitrile, ethanol, glycerol ethanol solution with a volume percentage concentration of 85% and glycerol are shown.

[0163] from Figure 1 and Figure 2 It can be seen from this:

[0164] The PPAC-C4 provided in Example 1 of this invention exhibits fluorescence emission at 480 nm, 533 nm, and 628 nm and can form I... 480nm / I 628nm and I 533nm / I 628nm The dual-ratio fluorescence emission group can therefore be used as a probe for viscosity detection.

[0165] Example 2: Fluorescence spectrum of PPAC-C4 as a function of viscosity

[0166] Take 30 μL of the PPAC-C4 / dimethyl sulfoxide solution containing 1 mM PPAC-C4 prepared in Example 1 above into multiple 5 mL centrifuge tubes, and then dilute each tube to 3 mL with solutions of different viscosities obtained by mixing ethanol and glycerol in different volume ratios. Then, perform fluorescence spectroscopy tests on each tube to obtain the following results: Figure 3The fluorescence spectra of PPAC-C4 provided by the present invention in solutions of different viscosities obtained by mixing ethanol and glycerol in different volume ratios are shown, wherein the excitation wavelength is 365 nm and the concentration of PPAC-C4 is 10 μM.

[0167] Based on the above fluorescence spectrum test results, the following can be plotted:

[0168] Figure 4 The PPAC-C4 provided by the present invention shown is I 480nm / I 628nm with I 533nm / I 628nm The graph showing the relationship between the emission spectra of the fluorescence emission group and the ratios of the emission spectra to low-viscosity solutions composed of ethanol-glycerol mixtures at different volume ratios; and

[0169] Figure 5 The PPAC-C4 provided by the present invention shown is I 480nm / I 628nm and I 533nm / I 628nm The graph shows the relationship between the emission spectrum of the fluorescence emission group and the ratio of the emission spectrum to the high viscosity range of solutions composed of ethanol-glycerol mixtures with different volume ratios.

[0170] from Figure 3 and Figure 4 , Figure 5 It can be seen from this:

[0171] As viscosity increases, the fluorescence intensity of PPAC-C4 provided in this embodiment of the invention increases at 480 nm and 533 nm, while the fluorescence intensity decreases at 628 nm; and the fluorescence emission group I 480nm / I 628nm with I 533nm / I 628nm The fluorescence intensity increases with increasing viscosity.

[0172] Example 3: PPAC-C4's selectivity for viscosity

[0173] Take 30 μL of the PPAC-C4 / dimethyl sulfoxide solution containing 1 mM PPAC-C4 prepared in Example 1 above into multiple 5 mL centrifuge tubes. Dilute each tube with a 30% (v / v) glycerol-ethanol mixture to 3 mL. Then, add K+ to each centrifuge tube. + Ca 2+ Mg 2+ Al 3+ Na + ,Br - I - F - ,ClO -Solutions containing ions, as well as solutions containing DTT (dithiothreitol), H₂O₂, Hcy (homocysteine), Cys (cysteine), and GSH (glutathione), were subjected to emission spectroscopy tests, yielding results such as:

[0174] Figure 6 The diagram shows the use of PPAC-C4 provided by this invention. 480nm / I 628nm and I 533nm / I 628nm The graph shows the relative ratios of the selectivity of three-emission dual-ratio emission spectra to the viscosity of ions or molecules, where the excitation wavelength is 365 nm, the final concentration of PPAC-C4 is 10 μM, and the final concentration of each selective ion or molecule is 0.2 mM.

[0175] from Figure 6 It can be seen from this:

[0176] The PPAC-C4 provided in Example 1 exhibits selectivity in viscosity detection and is not affected by K. + Ca 2+ Mg 2+ Al 3 + Na + ,Br - I - F - ,ClO - The effects of plasma and molecules such as DTT (dithiothreitol), H2O2, Hcy (homocysteine), Cys (cysteine), and GSH (glutathione).

[0177] Example 4: Confocal fluorescence microscopy of PPAC-C4 and Mito-Tracker Deep Red stained mitochondria in HeLa cells.

[0178] HeLa cell culture:

[0179] First, HeLa was inoculated into a culture dish containing 10% fetal bovine serum and 1% double antibiotic H-DMEM culture medium. Then, the culture dish was placed in an incubator at 37°C and 5% CO2 for adherent culture.

[0180] One day before imaging, cells were seeded into small dishes specifically for confocal imaging and allowed to adhere for 24 hours. In the experiment, cells treated with 1% DMSO (dimethyl sulfoxide) solution served as control cells.

[0181] HeLa cells seeded in a confocal microplate were first incubated with 5 μM PPAC-C4 / dimethyl sulfoxide solution at 37°C for 30 min, then washed three times with PBS buffer, and finally incubated with 0.2 μM Mito-Tracker Deep Red commercial probe at 37°C for 15 min, followed by three washes with PBS buffer. Confocal imaging was then performed, yielding results such as:

[0182] Figure 7 The image set shown is an image set obtained by co-localization experiments of HeLa cells seeded on a confocal microplate using PPAC-C4 and the commercial mitochondrial red fluorescent dye Mito-Tracker DeepRed provided by this invention. (The image shows images of these cells.)

[0183] Photo A1 is a fluorescence image of PPAC-C4 provided by the present invention under 405nm excitation irradiation, with λem = 520-620nm and scale bar = 20μm;

[0184] Photo B1 is a fluorescence image of the red fluorescent dye Mito-Tracker Deep Red under 633nm laser irradiation, with λem = 650-650nm and scale bar = 20μm;

[0185] Photo C1 is a superimposed image of Photo A1 and Photo B1, with a scale bar of 20 μm.

[0186] from Figure 7 It can be seen from this:

[0187] The PPAC-C4 provided in Example 1 can be used as a fluorescent probe to target mitochondria in living cells.

[0188] Example 5: Staining experiment of PPAC-C4 on mitochondria in different cells

[0189] Culture of HeLa, A549 and HUVEC cells:

[0190] First, HeLa, A549 and HUVEC cell lines were seeded into culture dishes containing 10% fetal bovine serum and 1% double antibiotic H-DMEM medium, respectively. Then, each culture dish was placed in an incubator at 37°C and 5% CO2 for adherent culture.

[0191] One day before imaging, HeLa, A549 and HUVEC cells were seeded into small dishes for confocal imaging and allowed to adhere for 24 hours. During the experiment, cells treated with 1% DMSO (dimethyl sulfoxide) solution were used as control cells.

[0192] Different cells seeded in a confocal microplate were added to a 5 μM PPAC-C4 / dimethyl sulfoxide solution (PPAC-C4) as a probe, incubated at 37°C for 30 min, washed three times with PBS buffer, and then subjected to confocal imaging to obtain the following results:

[0193] Figure 8 The image set shown is a collection of photographs obtained by staining different cells with PPAC-C4 provided by the present invention, wherein:

[0194] Column A2 contains images of A549 cells, column B2 contains images of HeLa cells, and column C2 contains images of HUVEC cells.

[0195] Photo a 21 -a 23 The image is an image captured using the blue channel, with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0196] Photo b 21 -b 23 The image is an image captured using the green channel, with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0197] Photo c 21 -c 23 This is an image captured using the red channel, with imaging conditions of λex = 405 nm, λem = 620-700 nm, and scale bar = 20 μm.

[0198] from Figure 8 It can be seen from this:

[0199] The PPAC-C4 provided in Example 1 can be used as a fluorescent probe for imaging analysis of various cells.

[0200] Example 6: PPAC-C4 was used to quantitatively analyze the viscosity changes in HeLa cells induced by nystatin treatment.

[0201] HeLa cells seeded in a confocal microplate were treated with 10 μM nystatin and incubated at 37°C for 0, 1, and 2 hours. They were then washed three times with PBS buffer, followed by incubation with 5 μM PPAC-C4 at 37°C for 30 minutes. After washing three more times with PBS buffer, confocal imaging was performed, yielding results as shown below:

[0202] Figure 9The image set shown is a collection of confocal fluorescence and ratio images of HeLa cells treated with nystatin at different time points, including:

[0203] Column A3 shows photos of the control group, column B3 shows photos of the group treated with nystatin for 1 hour, and column C3 shows photos of the group treated with nystatin for 2 hours.

[0204] Photo a 31 -a 33 The image is a Blue channel image with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0205] Photo b 31 -b 33 The image is a Green channel image with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0206] Photo c 31 -c 33 The image is a Red channel image with imaging conditions of λex = 405 nm, λem = 620-700 nm, and scale bar = 20 μm.

[0207] Photo d 31 -d 33 In Matlab software, use (a 31 -a 33 ) divided by (c 31 -c 33 The resulting overlay image has a scale bar of 20 μm.

[0208] Photo e 31 -e 33 In Matlab software, use (b 31 -b 33 ) divided by (c 31 -c 33 The resulting overlay image has a scale bar of 20 μm.

[0209] And such as:

[0210] Figure 10 The image shows the corresponding biproportional plots of HeLa cells after nystatin treatment at different time points, calculated using Matlab software. Where:

[0211] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I RedThe value is obtained by dividing the green channel by the red channel.

[0212] Other examples include:

[0213] Figure 11 The bar chart shown represents the mitochondrial viscosity values ​​of HeLa cells treated with nystatin at different time points, calculated using Matlab software in a dual-ratio manner. (The bar chart is shown below.)

[0214] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0215] from Figure 9 , Figure 10 and Figure 11 From this, we can see that:

[0216] According to the dual-scale imaging results of PPAC-C4 provided in Example 1, the mitochondrial viscosity of HeLa cells was 34 cP before treatment with nystatin. After 1 hour of treatment with nystatin, the mitochondrial viscosity increased to 60 cP, and after 2 hours of treatment with nystatin, it further increased to 92 cP. Therefore, it can be seen that PPAC-C4 provided in Example 1 can be used as a fluorescent probe to detect changes in mitochondrial viscosity of HeLa cells caused by nystatin.

[0217] Example 7: PPAC-C4 was used to quantitatively analyze changes in mitochondrial viscosity during ferroptosis in A549 cells.

[0218] In Example 5 above, A549 cells seeded in a confocal microarray were first treated with 10 μM of the ferroptosis inducer FIN56, then incubated at 37°C for 0, 0.5, and 4.5 hours. After washing three times with PBS buffer, 5 μM of PPAC-C4 was added, and the cells were incubated at 37°C for another 30 minutes. After washing three times with PBS, confocal imaging was performed. The control group was identical to the control group except that 10 μM of ferroptosis inducer FIN56 was replaced with 10 μM of DMSO, and FIN56 was dissolved in DMSO. The results were as follows:

[0219] Figure 12 The image set shown contains confocal fluorescence and ratio images of A549 cells treated with the ferroptosis inducer FIN56 at different time points.

[0220] Column A4 contains photos taken at 0 hours, column B4 contains photos taken at 0.5 hours, and column C4 contains photos taken at 4.5 hours.

[0221] Photo a 41 -a 43 The image is a Blue channel image with imaging conditions of λex = 405 nm, λem = 420-520 nm, and scale bar = 20 μm.

[0222] Photo b 41 -b 43 The image is a Green channel image with imaging conditions of λex = 405 nm, λem = 520-620 nm, and scale bar = 20 μm.

[0223] Photo c 41 -c 43 The image is a Red channel image with imaging conditions of λex = 405 nm, λem = 620-700 nm, and scale bar = 20 μm.

[0224] Photo d 41 -d 43 In Matlab software, use (a 41 -a 43 ) divided by (c 41 -c 43 The resulting overlay image has a scale bar of 20 μm.

[0225] Photo e 41 -e 43 In Matlab software, use (b 31 -b 33 ) divided by (c 41 -c 43 The resulting overlay image has a scale bar of 20 μm.

[0226] And such as:

[0227] Figure 13 The figure shows a dual proportion plot calculated using Matlab software for A549 cells treated with the ferroptosis inducer FIN56 at different time points and the control group. (The figure is incomplete in the original text.)

[0228] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0229] Other examples include:

[0230] Figure 14The bar chart shown presents the mitochondrial viscosity values ​​of A549 cells treated with the ferroptosis inducer FIN56 at different time points, as well as the control group, calculated using Matlab software.

[0231] I Blue / I Red The value is obtained by dividing the blue channel by the red channel, I Green / I Red The value is obtained by dividing the green channel by the red channel.

[0232] from Figure 12 , Figure 13 and Figure 14 From this, we can see that:

[0233] According to the results of dual-scale imaging, the mitochondrial viscosity of normal A549 cells is 43 cP, while the mitochondrial viscosity increases to 152 cP after ferroptosis. This shows that ferroptosis is indeed accompanied by an increase in mitochondrial viscosity. The PPAC-C4 provided in Example 1 of this invention can be used as a probe to detect this process and perform precise quantitative analysis.

[0234] In conclusion, it can be seen that:

[0235] The three-emission dual-ratio organic fluorescent compound provided in this invention is a three-emission dual-ratio organic fluorescent compound with ultra-high precision. It has a highly selective response to viscosity changes and is not affected by environmental pH, polarity, or other bioanalytes. Therefore, it can be used as a highly sensitive fluorescent probe for precise quantitative analysis of mitochondrial viscosity changes during ferroptosis. Compared with existing technologies, it has substantial features and significant progress, broad application prospects, and great promotion and application value.

[0236] In the description process of the above instruction manual:

[0237] The terms “this embodiment,” “an embodiment of the present invention,” “as shown,” “further,” etc., are used to indicate that the specific features, structures, materials, or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention.

[0238] In this specification, the illustrative expressions of the above terms are not necessarily directed to the same embodiments or examples, and the specific features, structures, materials or characteristics described may be combined or combined in any suitable manner in one or more embodiments or examples;

[0239] Furthermore, without creating contradictions, those skilled in the art can combine or integrate the different embodiments or examples described in this specification, as well as the features of the different embodiments or examples.

[0240] Finally, it should be noted that:

[0241] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A three-emission, dual-ratio organic fluorescent compound, characterized in that, It has the chemical structure shown in Formula I:

2. A method for preparing the three-emission dual-ratio organic fluorescent compound according to claim 1, characterized in that, This includes steps a to h in the following synthesis route, namely steps a to b to c to d to e to f to g to h:

3. The preparation method according to claim 2, characterized in that, Step a includes: Pyrene and ruthenium trichloride hydrate were dissolved in a mixed solution of dichloromethane, acetonitrile and water. Sodium periodate was then added in batches to the mixed solution to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected and evaporated to dryness. The filtrate was then purified by silica gel column chromatography to obtain an orange-red solid, namely compound 1.

4. The preparation method according to claim 2, characterized in that, Step b includes: Compound 1 was added to a reactor, and ultra-dry dichloromethane was added to the reactor under nitrogen protection. Aniline, pyridine and titanium tetrachloride solutions were added sequentially under ice bath conditions to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, and then the filtrate was evaporated to dryness and slurried with ethanol to obtain a dark red solid, namely compound 2.

5. The preparation method according to claim 2, characterized in that, Step c includes: Compound 2 was added to a reactor, and tetrahydrofuran was added to the reactor under nitrogen protection at 0°C to carry out the reaction. After the reaction was completed, the mixture was first filtered and the filter cake was washed with dichloromethane. The filtrate was collected, evaporated to dryness, and purified by silica gel column chromatography to obtain a bright yellow-green solid, namely compound 3.

6. The preparation method according to claim 2, characterized in that, Step d includes: Compound 3, copper sulfate, potassium carbonate, and m-trichlorobenzene were added to a reactor, heated to 170°C for reaction, then heated to 210°C, and iodobenzene was added for further reaction. After the reaction was completed, the mixture was cooled to room temperature, and m-trichlorobenzene was removed. The mixture was then purified by silica gel column chromatography to obtain a light yellow-green solid, namely compound 4.

7. The preparation method according to claim 2, characterized in that, Step e includes: Compound 4 was dissolved in N,N-dimethylformamide and added to a reactor. Phosphorus oxychloride was added dropwise under nitrogen protection at 0°C with stirring. The reaction mixture was then heated to 70°C and stirring continued. The resulting solution was then cooled to room temperature and poured into an ice-water mixture. The precipitate was then filtered out and purified by silica gel column chromatography to obtain a light yellow solid, namely compound 5.

8. The preparation method according to claim 2, characterized in that, Step f includes: Compound 5 was dissolved in tetrahydrofuran, and sodium borohydride was added at 0°C to carry out the reaction. The reaction was then stirred at room temperature, and the reaction mixture was poured into ice water. The crude product was filtered out and recrystallized from toluene to obtain a light yellow solid, namely compound 6.

9. The preparation method according to claim 2, characterized in that: Step g includes: Compound 6 and 4-dimethylaminopyridine were dissolved together in N,N-dimethylformamide and then added to a reactor. The mixture was stirred at 0°C, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added. The mixture was stirred at 0°C, and then 4-bromobutyric acid was added to the reaction mixture. The reaction mixture was then poured into a separatory funnel, extracted with water and dichloromethane, and the organic phase was collected. The solvent was then removed to obtain a pale yellow crude product. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid, namely compound 7. Step h includes: Compound 7 was dissolved in tetrahydrofuran and added to a reactor. 4-methylpyridine was then added, and the temperature was raised to 70°C. The mixture was refluxed and stirred. The reaction mixture was then cooled to room temperature, the precipitate was filtered off, and the precipitate was purified by silica gel column chromatography to obtain a light yellow solid, namely compound I.

10. An application of the three-emission dual-ratio organic fluorescent compound according to claim 1, characterized in that: The application involves using the tri-emission, dual-ratio organic fluorescent compound of claim 1 as a fluorescent probe in mitochondrial imaging within live cells, thereby accurately and quantitatively analyzing changes in mitochondrial viscosity within the cells, wherein: The living cells are immortalized cells or normal cells, and the immortalized cells are HeLa cells or A549 cells, while the normal cells are HUVEC cells. This application is not for the treatment or diagnosis of diseases.

Images