A dual-responsive fluorescent compound, its preparation method and applications

Abstract

The present invention relates to the field of fluorescence imaging technology, and discloses a dual-responsive fluorescent compound, a preparation method and an application thereof. The dual-responsive fluorescent compound provided by the present invention can simultaneously target RNA and lysosomes in living cells and emit fluorescence of different colors, realizing dual-color imaging of RNA and lysosomes in living cells and promoting the development of research on the interaction between lysosomes and RNA; the preparation method of the dual-responsive fluorescent compound has simple steps and high yield, and is suitable for industrial application; the fluorescent probe including the dual-responsive fluorescent compound can monitor RNA and lysosomes in living biological samples in real time, overcoming the problems of mutual interference and harsh staining conditions existing when commercial RNA probes and commercial lysosome probes are used to separately label RNA and lysosomes. The preparation process of the fluorescent probe is simple, the cost is low, the structure is stable, and it is convenient for storage, and it has broad application space in the research on the biological functions of RNA and lysosomes.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence imaging, and particularly relates to a dual-responsive fluorescent compound, a preparation method thereof, and an application thereof. Background Art

[0002] Lysosomes and RNA are two key functional units in cells, which are respectively involved in the degradation metabolism and gene expression regulation of cells. Among them, lysosomes, as the "recycling center" of cells, degrade damaged organelles, proteins, and foreign substances through the acidic hydrolases inside them to maintain the homeostasis of the intracellular environment. In recent years, studies have found that lysosomes are not only degradation centers but also participate in processes such as cell signal transduction, metabolic regulation, and immune response. For example, lysosomes regulate cell growth and metabolism through the mTORC1 signaling pathway and play a core role in the autophagy process. RNA (including mRNA, lncRNA, snRNA, etc.) plays a crucial role in gene expression regulation: mRNA is the template for protein synthesis, lncRNA participates in chromatin remodeling and gene expression regulation, snRNA is the core component of the spliceosome and is responsible for the splicing and processing of pre-mRNA, and the dynamic changes of RNA directly affect the physiological state and stress response of cells.

[0003] Although the functions of lysosomes and nuclear RNA in cells seem to be independent, more and more studies have shown that there are complex interactions between the two. For example: Some nuclear RNAs may enter lysosomes through autophagy or specific transport mechanisms and be degraded, thereby regulating gene expression; lysosomes regulate the transcription and processing of ribosomal RNA (rRNA) through signaling pathways such as mTORC1, affecting the protein synthesis ability of cells; some lncRNAs may affect the function and stability of lysosomes by regulating the expression of lysosome-related genes or directly interacting with lysosome proteins. And these interactions are of great significance in cell homeostasis, stress response, and disease occurrence. For example, in neurodegenerative diseases, abnormal lysosome function leads to RNA metabolism disorders, which further exacerbates neuronal damage; in cancer, the interaction between lysosomes and RNA may affect the metabolism and invasion ability of tumor cells. However, although significant progress has been made in the functional research of lysosomes and RNA, the research on the interaction between the two still faces technical challenges.

[0004] Existing fluorescence labeling tools (such as Lyso-Tracker Green for labeling lysosomes and SYTO RNASelect for labeling RNA) can usually only label lysosomes or RNA separately, making it difficult to simultaneously observe the spatial distribution and dynamic changes of both in the same cell. In addition, traditional labeling methods also have problems such as fluorescence signal overlap, high background noise, and poor photostability, which greatly limit their application in live cell dynamic imaging. Existing two-color imaging techniques, such as photooxidation-driven pure organic room temperature phosphorescence supramolecular systems, can achieve dual-targeted imaging of the nucleus and lysosomes; Okra505 fluorescent RNA technology provides a new tool for long-term continuous imaging and super-resolution imaging of live cell RNA. However, these technologies still have potential problems such as limited fluorescence colors, labeling efficiency, imaging depth, photobleaching, and cell environment dependence.

[0005] Therefore, developing a two-color imaging compound that can specifically label lysosomes and RNA simultaneously is of great significance for in-depth study of the interaction between lysosomes and nuclear RNA. Summary of the Invention

[0006] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, one of the objectives of the present invention is to provide a dual-responsive fluorescent compound.

[0007] Another objective of the present invention is to provide a preparation method for this dual-responsive fluorescent compound.

[0008] Another objective of the present invention is to provide a fluorescent probe.

[0009] Another objective of the present invention is to provide the application of the dual-responsive fluorescent compound or the fluorescent probe.

[0010] Another objective of the present invention is to provide a method for monitoring the interaction between live cell RNA and lysosomes.

[0011] To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

[0012] The first aspect of the present invention provides a dual-responsive fluorescent compound, the structural formula of which is shown in formula (Ⅰ):

[0013] Formula (Ⅰ);

[0014] Wherein, R - is selected from halide ions, p-toluenesulfonate, tetrafluoroborate, hexafluorophosphate, bis(trifluoromethanesulfonyl)imide or trifluoromethanesulfonate.

[0015] In some embodiments of the present invention, the R -Selected from iodide ion, tosylate ion or trifluoromethanesulfonate ion.

[0016] In some embodiments of the present invention, the dual-responsive fluorescent compound is shown as formula (A):

[0017] Formula (A).

[0018] In some embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in the acidic environment without binding to RNA is 555 - 569 nm.

[0019] In some embodiments of the present invention, the pH of the acidic environment without binding to RNA is 4.5 - 5.5.

[0020] In some embodiments of the present invention, the dual-responsive fluorescent compound emits yellow fluorescence at an excitation wavelength of 555 - 569 nm.

[0021] In some specific embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in the acidic environment without binding to RNA is 561 nm.

[0022] In some embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in the neutral environment binding to RNA is 635 - 645 nm.

[0023] In some embodiments of the present invention, the dual-responsive fluorescent compound emits red fluorescence at an excitation wavelength of 635 - 645 nm.

[0024] In some specific embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in the neutral environment binding to RNA is 640 nm.

[0025] The dual-responsive fluorescent compound provided by the present invention, due to the N cation in its quinoline parent structure, makes the molecule carry a certain positive charge, thus having the ability to target and bind to RNA; the un-methylated N atom in the amino side chain 9-formyljulolidine makes the molecule have a certain basicity and can selectively distribute in a weakly acidic environment such as lysosomes, thus having the ability to target lysosomes.

[0026] When the dual-responsive fluorescent compound enters the cell, it can target both RNA and lysosomes at the same time. Under normal physiological conditions, the dual-responsive fluorescent compound is distributed in acidic lysosomes (pH 4.5 - 5.5) in the form of monomers and produces yellow fluorescence ( λ ex = 561 nm, λ em≈610 nm); However, due to the large conjugated system with alternating single and double bonds in its structure, when the dual-responsive fluorescent compound binds to RNA in the nucleus, the dual-responsive fluorescent compound will aggregate, resulting in a red shift of fluorescence and generating red fluorescence ( λ ex = 640 nm, λ em ≈670 nm). Due to its good fluorescence performance, low cytotoxicity, and strong anti-photobleaching effect, it has the ability to visually monitor the interaction between RNA and lysosomes based on microscopy imaging, as well as the mutual changes between the two types of organelles during the process of cuproptosis.

[0027] The second aspect of the present invention provides a preparation method of the dual-responsive fluorescent compound described in the first aspect of the present invention, comprising the following steps:

[0028] S1. React 3,4-difluoroaniline with ethyl acetoacetate to obtain intermediate (a) ;

[0029] S2. React intermediate (a) with phosphorus oxychloride to obtain intermediate (b) ;

[0030] S3. Carry out a catalytic reaction on intermediate (b) to obtain intermediate (c) ;

[0031] S4. React intermediate (c) with a methylation reagent to obtain intermediate (d) ;

[0032] S5. Carry out a condensation reaction on intermediate (d) with 9-formyljulolidine and 1-methylpiperazine to obtain the described dual-responsive fluorescent compound;

[0033] Wherein, the definition of R - is as described in the first aspect of the present invention.

[0034] In some embodiments of the present invention, in step S1, the mass ratio of the 3,4-difluoroaniline to the ethyl acetoacetate is 1: (0.8 - 1.5).

[0035] In some specific embodiments of the present invention, in step S1, the mass ratio of the 3,4-difluoroaniline to the ethyl acetoacetate is 1: (0.8 - 1.2).

[0036] In some embodiments of the present invention, step S1 further includes using a catalyst, and the mass ratio of the catalyst to the 3,4-difluoroaniline is (10 - 15): 1.

[0037] In some specific embodiments of the present invention, the catalyst used in step S1 includes polyphosphoric acid (PPA).

[0038] In some embodiments of the present invention, in step S1, the reaction temperature of the reaction is 120 - 140 °C, and the reaction time is 3 - 8 h.

[0039] In some specific embodiments of the present invention, in step S1, the reaction temperature of the reaction is 125 - 135 °C, and the reaction time is 5 - 6 h.

[0040] In some embodiments of the present invention, in step S1, after the reaction, it further includes the operations of cooling the reaction solution to room temperature, pouring it into ice water, adjusting the pH of the solution to neutral with sodium hydroxide, performing vacuum filtration, drying, to obtain the intermediate (a).

[0041] In some embodiments of the present invention, in step S1, the yield of the intermediate (a) is 75% - 85%.

[0042] In some embodiments of the present invention, in step S2, the solid - liquid ratio of the intermediate (a) to phosphorus oxychloride (POCl 3 ) is 1 g : (4 - 6) mL.

[0043] In some specific embodiments of the present invention, in step S2, the solid - liquid ratio of the intermediate (a) to phosphorus oxychloride is 1 g : (4 - 5) mL.

[0044] In some embodiments of the present invention, in step S2, the reaction temperature of the reaction is 100 - 120 °C, and the reaction time is 3 - 8 h.

[0045] In some specific embodiments of the present invention, in step S2, the reaction temperature of the reaction is 105 - 115 °C, and the reaction time is 4 - 6 h.

[0046] In some embodiments of the present invention, in step S2, after the reaction, it further includes the operations of cooling the reaction solution to room temperature, pouring it into ice water, adjusting the pH of the solution to weakly alkaline with sodium hydroxide, performing vacuum filtration, drying the crude product, and purifying by column chromatography to obtain the intermediate (b).

[0047] In some embodiments of the present invention, in step S2, the yield of the intermediate (b) is 55% - 65%.

[0048] In some embodiments of the present invention, in step S3, the catalytic reaction is carried out under a protective atmosphere; the protective atmosphere includes hydrogen.

[0049] In some embodiments of the present invention, in step S3, the catalytic reaction includes using a catalyst; the mass ratio of the catalyst to intermediate (b) is 1: (5 - 10).

[0050] In some specific embodiments of the present invention, the catalyst used in step S3 includes palladium on carbon.

[0051] In some embodiments of the present invention, in step S3, an alcohol solvent is also used, and the solid - liquid ratio of intermediate (b) to the alcohol solvent is 1 g: (13 - 17) mL.

[0052] In some embodiments of the present invention, in step S3, the reaction time of the reaction is 10 - 15 h.

[0053] In some specific embodiments of the present invention, in step S3, the reaction time of the reaction is 10 - 12 h.

[0054] In some embodiments of the present invention, in step S3, after the catalytic reaction, operations including suction filtration and drying are also included to obtain intermediate (c).

[0055] In some embodiments of the present invention, in step S3, the yield of intermediate (c) is 65% - 75%.

[0056] In some embodiments of the present invention, in step S4, the solid - liquid ratio of intermediate (c) to the methylation reagent is 1 g: (2 - 4) mL.

[0057] In some specific embodiments of the present invention, in step S4, the solid - liquid ratio of intermediate (c) to the methylation reagent is 1 g: (2 - 3) mL.

[0058] In some embodiments of the present invention, in step S4, an organic solvent is also used, and the solid - liquid ratio of intermediate (c) to the organic solvent is 1 g: (2 - 3) mL.

[0059] In some embodiments of the present invention, in step S4, the reaction temperature of the reaction is 80 - 100 °C, and the reaction time is 20 - 30 h.

[0060] In some specific embodiments of the present invention, in step S4, the reaction temperature of the reaction is 80 - 90 °C, and the reaction time is 20 - 25 h.

[0061] In some embodiments of the present invention, in step S4, after the reaction, operations including cooling, suction filtration, washing, and drying are also included to obtain intermediate (d).

[0062] In some embodiments of the present invention, in step S4, the yield of the intermediate (d) is 60%-70%.

[0063] In some embodiments of the present invention, in step S5, the molar ratio of the intermediate (d), 9-formyljulolidine, and 1-methylpiperazine is 1: (1-3): (3-5).

[0064] In some specific embodiments of the present invention, in step S5, the molar ratio of the intermediate (d), 9-formyljulolidine, and 1-methylpiperazine is 1: (1.5-2.5): (3.5-4.5).

[0065] In some embodiments of the present invention, in step S5, the reaction temperature of the reaction is 80-100 °C, and the reaction time is 10-15 h.

[0066] In some specific embodiments of the present invention, in step S5, the reaction temperature of the reaction is 85-95 °C, and the reaction time is 10-12 h.

[0067] In some embodiments of the present invention, in step S5, after the reaction, it further includes operations of cooling, drying, and column chromatography purification to obtain the dual-responsive fluorescent compound.

[0068] In some embodiments of the present invention, in step S5, the yield of the dual-responsive fluorescent compound is 40%-50%.

[0069] The third aspect of the present invention provides a fluorescent probe, comprising the dual-responsive fluorescent compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0070] In some embodiments of the present invention, the fluorescent probe targets RNA and lysosomes.

[0071] The fourth aspect of the present invention provides the use of the dual-responsive fluorescent compound described in the first aspect of the present invention, or the fluorescent probe described in the third aspect, in dual-color imaging of live cell RNA and lysosomes.

[0072] In some embodiments of the present invention, the live cells include human osteosarcoma cells (U-2OS) and human cervical cancer cells (Hela).

[0073] In some embodiments of the present invention, the fluorescent probe further includes pharmaceutically acceptable excipients.

[0074] In some specific embodiments of the present invention, the pharmaceutically acceptable excipients include buffer solutions, antifreeze agents, or preservatives.

[0075] The fifth aspect of the present invention provides a method for monitoring the interaction between live cell RNA and lysosomes, comprising the following steps:

[0076] Prepare the fluorescent probe described in the third aspect of the present invention into a solution, stain live cells, and place the washed live cells under a microscope to monitor the interaction between RNA and lysosomes.

[0077] In some embodiments of the present invention, the concentration of the fluorescent probe solution is 5 - 15 μmol / L.

[0078] In some specific embodiments of the present invention, the concentration of the fluorescent probe solution is 5 - 10 μmol / L.

[0079] In some embodiments of the present invention, the staining time is 3 - 6 h.

[0080] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0081] 1) The dual-responsive fluorescent compound provided by the present invention can simultaneously target RNA and lysosomes in live cells and emit fluorescence of different colors, realizing dual-color imaging of RNA and lysosomes in live cells and promoting the development of research on the interaction between lysosomes and RNA;

[0082] 2) The preparation method of the dual-responsive fluorescent compound provided by the present invention has simple steps, high yield, and is suitable for industrial application;

[0083] 3) The fluorescent probe provided by the present invention, including the dual-responsive fluorescent compound, can perform real-time monitoring of RNA and lysosomes in live biological samples, overcoming the problems of mutual interference, harsh staining conditions, and long time consumption when commercial RNA probes and commercial lysosome probes are used for separate labeling of RNA and lysosomes. The preparation process of the fluorescent probe is simple, the cost is low, the structure is stable, and it is convenient for storage, having broad application prospects in the research of the biological functions of RNA and lysosomes. Description of the Drawings

[0084] Figure 1 1H NMR spectrum of the dual-responsive fluorescent compound of formula (A) in the example;

[0085] Figure 2 Detection results of the ultraviolet absorption characteristics of the dual-responsive fluorescent compound of formula (A) in Test Example 1;

[0086] Figure 3 Effect of RNA content on the fluorescence emission spectrum of the dual-responsive fluorescent compound of formula (A) in Test Example 1;

[0087] Figure 4 Confocal imaging diagram of the dual-responsive fluorescent compound of formula (A) in Test Example 2;

[0088] Figure 5 Results of the co-localization experiment of the dual-responsive fluorescent compound of formula (A) and a commercially available lysosome probe in Test Example 2;

[0089] Figure 6 Results of the in-situ observation of the interaction between RNA and lysosomes in living cells in Test Example 3;

[0090] Figure 7 Interaction sites between RNA and lysosomes in living cells in Test Example 3. Detailed implementation manners

[0091] The content of the present invention will be further described in detail below through specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial channels or can be obtained by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0092] Example: In this example, a dual-responsive fluorescent compound was prepared. The steps and synthetic route are shown as follows:

[0093] S11: Add 10 g of 3,4-difluoroaniline and 10 g of ethyl acetoacetate into a round-bottom flask, add 120 g of polyphosphoric acid, react at 130 °C for 5 h, cool to room temperature, pour the reaction solution into 500 mL of ice water, adjust the pH of the solution to neutral with saturated sodium hydroxide, perform suction filtration under reduced pressure, and dry to obtain the intermediate (a) as a yellow solid with a yield of 78.1%; ;

[0094] S21: Dissolve 10 g of intermediate (a) in 50 mL of phosphorus oxychloride, reflux and react at 110 °C for 5 h, cool to room temperature, pour into 500 mL of ice water, adjust the pH of the solution to weakly alkaline with saturated sodium hydroxide solution, perform suction filtration under reduced pressure, and dry to obtain the crude product. Purify by silica gel column chromatography using petroleum ether / dichloromethane (2:1, v / v) as the eluent to obtain the intermediate (b) as a white solid with a yield of 57.9%;

[0095] ;

[0096] S31: Dissolve 7 g of intermediate (b) in 100 mL of methanol, add 1 g of palladium-carbon for catalysis, stir at room temperature for 12 h in a hydrogen environment, perform suction filtration, and evaporate the filtrate to dryness to obtain the intermediate (c) as a white solid with a yield of 71.4%;

[0097] ;

[0098] S41. Dissolve 4 g of intermediate (c) in 8 mL of acetonitrile, add 10 mL of methyl iodide, react at 85 °C for 24 h, cool to room temperature, filter by suction, wash with anhydrous ether and then dry under vacuum to obtain the intermediate (d) as a light yellow solid with a yield of 66.0%;

[0099] ;

[0100] S51. Dissolve 1 mmol of intermediate (d) in 5 mL of absolute ethanol, then add 2 mmol of 9-formyljulolidine and 4 mmol of 1-methylpiperazine, heat and react at 90 °C for 12 h, cool to room temperature, rotary evaporate and dry, and purify by silica gel column chromatography using methanol / dichloromethane (1:30, v / v) as the eluent to obtain the dual-responsive fluorescent compound of formula (A) as a blue-black solid with a yield of 42.8%;

[0101] 。

[0102] The dual-responsive fluorescent compound of formula (A) in the examples was characterized by nuclear magnetic resonance 1 1H NMR, Figure 1 which is the 1H NMR spectrum of the dual-responsive fluorescent compound of formula (A) in the examples, 1 and the 1H NMR data results are as follows:

[0103] 1 1H NMR (400 MHz, DMSO) δ 8.49 (d, J J = 8.9 Hz, 1H), 8.20 (d, J J = 9.1 Hz, 1H), 7.96 (dd, J J = 14.2, 5.9 Hz, 2H), 7.43 (d, J J = 7.5 Hz, 1H), 7.39–7.31 (m, 3H), 4.32 (s, 3H), 3.41 (t, J J = 4.9 Hz, 4H), 3.31 (d, J J = 5.7 Hz, 4H), 2.73 (t, J J = 6.2 Hz, 4H), 2.54 (t, J J = 4.8 Hz, 4H), 2.27 (s, 3H), 1.89 (p, J J = 6.2 Hz, 4H).

[0104] The dual-responsive fluorescent compound of formula (A) in the examples was characterized by high-resolution mass spectrometry, and the results were as follows:

[0105] HRMS(ESI): calcd for (M-I) + (C 29 H 34F N 4 + ) 457.2832, found 457.2829.

[0106] Experimental Example 1. This experimental example verifies the dual-color luminescence characteristics of the dual-responsive fluorescent compound of formula (A) prepared in the examples:

[0107] 1. Add the dual-responsive fluorescent compound of formula (A) prepared in the examples to a Tris-HCl buffer solution (20 mmol / L NaCl, 20 mmol / L Tris) with pH = 4.5 to prepare a stock solution with a concentration of 2 μmol / L. Take 400 μL of the stock solution and test the ultraviolet absorption spectrum with a UV spectrophotometer;

[0108] Add RNA and the dual-responsive fluorescent compound of formula (A) prepared in the examples to a Tris-HCl buffer solution (20 mmol / L NaCl, 20 mmol / L Tris) with pH = 7.0 to prepare a stock solution with an RNA concentration of 1 μmol / L and a concentration of the dual-responsive fluorescent compound of formula (A) of 2 μmol / L. Take 400 μL of the stock solution and test the ultraviolet absorption spectrum with a UV spectrophotometer;

[0109] Figure 2 Table [ID number] shows the test results of the ultraviolet absorption characteristics of the dual-responsive fluorescent compound of formula (A) in Experimental Example 1. It can be seen that Figure 2 under weakly acidic (pH = 4.5) conditions without binding to RNA, the maximum absorption wavelength of the dual-responsive fluorescent compound of formula (A) is around 550 nm. In a neutral environment with bound RNA, the maximum absorption wavelength of the dual-responsive fluorescent compound of formula (A) is around 640 nm, and there is a red shift in the maximum ultraviolet absorption wavelength.

[0110] 2. Add RNA and the dual-responsive fluorescent compound of formula (A) prepared in the examples to a Tris-HCl buffer solution (20 mmol / L NaCl, 20 mmol / L Tris) with pH = 7.0 to prepare stock solutions with RNA concentrations of 1 μmol / L, 2 μmol / L, and 3 μmol / L, respectively, and a concentration of the dual-responsive fluorescent compound of formula (A) of 2 μmol / L. Take 400 μL of each stock solution and test the ultraviolet absorption spectrum with a UV spectrophotometer.

[0111] Figure 3 Table [ID number] shows the effect of the RNA content on the fluorescence emission spectrum of the dual-responsive fluorescent compound of formula (A) in Experimental Example 1. It can be seen from Figure 3It can be seen that in a neutral environment, after adding RNA, the fluorescence spectrum of the dual-responsive fluorescent compound of formula (A) changes significantly. The intensity of the absorption peak at 595 nm gradually weakens with the increase of the RNA concentration, and a new emission peak appears at 670 nm, and the peak value increases with the increase of the RNA concentration.

[0112] The above results show that the dual-responsive fluorescent compound of formula (A) prepared in the examples exhibits fluorescence emission at a shorter wavelength (monomer luminescence) in a weakly acidic environment. In a neutral environment, with the addition of RNA, the dual-responsive fluorescent compound of formula (A) aggregates, and both the maximum ultraviolet absorption wavelength and the emission wavelength are red-shifted, showing longer wavelength emission (aggregate-state luminescence), indicating that the dual-responsive fluorescent compound of formula (A) prepared in the examples has the characteristic of dual-color luminescence and has the potential to specifically label lysosomes and RNA simultaneously.

[0113] Experimental Example 2: This experimental example verifies the live cell fluorescence imaging performance of the dual-responsive fluorescent compound of formula (A) prepared in the examples:

[0114] 1. Place human cervical cancer cells Hela in a culture medium (DMEM culture solution and 10 v / v% fetal bovine serum), and culture them in an incubator at 37 °C, 5% CO 2 and 20% O 2 for 48 h; inoculate the cells in a confocal 96-well plate, and culture them in an incubator at 37 °C, 5% CO 2 and 20% O 2 for 24 h; take the small molecule fluorescent probe compound of formula (A) prepared in the examples, dilute it with Hela cell culture medium to a final concentration of 5 μmol / L, add the prepared probe to the 96-well plate inoculated with Hela cells, 100 μL per well, continue to culture for 5 h, then wash the samples 3 times with PBS buffer, and perform confocal microscopy imaging.

[0115] Figure 4 Figure 21 is the confocal imaging diagram of the dual-responsive fluorescent compound of formula (A) in Experimental Example 2. Among them, Figure 4 in (a) is the signal of the small molecule fluorescent probe compound of formula (A) in the channel with an excitation wavelength of 561 nm, Figure 4 in (b) is the signal of the small molecule fluorescent probe compound of formula (A) in the channel with an excitation wavelength of 640 nm, Figure 4 in (c) is the merged image. As can be seen from Figure 4 , in the channel with an excitation wavelength of 561 nm, yellow dot-like fluorescence signals are obtained in the cytoplasm, and in the channel with an excitation wavelength of 640 nm, red aggregated fluorescence signals are obtained in the nucleus, indicating that the dual-responsive fluorescent compound of formula (A) prepared in the examples can perform dual-color imaging.

[0116] 2. The co-localization experiment was carried out using the commercially available lysosome probe LysoTracker DeepRed (0.1 μmol / L) to examine whether the yellow punctate fluorescence signal was lysosome.

[0117] Figure 5 For the co-localization test results of the dual-responsive fluorescent compound of formula (A) in Test Example 2 and the commercially available lysosome probe, where Figure 5 (a) in is the signal of the small molecule fluorescent probe compound of formula (A), with an excitation wavelength of 561 nm, Figure 5 (b) in is the signal of the commercially available lysosome probe LysoTracker DeepRed, with an excitation wavelength of 404 nm, Figure 5 (c) in is the merged image. It can be seen from Figure 5 that the dual-responsive fluorescent compound of formula (A) shows yellow punctate fluorescence signals in the classical lysosome morphology, which overlaps with the localization of the commercially available lysosome probe. After being processed by Image J software, the co-localization coefficient (R) of the two is as high as 0.934, indicating that the dual-responsive fluorescent compound of formula (A) prepared in the example can target lysosomes in living cells.

[0118] Test Example 3: The adherent human cervical cancer cells Hela were incubated with the dual-responsive fluorescent compound of formula (A) prepared in the example (final concentration of 10 μmol / L) in an incubator at 37 °C, 5% CO 2 and 20% O 2 for 2 h, and then the interaction between RNA and lysosomes was observed in situ under a laser confocal microscope.

[0119] Figure 6 For the results of in-situ observation of the interaction between RNA and lysosomes in living cells in Test Example 3, where Figure 6 (a) in is the signal of the small molecule fluorescent probe compound of formula (A) in the channel with an excitation wavelength of 561 nm, used to observe lysosomes, Figure 6 (b) in is the signal of the small molecule fluorescent probe compound of formula (A) in the channel with an excitation wavelength of 640 nm, used to observe RNA, Figure 6 (c) in is the merged image; Figure 7 For the interaction sites between RNA and lysosomes in living cells in Test Example 3, where Figure 7 (a) in is all the interaction sites, Figure 7 (b) in are interaction sites 1 and 2, Figure 7 (c) in is interaction site 3, Figure 7 (d) in is interaction site 4; It can be seen from Figure 6 and Figure 7It can be seen that the dual-responsive fluorescent compound of formula (A) provided by the present invention has ultra-high selectivity for RNA and lysosomes in living cells, can well stain RNA and lysosomes in living cells, and visually monitor the interaction between the two.

Claims

1. A dual-response fluorescent compound, characterized in that: Its structural formula is shown in formula (I): Formula (I); Among them, R - Selected from halide, p-toluenesulfonate, tetrafluoroborate, hexafluorophosphate or trifluoromethanesulfonate.

2. The dual-response fluorescent compound according to claim 1, characterized in that: The R - Selected from iodide, p-toluenesulfonate or trifluoromethanesulfonate.

3. The method for preparing the dual-responsive fluorescent compound according to claim 1 or 2, characterized in that: The following steps are involved: S1, reacting 3,4-difluoroaniline with ethyl acetoacetate to obtain intermediate (a) ; S2. reacting the intermediate (a) with phosphorus oxychloride to obtain the intermediate (b) ; S3, catalyzing the intermediate (b) to obtain the intermediate (c) ; S4, reacting the intermediate (c) with a methylating agent to obtain the intermediate (d) ; S5, subjecting the intermediate (d) to a condensation reaction with 9-aldehyde julolidine and 1-methylpiperazine to obtain the dual-response fluorescent compound; Among them, R - The definition as claimed in claim 1 or 2.

4. The preparation method according to claim 3, characterized in that: In step S1, the mass ratio of 3,4-difluoroaniline to ethyl acetoacetate is 1: (0.8-1.5); In step S2, the solid-liquid ratio of the intermediate (a) to phosphorus oxychloride is 1 g: (4-6) mL; In step S4, the solid-liquid ratio of the intermediate (c) to the methylating agent is 1 g: (2-4) mL; In step S5, the molar ratio of the intermediate (d), 9-aldehyde julolidine and 1-methylpiperazine is 1: (1-3): (3-5).

5. The preparation method according to claim 3, characterized in that: In step S1, the reaction temperature is 120-140°C and the reaction time is 3-8h; In step S2, the reaction temperature is 100-120°C and the reaction time is 3-8h; In step S3, the reaction time of the reaction is 10-15h; In step S4, the reaction temperature is 80-100°C and the reaction time is 20-30h; In step S5, the reaction temperature is 80-100° C. and the reaction time is 10-15 h.

6. A fluorescent probe, characterized in that It comprises the dual-response fluorescent compound or a pharmaceutically acceptable salt thereof as described in claim 1 or 2.

7. The fluorescent probe according to claim 6, characterized in that The fluorescent probe targets RNA and lysosomes.

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