Double-response fluorescent compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescence imaging, and discloses a dual-response fluorescent compound as well as a preparation method and application thereof. The double-response fluorescent compound provided by the invention can target RNA and lysosome in living cells at the same time and emit fluorescence of different colors, so that double-color imaging of the RNA and the lysosome in the living cells is realized, and development of research on interaction between the lysosome and the RNA is promoted; the preparation method of the double-response fluorescent compound is simple in steps, high in yield and suitable for industrial application; comprising the double-response fluorescent compound, RNA and lysosome in a living biological sample can be monitored in real time, the problems of mutual interference, harsh dyeing conditions and the like when a commercial RNA probe and a commercial lysosome probe independently mark RNA and lysosome are solved, and the fluorescent probe is simple in preparation process, low in cost, stable in structure and easy to implement. And the method is convenient to store and has a wide application space in biological function research of RNA and lysosome.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence imaging technology, and in particular to a dual-response fluorescent compound and a preparation method and application thereof. Background Art

[0002] Lysosomes and RNA are two key functional units in cells, which are involved in cell degradation metabolism and gene expression regulation respectively. Among them, lysosomes, as the "recycling center" of cells, degrade damaged organelles, proteins and foreign substances through their internal acid hydrolases 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 vital role in gene expression regulation: mRNA is a template for protein synthesis, lncRNA participates in chromatin remodeling and gene expression regulation, snRNA is the core component of the spliceosome, responsible for the splicing processing of mRNA precursors, 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 independent, more and more studies have shown that there are complex interactions between the two. For example, some nuclear RNA may enter lysosomes and be degraded through autophagy or specific transport mechanisms, thereby regulating gene expression; lysosomes regulate the transcription and processing of ribosomal RNA (rRNA) through signaling pathways such as mTORC1, affecting the protein synthesis capacity of cells; some lncRNAs may affect the function and stability of lysosomes by regulating the expression of lysosome-related genes or directly interacting with lysosomal proteins. These interactions are of great significance in cell homeostasis, stress response and disease occurrence. For example, in neurodegenerative diseases, abnormal lysosomal function leads to RNA metabolic disorders, which in turn aggravates neuronal damage; in cancer, the interaction between lysosomes and RNA may affect the metabolism and invasive ability of tumor cells. However, although significant progress has been made in the study of the functions of lysosomes and RNA, research on the interaction between the two still faces technical challenges.

[0004] Existing fluorescent 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 limits their application in dynamic imaging of living cells. Existing two-color imaging technologies, such as pure organic room temperature phosphorescent supramolecular systems driven by photooxidation, can achieve dual-targeted imaging of cell nuclei and lysosomes; Okra505 fluorescent RNA technology provides new tools for long-term continuous imaging and super-resolution imaging of RNA in living cells. However, these technologies still have potential problems such as fluorescence color limitations, labeling efficiency, imaging depth, photobleaching, and cell environment dependence.

[0005] Therefore, developing a dual-color imaging compound that can simultaneously and specifically label lysosomes and RNA 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 solve at least one of the above-mentioned technical problems existing in the prior art. To this end, one of the purposes of the present invention is to provide a dual-response fluorescent compound.

[0007] The second object of the present invention is to provide a method for preparing the dual-response fluorescent compound.

[0008] A third object of the present invention is to provide a fluorescent probe.

[0009] A fourth object of the present invention is to provide applications of dual-response fluorescent compounds or fluorescent probes.

[0010] A fifth object of the present invention is to provide a method for monitoring the interaction between RNA and lysosomes in living cells.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is: The first aspect of the present invention provides a dual-response fluorescent compound, the structural formula of which is shown in formula (I): Formula (I); Among them, R - Selected from halide, p-toluenesulfonate, tetrafluoroborate, hexafluorophosphate, bis(trifluoromethanesulfonyl)imide or trifluoromethanesulfonate.

[0012] In some embodiments of the present invention, the R - Selected from iodide, p-toluenesulfonate or trifluoromethanesulfonate.

[0013] In some embodiments of the present invention, the dual-response fluorescent compound is as shown in formula (A): Formula (A).

[0014] In some embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in an acidic environment without binding to RNA is 555-569 nm.

[0015] In some embodiments of the present invention, the pH of the acidic environment to which the RNA is not bound is 4.5-5.5.

[0016] In some embodiments of the present invention, the dual-responsive fluorescent compound emits yellow fluorescence at an excitation wavelength of 555-569 nm.

[0017] In some specific embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in an acidic environment without binding to RNA is 561 nm.

[0018] In some embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in a neutral environment when bound to RNA is 635-645 nm.

[0019] In some embodiments of the present invention, the dual-responsive fluorescent compound emits red fluorescence at an excitation wavelength of 635-645 nm.

[0020] In some specific embodiments of the present invention, the excitation wavelength of the dual-responsive fluorescent compound in a neutral environment when combined with RNA is 640 nm.

[0021] The dual-response fluorescent compound provided by the present invention has a quinoline parent structure containing N cations, so that the molecule has a certain positive charge, thereby having the ability to target and bind to RNA; the unmethylated N atom in the amino side chain 9-aldehyde julolidine makes the molecule have a certain alkalinity, and can be selectively distributed in a weakly acidic environment such as a lysosome, thereby having the ability to target the lysosome.

[0022] When the dual-responsive fluorescent compound enters the cell, it can simultaneously target RNA and lysosomes. Under normal physiological conditions, the dual-responsive fluorescent compound is distributed in the acidic lysosome in the form of a monomer (pH 4.5-5.5) and produces yellow fluorescence ( λ ex =561nm, λ em ≈610nm); however, due to its large conjugated system with alternating single and double bonds in its structure, when the dual-responsive fluorescent compound binds to RNA in the cell nucleus, the dual-responsive fluorescent compound will aggregate, causing the fluorescence to red-shift and produce red fluorescence ( λex =640nm, λ em ≈670nm). Due to its good fluorescence properties, low cytotoxicity, and strong resistance to photobleaching, it has the ability to visualize and monitor the interaction between RNA and lysosomes based on microscopy imaging, as well as the mutual changes between the two types of organelles during copper death.

[0023] The second aspect of the present invention provides a method for preparing the dual-response fluorescent compound according to the first aspect of the present invention, comprising the following steps: 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 - is as defined in the first aspect of the present invention.

[0024] In some embodiments of the present invention, in step S1, the mass ratio of 3,4-difluoroaniline to ethyl acetoacetate is 1: (0.8-1.5).

[0025] In some specific embodiments of the present invention, in step S1, the mass ratio of 3,4-difluoroaniline to ethyl acetoacetate is 1: (0.8-1.2).

[0026] In some embodiments of the present invention, step S1 further comprises using a catalyst, wherein the mass ratio of the catalyst to 3,4-difluoroaniline is (10-15): 1.

[0027] In some specific embodiments of the present invention, the catalyst used in step S1 includes polyphosphoric acid (PPA).

[0028] 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.

[0029] 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.

[0030] In some embodiments of the present invention, in step S1, after the reaction is completed, the reaction solution is cooled to room temperature, poured into ice water, the pH of the solution is adjusted to neutral with sodium hydroxide, filtered under reduced pressure, and dried to obtain the intermediate (a).

[0031] In some embodiments of the present invention, in step S1, the yield of the intermediate (a) is 75%-85%.

[0032] In some embodiments of the present invention, in step S2, the solid-liquid ratio of the intermediate (a) to phosphorus oxychloride (POCl3) is 1 g: (4-6) mL.

[0033] In some specific embodiments of the present invention, in step S2, the solid-to-liquid ratio of the intermediate (a) to phosphorus oxychloride is 1 g: (4-5) mL.

[0034] 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.

[0035] 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.

[0036] In some embodiments of the present invention, in step S2, after the reaction is completed, the reaction solution is cooled to room temperature, poured into ice water, the pH of the solution is adjusted to weak alkalinity with sodium hydroxide, filtered under reduced pressure, the crude product is dried, and purified by column chromatography to obtain the intermediate (b).

[0037] In some embodiments of the present invention, in step S2, the yield of the intermediate (b) is 55%-65%.

[0038] 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.

[0039] In some embodiments of the present invention, in step S3, the catalytic reaction includes using a catalyst; the mass ratio of the catalyst to the intermediate (b) is 1: (5-10).

[0040] In some specific embodiments of the present invention, the catalyst used in step S3 includes palladium on carbon.

[0041] In some embodiments of the present invention, step S3 further comprises using an alcohol solvent, and the solid-liquid ratio of the intermediate (b) to the alcohol solvent is 1 g: (13-17) mL.

[0042] In some embodiments of the present invention, in step S3, the reaction time of the reaction is 10-15 hours.

[0043] In some specific embodiments of the present invention, in step S3, the reaction time of the reaction is 10-12 hours.

[0044] In some embodiments of the present invention, in step S3, after the catalytic reaction is completed, the steps further include filtering and drying to obtain the intermediate (c).

[0045] In some embodiments of the present invention, in step S3, the yield of the intermediate (c) is 65%-75%.

[0046] In some embodiments of the present invention, in step S4, the solid-liquid ratio of the intermediate (c) to the methylating agent is 1 g: (2-4) mL.

[0047] In some specific embodiments of the present invention, in step S4, the solid-liquid ratio of the intermediate (c) to the methylating agent is 1 g: (2-3) mL.

[0048] In some embodiments of the present invention, step S4 further comprises using an organic solvent, and the solid-liquid ratio of the intermediate (c) to the organic solvent is 1 g: (2-3) mL.

[0049] 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.

[0050] 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.

[0051] In some embodiments of the present invention, in step S4, after the reaction is completed, the steps of cooling, filtering, washing and drying are further performed to obtain the intermediate (d).

[0052] In some embodiments of the present invention, in step S4, the yield of the intermediate (d) is 60%-70%.

[0053] In some embodiments of the present invention, in step S5, the molar ratio of the intermediate (d), 9-aldehyde julolidine and 1-methylpiperazine is 1: (1-3): (3-5).

[0054] In some specific embodiments of the present invention, in step S5, the molar ratio of the intermediate (d), 9-aldehyde julolidine and 1-methylpiperazine is 1: (1.5-2.5): (3.5-4.5).

[0055] 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.

[0056] 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.

[0057] In some embodiments of the present invention, in step S5, after the reaction is completed, the steps further include cooling, drying, and column chromatography purification to obtain the dual-responsive fluorescent compound.

[0058] In some embodiments of the present invention, in step S5, the yield of the dual-responsive fluorescent compound is 40%-50%.

[0059] The third aspect of the present invention provides a fluorescent probe, comprising the dual-response fluorescent compound or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention.

[0060] In some embodiments of the invention, the fluorescent probe targets RNA and lysosomes.

[0061] The fourth aspect of the present invention provides the use of the dual-response 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 RNA and lysosomes in living cells.

[0062] In some embodiments of the present invention, the living cells include human osteosarcoma cells (U-2OS) and human cervical cancer cells (Hela).

[0063] In some embodiments of the present invention, the fluorescent probe further comprises a pharmaceutically acceptable excipient.

[0064] In some specific embodiments of the present invention, the pharmaceutically acceptable excipients include buffers, antifreeze agents or preservatives.

[0065] A fifth aspect of the present invention provides a method for monitoring the interaction between RNA and lysosomes in living cells, comprising the following steps: The fluorescent probe described in the third aspect of the present invention is prepared into a solution, used to stain living cells, and the washed living cells are placed under a microscope to monitor the interaction between RNA and lysosomes.

[0066] In some embodiments of the present invention, the concentration of the fluorescent probe solution is 5-15 µmol / L.

[0067] In some specific embodiments of the present invention, the concentration of the fluorescent probe solution is 5-10 µmol / L.

[0068] In some embodiments of the present invention, the staining time is 3-6 hours.

[0069] Compared with the prior art, the present invention has the following beneficial effects: 1) The dual-response fluorescent compound provided by the present invention can simultaneously target RNA and lysosomes in living cells and emit fluorescence of different colors, thereby realizing dual-color imaging of RNA and lysosomes in living cells and promoting the development of research on the interaction between lysosomes and RNA; 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 use; 3) The fluorescent probe provided by the present invention includes a dual-response fluorescent compound, which can monitor RNA and lysosomes in living biological samples in real time, overcoming the problems of mutual interference, harsh staining conditions, and long time consumption when commercial RNA probes and commercial lysosomal 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 easy to store. It has a broad application space in the study of RNA and lysosomal biological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 is a hydrogen nuclear magnetic resonance spectrum of the dual-responsive fluorescent compound of formula (A) in the embodiment; Figure 2 The ultraviolet absorption characteristic test results of the dual-response fluorescent compound of formula (A) in Experimental Example 1; Figure 3 The effect of RNA content on the fluorescence emission spectrum of the dual-response fluorescent compound of formula (A) in Experimental Example 1; Figure 4 This is the confocal imaging image of the dual-response fluorescent compound of formula (A) in Experimental Example 2; Figure 5 The co-localization test results of the dual-response fluorescent compound of formula (A) and the commercially available lysosomal probe in Experimental Example 2; Figure 6 The results of in situ observation of the interaction between RNA and lysosomes in living cells in Experimental Example 3; Figure 7 This is the interaction site between RNA and lysosome in living cells in Experimental Example 3. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0072] Example: This example prepares a dual-response fluorescent compound, and the steps and synthesis route are shown as follows: 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, filter under reduced pressure, and dry to obtain intermediate (a) as a yellow solid with a yield of 78.1%; ; S21, dissolving 10 g of intermediate (a) in 50 mL of phosphorus oxychloride, reflux at 110° C. for 5 h, cooling to room temperature, pouring into 500 mL of ice water, adjusting the pH of the solution to be weakly alkaline with saturated sodium hydroxide solution, filtering under reduced pressure, and drying to obtain a crude product, which was purified by silica gel column chromatography using petroleum ether / dichloromethane (2: 1, v / v) as an eluent to obtain intermediate (b) as a white solid with a yield of 57.9%; ; S31, dissolve 7 g of intermediate (b) in 100 mL of methanol, add 1 g of palladium carbon catalyst, stir at room temperature in a hydrogen environment for 12 h, filter with suction, evaporate the filtrate to obtain intermediate (c) as a white solid, with a yield of 71.4%; ; S41, 4 g of intermediate (c) was dissolved in 8 mL of acetonitrile, 10 mL of iodomethane was added, the mixture was reacted at 85°C for 24 h, cooled to room temperature, filtered, washed with anhydrous ether and dried in vacuo to obtain intermediate (d) as a light yellow solid, with a yield of 66.0%; ; S51, 1 mmol of intermediate (d) was dissolved in 5 mL of anhydrous ethanol, and then 2 mmol of 9-aldehyde julolidine and 4 mmol of 1-methylpiperazine were added, and the mixture was heated at 90°C for 12 h, cooled to room temperature, and dried by rotary evaporation. The mixture was purified by silica gel column chromatography using methanol / dichloromethane (1:30, v / v) as the eluent to obtain a blue-black solid dual-responsive fluorescent compound of formula (A), and the yield was 42.8%; .

[0073] The dual-response fluorescent compound of formula (A) in the embodiment was subjected to nuclear magnetic resonance. 1 H NMR characterization, Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the dual-response fluorescent compound of formula (A) in the embodiment, 1 The H NMR data results are: 1 H NMR (400MHz, DMSO) δ 8.49 (d, J =8.9Hz, 1H), 8.20 (d,J =9.1Hz, 1H),7.96 (dd, J =14.2, 5.9Hz, 2H), 7.43 (d, J =7.5Hz, 1H), 7.39–7.31 (m, 3H), 4.32(s, 3H), 3.41 (t, J =4.9 Hz, 4H), 3.31 (d, J =5.7Hz, 4H), 2.73 (t, J =6.2Hz,4H), 2.54 (t, J =4.8Hz, 4H), 2.27 (s, 3H), 1.89 (p, J =6.2 Hz, 4H).

[0074] The dual-response fluorescent compound of formula (A) in the embodiment was characterized by high-resolution mass spectrometry, and the results were: HRMS(ESI):calcd for (MI) + (C 29 H 34F N4 + ) 457.2832, found 457.2829.

[0075] Experimental Example 1: This experimental example verifies the dual-color luminescence characteristics of the dual-response fluorescent compound of formula (A) prepared in the example: 1. Add the dual-responsive fluorescent compound of formula (A) prepared in the example to a Tris-HCl buffer (20 mmol / L NaCl, 20 mmol / L Tris) at pH = 4.5 to prepare a mother solution with a concentration of 2 μmol / L. Take 400 μL of the mother solution and test the ultraviolet absorption spectrum in an ultraviolet spectrophotometer; RNA and the dual-responsive fluorescent compound of formula (A) prepared in the embodiment were added to a Tris-HCl buffer (20 mmol / L NaCl, 20 mmol / L Tris) at pH = 7.0 to prepare a mother solution with an RNA concentration of 1 μmol / L and a dual-responsive fluorescent compound of formula (A) concentration of 2 μmol / L. 400 μL of the mother solution was taken and tested for ultraviolet absorption spectrum in an ultraviolet spectrophotometer; Figure 2 is the ultraviolet absorption characteristic test result of the dual-response fluorescent compound of formula (A) in Experimental Example 1, Figure 2It can be seen that under weakly acidic conditions (pH=4.5) without RNA binding, the maximum absorption wavelength of the dual-responsive fluorescent compound of formula (A) is around 550nm. Under a neutral environment with RNA binding, the maximum absorption wavelength of the dual-responsive fluorescent compound of formula (A) is around 640nm, and the maximum ultraviolet absorption wavelength shows a red shift phenomenon.

[0076] 2. RNA and the dual-responsive fluorescent compound of formula (A) prepared in the example were added to Tris-HCl buffer (20 mmol / L NaCl, 20 mmol / L Tris) at pH 7.0 to prepare mother solutions with RNA concentrations of 1 μmol / L, 2 μmol / L and 3 μmol / L, respectively, and dual-responsive fluorescent compound concentrations of formula (A) of 2 μmol / L. 400 μL of each mother solution was taken and tested for UV absorption spectrum in a UV spectrophotometer.

[0077] Figure 3 The effect of RNA content on the fluorescence emission spectrum of the dual-response fluorescent compound of formula (A) in Experimental Example 1 is shown in FIG. Figure 3 It can be seen that under a neutral environment, after adding RNA, the fluorescence spectrum of the dual-response fluorescent compound of formula (A) changes significantly. The absorption peak intensity at 595 nm gradually weakens with the increase of RNA concentration, and a new emission peak appears at 670 nm, and the peak value increases with the increase of RNA concentration.

[0078] The above results show that the dual-responsive fluorescent compound of formula (A) prepared in the example exhibits a shorter wavelength fluorescence emission (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, exhibiting a longer wavelength emission (aggregate luminescence), indicating that the dual-responsive fluorescent compound of formula (A) prepared in the example has a dual-color luminescence characteristic and has the potential to simultaneously and specifically label lysosomes and RNA.

[0079] Experimental Example 2: This experimental example verifies the live cell fluorescence imaging performance of the dual-response fluorescent compound of formula (A) prepared in the example: 1. Human cervical cancer cells Hela were placed in a culture medium (DMEM culture medium and 10v / v% embryonic bovine serum) and cultured in an incubator at 37°C, 5% CO2 and 20% O2 for 48h; the cells were inoculated in a laser confocal 96-well plate and cultured in an incubator at 37°C, 5% CO2 and 20% O2 for 24h; the small molecule fluorescent probe compound of formula (A) prepared in the embodiment was diluted with Hela cell culture medium to a final concentration of 5μmol / L, and the prepared probe was added to a 96-well plate inoculated with Hela cells, 100μL per well, and after continuing to culture for 5h, the sample was washed 3 times with PBS buffer and imaged under a laser confocal microscope.

[0080] Figure 4 This is a confocal imaging diagram of the dual-response fluorescent compound of formula (A) in Experimental Example 2, wherein: Figure 4 (a) is the signal of the small molecule fluorescent probe compound of formula (A) at an excitation wavelength of 561 nm. Figure 4 (b) is the signal of the small molecule fluorescent probe compound of formula (A) at an excitation wavelength of 640 nm. Figure 4 (c) in the figure is the fusion graph, which is Figure 4 It can be seen that the channel with an excitation wavelength of 561 nm obtains a yellow dot-like fluorescence signal in the cytoplasm, and the channel with an excitation wavelength of 640 nm obtains a red aggregated fluorescence signal in the cell nucleus, indicating that the dual-responsive fluorescent compound of formula (A) prepared in the example can perform dual-color imaging.

[0081] 2. A co-localization test was performed using the commercially available lysosomal probe LysoTracker DeepRed (0.1 μmol / L) to test whether the yellow dot-like fluorescent signal was a lysosome.

[0082] Figure 5 The co-localization test results of the dual-response fluorescent compound of formula (A) and the commercially available lysosomal probe in Experimental Example 2, wherein: Figure 5 (a) is the signal of the small molecule fluorescent probe compound of formula (A), and the excitation wavelength is 561 nm. Figure 5 (b) is the signal of the commercially available lysosomal probe LysoTracker DeepRed, with an excitation wavelength of 404 nm. Figure 5 (c) in the figure is the fusion graph, which is Figure 5 It can be seen that the dual-responsive fluorescent compound of formula (A) presents a yellow dot-shaped fluorescent signal in the classic lysosomal morphology, which overlaps with the positioning of the commercially available lysosomal probe. After processing with image J software, the colocalization coefficient (R) of the two is as high as 0.934, indicating that the dual-responsive fluorescent compound of formula (A) prepared in the embodiment can target lysosomes in living cells.

[0083] Experimental Example 3: After incubating adherent human cervical cancer cells Hela 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% CO2 and 20% O2 for 2 h, the interaction between RNA and lysosomes was observed in situ under a laser confocal microscope.

[0084] Figure 6 The results of in situ observation of the interaction between RNA and lysosomes in living cells in Experimental Example 3, wherein: Figure 6 (a) is the signal of the small molecule fluorescent probe compound of formula (A) at an excitation wavelength of 561 nm, which is used to observe lysosomes. Figure 6 (b) is the signal of the small molecule fluorescent probe compound of formula (A) at an excitation wavelength of 640 nm, which is used to observe RNA. Figure 6 (c) in the figure is the fusion image; Figure 7 is the interaction site between RNA and lysosome in living cells in Experimental Example 3, wherein, Figure 7 (a) in the figure represents all interaction sites. Figure 7 (b) in the figure is the interaction sites 1 and 2. Figure 7 (c) in the figure is interaction site 3, Figure 7 (d) in the figure is interaction site 4; Figure 6 and Figure 7 It 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, bis(trifluoromethanesulfonyl)imide 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); And / or, in step S2, the solid-liquid ratio of the intermediate (a) to phosphorus oxychloride is 1 g: (4-6) mL; and / or, in step S4, the solid-liquid ratio of the intermediate (c) to the methylating agent is 1 g: (2-4) mL; And / or, 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; And / or, in step S2, the reaction temperature of the reaction is 100-120° C., and the reaction time is 3-8 h; And / or, in step S3, the reaction time of the reaction is 10-15h; And / or, in step S4, the reaction temperature of the reaction is 80-100° C., and the reaction time is 20-30 h; And / or, in step S5, the reaction temperature of the reaction 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.

8. Use of the dual-response fluorescent compound according to claim 1 or 2, or the fluorescent probe according to claim 6 or 7 in dual-color imaging of RNA and lysosomes in living cells.

9. A method for monitoring the interaction between RNA and lysosome in living cells, characterized in that: The following steps are involved: The fluorescent probe according to claim 6 or 7 is prepared into a solution, used to stain living cells, and the washed living cells are placed under a microscope to monitor the interaction between RNA and lysosome.

10. The monitoring method according to claim 9, characterized in that: The concentration of the fluorescent probe solution is 5-15 µmol / L; And / or, the dyeing time is 3-6h.

Images