Lipid droplet and hypochlorous acid sequence response type fluorescent probe as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of organic fluorescent probes, and particularly discloses a lipid droplet and hypochlorous acid sequence response type fluorescent probe, the structural formula is # imgabs0 #, and R is cyano, benzothiazole, benzimidazole, amido, ethoxycarbonyl or acetyl. According to the precise imaging lipid droplet and hypochlorous acid sequence response fluorescent probe, a phenothiazine and coumarin dye bracket is used as a parent body, bis (trifluoromethylbenzyl) is used as a lipid droplet targeting group, and different electron withdrawing groups are introduced to adjust the emission wavelength of the dye, so that a series of probes are synthesized; and high-targeting sequence imaging of the lipid droplets and hypochlorous acid in the atherosclerotic plaque is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of organic fluorescent probes, and in particular to a lipid droplet and hypochlorous acid sequence responsive fluorescent probe, and a synthesis method and application thereof. Background Art

[0002] Atherosclerosis is the main cause of cardiovascular disease. Accurately identifying atherosclerotic plaques can effectively prevent the aggravation of cardiovascular disease. The formation and evolution of atherosclerotic plaques can narrow the arteries, thereby hindering blood flow and leading to fatal cardiovascular events such as myocardial infarction, stroke, and even sudden death. Given that the morphology and distribution of plaques determine the severity of atherosclerotic lesions, accurate identification of atherosclerotic plaques is crucial for doctors to make timely clinical decisions and take proactive interventions as early as possible.

[0003] Phenothiazine is a "butterfly-shaped" non-planar heterocyclic structure, a classic fluorophore with a large Stokes shift and multiple functional group modification sites. The non-planar structure of phenothiazine effectively prevents the π-π stacking within the dye molecule. When the heteroatoms on the phenothiazine fluorophore are replaced, a non-planar bending structure will further occur. This bending structure can hinder the aggregation between molecules and increase the solubility of the fluorophore. In addition, phenothiazine itself is a very good electron-donating group. In recent years, it has gradually become a hot topic in the design of intramolecular charge transfer fluorescent probes. In addition, coumarin has excellent photophysical properties, high fluorescence quantum yield, large Stokes shift, good biocompatibility and easy functional group modification. In addition, it can also form a stable push-pull electronic structure with the coumarin parent by modifying strong electron-withdrawing groups or strong electron-donating groups to promote charge transfer within the molecule, thereby emitting stronger fluorescence. The Chinese patent with the announcement number CN 114163456 B discloses a phenothiazine coumarin-based pyridinium salt compound for detecting endogenous HOCl in cells and distinguishing normal cells from tumor cells.

[0004] Abnormal lipid droplet concentration and elevated hypochlorous acid levels are two major signs of atherosclerotic plaque formation. Accurate detection of lipid droplet concentration and hypochlorous acid levels is of great significance for accurately identifying atherosclerotic plaques and effectively preventing cardiovascular diseases. Chinese patent No. CN 108997289 B discloses a hypochlorous acid ratio fluorescent probe targeting lipid droplets, which uses coumarin fluorophore as energy donor, (E)-2-(3-(4-(disubstituted amino)phenylvinyl)-(5,5-dimethyl)cyclohex-2-ene-1-ylidene)malononitrile as energy receptor, and acylpiperazine as linking group. The probe can selectively react with hypochlorous acid to detect low concentrations of hypochlorous acid and can be used for ratio imaging in cells. The fluorescent probe is only used for hypochlorous acid detection, and the imaging of lipid concentration and atherosclerotic plaques is not explored. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a lipid droplet and hypochlorous acid sequence response type fluorescent probe for accurately imaging lipid droplets and hypochlorous acid sequence response in atherosclerotic plaques. The specific technical scheme is as follows: A lipid droplet and hypochlorous acid sequence responsive fluorescent probe, the structural formula of the fluorescent probe is: Wherein, R is cyano, benzothiazole, benzimidazole, amido, ethoxycarbonyl or acetyl.

[0006] Preferably, in the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probe, R is cyano.

[0007] The present invention also provides a method for synthesizing the above-mentioned lipid droplet and hypochlorous acid sequence responsive fluorescent probe, comprising the following steps: (1) Under protective atmosphere conditions, the compound 2-methoxyphenothiazine is dissolved in an organic solvent, sodium hydroxide and potassium iodide are added in sequence and stirred, 3,5-bis(trifluoromethylbenzyl bromide) is added, and the mixture is heated under reflux for reaction. After the reaction is completed, the organic phase is extracted and washed, and distilled under reduced pressure to obtain intermediate 1; (2) Mix anhydrous N,N-dimethylformamide and anhydrous dichloromethane, add phosphorus oxychloride dropwise at 0-5°C under a protective atmosphere, stir at 0-5°C for 0.5-1h, and then add a dichloromethane mixture containing intermediate 1 dropwise; heat to reflux for reaction. After the reaction is completed, extract and wash the organic phase, and distill under reduced pressure to obtain intermediate 2; (3) Dissolve the intermediate 2 in dichloromethane, add boron tribromide dropwise at 0-5°C, and react by stirring at 0-5°C. After the reaction is completed, extract and wash the organic phase, perform vacuum distillation, and perform gradient elution to obtain the intermediate 3; (4) The intermediate 3 and the corresponding cyanide or acetyl ester compound are dissolved in ethanol, piperidine is added, and the mixture is heated under reflux under a protective atmosphere. The fluorescent probe is then extracted and purified to obtain the fluorescent probe.

[0008] Preferably, in the above-mentioned method for synthesizing lipid droplets and hypochlorous acid sequence-responsive fluorescent probes, in step (1), the molar volume ratio of 2-methoxyphenothiazine to the organic solvent is 1 mmol:1~1.5 mL, the molar ratio of 2-methoxyphenothiazine to sodium hydroxide is 1:1.5~2, the molar ratio of 2-methoxyphenothiazine to 3,5-bis(trifluoromethyl)benzyl bromide is 1:1.5~2, the heating reflux reaction temperature is 85~110°C, and the reaction time is 3~5h.

[0009] Preferably, in the above-mentioned method for synthesizing lipid droplets and hypochlorous acid sequence-responsive fluorescent probes, in the step (2), the volume ratio of anhydrous N,N-dimethylformamide to anhydrous dichloromethane is 1:1-1.2, the molar ratio of phosphorus oxychloride to anhydrous N,N-dimethylformamide is 1:1.2-1.5, the molar ratio of intermediate 1 to phosphorus oxychloride is 1:3-6, the heating reflux reaction temperature is 50-65°C, and the reaction time is 2-3h.

[0010] Preferably, in the above-mentioned method for synthesizing lipid droplets and hypochlorous acid sequence-responsive fluorescent probes, in step (3), the molar volume ratio of intermediate 2 to dichloromethane solvent is 1 mmol:2~2.5 mL, and the molar ratio of intermediate 2 to boron tribromide is 1:2~3.

[0011] Preferably, in the above-mentioned method for synthesizing lipid droplets and hypochlorous acid sequence-responsive fluorescent probes, in step (4), the molar ratio of intermediate 3 to the corresponding cyanide or acetyl ester compound is 1:1.1-1.8, the molar ratio of intermediate 3 to piperidine is 1:1.3-1.6, and the molar volume ratio of intermediate 3 to ethanol solvent is 1 mmol:20-25 mL.

[0012] On the other hand, the present invention also provides the use of the above-mentioned lipid droplet and hypochlorous acid sequence responsive fluorescent probe in detecting lipids and hypochlorous acid.

[0013] On the other hand, the present invention also provides the use of the above-mentioned lipid droplet and hypochlorous acid sequence responsive fluorescent probe in cell imaging.

[0014] On the other hand, the present invention also provides the use of the above-mentioned lipid droplet and hypochlorous acid sequence responsive fluorescent probe in the imaging of mouse arteriosclerotic plaque model.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The lipid droplet and hypochlorous acid sequence response type fluorescent probe of the present invention uses the phenothiazine-coumarin skeleton as the matrix, and realizes the sequence targeted imaging of lipid droplets in atherosclerotic plaques by introducing bis-trifluoromethylbenzyl. The sulfur atom in the phenothiazine-coumarin matrix is ​​used as the hypochlorous acid detection response site, and a series of fluorescent probes for detecting HClO are designed to realize the directional targeting of lipids in atherosclerotic plaques and the sequential response to lipid hypochlorous acid. It has excellent lipid droplet response ability, achieves good ratio fluorescence signal response to hypochlorous acid, has good targeting and strong anti-interference ability, and performs in vitro biological imaging on mouse atherosclerosis model, showing good targeting and sequential response.

[0016] 2. In the present invention, the single fluorophore of phenothiazine substituted by trifluorophenyl is converted into a phenothiazine-coumarin integrated fluorescent probe through ring closure, which inherits the excellent properties of coumarin dyes such as good photostability and good biocompatibility to a certain extent. More importantly, the phenothiazine-coumarin integrated fluorophore not only extends the conjugated π system and increases the emission wavelength of the probe, but also obtains a fluorescent probe with a sulfur atom reaction site that can respond to hypochlorous acid. This series of probes has excellent fluorescence properties such as large ultraviolet absorption and fluorescence emission, large Stokes shift, and has broad application prospects in the field of biological fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is the H NMR spectrum of the fluorescent probe NOR1 in Example 1 of the present invention.

[0019] Figure 2 It is the NMR carbon spectrum of the fluorescent probe NOR1 in Example 1 of the present invention.

[0020] Figure 3 It is the H NMR spectrum of the fluorescent probe NOR2 in Example 2 of the present invention.

[0021] Figure 4 It is the NMR carbon spectrum of the fluorescent probe NOR2 in Example 2 of the present invention.

[0022] Figure 5 This is the H NMR spectrum of the fluorescent probe NOR3 in Example 3 of the present invention.

[0023] Figure 6 It is the NMR carbon spectrum of the fluorescent probe NOR3 in Example 3 of the present invention.

[0024] Figure 7 It is the H NMR spectrum of the fluorescent probe NOR4 in Example 4 of the present invention.

[0025] Figure 8 It is the NMR carbon spectrum of the fluorescent probe NOR4 in Example 4 of the present invention.

[0026] Fig. 9 This is the H NMR spectrum of the fluorescent probe NOR5 in Example 5 of the present invention.

[0027] Fig.10 This is the NMR carbon spectrum of the fluorescent probe NOR5 in Example 5 of the present invention.

[0028] Fig.11 This is the H NMR spectrum of the fluorescent probe NOR6 in Example 6 of the present invention.

[0029] Fig.12 It is the NMR carbon spectrum of the fluorescent probe NOR6 in Example 6 of the present invention.

[0030] Fig.13 It is the ultraviolet absorption spectrum of the fluorescent probes NOR1-6 in Application Example 1 of the present invention.

[0031] Fig.14 It is the fluorescence emission spectrum of the fluorescent probes NOR1-6 in Application Example 2 of the present invention.

[0032] Fig.15 This is the fluorescence spectrum of the fluorescent probe NOR1 in Application Example 3 of the present invention in response to lipids.

[0033] Fig.16 This is the ultraviolet absorption spectrum of the fluorescent probe NOR1 in Application Example 4 of the present invention in response to different concentrations of hypochlorous acid in lipids.

[0034] Fig.17 This is the fluorescence emission spectrum of the fluorescent probe NOR1 in Application Example 5 of the present invention in response to different concentrations of hypochlorous acid in lipids.

[0035] Fig.18 This is a fluorescence curve diagram of the time response of the fluorescent probe NOR1 in Application Example 6 of the present invention to hypochlorous acid in lipids.

[0036] Fig.19 This is a fluorescence curve diagram of the fluorescent probe NOR1 and lipid time response in Application Example 7 of the present invention.

[0037] Fig. 20 This is a bar graph showing the selectivity of the fluorescent probe NOR1 in Application Example 8 of the present invention to various interferents.

[0038] Fig.21 This is the lipid co-localization diagram of the fluorescent probe NOR1 cell imaging in Application Example 9 of the present invention.

[0039] Fig. 22 This is a cell imaging diagram of the concentration gradient of oxidized low-density lipoprotein in cells by the fluorescent probe NOR1 in Application Example 10 of the present invention.

[0040] Fig.23 This is the fluorescence imaging diagram of the mouse arteriosclerosis model using the fluorescent probe NOR1 in Application Example 11 of the present invention.

[0041] Fig.24 This is the fluorescence imaging image of frozen sections of the thoracic aorta of a mouse arteriosclerosis model using the fluorescent probe NOR1 in Application Example 12 of the present invention. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention, etc. can be purchased from the market or can be prepared by existing methods.

[0043] The synthesis route of lipid droplet and hypochlorous acid sequence-responsive fluorescent probe is as follows: .

[0044] Example 1 A lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR1, the structural formula is: .

[0045] This embodiment also provides a method for synthesizing a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe, comprising the following steps: (1) Synthesis of intermediate 1 The synthesis route is: .

[0046] The synthesis steps are as follows: under nitrogen protection, 10 mmol of 2-methoxyphenothiazine is placed in a reaction bottle, 10 mL of dimethyl sulfoxide is added to dissolve, 20 mmol of sodium hydroxide and 2 mmol of potassium iodide are added in sequence, and the mixture is stirred at room temperature for 3 min; 20 mmol of 3,5-bis(trifluoromethyl)benzyl bromide is added to the above reaction system and the reaction system is placed at 90° C. for reaction reflux for 4 h; TLC monitors that the reaction has been completed, the reaction is stopped, and after natural cooling, the reaction solution is poured into 200 mL of water, and extracted repeatedly with ethyl acetate to obtain an organic phase, which is dried over anhydrous sodium sulfate, filtered, and the solvent is removed by distillation under reduced pressure to obtain a crude product, which is separated by column chromatography to obtain intermediate 1 as a light-colored oily liquid.

[0047] (2) Synthesis of intermediate 2 The synthesis route is: The synthesis steps are as follows: 66 mmol of anhydrous N,N-dimethylformamide is placed in a 100 mL round-bottom flask, 5 mL of anhydrous dichloromethane is added to dissolve, and the mixture is stirred at 0°C for 15 min under nitrogen protection; 52.7 mmol of phosphorus oxychloride is slowly added dropwise to the round-bottom flask and stirred at 0°C for 1 h; then a mixture of 10 mmol of intermediate 1 and dichloromethane (10 mL of dichloromethane) is added dropwise, and after the addition is complete, the reaction is naturally restored to room temperature, and the mixture is transferred to 50°C and heated under reflux for 2 h; the reaction is stopped after TLC monitoring of the completion of the reaction, and the reaction solution is poured into 200 mL of saturated sodium bicarbonate solution, extracted with ethyl acetate, and the crude product obtained after removing the solvent is separated by column chromatography to obtain intermediate 2 as a yellow solid.

[0048] (3) Synthesis of intermediate 3 The synthesis route is: The synthesis steps are as follows: 4.14 mmol of intermediate 2 is placed in a 100 mL round-bottom flask, 10 mL of anhydrous dichloromethane is added to dissolve it, and the above system is stirred at 0°C for 15 min under nitrogen protection; 12.42 mmol of boron tribromide is slowly added dropwise to the round-bottom flask and stirred at 0°C for 1 h; the reaction is stopped, saturated sodium bicarbonate solution is used to quench the reaction until the pH of the mixed solution is alkaline, and ethyl acetate is used for extraction. After removing the solvent, the crude product is obtained and separated by column chromatography to obtain intermediate 3 as a yellow solid.

[0049] (4) Synthesis of fluorescent probe NOR1 The synthesis route is: The synthesis steps are as follows: 0.426 mmol of intermediate 3 is placed in a 50 mL round-bottom flask, 10 mL of anhydrous ethanol, 0.639 mmol of malononitrile, and 0.606 mmol of piperidine are added in sequence, and the mixture is stirred at room temperature under nitrogen protection for 5 min. The reaction is stopped after TLC monitoring shows that the reaction is complete. The solvent is removed by distillation under reduced pressure, dichloromethane is added to dissolve, 1 mol / L hydrochloric acid solution (200 µL) is added, and the reaction is heated to reflux at 60 °C for 30 min. Stop the reaction. The reaction is quenched with saturated sodium bicarbonate solution until the pH of the mixed solution is alkaline, extracted with ethyl acetate, and the crude product is obtained after removal of the solvent. The fluorescent probe NOR1 is separated by column chromatography as a red solid. The hydrogen and carbon nuclear magnetic spectra of the fluorescent probe NOR1-6 are as follows Figure 1 and Figure 2 shown.

[0050] Example 2 A lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR2, the structural formula is: The synthesis method of the lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR2 in this embodiment is the same as that in Example 1, except that benzothiazole-2-acetonitrile is used in place of malononitrile in step (4), and the nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the obtained fluorescent probe NOR2 are as follows: Figure 3 and Figure 4 shown.

[0051] Example 3 A lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR3, the structural formula is: The synthesis method of lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR3 in this embodiment is the same as that in embodiment 1, except that benzimidazole-2-acetonitrile is used to replace malononitrile in step (4), and the nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the obtained fluorescent probe NOR3 are as follows: Figure 5 and Figure 6 shown.

[0052] Example 4 A lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR4, the structural formula is: The synthesis method of lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR4 in this embodiment is the same as that in embodiment 1, except that ethyl carbamoyl acetate is used to replace malononitrile in step (4), and hydrochloric acid is not required for the catalytic reaction. The H NMR spectrum and C NMR spectrum of the obtained fluorescent probe NOR4 are as follows: Figure 7 and Figure 8 shown.

[0053] Example 5 A lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR5, the structural formula is: The synthesis method of the lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR5 in this embodiment is the same as that in Example 1, except that diethyl malonate is used to replace malononitrile in step (4), and hydrochloric acid is not required for the catalytic reaction. The nuclear magnetic hydrogen spectrum and carbon spectrum of the obtained fluorescent probe NOR5 are as follows: Fig. 9 and Fig.10 shown.

[0054] Example 6 A lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR6, the structural formula is: The synthesis method of lipid droplet and hypochlorous acid sequence responsive fluorescent probe NOR6 in this embodiment is the same as that in Example 1, except that ethyl acetoacetate is used to replace malononitrile in step (4), and hydrochloric acid is not required for the catalytic reaction. The H NMR spectrum and C NMR spectrum of the obtained fluorescent probe NOR6 are as follows: Fig.11 and Fig.12 shown.

[0055] Application Example 1: UV absorption spectrum of fluorescent probe NOR1-6 NOR1-6 were prepared with toluene, dichloromethane (DCM), ethanol (EtOH), acetonitrile (Acetonitrile), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) to a concentration of 10 -3 mol / L mother solution, and then take an appropriate amount of mother solution and use Toluene, DCM, EtOH, Acetonitrile, DMSO and THF to make the volume to 2mL respectively. At this time, the concentration is 10 - 5 mol / L, test its UV absorption value and obtain the UV absorption spectrum. The UV absorption spectrum of NOR1-6 fluorescent probe is as follows: Fig.13 The fluorescent probe NOR1-6 of the present invention exhibits similar ultraviolet absorption spectra in organic solvents such as toluene and dichloromethane, and the maximum absorption peak is located between 400nm and 500nm.

[0056] Application Example 2 Fluorescence Spectrum of Fluorescent Probe NOR1-6 NOR1-6 were prepared with toluene, dichloromethane (DCM), ethanol (EtOH), acetonitrile (Acetonitrile), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) to a concentration of 10 -3 mol / L mother solution, and then take an appropriate amount of mother solution and use Toluene, DCM, EtOH, Acetonitrile, DMSO and THF to make the volume to 2mL respectively. At this time, the concentration is 10 - 5 mol / L, and measured its fluorescence spectrum. The fluorescence spectrum of NOR1-6 fluorescent probe is as follows Fig.14 As shown, the fluorescent probes NOR1-6 of the present invention exhibit similar fluorescence spectra in organic solvents such as toluene and dichloromethane.

[0057] Application Example 3 Fluorescence Spectra of Fluorescent Probe NOR1-6 in Response to Lipids NOR1-6 were prepared with acetonitrile to a concentration of 10 -3mol / L mother solution, and then take an appropriate amount of probe mother solution and dilute it into 2mL of lipid solution (myristoyl phosphatidylcholine = 0~200 µg / mL, trilaurin = 0~400 µg / mL) with different concentrations (concentration (μg / mL) are: 0, 60, 150, 180, 240, 300, 450, 600) respectively. At this time, the probe concentration is 10 - 5 mol / L, and its fluorescence spectrum was measured. The fluorescence spectrum of NOR1-6 fluorescent probe in response to lipids was as follows Fig.15 shown.

[0058] Preparation of lipid solutions of different concentrations: Dimyristoyl phosphatidylcholine (2.0 mg) and trilaurin (4.0 mg) were dissolved in 2 mL of chloroform to obtain a stock solution. After the stock solution was evaporated by nitrogen flow, the residue was suspended in 10 mL of deionized water (containing 0.001 M sodium cholate) and treated under ultrasonic emulsification conditions for 10 minutes to obtain a lipid solution with a concentration of 0.6 mg / mL (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL). Lipid solutions of different concentrations were obtained by diluting the solution in proportion to the required concentration.

[0059] Application Example 4: UV absorption spectrum of fluorescent probe NOR1 in response to different concentrations of hypochlorous acid in lipids The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10 -3 mol / L of the mother solution, and then take an appropriate amount of the mother solution and dilute it to 2mL of lipid solution (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL). At this time, the probe concentration is 10 -5 mol / L, and measured the UV absorption spectra of hypochlorous acid at different concentrations (0-50 µM). The UV absorption spectra of NOR1 in lipids in response to hypochlorous acid at different concentrations are shown in Figure 2. Fig.16 shown.

[0060] Application Example 5: Fluorescence spectrum of fluorescent probe NOR1 in response to different concentrations of hypochlorous acid in lipids The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10 -3 mol / L of the mother solution, and then take an appropriate amount of the mother solution and dilute it to 2mL of lipid solution (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL). At this time, the probe concentration is 10 -5 mol / L, and the fluorescence spectra of NOR1 in lipids in response to different concentrations of hypochlorous acid (concentration (µM) is 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100) were measured. Fig.17 As shown in the figure, NOR1 has an obvious ratiometric fluorescence response to HClO in lipids.

[0061] Application Example 6 Time response curve of fluorescent probe NOR1 to hypochlorous acid in lipids The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10 -3 mol / L of the mother solution, and then take an appropriate amount of the mother solution and dilute it to 2mL of lipid solution (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL). At this time, the probe concentration is 10 -5 mol / L, hypochlorous acid was added, and the time response curve of the fluorescent probe NOR1 and hypochlorous acid (100 µM) was measured. The time response curve of NOR1 in lipids and hypochlorous acid is shown in Fig.18 As shown in the figure, it can be seen that the response time of NOR1 to hypochlorous acid in lipid is within 80s, and NOR1 exhibits a rapid response to hypochlorous acid in lipid.

[0062] Application Example 7 Time response curve of fluorescent probe NOR1 and lipids The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10 -3 mol / L stock solution, the probe was added to the lipid solution (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL), at which the probe concentration was 10 - 5 mol / L, the time response curve of the fluorescent probe NOR1 and the lipid solution was measured. The time response curve of NOR1 and the lipid solution is shown in Fig.19 As shown in the figure, it can be seen that the response time of NOR1 to lipids is within 70s, and NOR1 shows a rapid response to lipids.

[0063] Application Example 8 Selectivity bar graph of fluorescent probe NOR1 to various interferents The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10 -3 mol / L of the mother solution, and then take an appropriate amount of the mother solution and dilute it to 2mL of lipid solution (myristoyl phosphatidylcholine = 100 µg / mL, trilaurin = 200 µg / mL). At this time, the probe concentration is 10 -5 mol / L, add 15 kinds of interfering substances (100µM) such as metal ions, various anions, various active sulfurs, various active oxygens, etc. commonly found in the blood of organisms. Determine the change of the fluorescence peak of NOR1 as follows Fig. 20 As shown in the figure, it can be seen that NOR1 has an excellent selective response to hypochlorous acid.

[0064] Application Example 9: Fluorescent Probe NOR1 Cellular Lipid Localization After inducing lipid droplet-rich RAW264.7 cells with 100 μg / mL acetylated low-density lipoprotein (Ac-LDL), lipopolysaccharide (LPS) and phorbol myristate ester (PMA) (10 / 5 μg / mL) were added for 6 hours, followed by co-incubation with NOR1 (1 μM), Hoechst 33342 (1 μM) and commercial lipid droplet dye LD-Tracker (1 μM) for 2 hours before imaging (see Figure 2). Fig.21 ), it can be seen from the figure that the colocalization coefficient of NOR1 is 0.98, indicating that NOR1 has excellent lipid droplet targeting function. Blue channel: λex = 405 nm, signal collection: 425-475 nm; Green channel: λex = 488 nm, signal collection: 500-550 nm; Red channel: λex = 488 nm, signal collection: 570-620 nm. Compared with commercial lipid droplet dyes, the fluorescent probe of the present invention has a higher fluorescence intensity growth rate at the same lipid concentration and provides a lower signal-to-noise ratio. Better photostability and signal retention in cell tests.

[0065] Application Example 10 Fluorescent Probe NOR1 Imaging of Oxidized Lipid Concentration Gradient in Cells Fig. 22 (a) Fluorescence images of RAW264.7 cells treated with different concentrations (0-100 μg / mL) of oxidized low-density lipoprotein and then incubated with NOR1 (1 μM green channel, red channel) and Hoechst33342 (1 μM, blue channel). Fig. 22 (b) shows the average fluorescence intensity of each cell in the green and red channels. (c) shows the G / R ratio of NOR1 in the green channel and red channel. Fig. 22 As can be seen in the figure, NOR1 has an excellent selective response to oxidized low-density lipoprotein. Blue channel: λex = 405 nm, signal collection: 425-475 nm; Green channel: λex = 488 nm, signal collection: 500-550 nm; Red channel: λex = 488 nm, signal collection: 570-620 nm.

[0066] Application Example 11 Fluorescence imaging of mouse arteriosclerosis model using fluorescent probe NOR1 Through ApoE - / -Mice were fed a Western diet (42.7% carbohydrates, 42% fat, and 15.2% protein) for 14 weeks to establish an atherosclerosis mouse model. Fig.23 (a) ApoE expression at different time points after intravenous injection of 100 μg / mL NOR1 (30 μg / 25g mouse). - / - Fluorescence imaging of the carotid arch region of mice in the green channel (500 nm) and the red channel (600 nm). Fig.23 (b) Fig.23 (a) Mean radiant efficiency (MRE) of the red and green channels. Fig.23 (c) Fig.23 (b) G / R ratio of NOR1 in green and red channels. λex=488nm, Green Channel: λem = 500-550 nm;Red channel: λem = 570-620 nm. Data are shown as mean ± SD (n = 3). Fig.23 As can be seen, NOR1 has excellent selective fluorescence imaging of the mouse aortic arch in the mouse atherosclerosis model.

[0067] Application Example 12 Imaging of frozen sections of thoracic aorta in mouse atherosclerosis model using fluorescent probe NOR1 Fig.24 (a) For ApoE - / - Cryosections of thoracic aortas from mice and healthy C57BL / 6J mice were stained with Oil Red and imaged after 24 h of incubation with complexes of NOR1 and acetylated low-density lipoprotein. Fig.24 (b) Sections were subjected to fluorescence imaging (NOR1: red channel, green channel) and G / R ratio determination. Fig.24 (c) is the fluorescence intensity values ​​of the red and green channels obtained from (b). Fig.24 (d) is the ratio of green channel to red channel obtained from (c). λex=488nm, Green Channel: λem = 500-550 nm; Red channel: λem = 570-620 nm. Data are expressed as mean ± SD (n = 3). ***p<0.0001. Fig.24 As can be seen, NOR1 has excellent selective fluorescence response imaging of atherosclerotic plaques in the thoracic aorta in a mouse atherosclerosis model.

[0068] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A lipid droplet and hypochlorous acid sequence responsive fluorescent probe, characterized in that: The structural formula of the fluorescent probe is: Wherein, R is cyano, benzothiazole, benzimidazole, amido, ethoxycarbonyl or acetyl.

2. The lipid droplet and hypochlorous acid sequence-responsive fluorescent probe according to claim 1, characterized in that: The R is a cyano group.

3. A method for synthesizing a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Under protective atmosphere conditions, the compound 2-methoxyphenothiazine is dissolved in an organic solvent, sodium hydroxide and potassium iodide are added in sequence and stirred, 3,5-bis(trifluoromethyl)benzyl bromide is added, and the mixture is heated under reflux for reaction. After the reaction is completed, the organic phase is extracted and washed, and distilled under reduced pressure to obtain intermediate 1; (2) Mix anhydrous N,N-dimethylformamide and anhydrous dichloromethane, add phosphorus oxychloride dropwise at 0-5°C under a protective atmosphere, stir at 0-5°C for 0.5-1h, and then add a dichloromethane mixture containing intermediate 1 dropwise; heat to reflux for reaction, extract and wash the organic phase after the reaction is completed, and distill under reduced pressure to obtain intermediate 2; (3) Dissolve the intermediate 2 in dichloromethane, add boron tribromide dropwise at 0-5°C, and react by stirring at 0-5°C. After the reaction is completed, extract and wash the organic phase, perform vacuum distillation, and perform gradient elution to obtain the intermediate 3; (4) The intermediate 3 and the corresponding cyanide or acetyl ester compound are dissolved in ethanol, piperidine is added, and the mixture is heated under reflux under a protective atmosphere for reaction, and then extracted and purified to obtain a fluorescent probe.

4. The synthesis method according to claim 3, characterized in that In the step (1), the molar volume ratio of 2-methoxyphenothiazine to the organic solvent is 1 mmol:1-1.5 mL, the molar ratio of 2-methoxyphenothiazine to sodium hydroxide is 1:1.5-2, the molar ratio of 2-methoxyphenothiazine to 3,5-bis(trifluoromethyl)benzyl bromide is 1:1.5-2, the heating reflux reaction temperature is 85-110° C., and the reaction time is 3-5 h.

5. The synthesis method according to claim 3, characterized in that In the step (2), the volume ratio of anhydrous N,N-dimethylformamide to anhydrous dichloromethane is 1:1-1.2, the molar ratio of phosphorus oxychloride to anhydrous N,N-dimethylformamide is 1:1.2-1.5, the molar ratio of intermediate 1 to phosphorus oxychloride is 1:3-6, the heating reflux reaction temperature is 50-65°C, and the reaction time is 2-3h.

6. The synthesis method according to claim 3, characterized in that In the step (3), the molar volume ratio of the intermediate 2 to the dichloromethane solvent is 1 mmol:2-2.5 mL, and the molar ratio of the intermediate 2 to boron tribromide is 1:2-3.

7. The synthesis method according to claim 3, characterized in that In the step (4), the molar ratio of the intermediate 3 to the corresponding cyanide or acetyl ester compound is 1:1.2-1.5, the molar ratio of the intermediate 3 to piperidine is 1:1.3-1.6, and the molar volume ratio of the intermediate 3 to the ethanol solvent is 1 mmol:20-25 mL.

8. Use of the lipid droplet and hypochlorous acid sequence responsive fluorescent probe as claimed in claim 1 or 2 in detecting lipids and hypochlorous acid.

9. Use of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe as claimed in claim 1 or 2 in cell imaging.

10. Use of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe as claimed in claim 1 or 2 in imaging of mouse arteriosclerotic plaque model.

Images