Lipid droplet and hypochlorous acid sequence-responsive fluorescent probe and synthesis method and application thereof

Abstract

The application relates to the technical field of organic fluorescent probes, and particularly discloses a lipid droplet and hypochlorous acid sequence response type fluorescent probe, a structural formula of which is as shown in the figure; wherein R is a cyano group, a benzothiazole group, a benzimidazole group, an amido group, an ethoxycarbonyl group or an acetyl group. The precise imaging lipid droplet and hypochlorous acid sequence response fluorescent probe of the application takes a phenothiazine-coumarin dye support as a matrix, utilizes a double trifluoromethyl benzyl group as a lipid droplet targeting group, adjusts the dye emission wavelength by introducing different electron-withdrawing groups, synthesizes a series of probes, and realizes high-targeting sequence imaging of lipid droplets and hypochlorous acid in atherosclerotic plaques.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent probe technology, and in particular to a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe, its synthesis method, and its application. Background Technology

[0002] Atherosclerosis is a major cause of cardiovascular disease, and accurate identification of atherosclerotic plaques can effectively prevent the progression of cardiovascular disease. The formation and evolution of atherosclerotic plaques narrow arteries, obstructing 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 physicians to make timely clinical decisions and implement 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 π–π stacking within dye molecules. When heteroatoms on the phenothiazine fluorophore are substituted, a further non-planar bending structure emerges. This bending structure hinders intermolecular aggregation, thus increasing the solubility of the fluorophore. Furthermore, phenothiazine itself is a very good electron-donating group, and in recent years has become a research hotspot for designing intramolecular charge transfer fluorescent probes. In addition, coumarin possesses excellent photophysical properties, high fluorescence quantum yield, large Stokes shift, good biocompatibility, and ease of functional group modification. Moreover, by modifying it with strong electron-withdrawing or electron-donating groups, a stable push-pull electron structure can be formed with the coumarin parent compound, promoting intramolecular charge transfer and thus emitting stronger fluorescence. Chinese Patent Publication No. CN 114163456 B discloses a phenothiazine coumarin-based pyridine salt compound for the detection of endogenous HOCl in cells, used to distinguish between normal cells and tumor cells.

[0004] Abnormal lipid droplet concentration and elevated hypochlorous acid levels are two major markers of atherosclerotic plaque formation. Accurate detection of lipid droplet concentration and hypochlorous acid levels is crucial for precise identification of atherosclerotic plaques and effective prevention of cardiovascular disease. Chinese Patent Publication No. CN 108997289 B discloses a hypochlorous acid ratiometric fluorescent probe targeting lipid droplets. This probe uses a coumarin fluorophore as the energy donor, (E)-2-(3-(4-(disubstituted amino)styryl)-(5,5-dimethyl)cyclohex-2-en-1-yl)malononitrile as the energy acceptor, and an acylpiperazine as the linker group. This probe selectively interacts with hypochlorous acid to detect low concentrations of hypochlorous acid and can be used for intracellular ratio imaging. However, this fluorescent probe is only used for hypochlorous acid detection; its effects on lipid concentration and atherosclerotic plaque imaging have not been investigated. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe for precise imaging of lipid droplet and hypochlorous acid sequence responses in atherosclerotic plaques. The specific technical solution is as follows:

[0006] A lipid droplet and hypochlorous acid sequence-responsive fluorescent probe, the structural formula of which is:

[0007] Wherein, R is cyano, benzothiazole, benzimidazole, amide, ethoxycarbonyl, or acetyl.

[0008] Preferably, in the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probes, R is a cyano group.

[0009] This invention also provides a method for synthesizing the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probe, comprising the following steps:

[0010] (1) Under a protective atmosphere, compound 2-methoxyphenthiazide was dissolved in an organic solvent, sodium hydroxide and potassium iodide were added in sequence and stirred, 3,5-bis(trifluoromethyl)benzyl bromide was added, and the mixture was heated under reflux. After the reaction was completed, the organic phase was extracted and washed, and the mixture was distilled under reduced pressure to obtain intermediate 1.

[0011] (2) Anhydrous N,N-dimethylformamide and anhydrous dichloromethane were mixed, and phosphorus oxychloride was added dropwise under a protective atmosphere at 0~5℃. The mixture was stirred at 0~5℃ for 0.5~1h, and then a dichloromethane mixture containing intermediate 1 was added dropwise. The mixture was heated to reflux and reacted. After the reaction was completed, the organic phase was extracted and washed, and the mixture was distilled under reduced pressure to obtain intermediate 2.

[0012] (3) Dissolve intermediate 2 in dichloromethane, add boron tribromide dropwise at 0~5℃, stir the reaction at 0~5℃, after the reaction is completed, extract and wash the organic phase, distill under reduced pressure and elute with gradient to obtain intermediate 3;

[0013] (4) Dissolve intermediate 3 and the corresponding cyanide or acetyl ester compound in ethanol, add piperidine, heat under a protective atmosphere and reflux to react, extract and purify to obtain the fluorescent probe.

[0014] 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-methoxyphenthiazide to organic solvent is 1 mmol: 1~1.5 mL, the molar ratio of 2-methoxyphenthiazide to sodium hydroxide is 1:1.5~2, the molar ratio of 2-methoxyphenthiazide to 3,5-bis(trifluoromethyl)benzyl bromide is 1:1.5~2, the reflux reaction temperature is 85~110℃, and the reaction time is 3~5 h.

[0015] Preferably, in the above-mentioned method for synthesizing lipid droplets and hypochlorous acid sequence-responsive fluorescent probes, in 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 reflux reaction temperature is 50~65℃, and the reaction time is 2~3h.

[0016] 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.

[0017] 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 1mmol:20~25mL.

[0018] On the other hand, the present invention also provides the application of the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probes in the detection of lipids and hypochlorous acid.

[0019] On the other hand, the present invention also provides the application of the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probes in cell imaging.

[0020] On the other hand, the present invention also provides the application of the above-mentioned lipid droplet and hypochlorous acid sequence-responsive fluorescent probes in imaging a mouse atherosclerotic plaque model.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. The lipid droplet and hypochlorous acid sequence-responsive fluorescent probes of this invention use a phenothiazine-coumarin backbone as the parent structure. By introducing bis(trifluoromethyl)benzyl groups, sequence-targeted imaging of lipid droplets in atherosclerotic plaques is achieved. Using sulfur atoms in the phenothiazine-coumarin backbone as hypochlorous acid detection response sites, a series of fluorescent probes for detecting HClO are designed, achieving targeted lipid targeting and sequential response to hypochlorous acid in atherosclerotic plaques. It exhibits excellent lipid droplet response, good ratiometric fluorescence signal response to hypochlorous acid, good targeting, and strong anti-interference ability. In vitro bioimaging of a mouse atherosclerosis model demonstrates good targeting and sequential response.

[0023] 2. In this invention, a single fluorophore of trifluorophenyl-substituted phenothiazine is transformed into a phenothiazine-coumarin integrated fluorescent probe through cyclization. This inherits, to some extent, the excellent properties of coumarin dyes, such as good photostability and biocompatibility. More importantly, the phenothiazine-coumarin integrated fluorophore not only extends the conjugated π-system, increasing the emission wavelength of the probe, but also yields a fluorescent probe with a sulfur atom reaction site responsive to hypochlorous acid. This series of probes exhibits excellent fluorescence properties, including large ultraviolet absorption and fluorescence emission, and a large Stokes shift, showing broad application prospects in the field of biofluorescence imaging. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is the 1H NMR spectrum of the fluorescent probe NOR1 in Example 1 of this invention.

[0026] Figure 2 This is the carbon NMR spectrum of the fluorescent probe NOR1 in Example 1 of this invention.

[0027] Figure 3 This is the 1H NMR spectrum of the fluorescent probe NOR2 in Example 2 of this invention.

[0028] Figure 4 This is the carbon NMR spectrum of the fluorescent probe NOR2 in Example 2 of this invention.

[0029] Figure 5 This is the hydrogen nuclear magnetic spectrum of the fluorescent probe NOR3 in Example 3 of this invention.

[0030] Figure 6 This is the carbon NMR spectrum of the fluorescent probe NOR3 in Example 3 of this invention.

[0031] Figure 7 This is the 1H NMR spectrum of the fluorescent probe NOR4 in Example 4 of this invention.

[0032] Figure 8 This is the carbon NMR spectrum of the fluorescent probe NOR4 in Example 4 of this invention.

[0033] Figure 9 This is the 1H NMR spectrum of the fluorescent probe NOR5 in Example 5 of this invention.

[0034] Figure 10 This is the carbon NMR spectrum of the fluorescent probe NOR5 in Example 5 of this invention.

[0035] Figure 11 This is the 1H NMR spectrum of the fluorescent probe NOR6 in Example 6 of this invention.

[0036] Figure 12 This is the carbon NMR spectrum of the fluorescent probe NOR6 in Example 6 of this invention.

[0037] Figure 13 This is the ultraviolet absorption spectrum of the fluorescent probes NOR1~6 in Application Example 1 of this invention.

[0038] Figure 14 This is the fluorescence emission spectrum of the fluorescent probes NOR1~6 in Application Example 2 of this invention.

[0039] Figure 15 This is the fluorescence spectrum of the fluorescent probe NOR1 in application example 3 of the present invention in response to lipids.

[0040] Figure 16 This is the UV absorption spectrum of the fluorescent probe NOR1 in lipids in application example 4 of this invention in response to different concentrations of hypochlorous acid.

[0041] Figure 17 This is the fluorescence emission spectrum of the fluorescent probe NOR1 in lipids in application example 5 of the present invention in response to different concentrations of hypochlorous acid.

[0042] Figure 18 This is a time-response fluorescence curve of the fluorescent probe NOR1 in lipids and hypochlorous acid in Application Example 6 of the present invention.

[0043] Figure 19 This is a fluorescence curve of the fluorescent probe NOR1 and lipid time response in Application Example 7 of the present invention.

[0044] Figure 20 This is a bar chart showing the selectivity of the fluorescent probe NOR1 for various interfering substances in Application Example 8 of this invention.

[0045] Figure 21 This is a lipid colocalization map of the fluorescent probe NOR1 in cell imaging, as shown in Application Example 9 of this invention.

[0046] Figure 22 This is a cell imaging image of the concentration gradient of oxidized low-density lipoprotein in cells, obtained by the fluorescent probe NOR1 in Application Example 10 of the present invention.

[0047] Figure 23 This is a fluorescence imaging image of a mouse arteriosclerosis model using the fluorescent probe NOR1 from Application Example 11 of this invention.

[0048] Figure 24 This is a fluorescence imaging image of a frozen section of the thoracic aorta of a mouse arteriosclerosis model, obtained using the fluorescent probe NOR1 from Example 12 of this invention. Detailed Implementation

[0049] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0050] The synthesis route for lipid droplet and hypochlorous acid sequence-responsive fluorescent probes is as follows:

[0051] .

[0052] Example 1

[0053] NOR1 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0054] .

[0055] This embodiment also provides a method for synthesizing lipid droplet and hypochlorous acid sequence-responsive fluorescent probes, including the following steps:

[0056] (1) Synthesis of intermediate 1

[0057] The synthesis circuit is as follows:

[0058] .

[0059] The synthesis steps were as follows: Under nitrogen protection, 10 mmol of 2-methoxyphenthiazide was placed in a reaction flask, dissolved in 10 mL of dimethyl sulfoxide, followed by the addition of 20 mmol of sodium hydroxide and 2 mmol of potassium iodide. The mixture was stirred at room temperature for 3 min. 20 mmol of 3,5-bis(trifluoromethyl)benzyl bromide was added to the above reaction system, and the reaction system was refluxed at 90 °C for 4 h. The reaction was stopped by TLC monitoring and allowed to cool naturally. The reaction solution was poured into 200 mL of water and extracted repeatedly with ethyl acetate to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was separated by column chromatography to obtain intermediate 1, which was a light-colored oily liquid.

[0060] (2) Synthesis of intermediate 2

[0061] The synthesis circuit is as follows:

[0062]

[0063] The synthesis steps were as follows: 66 mmol of anhydrous N,N-dimethylformamide was placed in a 100 mL round-bottom flask and dissolved in 5 mL of anhydrous dichloromethane. The mixture was stirred at 0 °C for 15 min under nitrogen protection. 52.7 mmol of phosphorus oxychloride was slowly added dropwise to the round-bottom flask and stirred at 0 °C for 1 h. Then, 10 mmol of a mixture of intermediate 1 and dichloromethane (10 mL of dichloromethane) was added dropwise. After the addition was complete, the reaction was allowed to return to room temperature and then heated under reflux at 50 °C for 2 h. The reaction was monitored by TLC until it was complete. The reaction was then stopped, and the reaction solution was poured into 200 mL of saturated sodium bicarbonate solution. The solution was extracted with ethyl acetate, and the crude product obtained after solvent removal was separated by column chromatography to obtain intermediate 2 as a yellow solid.

[0064] (3) Synthesis of intermediate 3

[0065] The synthesis circuit is as follows:

[0066]

[0067] The synthesis steps were as follows: 4.14 mmol of intermediate 2 was placed in a 100 mL round-bottom flask and dissolved in 10 mL of anhydrous dichloromethane. The system was stirred at 0 °C for 15 min under nitrogen protection. 12.42 mmol of boron tribromide was slowly added dropwise to the round-bottom flask and stirred at 0 °C for 1 h. The reaction was stopped and quenched with saturated sodium bicarbonate solution until the pH of the mixture was alkaline. The mixture was extracted with ethyl acetate to remove the solvent and the crude product was separated by column chromatography to obtain intermediate 3 as a yellow solid.

[0068] (4) Synthesis of fluorescent probe NOR1

[0069] The synthesis circuit is as follows:

[0070]

[0071] The synthesis steps were as follows: 0.426 mmol of intermediate 3 was placed in a 50 mL round-bottom flask, and 10 mL of anhydrous ethanol, 0.639 mmol of malononitrile, and 0.606 mmol of piperidine were added sequentially. The mixture was stirred at room temperature for 5 min under nitrogen protection. The reaction was stopped after TLC monitoring showed completion. The solvent was removed by vacuum distillation, and the mixture was dissolved in dichloromethane. 200 µL of 1 mol / L hydrochloric acid solution was added, and the reaction was heated under reflux at 60 °C for 30 min. The reaction was then stopped. The reaction was quenched with saturated sodium bicarbonate solution until the pH of the mixture was alkaline. The mixture was extracted with ethyl acetate to remove the solvent, and the crude product was separated by column chromatography to obtain the fluorescent probe NOR1 as a red solid. The 1H and 1C NMR spectra of the fluorescent probe NOR1-6 are shown below. Figure 1 and Figure 2 As shown.

[0072] Example 2

[0073] NOR2 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0074]

[0075] The synthesis method of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR2 in this embodiment is the same as in Example 1, except that benzothiazole-2-acetonitrile is used instead of malononitrile in step (4). The 1H and 1C NMR spectra of the obtained fluorescent probe NOR2 are as follows: Figure 3 and Figure 4 As shown.

[0076] Example 3

[0077] NOR3 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0078]

[0079] The synthesis method of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR3 in this embodiment is the same as that in Example 1, except that in step (4), benzimidazole-2-acetonitrile is used instead of malononitrile. The 1H and 1C NMR spectra of the obtained fluorescent probe NOR3 are as follows: Figure 5 and Figure 6 As shown.

[0080] Example 4

[0081] NOR4 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0082]

[0083] The synthesis method of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR4 in this embodiment is the same as in Example 1, except that ethyl carbamoyl is used instead of malononitrile in step (4), eliminating the need for hydrochloric acid catalysis. The 1H and 1C NMR spectra of the obtained fluorescent probe NOR4 are shown below. Figure 7 and Figure 8 As shown.

[0084] Example 5

[0085] NOR5 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0086]

[0087] The synthesis method of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR5 in this embodiment is the same as in Example 1, except that diethyl malonate is used instead of malononitrile in step (4), eliminating the need for hydrochloric acid catalysis. The 1H and 1C NMR spectra of the obtained fluorescent probe NOR5 are shown below. Figure 9 and Figure 10 As shown.

[0088] Example 6

[0089] NOR6 is a lipid droplet and hypochlorous acid sequence-responsive fluorescent probe with the following structural formula:

[0090]

[0091] The synthesis method of the lipid droplet and hypochlorous acid sequence-responsive fluorescent probe NOR6 in this embodiment is the same as in Example 1, except that ethyl acetoacetate is used instead of malononitrile in step (4), eliminating the need for hydrochloric acid catalysis. The 1H and 1C NMR spectra of the obtained fluorescent probe NOR6 are shown below. Figure 11 and Figure 12 As shown.

[0092] Application Example 1: Ultraviolet Absorption Spectroscopy of Fluorescent Probe NOR1-6

[0093] NOR1-6 was prepared into concentrations of 10% using toluene, dichloromethane (DCM), ethanol (EtOH), acetonitrile, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). -3 The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL with Toluene, DCM, EtOH, Acetonitrile, DMSO, and THF, respectively. The concentration at this point was 10 mol / L. - 5 The UV absorbance of the NOR1-6 fluorescent probe was measured using mol / L, and the UV absorption spectrum was obtained. The UV absorption spectrum of the NOR1-6 fluorescent probe is shown below. Figure 13 As shown, the fluorescent probe NOR1-6 of this invention exhibits similar ultraviolet absorption spectra in organic solvents such as toluene and dichloromethane, with the maximum absorption peak located between 400 nm and 500 nm.

[0094] Application Example 2: Fluorescence Spectroscopy of Fluorescent Probe NOR1-6

[0095] NOR1-6 was prepared into concentrations of 10% using toluene, dichloromethane (DCM), ethanol (EtOH), acetonitrile, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). -3The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL with Toluene, DCM, EtOH, Acetonitrile, DMSO, and THF, respectively. The concentration at this point was 10 mol / L. - 5 mol / L, its fluorescence spectrum was measured, and the fluorescence spectrum of the NOR1-6 fluorescent probe is as follows: Figure 14 As shown, the fluorescent probe NOR1-6 of the present invention exhibits similar fluorescence spectra in organic solvents such as toluene and dichloromethane.

[0096] Application Example 3: Fluorescent probe NOR1-6 responds to lipid fluorescence spectra

[0097] NOR1-6 was prepared with acetonitrile to a concentration of 10. -3 The stock solution was prepared at mol / L. An appropriate amount of the probe stock solution was then diluted to 2 mL of lipid solutions of different concentrations (μg / mL: 0, 60, 150, 180, 240, 300, 450, 600) (myristoyl lecithin = 0~200 µg / mL, glyceryl trilaurate = 0~400 µg / mL). At this point, the probe concentration was 10. - 5 mol / L, its fluorescence spectrum was measured, and the fluorescence spectrum of the NOR1-6 fluorescent probe in response to lipids is as follows. Figure 15 As shown.

[0098] To prepare lipid solutions of different concentrations, dimyristic lecithin (2.0 mg) and glyceryl trilaurate (4.0 mg) were dissolved in 2 mL of chloroform to obtain a stock solution. After evaporation of the stock solution using a nitrogen stream, the residue was suspended in 10 mL of deionized water (containing 0.001 M sodium cholate) and treated under ultrasonic emulsification for 10 minutes to obtain a lipid solution with a concentration of 0.6 mg / mL (myristic lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL). Different concentration lipid solutions could be obtained by proportional dilution according to the desired concentration.

[0099] Application Example 4: UV absorption spectra of the fluorescent probe NOR1 in lipids in response to different concentrations of hypochlorous acid.

[0100] The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10. -3 The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL of lipid solution (myristoyl lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL). At this point, the probe concentration was 10 mol / L. -5 The UV absorption spectra of NOR1 in lipids at different concentrations of hypochlorous acid (0–50 µM) were measured using mol / L. Figure 16 As shown.

[0101] Application Example 5: Fluorescence spectra of the fluorescent probe NOR1 in lipids in response to different concentrations of hypochlorous acid.

[0102] The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10. -3 The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL of lipid solution (myristoyl lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL). At this point, the probe concentration was 10 mol / L. -5 Fluorescence spectra of NOR1 in lipids were measured at different concentrations of hypochlorous acid (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µM) using mol / L. The fluorescence spectra of NOR1 in lipids in response to different concentrations of hypochlorous acid are shown below. Figure 17 As shown in the figure, NOR1 exhibits a significant ratiometric fluorescence response to HClO in lipids.

[0103] Application Example 6: Time-response curve of fluorescent probe NOR1 with hypochlorous acid in lipids

[0104] The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10. -3 The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL of lipid solution (myristoyl lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL). At this point, the probe concentration was 10 mol / L. -5 Add hypochlorous acid at a concentration of mol / L, and measure the time response curves of the fluorescent probe NOR1 with hypochlorous acid (100 µM). The time response curves of NOR1 with hypochlorous acid in lipids are shown below. Figure 18 As shown in the figure, NOR1 responds to hypochlorous acid in lipids within 80 seconds, demonstrating a rapid response to hypochlorous acid in lipids.

[0105] Application Example 7: Time-response curves of fluorescent probe NOR1 with lipids

[0106] The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10. -3 The probe was added to a lipid solution (myristoyl lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL) using a stock solution of mol / L. The probe concentration at this point was 10 mol / L. - 5 The time response curves of the fluorescent probe NOR1 with the lipid solution were measured using mol / L. The time response curves of NOR1 with the lipid solution are shown below. Figure 19 As shown in the figure, NOR1 responds to lipids within 70 seconds, demonstrating a rapid response to lipids.

[0107] Application Example 8: Selectivity of the fluorescent probe NOR1 for various interfering substances

[0108] The NOR1 fluorescent probe was prepared with acetonitrile to a concentration of 10. -3 The stock solution was prepared at mol / L. An appropriate amount of the stock solution was then diluted to 2 mL of lipid solution (myristoyl lecithin = 100 µg / mL, glyceryl trilaurate = 200 µg / mL). At this point, the probe concentration was 10 mol / L. -5 Fifteen interfering substances (100 µM) commonly found in biological blood, including metal ions, various anions, various reactive sulfur compounds, and various reactive oxygen species, were added at a concentration of mol / L. The changes in the fluorescence peak of NOR1 were measured as follows: Figure 20 As shown in the figure, NOR1 exhibits excellent selective response to hypochlorous acid.

[0109] Application Example 9: Fluorescent probe NOR1 cell lipid localization

[0110] RAW264.7 cells rich in lipid droplets were induced with 100 μg / mL acetylated low-density lipoprotein (Ac-LDL), then co-cultured with lipopolysaccharide (LPS) and phorbol ester (PMA) (10 / 5 μg / mL) for 6 hours. Following this, NOR1 (1 μM), Hoechst 33342 (1 μM), and the commercial lipid droplet dye LD-Tracker (1 μM) were added and incubated for 2 hours before imaging (e.g., ...). Figure 21 As shown in the figure, 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 this invention has a higher fluorescence intensity fold increase and provides a lower signal-to-noise ratio at the same lipid concentration. It also exhibits better photostability and signal retention in cell assays.

[0111] Application Example 10: Imaging of Oxidized Lipid Concentration Gradients in Cells Using the Fluorescent Probe NOR1

[0112] Figure 22 In the middle (a), RAW264.7 cells were 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) fluorescence images. Figure 22 (b) represents the average fluorescence intensity of each cell in the green and red channels. Figure 22 In the middle (c), the G / R ratio of the NOR1 green channel to the red channel is shown. From... Figure 22 As can be seen, NOR1 exhibits 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.

[0113] Application Example 11: Fluorescence Imaging of a Mouse Arteriosclerosis Model Using the Fluorescent Probe NOR1

[0114] Through ApoE - / - A mouse model of arteriosclerosis was established by feeding mice a Western diet (42.7% carbohydrates, 42% fat and 15.2% protein) for 14 weeks. Figure 23 (a) shows the ApoE results at different time points after intravenous injection of 100 μg / mL NOR1 (30 μg / 25g mice). - / - Fluorescence imaging of the green channel (500 nm) and red channel (600 nm) in the carotid arch region of a mouse. Figure 23 (b) is Figure 23 (a) Average radiation efficiency (MRE) of the red and green channels. Figure 23 (c) is Figure 23 (b) G / R ratios of the green and red channels of NOR1. λex = 488 nm, Green Channel: λem = 500-550 nm; Red channel: λem = 570-620 nm. Data are presented as mean ± standard deviation (n = 3). From Figure 23 As can be seen, NOR1 exhibits excellent selective fluorescence imaging of the mouse arterial arch in a mouse model of arteriosclerosis.

[0115] Application Example 12: Imaging of frozen sections of the thoracic aorta using the fluorescent probe NOR1 in a mouse model of arteriosclerosis.

[0116] Figure 24 (a) ApoE - / - Frozen sections of the thoracic aorta from mice and healthy C57BL / 6J mice were stained with Oil Red and imaged after incubation with NOR1 and acetylated low-density lipoprotein complex for 24 hours. Figure 24In (b), the slides were subjected to fluorescence imaging (NOR1: red channel, green channel) and G / R ratio determination. Figure 24 (c) is from Figure 24 In (b), the fluorescence intensity values ​​of the red and green channels are obtained. Figure 24 (d) is from Figure 24 The ratio of the green channel to the red channel obtained in (c). λex = 488 nm, Green Channel: λem = 500-550 nm; Red channel: λem = 570-620 nm. Data are expressed as mean ± standard deviation (n = 3). p<0.0001. From Figure 24 As can be seen, NOR1 exhibits excellent selective fluorescence response imaging of atherosclerotic plaques in the thoracic aorta of a mouse model of arteriosclerosis.

[0117] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined 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: R is any one of the following: 。 2. A method of synthesizing a lipid droplet and hypochlorous acid sequence- responsive fluorescent probe according to claim 1, characterized in that, The method comprises the following steps: (1) Under the condition of a protective atmosphere, compound 2-methoxy phenothiazine is dissolved in an organic solvent, sodium hydroxide and potassium iodide are added in sequence and stirred, 3,5-bistrifluoromethyl benzyl bromide is added, heated and refluxed, after the reaction is completed, the organic phase is extracted and washed, and vacuum distillation is performed to obtain an intermediate 1; (2) Anhydrous N,N-dimethylformamide and anhydrous dichloromethane are mixed, under the condition of a protective atmosphere and at 0-5℃, phosphorus oxychloride is added dropwise, stirred at 0-5℃ for 0.5-1h, then a dichloromethane mixture containing the intermediate 1 is added dropwise, heated and refluxed, after the reaction is completed, the organic phase is extracted and washed, and vacuum distillation is performed to obtain an intermediate 2; (3) The intermediate 2 is dissolved in dichloromethane, at 0-5℃, boron tribromide is added dropwise, stirred at 0-5℃, after the reaction is completed, the organic phase is extracted and washed, vacuum distillation and gradient elution are performed to obtain an intermediate 3; (4) The intermediate 3 and the corresponding cyanide or acetoacetate compound are dissolved in ethanol, piperidine is added, heated and refluxed under the condition of a protective atmosphere, extracted and purified to obtain the lipid droplet and hypochlorous acid sequence responsive fluorescent probe according to claim 1; The structural formula of the intermediate 1 is: ; The structural formula of the intermediate 2 is: ; The structural formula of the intermediate 3 is: ; The structural formula of the cyanide is: ； The structural formula of the acetoacetate compound is: 。 3. The method of synthesis of claim 2, wherein, In the step (1), the molar volume ratio of 2-methoxy phenothiazine to the organic solvent is 1mmol:1-1.5mL, the molar ratio of 2-methoxy phenothiazine to sodium hydroxide is 1:1.5-2, the molar ratio of 2-methoxy phenothiazine to 3,5-bistrifluoromethyl benzyl bromide is 1:1.5-2, the heating reflux reaction temperature is 85-110℃, and the reaction time is 3-5h.

4. The method of synthesis of claim 2, wherein, 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 the intermediate 1 to phosphorus oxychloride is 1:3-6, the heating reflux reaction temperature is 50-65℃, and the reaction time is 2-3h.

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

6. The method of synthesis of claim 2, wherein, In the step (4), the molar ratio of the intermediate 3 to the corresponding cyanide or acetoacetate 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 1mmol:20-25mL.

7. The lipid droplet and hypochlorous acid sequence responsive fluorescent probe according to claim 1 is applied to the preparation of a reagent for detecting lipids and hypochlorous acid.

8. The lipid droplet and hypochlorous acid sequence responsive fluorescent probe according to claim 1 is applied to the preparation of a reagent for cell imaging.

9. The lipid droplet and hypochlorous acid sequence responsive fluorescent probe according to claim 1 is applied to the preparation of a reagent for imaging a mouse arteriosclerosis plaque model.

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