Pyrrole derivative fluorescent dye, polar sensitive fluorescent probe for lipid droplet imaging based on fluorescent dye, and preparation method and application of polar sensitive fluorescent probe

Abstract

The invention discloses a pyrrole derivative fluorescent dye, a polar sensitive fluorescent probe for lipid droplet imaging based on the fluorescent dye, and a preparation method and application of the polar sensitive fluorescent probe, and belongs to the field of small organic molecule fluorescent probes. The preparation method of the fluorescent dye comprises the following steps: mixing 1, 4-naphthoquinone, benzaldehyde or a derivative thereof, ammonium iodide and a solvent, carrying out stirring reaction at 120-140 DEG C, and carrying out post-treatment, the preparation method of the fluorescent probe comprises the following steps: adding the fluorescent dye into a KOH-containing solvent, uniformly stirring, adding CH3I, heating to 45-55 DEG C, stirring and reacting at 45-55 DEG C, and carrying out post-treatment. A series of novel fluorescent dyes are successfully synthesized through substituent group modification, a polarity sensitive fluorescent probe is further developed, and the prepared polarity sensitive fluorescent probe not only shows high polarity sensitivity and solvent discoloration, but also shows good fluorescence stability, large Stokes shift and biocompatibility.

Description

Technical Field

[0001] The present invention relates to a pyrrole derivative fluorescent dye, a polarity-sensitive fluorescent probe for lipid droplet imaging based on the fluorescent dye, a preparation method and an application, and belongs to the field of organic small molecule fluorescent probes. Background Art

[0002] Lipid droplets are important organelles for intracellular lipid storage. Lipids mainly consist of neutral fats and cholesterol esters, and are wrapped by a phospholipid membrane and specific proteins. Lipid droplets play important roles in energy metabolism, lipid metabolism and cell signal transduction. Under normal physiological conditions, lipid droplets can help cells store excess energy and release lipids as energy when needed. However, under pathological conditions, such as obesity, diabetes and certain cardiovascular diseases, the accumulation and metabolic disorders of lipid droplets may lead to various health problems. At the same time, the number and size of lipid droplets vary greatly among different types of cells. For example, cancer cells need more energy to grow rapidly, so they usually have more lipid droplets than normal cells. By monitoring the changes of intracellular lipid droplets, the physiological state of cells can be better understood, and whether the cells are healthy can be judged. As a highly promising practical detection tool, fluorescent probes play an important role in specific detection, especially in cell imaging in vivo. Compared with other analytical detection methods, the fluorescent probe technology has significant advantages such as low cost, strong real-time performance, high sensitivity and high detection accuracy. In recent years, a variety of fluorescent probes for specific lipid droplet imaging have been reported, successfully realizing the specific labeling and dynamic monitoring of lipid droplets in living cells. Therefore, developing more new fluorescent probes to monitor the changes of intracellular lipid droplets is of great significance for understanding the physiological state of cells and early cancer diagnosis.

[0003] In recent years, organic small molecule dyes have been widely studied and applied in biological imaging and other aspects due to their advantages such as simple operation, rapid response, high sensitivity and easy manufacture. Although many fluorescent probes for lipid droplet imaging have been reported, with the progress of fluorescence technology and the in-depth development of biomedical research, more new fluorescent sensors with better luminescence properties are needed. Compared with the further development of traditional fluorophores, the research and development of new organic fluorescent molecules with good fluorescence phenomena show greater development prospects. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a pyrrole derivative fluorescent dye, a polarity-sensitive fluorescent probe for lipid droplet imaging based on this fluorescent dye, a preparation method and an application. A series of novel fluorescent dyes are successfully synthesized by substituent modification, and based on the research of the novel fluorescent dyes, a polarity-sensitive fluorescent probe is further developed. The prepared polarity-sensitive fluorescent probe not only exhibits high polarity sensitivity and solvatochromism, but also exhibits good fluorescence stability, large Stokes shift and biocompatibility.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] A pyrrole derivative fluorescent dye has the following molecular structural formula: ; Among them, R is H, Br, OH, CH3 or N(CH3)2.

[0007] A preparation method of the above-mentioned pyrrole derivative fluorescent dye includes the following steps: Mix 1,4-naphthoquinone, benzaldehyde or its derivative, ammonium iodide and a solvent, stir and react at 120-140 °C, and perform post-treatment to obtain fluorescent dyes 3a-3e; Preferably, the reaction route of the above-mentioned preparation method of the fluorescent dye is as follows: ; The molecular structural formulas of fluorescent dyes 3a-3e are as follows: .

[0008] Preferably, the benzaldehyde or its derivative is benzaldehyde, 4-bromobenzaldehyde, 4-hydroxybenzaldehyde, 4-methylbenzaldehyde, 4-dimethylaminobenzaldehyde; Preferably, the molar ratio of 1,4-naphthoquinone, benzaldehyde or its derivative, and ammonium iodide is 1:3.8-4.2:2.8-3.2; Preferably, the solvent is toluene; Preferably, the dosage ratio of 1,4-naphthoquinone to the solvent is 316.4 mg:9.5-10.5 mL; Preferably, the stirring reaction time is 22-26 hours; Preferably, the post-treatment is to cool to room temperature, remove volatiles, and purify by chromatography; Furthermore, neutral alumina is used in the chromatography purification, and petroleum ether / ethyl acetate is used as the eluent, and the ratio of petroleum ether to ethyl acetate is 10:1.

[0009] An application of the above-mentioned pyrrole derivative fluorescent dye in the preparation of a fluorescent probe.

[0010] A polarity-sensitive fluorescent probe for lipid droplet imaging based on the aforementioned fluorescent dye has the following molecular structural formula: .

[0011] A preparation method of a polarity-sensitive fluorescent probe for lipid droplet imaging based on the aforementioned fluorescent dye includes the following steps: respectively test the ultraviolet absorption spectra and fluorescence emission spectra of fluorescent dyes 3a-3e, select the fluorescent dye 3e with the maximum ultraviolet absorption wavelength and fluorescence emission wavelength, add the fluorescent dye 3e to a solvent containing KOH, stir evenly, add CH3I, heat to 45-55 °C, stir and react at 45-55 °C to obtain a reaction solution, and perform post-treatment on the reaction solution to obtain a fluorescent probe; Preferably, the reaction route of the aforementioned preparation method of the fluorescent probe is as follows: .

[0012] Preferably, the molar ratio of the fluorescent dye 3e, KOH, and CH3I is 1:6-6.5:2.3-2.7; Preferably, the solvent is DMF; Preferably, the dosage ratio of the fluorescent dye 3e to the solvent is 87.3 mg:4.7-5.3 mL; Preferably, the stirring time for even stirring is 1-1.5 h; Preferably, the stirring reaction time is overnight; Preferably, the post-treatment is to pour the reaction solution into water, extract with dichloromethane, take the organic layer, wash with water, dry with anhydrous sodium sulfate, filter, remove the solvent under vacuum, and purify by chromatography to obtain a fluorescent probe; Furthermore, neutral alumina is used in the chromatography purification, and petroleum ether / ethyl acetate is used as the eluent, and the ratio of petroleum ether to ethyl acetate is 10:1.

[0013] An application of the aforementioned fluorescent probe in one of the following: detecting the change of system polarity, lipid droplet detection, zebrafish bioimaging.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1) The synthesis steps of the fluorescent probe of the present invention are simple, capable of constructing a novel fluorescent platform, and can achieve the modification of substituents on the fluorescent platform by replacing the benzaldehyde raw material, thereby constructing a series of fluorescent dyes. Utilizing the unique chemical structure of the fluorescent platform, a D-A-D type polar fluorescent probe is prepared. The prepared fluorescent probe exhibits high polarity sensitivity, can be used to detect the polarity change of the environmental system, has a good response effect on lipid droplets, can effectively enter living cells to label lipid droplets, enters cells quickly, has a good targeting effect, has low cytotoxicity in cell imaging experiments, can label the lipid droplets in cells, and is further used for zebrafish bioimaging. In addition, the fluorescent probe of the present invention also exhibits satisfactory large Stokes shift, fluorescence stability, and biocompatibility.

[0015] (2) The yield of the fluorescent dye prepared by the present invention is 19 - 68%, and the yield of the prepared fluorescent probe is 80%. Brief Description of the Drawings

[0016] Figure 1 1H NMR spectrum of the fluorescent dye 3a prepared in Example 1 of the present invention 1 1H NMR spectrum.

[0017] Figure 2 13C NMR spectrum of the fluorescent dye 3a prepared in Example 1 of the present invention 13 13C NMR spectrum.

[0018] Figure 3 1H NMR spectrum of the fluorescent dye 3b prepared in Example 1 of the present invention 1 1H NMR spectrum.

[0019] Figure 4 13C NMR spectrum of the fluorescent dye 3b prepared in Example 1 of the present invention 13 13C NMR spectrum.

[0020] Figure 5 1H NMR spectrum of the fluorescent dye 3c prepared in Example 1 of the present invention 1 1H NMR spectrum.

[0021] Figure 6 1H NMR spectrum of the fluorescent dye 3d prepared in Example 1 of the present invention 1 1H NMR spectrum.

[0022] Figure 7 13C NMR spectrum of the fluorescent dye 3d prepared in Example 1 of the present invention 13 13C NMR spectrum.

[0023] Figure 8 1H NMR spectrum of the fluorescent dye 3e prepared in Example 1 of the present invention 1 1H NMR spectrum.

[0024] Figure 9 13C NMR spectrum of the fluorescent dye 3e prepared in Example 1 of the present invention 13 13C NMR spectrum

[0025] Figure 10 1H NMR spectrum of the fluorescent probe Py-LD prepared in Example 2 of the present invention 1 1H NMR spectrum

[0026] Figure 11 13C NMR spectrum of the fluorescent probe Py-LD prepared in Example 2 of the present invention 13 13C NMR spectrum

[0027] Figure 12 HRMS spectrum of the fluorescent probe Py-LD prepared in Example 2 of the present invention

[0028] Figure 13 UV absorption spectra and fluorescence emission spectra of the fluorescent dye 3a (10 μM) prepared in Example 1 of the present invention in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent dye 3a (10 μM) prepared in Example 1 of the present invention in different solvents; (b) is the fluorescence emission spectrum of the fluorescent dye 3a (10 μM) prepared in Example 1 of the present invention in different solvents Figure 14 UV absorption spectra and fluorescence emission spectra of the fluorescent dye 3b (10 μM) prepared in Example 1 of the present invention in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent dye 3b (10 μM) prepared in Example 1 of the present invention in different solvents; (b) is the fluorescence emission spectrum of the fluorescent dye 3b (10 μM) prepared in Example 1 of the present invention in different solvents Figure 15 UV absorption spectra and fluorescence emission spectra of the fluorescent dye 3c (10 μM) prepared in Example 1 of the present invention in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent dye 3c (10 μM) prepared in Example 1 of the present invention in different solvents; (b) is the fluorescence emission spectrum of the fluorescent dye 3c (10 μM) prepared in Example 1 of the present invention in different solvents Figure 16 UV absorption spectra and fluorescence emission spectra of the fluorescent dye 3d (10 μM) prepared in Example 1 of the present invention in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent dye 3d (10 μM) prepared in Example 1 of the present invention in different solvents; (b) is the fluorescence emission spectrum of the fluorescent dye 3d (10 μM) prepared in Example 1 of the present invention in different solvents Figure 17The UV absorption spectra and fluorescence emission spectra of the fluorescent dye 3e (10 μM) prepared in Example 1 of the present invention in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent dye 3e (10 μM) prepared in Example 1 of the present invention in different solvents; (b) is the fluorescence emission spectrum of the fluorescent dye 3e (10 μM) prepared in Example 1 of the present invention in different solvents; Figure 18 For the UV absorption spectrum, fluorescence emission spectrum, normalized fluorescence spectrum, and photos under UV light of the fluorescent probe Py-LD in different solvents; in the figure, (a) is the UV absorption spectrum of the fluorescent probe Py-LD (10 μM) in different solvents; (b) is the fluorescence emission spectrum of the fluorescent probe Py-LD (10 μM) in different solvents; (c) is the normalized fluorescence spectrum of the fluorescent probe Py-LD (10 μM) in different solvents; (d) is a photo of the fluorescent probe Py-LD in different polar solvents under 365 nm UV light.

[0029] Figure 19 For the polarity sensitivity study experiment of the fluorescent probe Py-LD; in the figure, (a) is the fluorescence emission spectrum of the fluorescent probe Py-LD (10 μM) in different ratios of 1,4-dioxane / methanol; (b) is a line graph of the volume concentration of 1,4-dioxane and the fluorescence emission intensity of the fluorescent probe Py-LD (10 μM) in different ratios of 1,4-dioxane / methanol.

[0030] Figure 20 For the stability experiment of the fluorescent probe Py-LD; in the figure, (a) is a fluorescence stability test chart of the fluorescent probe Py-LD (10 μM) in toluene, 1,4-dioxane, and methanol solutions; (b) is a selectivity test chart of the fluorescent probe Py-LD (10 μM) for different ions; (c) is a fluorescence emission intensity test chart of the fluorescent probe Py-LD (10 μM) in PBS buffer solutions with different pH values (1,4-dioxane / PBS = 1:1).

[0031] Figure 21 For the co-localization cell images of the fluorescent probe Py-LD (10 μM) in live HeLa cells; in the figure, (a) is the red channel stained with the fluorescent probe Py-LD (λex = 405 nm, collection: 550 - 700 nm); (b) is the green channel stained with BODIPY (λex = 493 nm, collection: 480 - 530 nm, scale bar: 10 μm); (d) is the merged image of (a) and (b); (d) is the bright field of HeLa cells; (e) is the fluorescence intensity distribution chart of the ROI line.

[0032] Figure 22It is the cellular fluorescence imaging diagram of the fluorescent probe Py-LD in oleic acid-stimulated HeLa cells; in the figure, (a) are the fluorescence images of the fluorescent probe Py-LD of HeLa cells pretreated with different concentrations of oleic acid (0 μM, 50 μM, 100 μM); (b) is the fluorescence intensity histogram of the red channel in (a) (λex = 405 nm, collection: 550 - 700 nm, scale bar: 10 μm).

[0033] Figure 23 It is the fluorescence image of the live zebrafish treated with the fluorescent probe Py-LD; in the figure, (a) is the bright field, (b) is the red channel (λex = 405 nm, collection: 550 - 700 nm); (c) is the merged image of (a) and (b) (scale bar: 250 μm). Detailed implementation manners

[0034] For a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed implementation manners of the present invention are now described.

[0035] Py-LD in each embodiment represents a polarity-sensitive fluorescent probe for lipid droplet imaging based on pyrrole derivative fluorescent dyes, and the numbers of the compounds in the embodiments correspond to the compound numbers in the synthesis route of the above-mentioned invention content.

[0036] Example 1 Preparation of pyrrole derivative fluorescent dyes 3a - 3e This example provides a kind of pyrrole derivative fluorescent dye, including five types of 3a - 3e, and 3a - 3e have the following general molecular structural formula:

[0037] When R = H in the above-mentioned general molecular structural formula, it is the pyrrole derivative fluorescent dye 3a; When R = Br in the above-mentioned general molecular structural formula, it is the pyrrole derivative fluorescent dye 3b; When R = OH in the above-mentioned general molecular structural formula, it is the pyrrole derivative fluorescent dye 3c; When R = CH3 in the above-mentioned general molecular structural formula, it is the pyrrole derivative fluorescent dye 3d; When R = N(CH3)2 in the above-mentioned general molecular structural formula, it is the pyrrole derivative fluorescent dye 3e; That is, 3a - 3e have the following molecular structural formulas respectively: .

[0038] This example also provides a preparation method of the above-mentioned pyrrole derivative fluorescent dye, and the specific synthesis route is as follows: .

[0039] The specific preparation method of the aforementioned pyrrole derivative fluorescent dyes is as follows: 1. Preparation of fluorescent dye 3a Add 1,4-naphthoquinone (316.4 mg, 2 mmol, 1.0 equiv), benzaldehyde (848.8 mg, 8 mmol, 4.0 equiv), ammonium iodide (869.64 mg, 6 mmol, 3.0 equiv) and toluene (10 mL) into a 100 mL reaction flask. Stir and react at 130 °C for 24 hours. After cooling to room temperature, remove the volatiles under reduced pressure. Purify the residue by column chromatography on neutral alumina (petroleum ether / ethyl acetate = 10:1) to obtain fluorescent dye 3a (129.7 mg, yield: 19%).

[0040] For fluorescent dye 3a 1 The Figure 1 H NMR spectrum is as Figure 1 shown. It can be seen that the results of the 1H NMR spectrum of fluorescent dye 3a are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.13 (dd, J = 5.8, 3.4 Hz, 2H), 7.99–7.90 (m, 4H), 7.81 (dd, J = 5.8, 3.3 Hz, 2H), 7.55–7.47 (m, 6H). For fluorescent dye 3a 13 The Figure 2 C NMR spectrum is as Figure 2 shown. It can be seen that the results of the 13C NMR spectrum of fluorescent dye 3a are as follows: 13 C NMR (101 MHz, DMSO-d6) δ 179.80, 138.41, 135.94, 133.73, 130.41, 130.13, 129.74, 128.35, 126.85, 118.04. The molecular formula of fluorescent dye 3a is: C 24 H 15 NO2.

[0041] 2. Synthesis of fluorescent dye 3b The synthesis route of 3b is similar to that of 3a. Specifically, on the basis of the first step, replace benzaldehyde with an equimolar amount of 4-bromobenzaldehyde, and keep the other operation steps unchanged.

[0042] The yield of fluorescent dye 3b is 32%.

[0043] For fluorescent dye 3b1 The 1H NMR spectrum is as Figure 3 shown. It can be seen from Figure 3 that the results of the 1H NMR spectrum of the fluorescent dye 3b are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 8.07 (dd, J = 5.8, 3.3 Hz, 2H), 7.89 – 7.84 (m, 4H), 7.77 (dd, J = 5.8, 3.3 Hz, 2H), 7.72–7.66 (m, 4H). For the fluorescent dye 3b 13 the 13C NMR spectrum is as Figure 4 shown. It can be seen from Figure 4 that the results of the 13C NMR spectrum of the fluorescent dye 3b are as follows: 13 13C NMR (101 MHz, CDCl3) δ 180.56, 136.31, 133.40, 131.82, 130.23, 128.62, 126.99, 124.25, 118.84. The molecular formula of the probe 3b is: C 24 H 13 Br2NO2.

[0044] 3. Synthesis of the fluorescent dye 3c The synthesis route of 3c is similar to that of 3a. Specifically, on the basis of the first step, benzaldehyde is replaced with an equimolar amount of 4-hydroxybenzaldehyde, and the remaining operation steps remain unchanged.

[0045] The yield of the fluorescent dye 3c is 47%.

[0046] For the fluorescent dye 3c 1 the 1H NMR spectrum is as Figure 5 shown. It can be seen from Figure 5 that the results of the 1H NMR spectrum of the fluorescent dye 3c are as follows: 1 1H NMR (400 MHz, CDCl3) δ 12.39 (s, 1H), 10.10 (s, 2H), 8.12 (dd, J = 5.8, 3.3 Hz, 2H), 7.82 (s, 2H), 7.81 – 7.75 (m, 4H), 6.90 (d, J = 8.7 Hz, 4H). The molecular formula of the fluorescent dye 3c is: C 24 H 15 NO4.

[0047] 4. Synthesis of Fluorescent Dye 3d The synthesis route of 3d is similar to that of 3a. Specifically, based on the first step, benzaldehyde is replaced with an equimolar amount of 4-methylbenzaldehyde, and the remaining operation steps remain unchanged.

[0048] The yield of fluorescent dye 3d is 68%.

[0049] For fluorescent dye 3d 1 The 1H NMR spectrum is as follows Figure 6 As can be seen from Figure 6 the results of the 1H NMR spectrum of fluorescent dye 3d are as follows 1 1H NMR (400 MHz, CDCl3) δ 9.25 (s, 1H), 8.22 (dd, J = 5.8, 3.4 Hz, 2H), 7.74–7.69 (m, 4H), 7.67 (dd, J = 5.8, 3.3 Hz, 2H), 7.16 (d, J = 7.9 Hz, 4H), 2.33 (s, 6H). For fluorescent dye 3d 13 The 13C NMR spectrum is as follows Figure 7 As can be seen from Figure 7 the results of the 13C NMR spectrum of fluorescent dye 3d are as follows 13 13C NMR (101 MHz, CDCl3) δ 180.65, 139.84, 137.96, 136.07, 132.94, 129.05, 128.58, 126.99, 126.78, 118.02, 21.55. The molecular formula of probe 3d is: C 26 H 19 NO2.

[0050] 5. Synthesis of Fluorescent Dye 3e The synthesis route of 3e is similar to that of 3a. Specifically 1,4-Naphthoquinone (316.4 mg, 2 mmol, 1.0 equiv), 4-dimethylaminobenzaldehyde (1193.52 mg, 8 mmol, 4.0 equiv) and ammonium iodide (869.64 mg, 6 mmol, 3.0 equiv) were placed in a 100 mL reaction flask, and 10 mL of toluene was added as a solvent. The reaction was stirred at 130 °C for 24 hours. At the end of the reaction, it was cooled to room temperature, and the volatiles were removed under reduced pressure. The residue was purified by column chromatography on neutral alumina (petroleum ether / ethyl acetate = 10:1) to obtain the desired product 3e (305 mg, yield: 35%).

[0051] 1H NMR spectrum of fluorescent dye 3e 1 is as follows Figure 8 as shown, from Figure 8 it can be seen that the results of the 1H NMR spectrum of fluorescent dye 3e are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 12.18 (s, 1H), 8.14 (dd, J = 5.8, 3.3 Hz, 2H), 7.93–7.86 (m, 4H), 7.78 (dd, J = 5.8, 3.3 Hz, 2H), 6.80 (d, J = 9.0 Hz, 4H), 3.01 (s, 12H). 13C NMR spectrum of fluorescent dye 3e 13 is as follows Figure 9 as shown, from Figure 9 it can be seen that the results of the 1H NMR spectrum of fluorescent dye 3e are as follows: 13 13C NMR (101 MHz, DMSO-d6) δ 179.13, 151.34, 139.88, 136.34, 133.20, 131.02, 126.71, 117.63, 116.53, 111.41, 40.41. The molecular formula of probe 3e is: C 28 H 25 N3O2.

[0052] Example 2 Preparation of a polarity-sensitive fluorescent probe for lipid droplet imaging based on pyrrole derivative fluorescent dyes This example provides a polarity-sensitive fluorescent probe for lipid droplet imaging based on pyrrole derivative fluorescent dyes, having the following molecular structural formula: .

[0053] This example also provides a preparation method of the foregoing polarity-sensitive fluorescent probe for lipid droplet imaging based on pyrrole derivative fluorescent dyes, and the specific synthesis route is as follows: .

[0054] The specific preparation method of the foregoing polarity-sensitive fluorescent probe for lipid droplet imaging based on pyrrole derivative fluorescent dyes is: The fluorescent dye 3e (87.3 mg, 0.2 mmol, 1.0 equiv) prepared in Example 1 was added to a suspension of KOH (70.0 mg, 1.25 mmol, 6.25 equiv) in DMF (5 mL). After stirring for 1 h, CH3I (31 μL, 0.5 mmol, 2.5 equiv) was added, and then the mixture was heated to 50 °C and stirred overnight at 50 °C. The resulting suspension was poured into water and extracted with dichloromethane. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. The crude product was purified by column chromatography on neutral alumina (petroleum ether / ethyl acetate = 10:1) to obtain Py-LD (72.7 mg, yield: 80%).

[0055] The 1 1H NMR spectrum of Py-LD is as Figure 10 shown. It can be seen from Figure 10 that the results of the 1H NMR spectrum of the fluorescent dye 3e are as follows: 1 1H NMR (400 MHz, DMSO- d6 ) δ 8.04 (dd, J = 5.8, 3.3 Hz, 2H), 7.75 (dd, J = 5.8, 3.3 Hz, 2H), 7.51 – 7.42 (m, 4H), 6.87 – 6.79 (m, 4H), 3.34 (s, 3H), 3.01 (s, 12H). The 13 13C NMR spectrum of Py-LD is as Figure 11 shown. It can be seen from Figure 11 that the results of the 1H NMR spectrum of the fluorescent dye 3e are as follows: 13 13C NMR (101 MHz, CDCl3) δ 179.93, 150.84, 140.37, 136.34, 132.55, 131.68, 126.64, 117.84, 111.66, 40.36, 33.80. HRMS (ESI) Found: 450.2174 [M+H] + ; Molecular formula C 29 H 27 N3O2 requires [M+H] + 450.2177. The HRMS spectrum of Py-LD is as Figure 12 shown.

[0056] The molecular formula of Py-LD is: C29 H 27 N3O2。

[0057] Test Example 1: Spectral Property Test of Fluorescent Dyes 3a - 3e In Example 1, a series of novel fluorescent dyes (3a - 3e) were prepared by a one - step method using benzaldehydes (2a - 2e) with different pairs of substituents as raw materials. This fluorescent structure based on pyrrole derivatives is a typical polar conjugated system, which prompted us to study its luminescence properties. First, the ultraviolet absorption spectra and fluorescence emission spectra of the fluorescent dyes 3a - 3e prepared in Example 1 in different solvents were tested. Specifically, the fluorescent dyes 3a - 3e were respectively dissolved in DMSO to prepare 1 mM stock solutions, and then 20 μL of the stock solution was added to 2 mL of different polar solvents to obtain a fluorescence probe test solution with a concentration of 10 μM, and the ultraviolet absorption spectra and fluorescence emission spectra were tested. The obtained ultraviolet absorption spectra and fluorescence emission spectra are as Figures 13 - 17 shown. It can be seen from Figures 13 - 17 that introducing different substituents can significantly change the luminescence properties of the fluorophore. The fluorescence emission of the fluorescent dyes 3a - 3e changes significantly in a wide wavelength range of 500 - 650 nm. As expected, introducing strong electron - donating groups into the fluorophore leads to the formation of a D - A - D structure, thereby producing excellent fluorescence properties, with a larger emission wavelength and a large Stokes shift. After further analysis, it was found that the dimethylamino group in the fluorescent dye 3e has a good electron - donating effect, so the fluorescent dye 3e has a longer ultraviolet absorption wavelength and fluorescence emission wavelength. It has been reported that fluorescent probes with longer emission wavelengths have the advantages of less background interference, lower light source energy, stronger tissue penetration, and less tissue damage. Therefore, in Example 2, the fluorescent dye 3e was selected from the fluorescent dyes 3a - 3e to construct a fluorescent probe Py - LD with excellent photophysical properties, and further tests were carried out on the fluorescent probe Py - LD.

[0058] Test Example 2: Spectral Property Test of Fluorescent Probe Py - LD First, the fluorescent probe Py - LD was dissolved in DMSO to prepare a 1 mM stock solution. By adding 20 μL of the stock solution to 2 mL of different polar solvents, a fluorescence probe with a concentration of 10 μM was obtained, and the fluorescence probe with a concentration of 10 μM was used in all subsequent spectral experiments. The optical properties of the fluorescent probe Py - LD were studied, and the ultraviolet absorption spectra and fluorescence emission spectra of the fluorescent probe Py - LD in different solvents were tested, as Figure 18 shown. It can be seen from Figure 18It can be seen that in different solvents, the maximum absorption wavelength of the ultraviolet absorption spectrum remains relatively constant; as can be seen from 18b, in the fluorescence emission spectrum, the maximum emission wavelength of the fluorescent probe Py-LD shows a significant red shift with the increase of solvent polarity, and this trend can be more clearly observed from Figure 18 c in the normalized fluorescence spectra of the fluorescent probe Py-LD in different solvents; from Figure 18 d, it can be seen that under 365 nm ultraviolet irradiation, the solvent color of Py-LD also changes from yellow to red with the increase of solvent polarity, which is consistent with the spectral test results, showing a positive solvatochromic effect. It should be noted that the fluorescent probe Py-LD shows a polarity quenching effect in high-polarity solvents (acetone, DMF), resulting in the inhibition of fluorescence emission and the inability to observe the fluorescence changes in the solvent. All these results indicate that this probe has good polarity response characteristics and the potential to become a polarity-sensitive probe.

[0059] Test Example 3 Polarity Sensitivity Experiment of Fluorescent Probe Py-LD Take 20 μL of the probe stock solution prepared in Test Example 2 and add it to 2 mL of 1,4-dioxane / methanol containing different volume fractions of 1,4-dioxane to study the sensitivity of the probe Py-LD to solvent polarity detection, as shown in Figure 19 shown. As can be seen from Figure 19 a and 19b, with the increase of the 1,4-dioxane content in the mixed solvent, the fluorescence emission intensity of the probe Py-LD increases significantly, and the maximum emission wavelength shows an obvious blue shift, which fully indicates that the probe Py-LD has excellent sensitivity to the polar environment and good fluorescence emission intensity.

[0060] Test Example 4 Stability Experiment of Fluorescent Probe Py-LD Take 20 μL of the probe stock solution prepared in Test Example 2 and add it to 2 mL of toluene, 1,4-dioxane, and methanol solutions respectively, which are used as solvents for detecting the time stability of the fluorescent probe to determine the fluorescence stability of the fluorescent probe in different polar environments, as shown in Figure 20 a. As can be seen from Figure 20 a, the fluorescent probe has good stability in different polar environments, indicating that the probe can maintain good stability in different polar environments.

[0061] Take 20 μL of the probe stock solution prepared in Test Example 2, add it to 2 mL of a 1,4-dioxane / PBS (V / V = 1:1) mixed solution, and then add 20 μL of 1 mmol / L of different interfering solutions, which are MgSO4 solution, K3PO4 solution, HClO4 solution, K2CO3 solution, L-cysteine solution, NaHSO4 solution, H2O2 solution, NaNO2 solution, NaCl solution, Na2SO4 solution, FeCl3 solution, Hg(NO3)2 solution, MgSO4 solution, K3PO4 solution, CuCl2 solution, and CaCO3 solution respectively. The selectivity test graph of the obtained fluorescent probe Py-LD (10 μM) for different ions is as shown in Figure 20 Figure b, as can be seen from Figure 20 Figure b, when different ions are added, the fluorescence intensity of the fluorescent probe hardly changes, indicating that the influence of the analyte on the fluorescence intensity can be ignored. The results show that this probe has good anti-interference performance and can be used for fluorescence testing in complex environments.

[0062] Take 20 μL of the probe stock solution prepared in Example 2 and add it to 2 mL of 1,4-dioxane / PBS buffer solution with different pH values (V = 1:1) as the solvent for detecting the pH stability of the probe. The fluorescence emission intensity test graph of the fluorescent probe Py-LD (10 μM) in PBS buffer solutions with different pH values is as shown in Figure 20 Figure c, as can be seen from Figure 20 Figure c, the fluorescence intensity of the fluorescent probe Py-LD remains relatively stable in different pH environments, indicating that the probe is less affected by pH. The fluorescent probe Py-LD shows good fluorescence stability in the biological pH range.

[0063] Test Example 5 Lipid droplet co-localization imaging experiment of fluorescent probe Py-LD Use the fluorescent probe Py-LD for cell co-localization imaging of lipid droplets. The specific operation is as follows: Incubate HeLa cells, BODIPY (500 nM) and Py-LD (10 μM) for 20 minutes, then wash the cells three times with sterile PBS buffer solution, and then perform cell co-localization imaging. The obtained results are as shown in Figure 21 Figure.

[0064] As can be seen from Figure 21 Figure a, there is obvious red fluorescence (λ ex = 405 nm, collection: 550 - 700 nm) on the cell lipid droplets. As can be seen from Figure 21 Figure b, the fluorescence imaging is collected by the commercial organic dye BODIPY (λ ex = 488 nm, collection: 500 - 530 nm), showing obvious green fluorescence. As can be seen from Figure 21From the overlay channel in c, it can be seen that the green and red light spots are basically overlapped, and the Pearson correlation coefficient is as high as 0.92, indicating a high degree of colocalization between the two. Figure 21 d shows the cell morphology and lipid droplet status, and is compared with the fluorescence channel. Figure 21 e is the colocalization curve of two cell images in the region of interest (ROI). From the colocalization curve, it can be seen that the two probes show good colocalization effects in the same cell. It shows that the fluorescent probe Py-LD, like the commercial lipid droplet probe BODIPY, can target cell lipid droplets well and has good live cell lipid droplet imaging effects.

[0065] All of the above features indicate that the fluorescent probe Py-LD can be used for cell lipid droplet labeling.

[0066] Test Example 6 Monitoring experiment of the changes of intracellular lipid droplets by the fluorescent probe Py-LD The changes of intracellular lipid droplets stimulated by oleic acid with the fluorescent probe Py-LD were monitored. The specific operation was to incubate lipid droplet inducer oleic acid (OA) at different concentrations (0 μM, 50 μM, 100 μM), Py-LD (10 μM) with HeLa cells for 20 minutes, then wash the cells three times with sterile PBS buffer, and then perform cell fluorescence imaging. The results are as Figure 22 shown. From Figure 22 it can be seen that there are fewer and more dispersed lipid droplets in the cells without OA treatment. With the increase of OA concentration, the size and number of intracellular lipid droplets increase significantly. The more lipid droplets, the stronger the fluorescence signal of Py-LD, and the fluorescence intensity is positively correlated with the OA concentration. These experimental results indicate that Py-LD can effectively monitor the changes of intracellular lipid droplets.

[0067] Test Example 7 Bioimaging experiment of the fluorescent probe Py-LD on zebrafish To evaluate the luminescence performance of the fluorescent probe Py-LD in bioimaging, zebrafish was used as a model organism to evaluate its imaging ability. During the development of zebrafish, the yolk sac is an important structure providing nutritional support and is composed of about 70% neutral lipids. After treating live zebrafish with the fluorescent probe Py-LD and incubating for 30 minutes, the obtained fluorescence images are shown in Figure 23 . From Figure 23 it can be seen that after incubating with the fluorescent probe Py-LD for 30 minutes, the yolk sac of zebrafish is significantly stained red in the red channel. This indicates that the fluorescent probe Py-LD has successfully labeled the lipid structure in the yolk sac of zebrafish. Therefore, the fluorescent probe Py-LD has great application potential in bioimaging.

Claims

1. A pyrrole derivative fluorescent dye, characterized in that, It has the following molecular structural formula: ； Wherein, R is H, Br, OH, CH3 or N(CH3)2.

2. A method for preparing a pyrrole derivative-based fluorescent dye as described in claim 1, characterized in that, It includes the following steps: Mix 1,4-naphthoquinone, benzaldehyde or its derivative, ammonium iodide and a solvent, stir and react at 120 - 140 °C, and perform post-treatment to obtain a fluorescent dye.

3. The preparation method of the pyrrole derivative-based fluorescent dye according to claim 2, characterized in that, The benzaldehyde or its derivative is benzaldehyde, 4-bromobenzaldehyde, 4-hydroxybenzaldehyde, 4-methylbenzaldehyde, 4-dimethylaminobenzaldehyde; The molar ratio of 1,4-naphthoquinone, benzaldehyde or its derivative, and ammonium iodide is 1:3.8 - 4.2:2.8 - 3.2; The solvent is toluene; The dosage ratio of 1,4-naphthoquinone to the solvent is 316.4 mg:9.5 - 10.5 mL; The stirring reaction time is 22 - 26 hours.

4. The preparation method of the pyrrole derivative-based fluorescent dye according to claim 2, characterized in that, The post-treatment is to cool to room temperature, remove volatiles, and perform column chromatography purification; Neutral alumina is used in the column chromatography purification, and petroleum ether / ethyl acetate is used as the eluent, and the ratio of petroleum ether to ethyl acetate is 10:

1.

5. Use of a pyrrole derivative fluorescent dye as described in claim 1 in the preparation of a fluorescent probe.

6. A polarity-sensitive fluorescent probe for lipid droplet imaging based on the fluorescent dye described in claim 1, characterized in that, It has the following molecular structural formula: 。 7. A method for preparing a polarity-sensitive fluorescent probe for lipid droplet imaging according to claim 6, characterized in that, It includes the following steps: Add the fluorescent dye when R is N(CH3)2 to a solvent containing KOH, stir evenly, add CH3I, heat to 45 - 55 °C, stir and react at 45 - 55 °C to obtain a reaction solution, and perform post-treatment on the reaction solution to obtain a fluorescent probe.

8. The preparation method of the polarity-sensitive fluorescent probe for lipid droplet imaging according to claim 7, characterized in that, The molar ratio of the fluorescent dye, KOH, and CH3I is 1:6 - 6.5:2.3 - 2.7; The solvent is DMF; The dosage ratio of the fluorescent dye to the solvent is 87.3 mg:4.7 - 5.3 mL; The stirring time for even mixing is 1 - 1.5 h; The stirring reaction time is overnight.

9. The preparation method of the polarity-sensitive fluorescent probe for lipid droplet imaging according to claim 7, wherein, The post-treatment is to pour the reaction solution into water, extract with dichloromethane, take the organic layer, wash with water, dry with anhydrous sodium sulfate, filter, remove the solvent under vacuum, and perform column chromatography purification to obtain a fluorescent probe; Neutral alumina is used in the column chromatography purification, and petroleum ether / ethyl acetate is used as the eluent, and the ratio of petroleum ether to ethyl acetate is 10:

1.

10. Use of a polarity-sensitive fluorescent probe for lipid droplet imaging as described in claim 6, characterized in that, Use of the fluorescent probe in one of the following: detecting the change of system polarity, lipid droplet detection, zebrafish bioimaging.

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