High-brightness fluorescent dye for lipid droplet labeling and synthesis and application thereof

Abstract

The application provides a high-brightness fluorescent dye for lipid droplet labeling and synthesis and application thereof, the fluorescent dye taking 1-acetyl pyrene as a parent body, and introducing a diphenylamine substituent group at 8, as shown in the following formula (1). The probe can specifically label lipid droplets in living cells, and has the advantages of high brightness, high signal-to-noise ratio, rapid labeling and the like. The lipid droplet dye has a very broad application prospect in dynamic imaging and real-time monitoring of lipid droplets.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent dye technology, specifically relating to a high-brightness fluorescent dye for lipid droplet-specific labeling, its synthesis method, and its application. Background Technology

[0002] Lipid droplets, also known as fat bodies or lipid bodies, are lipid-rich organelles within cells that regulate the storage of neutral lipids, including triglycerides and cholesterol esters. In recent years, research on lipid droplets has attracted significant attention because they have been found to participate in numerous physiological processes, including membrane synthesis and transport, protein degradation, inflammation and pathology, as well as obesity, diabetes, atherosclerosis, and cancer. Therefore, in-situ, real-time imaging and tracking of lipid droplets are crucial for studying their dynamic behavior and function within living cells.

[0003] Lipid droplet fluorescent dyes can locate lipid droplets and emit fluorescence within them, thus revealing the lipid droplet structure. Although various lipid droplet fluorescent dyes have been developed, they suffer from several drawbacks in bioimaging, such as poor photostability, non-specific labeling of lipid droplets, and low brightness, which significantly limit their application in lipid droplet imaging. Especially in the era of super-resolution fluorescence imaging, imaging technology places higher demands on the fluorescence stability and intensity, as well as the specificity of dye labeling. There is an urgent need for specifically labeled, high-brightness, and stable lipid droplet fluorescent dyes. Therefore, the development of high-brightness, specifically labeled fluorescent dyes for lipid droplet labeling has a very broad application prospect. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent dye for lipid droplet labeling, its synthesis method, and its applications. The method involved in the synthesis of this dye features inexpensive and readily available raw materials, simple synthesis procedures, and convenient purification methods. Furthermore, this dye can specifically label lipid droplets in living cells and exhibits high fluorescence intensity.

[0005] This invention provides a fluorescent probe for lipid droplet labeling. Using 1-acetylpyrene as the parent compound, an amino substituent is introduced at position 8, resulting in the design and synthesis of a high-brightness fluorescent probe capable of rapid and accurate labeling of lipid droplets. This fluorescent probe meets the requirements of high sensitivity, good selectivity, large Stokes shift, high fluorescence intensity, and high signal-to-noise ratio, making it a powerful tool for studying lipid droplets.

[0006] A type of high-brightness fluorescent probe for lipid droplet labeling has the following structure:

[0007]

[0008] A method for synthesizing a fluorescent dye for lipid droplet labeling, the synthetic route of which is as follows:

[0009]

[0010] The specific synthesis steps are as follows:

[0011] (1) Synthesis of intermediate 1-acetyl-8-bromopyrene

[0012] Under nitrogen atmosphere, 1-acetyl-8-bromopyrene and anhydrous aluminum chloride were dissolved in dichloromethane. Acetyl chloride was added dropwise to the solution at 0°C, and the mixture was stirred at room temperature for 10–30 h. After the reaction was complete, saturated sodium bicarbonate solution was added to quench the reaction. The reaction solution was extracted, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography to obtain a pale yellow powder, 1-acetyl-8-bromopyrene.

[0013] In step (1): the molar ratio of 1-bromopyrene to acetyl chloride is 1:1-1.5; the molar ratio of 1-bromopyrene to anhydrous aluminum chloride is 1:2.2-3.9; and the mass-volume ratio of 1-bromopyrene to dichloromethane is 0.004-0.008 g / mL.

[0014] (2) Synthesis of fluorescent probes

[0015] A mixture of 1-acetyl-8-bromopyrene, Pd2dba3, XPhos, Cs2CO3, and diphenylamine was placed in a two-necked flask and purged with nitrogen 3-4 times. Toluene was then added to the flask, and the reaction was stirred at 80-120°C for 12-36 hours. The solvent was removed under reduced pressure, and the residue was separated by silica gel column chromatography to obtain a yellow solid (fluorescent probe).

[0016] In step (2): the molar ratio of 1-acetyl-8-bromopyrene to diphenylamine is 1:4-8; Pd2dba3 is 1-20 mol% of 1-acetyl-8-bromopyrene; XPhos is 10-50 mol% of 1-acetyl-8-bromopyrene; the mass ratio of Cs2CO3 to 1-acetyl-8-bromopyrene is 1-5:1; the mass ratio of diphenylamine to toluene is 1:15-30 g / mL g / mL.

[0017] Application of a fluorescent dye for lipid droplet labeling in the field of fluorescence imaging of lipid droplets in live cells.

[0018] A fluorescent dye for lipid droplet labeling can specifically label lipid droplets in living cells and enable real-time fluorescence imaging.

[0019] The dye in this invention has advantages in terms of the availability and low cost of raw materials, the simplicity of the synthesis method, and the ease of purification. This dye can specifically label lipid droplets and exhibits very high brightness, with a measured quantum yield of 1, laying a solid foundation for its use as a high-brightness, high-specificity fluorescent dye for lipid droplet labeling.

[0020] This dye can be used as a fluorescent probe for the specific labeling and fluorescence imaging of lipid droplets in living cells. Attached Figure Description

[0021] Figure 1 The 1H NMR spectrum of the 182ph-lipid prepared in Example 1.

[0022] Figure 2 The carbon NMR spectrum of the 182ph-lipid prepared in Example 1.

[0023] Figure 3 The image shows the crystal of the 182ph-lipid prepared in Example 1.

[0024] Figure 4 The absorption spectra of the lipid droplet dye 182ph-lipid prepared in Example 1 in different solvents are shown. The horizontal axis represents wavelength, and the vertical axis represents absorbance. The concentration of the fluorescent probe is 10 μM.

[0025] Figure 5 The fluorescence spectra of the lipid droplet dye 182ph-lipid prepared in Example 1 in different solvents are shown. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity. The concentration of the fluorescent probe is 10 μM.

[0026] Figure 6 Fluorescence imaging of cellular lipid droplets labeled with the lipid droplet dye 182ph-lipid prepared in Example 1. Figure 7 This is a fluorescence imaging image showing the co-localization of cellular lipid droplets labeled with the lipid droplet dye 182ph-lipid prepared in Example 1 and a commercial lipid droplet dye. Detailed Implementation

[0027] This invention provides a class of lipid droplet-targeting fluorescent probes, their synthesis method, and their application in fluorescence imaging.

[0028] The solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The following three examples (Examples 1-3) illustrate the preparation of 182ph-lipid, and their reaction formulas are shown below:

[0030]

[0031] Example 1.

[0032] (1) Synthesis of intermediate 1-acetyl-8-bromopyrene

[0033]

[0034] Under nitrogen atmosphere, 1-bromopyrene (28.12 mg, 100 μmol) and anhydrous aluminum chloride (29.33 mg, 220 μmol) were dissolved in 2.52 mL of dichloromethane. At 0 °C, acetyl chloride (7.11 μL, 100 μmol) was added dropwise to the solution. The mixture was stirred at room temperature for 10 h. After the reaction was complete, 5 mL of saturated sodium bicarbonate solution was added to quench the reaction. The reaction mixture was extracted three times (the extract was dichloromethane / water = 1:2 V / V, with a total extract volume of 30 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether / dichloromethane = 10:1-2:1; V / V) to give 18.20 mg of pale yellow powder 1-acetyl-8-bromopyrene, with a yield of 44%.

[0035] Its 1H NMR, 1C NMR, and mass spectrometry data are as follows: 1 H NMR (400MHz, CDCl3) δ8.42(d,1H),8.22(d,1H),8.08(d,2H),8.00(d,1H),7.99(d,1H),7.70(d,2H),2.50(s,3H). 13 C NMR (101MHz, CDCl3) δ 30.5, 121.2, 124.3, 124.7, 125.4, 126.5, 126.7, 127.3, 127.5, 128.1, 129.0, 129.2, 130.4, 130.5, 130.6, 132.3, 133.9, 201.9. HRMS: m / z: theoretical value C 18 H 12 BrO + 323.0072, [M+H] + Actual value: 323.0075.

[0036] Its structure, as shown in the formula 1-acetyl-8-bromopyrene, was determined by testing.

[0037] (2) Synthesis of the fluorescent probe 1-acetyl-8-diphenylaminopyrene (182ph-lipid):

[0038]

[0039] A mixture of 1-acetyl-8-bromopyrene (271 mg, 0.84 mmol), Pd₂dba₃ (7.7 mg, 0.0084 mmol), XPhos (40 mg, 0.084 mmol), Cs₂CO₃ (271 mg, 0.84 mmol), and diphenylamine (568.58 mg, 3.36 mmol) was placed in a two-necked flask and purged with nitrogen 3–4 times. Then, toluene (8.53 mL) was added, and the reaction was stirred at 80 °C for 12 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (petroleum ether / dichloromethane = 10:1–4:1; V / V) to give 174.89 mg of a yellow solid, 85% yield.

[0040] Its NMR spectra (H1N and C1N) are as follows: Figure 1 , 2 The specific data for the 1H NMR, 1C NMR, and mass spectrometry are shown below: 1 H NMR (400MHz, CDCl3) δ8.86(d,J=9.7Hz,1H),8.33(dd,J=19.4,8.9Hz,2H),8.21(d,J=8.2Hz,1H),8.18–8.11(m,2H),8.04( d,J=8.8Hz,1H),7.87(d,J=8.2Hz,1H),7.20(tt,J=3.8,2.0Hz,4H),7.08–7.03(m,4H),6.96(t,J=7.3Hz,2H),2.85(s,3H). 13 C NMR (101MHz, CDCl3) δ202.00,148.76,142.30,134.16,132.05,129.43,129.32,129.27,127.9 8,127.48,127.36,127.31,126.67,126.12,125.88,125.27,125.23,124.15,122.42,122.10,

[0041] 30.32.HRMS:m / z:theoretical value C 30 H 22 NO + :412.1701,[M+H] + Actual value: 412.1710.

[0042] The data shows that its structure is as shown in the above formula for 182ph-lipid. The crystal diagram of 182ph-lipid is shown below. Figure 3 As shown, the crystal diagram also verifies the correctness of the structure.

[0043] The spectral testing of 182ph-lipid was performed as follows: The 182ph-lipid probe was dissolved in DMSO solution to prepare a 2mM 182ph-lipid solution. 20 μL of this 2mM solution was added to 4 mL of different solvents to prepare a 10 μM fluorescent probe test solution. Absorption and fluorescence spectra were then measured (excitation wavelength 410 nm). The absorption and fluorescence spectra of 182ph-lipid in different solvents are shown below. Figure 4 , 5 As shown, the 182ph-lipid exhibits high fluorescence intensity in chloroform, a highly lipid-soluble solvent, but very low fluorescence intensity in water. The quantum yield of this probe in chloroform is 1.

[0044] Example 2.

[0045] (1) Synthesis of intermediate 1-acetyl-8-bromopyrene

[0046]

[0047] Under nitrogen atmosphere, 1-bromopyrene (28.12 mg, 100 μmol) and anhydrous aluminum chloride (40.00 mg, 300 μmol) were dissolved in 4.69 mL of dichloromethane. At 0 °C, acetyl chloride (8.53 μL, 120 μmol) was added dropwise to the solution. The mixture was stirred at room temperature for 20 h. After the reaction was complete, 6 mL of saturated sodium bicarbonate solution was added to quench the reaction. The reaction mixture was extracted three times (the extract was dichloromethane / water = 1:2 V / V, with a total extract volume of 30 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether / dichloromethane = 10:1-2:1; V / V) to give 19.03 mg of pale yellow powder 1-acetyl-8-bromopyrene, with a yield of 46%.

[0048] (2) Synthesis of the fluorescent probe 1-acetyl-8-diphenylaminopyrene (182ph-lipid):

[0049]

[0050] A mixture of 1-acetyl-8-bromopyrene (161.59 mg, 0.50 mmol), Pd2dba3 (45.79 mg, 0.05 mmol, 10% mmol), XPhos (71.51 mg, 0.15 mmol, 30% mmol), Cs2CO3 (488.73 mg, 1.50 mmol), and diphenylamine (507.66 mg, 3 mmol) was placed in a two-necked flask. The flask was sealed and purged three times with nitrogen. Then, toluene (11.42 mL) was added, and the reaction was stirred at 95 °C for 24 h. The solvent was removed under reduced pressure. The residue was then purified by silica gel chromatography (petroleum ether / dichloromethane = 10:1–4:1; V / V) to give 179.00 mg of solid, 87% yield.

[0051] Example 3.

[0052] (1) Synthesis of intermediate 1-acetyl-8-bromopyrene

[0053]

[0054] Under nitrogen atmosphere, 1-bromopyrene (28.12 mg, 100 μmol) and anhydrous aluminum chloride (52.00 mg, 390 μmol) were dissolved in 7.03 mL of dichloromethane. At 0 °C, acetyl chloride (10.67 μL, 150 μmol) was added dropwise to the solution. The mixture was stirred at room temperature for 30 h. After the reaction was complete, 7 mL of saturated sodium bicarbonate solution was added to quench the reaction. The reaction mixture was extracted three times (the extract was dichloromethane / water = 1:2 V / V, with a total extract volume of 30 mL). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (petroleum ether / dichloromethane = 10:1-2:1; V / V) to give 17.79 mg of pale yellow powder 1-acetyl-8-bromopyrene, with a yield of 43%.

[0055] (2) Synthesis of the fluorescent probe 1-acetyl-8-diphenylaminopyrene (182ph-lipid):

[0056]

[0057] A mixture of 1-acetyl-8-bromopyrene (161.59 mg, 0.50 mmol), Pd₂dba₃ (91.57 mg, 0.1 mmol), XPhos (119.18 mg, 0.25 mmol), Cs₂CO₃ (807.95 mg, 2.48 mmol), and diphenylamine (676.88 mg, 4 mmol) was placed in a two-necked flask. The flask was sealed and purged three times with nitrogen. Then, toluene (20.31 mL) was added, and the reaction was stirred at 120 °C for 36 h. The solvent was removed under reduced pressure. The residue was then purified by silica gel chromatography (petroleum ether / dichloromethane = 10:1–4:1; V / V) to give 176.95 mg of solid, in 86% yield.

[0058] Example 4

[0059] Confocal fluorescence imaging of live cell staining using the fluorescent probe 182ph-lipid. The probe 182ph-lipid was dissolved in DMSO solution to prepare a 2mM 182ph-lipid solution. 0.5 μL of this 2mM 182ph-lipid solution was added to 1 mL of HeLa cell culture medium. Cells were incubated at 37°C under 5% CO2 air for 20 minutes before confocal fluorescence imaging.

[0060] 182ph-lipid confocal fluorescence imaging after staining live cells, as shown Figure 6 As shown.

[0061] 182ph-lipid labeling of cellular lipid droplets and fluorescence imaging co-localization with the commercial dye LD540, as shown in... Figure 7 As shown, the Pearson correlation coefficient for co-localization was 0.993, indicating that 182ph-lipid highly specifically labels lipid droplets. The co-localization procedure was as follows: First, HeLa cells were stained with 182ph-lipid. 0.5 μL of 2 mM 182ph-lipid DMSO solution was added to 1 mL of HeLa cell culture medium. Then, 0.1 μL of 2 mM commercial lipid droplet dye LD540 was added to the above 1 mL of HeLa cell culture medium. The cells were incubated at 37°C under 5% CO2 air for 20 minutes, followed by confocal co-localization fluorescence imaging.

[0062] The lipid droplets labeled with 182ph-lipid were clearly visible, achieving high-brightness and specific fluorescent labeling of lipid droplets in living cells by 182ph-lipid.

Claims

1. A method for synthesizing a fluorescent probe for lipid droplet labeling, characterized in that, The structural formula of the fluorescent probe used for lipid droplet labeling is shown in formula (1). ； This method includes the following synthesis steps: (1) Synthesis of intermediate 1-acetyl-8-bromopyrene Under nitrogen atmosphere, 1-bromopyrene and anhydrous aluminum chloride were dissolved in dichloromethane. Acetyl chloride was added dropwise to the above solution at 0°C and stirred at room temperature for 10-30 h. After the reaction was completed, the solution was separated and purified to obtain a light yellow powder, 1-acetyl-8-bromopyrene. (2) Synthesis of lipid droplet fluorescent probes A mixture of 1-acetyl-8-bromopyrene, cesium carbonate, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (XPhos), tris(dibenzylacetone)dipalladium (Pd2dba3), and diphenylamine was placed in a container, and the atmosphere inside the container was purged with nitrogen 3-4 times. Toluene was then added to the container, and the reaction was stirred at 80-120°C for 12-36 hours. The target product was obtained by separation and purification.

2. The synthesis method according to claim 1, characterized in that: In step (1): the molar ratio of 1-bromopyrene to acetyl chloride is 1:1-1.5; the molar ratio of 1-bromopyrene to anhydrous aluminum chloride is 1:2.2-3.9; and the mass-volume ratio of 1-bromopyrene to dichloromethane is 0.004-0.008 g / mL.

3. The synthesis method according to claim 1, characterized in that: In step (2): the molar ratio of 1-acetyl-8-bromopyrene to diphenylamine is 1:4-8; Pd2dba3 is 1-20 mol% of 1-acetyl-8-bromopyrene; XPhos is 10-50 mol% of 1-acetyl-8-bromopyrene; the mass ratio of Cs2CO3 to 1-acetyl-8-bromopyrene is 1-5:1; the mass ratio of diphenylamine to toluene is 1:15-30 g / mL.

4. The application of the fluorescent probe for lipid droplet labeling as described in claim 1, not for the purpose of diagnosing or treating a disease, in intracellular lipid droplet fluorescence imaging.

Images