A lipid droplet-specific fluorescent probe and its preparation method and application

Abstract

The present invention discloses a lipid droplet-specific fluorescent probe, its preparation method and application, relating to the technical field of biomedicine; the present invention provides the structural formula of the lipid droplet-specific fluorescent probe, and during preparation, 2-formaldehyde-6-(diethylamino)benzofuran and a malononitrile derivative are used to prepare a class of fluorescent probes with an extended π-conjugated system under the action of an alkaline environment; these fluorescent probes all have the characteristics of intramolecular charge transfer (TICT), with extremely weak fluorescence in aqueous solution, while the fluorescence is significantly enhanced in sunflower oil, having a significant Stokes shift, excellent biocompatibility, excellent photostability and a high signal-to-noise ratio. At the same time, this fluorescent probe shows extremely high sensitivity and signal-to-noise ratio in cell lipid droplet imaging, and can easily achieve dynamic and precise targeted imaging of lipid droplets. Therefore, the fluorescent probe of the present invention is expected to become an alternative for the accurate detection of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a lipid droplet-specific fluorescent probe, a preparation method thereof, and an application thereof. Background Art

[0002] Lipid droplets, as dynamic intracellular organelles, play a key role in lipid storage and metabolism. Research shows that the abnormal accumulation of lipid droplets is closely related to various metabolic disorders, such as type II diabetes, fatty liver disease, and atherosclerosis. Among them, atherosclerosis is a chronic systemic progressive disease and has become the main cause of the morbidity and mortality of cardiovascular diseases globally. The development of atherosclerotic lesions is accompanied by continuous lipid deposition, which further promotes plaque formation and ultimately leads to acute cardiovascular events. Visualization of lipid droplets shows great potential in clinical intervention. By providing valuable information at the cellular and tissue levels, lipid droplet imaging will contribute to the early diagnosis and treatment of atherosclerosis, as well as the prevention of acute cardiovascular events related to atherosclerosis.

[0003] In recent years, fluorescence imaging for lipid droplets has received extensive attention due to its unique advantages such as sensitivity, rapidity, non-invasiveness, high spatio-temporal resolution, and real-time in-situ, showing broad application prospects in clinical translation. In recent years, various fluorescent dyes for lipid droplet imaging have been developed. Among them, Nile red and BODIPY 493 / 503 are currently widely used commercial fluorescent dyes. However, Nile red is limited by its low specificity and broad emission spectrum, with a relatively low signal-to-noise ratio. BODIPY 493 / 503 has a small Stokes shift, limited photostability, and insufficient specificity. In addition, most of the reported probes show strong emission in both aqueous and lipid droplet environments, with limited applications in bioimaging. Therefore, novel lipid droplet-specific fluorescent probes with high signal-to-noise ratio and large Stokes shift still need to be further developed. Summary of the Invention

[0004] To solve the above technical problems, the object of the present invention is to provide a lipid droplet-specific fluorescent probe, a preparation method thereof, and an application thereof. The fluorescent probe with an extended π-conjugated system shows extremely high sensitivity and signal-to-noise ratio in cellular lipid droplet imaging, can easily achieve dynamic and precise targeting imaging of lipid droplets, and effectively solves the problems of low signal-to-noise ratio and limited bioimaging applications of existing fluorescent probes.

[0005] The technical solution for the present invention to solve the above technical problems is as follows: Provide a lipid droplet-specific fluorescent probe, and its structural formula is one of the following:

[0006] 、 、 、 、 and 。

[0007] The present invention also provides a preparation method of the above-mentioned lipid droplet-specific fluorescent probe, which includes the following steps:

[0008] Dissolve 2-formaldehyde-6-(diethylamino)benzofuran and malononitrile derivative in a solvent, and then react under the action of a base, followed by concentration and purification to obtain the lipid droplet-specific fluorescent probe.

[0009] Furthermore, the molar ratio of 2-formaldehyde-6-(diethylamino)benzofuran to the malononitrile derivative is 1:5 - 5:1.

[0010] Furthermore, the molar ratio of the base to 2-formaldehyde-6-(diethylamino)benzofuran is 1:4000 - 1:200.

[0011] Furthermore, the malononitrile derivative is one of malononitrile, 1,3-indanedione, (3,5,5-trimethylcyclohex-2-enylidene)malononitrile, 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile, (E)-2-isocyano-2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)acetonitrile, and (E)-2-(3-(cyano(isocyano)methylene)-2,3-dihydro-1H-inden-1-ylidene)malononitrile.

[0012] Furthermore, the base is at least one of triethylamine, piperidine, diazabicycloundecene, potassium carbonate, sodium hydride, lithium hexamethyldisilazide, and sodium bicarbonate.

[0013] Furthermore, react at a temperature of 0 - 180 °C for 1 - 72 h, concentrate the reaction solution, and separate and purify by column chromatography.

[0014] Furthermore, react at a temperature of 25 °C for 24 h.

[0015] Furthermore, the solvent is at least one of water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, dichloromethane, chloroform, ether, dimethyl sulfoxide, benzene, toluene, xylene, mesitylene, tert-amyl alcohol, 1,4-dioxane, 1,2-dichloroethane, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0016] The present invention also provides the application of the above-mentioned lipid droplet-specific fluorescent probe in cell lipid droplet imaging and / or lipid droplet-specific labeling.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention prepares D-A type fluorescent molecules with push-pull electron characteristics by using a benzofuran fragment as a π-bridge as a power supply group and malononitrile and its derivatives as electron acceptor groups, which exhibit significant Stokes shift, excellent biocompatibility, excellent photostability, and high signal-to-noise ratio, contributing to reducing background fluorescence interference and improving the sensitivity of lipid droplet imaging.

[0019] 2. The fluorescent probe molecule of the present invention has the characteristics of twisted intramolecular charge transfer (TICT), has strong fluorescence in oils and fats, and very weak fluorescence in aqueous solutions, which is beneficial to obtaining a higher signal-to-noise ratio in biological detection applications.

[0020] 3. The preparation method of the present invention is simple, the raw materials are easily available, the cost-benefit is high, and it is easy to realize industrial production. Description of the Drawings

[0021] Figure 1 1H NMR spectrum of the product obtained in Example 1;

[0022] Figure 2 1H NMR spectrum of the product obtained in Example 2;

[0023] Figure 3 Fluorescence emission spectra of the product obtained in Example 1 in different solvents;

[0024] Figure 4 Fluorescence emission spectra of the product obtained in Example 2 in different solvents;

[0025] Figure 5 Fluorescence emission spectra of the product obtained in Example 1 in water and sunflower oil;

[0026] Figure 6 Fluorescence emission spectra of the product obtained in Example 2 in water and sunflower oil;

[0027] Figure 7 Laser confocal images of the product obtained in Example 1 for imaging lipids in cells;

[0028] Figure 8 Laser confocal images of the product obtained in Example 2 for imaging lipids in cells. Detailed Embodiments

[0029] The principles and characteristics of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] Example 1

[0031] A lipid droplet-specific fluorescent probe, and its preparation method comprises the following steps:

[0032] At room temperature, 2-formyl-6-(diethylamino)benzofuran (0.50 g, 2.30 mmol) and malononitrile (0.15 g, 2.30 mmol) were dissolved in 20 mL of dichloromethane (DCM), one drop of triethylamine was added dropwise, and the reaction was carried out under stirring for 24 h. The reaction solution was concentrated, and purified by flash column chromatography to obtain a lipid droplet-specific fluorescent probe (0.55 g, yield 90.1%); the 1H NMR spectrum of the obtained product is as Figure 1 shown.

[0033] The reaction process is as follows:

[0034] .

[0035] Example 2

[0036] At room temperature, 2-formyl-6-(diethylamino)benzofuran (0.50 g, 2.30 mmol) and 1,3-indanedione (0.34 g, 2.30 mmol) were dissolved in 20 mL of dichloromethane (DCM), one drop of triethylamine was added dropwise, and the reaction was carried out under stirring for 24 h. The reaction solution was concentrated, and purified by flash column chromatography to obtain a lipid droplet-specific fluorescent probe (0.71 g, yield 89.3%); the 1H NMR spectrum of the obtained product is as Figure 2 shown.

[0037] The reaction process is as follows:

[0038] .

[0039] Test Example 1

[0040] 1. The products obtained in Example 1 and Example 2 were respectively dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, and then diluted with different solvents to a 10 μM test solution for fluorescence testing. The results are respectively as Figure 3 and Figure 4 shown.

[0041] From Figure 3 and Figure 4 it can be seen that as the polarity of the solvent increases, the maximum emission wavelength of the fluorescent probe of the present invention shifts to a longer wavelength (i.e., the red end) in solvents with different polarities; this is probably attributed to the twisted intramolecular charge transfer (TICT) effect.

[0042] 2. The products obtained in Example 1 and Example 2 were respectively dissolved in dimethyl sulfoxide to prepare a 10 mM stock solution, and then diluted with water or sunflower oil to a 10 μM test solution for fluorescence testing. The results are respectively shown as Figure 5 and Figure 6 shown.

[0043] It can be seen from Figure 5 and Figure 6 that the fluorescent probe of the present invention has almost no fluorescence in aqueous solution, while the fluorescence intensity in sunflower oil increases significantly. This characteristic of fluorescence enhancement only in the oil environment greatly reduces the interference caused by the fluorescent probe itself, which is beneficial to improving the signal-to-noise ratio.

[0044] 3. HeLa cells were inoculated into a glass-bottom dish and then incubated for 2 h under the condition of containing 20 μM oleic acid; then the fluorescent probes obtained in Example 1 and Example 2 were respectively prepared into a 10 mM dimethyl sulfoxide stock solution and diluted with a culture medium to a 5 μM staining solution; the old culture medium was aspirated, the staining solution was added, and stained for 1 h; then the staining solution was aspirated, and further incubated with BODIPY 493 / 503 green fluorescent dye for 30 min to label lipid droplets; the unbound dye was removed by washing three times with PBS, and the lipid droplet targeting imaging ability of the fluorescent probe was observed by confocal laser scanning microscope (CLSM). The results are shown as Figure 7 and Figure 8 shown.

[0045] It can be seen from Figure 7 and Figure 8 that the red fluorescence emitted by the fluorescent probe of the present invention shows good overlap with the green fluorescence of BODIPY493 / 503; this phenomenon fully indicates that the fluorescent probe of the present invention is suitable for the biological imaging research of lipid droplets.

[0046] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A lipid droplet-specific fluorescent probe, characterized in that, Its structural formula is one of the following: 、 。 2. The preparation method of the lipid droplet-specific fluorescent probe according to claim 1, characterized in that, It includes the following steps: Dissolve 2-formaldehyde-6-(diethylamino)benzo[b]furan and malononitrile derivative in a solvent, then react under the action of a base, and successively carry out concentration and purification to obtain a lipid droplet-specific fluorescent probe; The malononitrile derivative is malononitrile or 1,3-indandione.

3. The preparation method of the lipid droplet-specific fluorescent probe according to claim 2, wherein The molar ratio of the 2-formaldehyde-6-(diethylamino)benzo[b]furan to the malononitrile derivative is 1:5 - 5:

1.

4. The preparation method of the lipid droplet-specific fluorescent probe according to claim 2, characterized in that, The molar ratio of the base to the 2-formaldehyde-6-(diethylamino)benzo[b]furan is 1:4000 - 1:

200.

5. The preparation method of the lipid droplet-specific fluorescent probe according to claim 2, wherein The base is at least one of triethylamine, piperidine, diazabicycloundecene, potassium carbonate, sodium hydride, lithium hexamethyldisilazide, and sodium bicarbonate.

6. The preparation method of the lipid droplet-specific fluorescent probe according to claim 2, wherein React at a temperature of 0 - 180 °C for 1 - 72 h, concentrate the reaction solution, and separate and purify by column chromatography.

7. The preparation method of the lipid droplet-specific fluorescent probe according to claim 2, characterized in that, The solvent is at least one of water, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, dichloromethane, chloroform, ether, dimethyl sulfoxide, benzene, toluene, xylene, mesitylene, tert-amyl alcohol, 1,4-dioxane, 1,2-dichloroethane, N,N-dimethylformamide, and N,N-dimethylacetamide.

8. Use of the lipid droplet-specific fluorescent probe according to claim 1 in the preparation of a cell lipid droplet imaging and / or lipid droplet-specific labeling reagent.

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