A lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, preparation method and application thereof

Abstract

The present invention provides a selenobenzothiadiazole-based lipid droplet-sensitive fluorescent probe, a preparation method, and applications thereof, relating to the field of organic small molecule fluorescent probes. The method for preparing the selenobenzothiadiazole-based lipid droplet-sensitive fluorescent probe comprises the following steps: preparing compound b, preparing compound 1b, and preparing the lipid droplet-sensitive fluorescent probe. The selenobenzothiadiazole-based lipid droplet-sensitive fluorescent probe of the present invention can accurately stain and track intracellular LDs with high fidelity, while improving its selectivity and cell imaging effect and reducing its cytotoxicity. In HepG2 cell imaging experiments, the probe exhibits low cytotoxicity and can accurately locate cells.

Description

Technical Field

[0001] The present invention relates to the field of organic small molecule fluorescent probes, and in particular to a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, a preparation method and an application thereof. Background Art

[0002] The applications of organic functional materials are becoming increasingly widespread. Among them, p-type polycyclic aromatic or heteroaromatic compounds, such as 2,1,3-benzothiadiazole and 2,1,3-benzoselenadiazole, are being actively studied as inexpensive alternatives to traditional inorganic semiconductors in the development of organic light-emitting diodes (OLEDs), organic photovoltaics (OPVs), and bioprobes for bioimaging and analysis. Fluorescence detection methods are diverse, with fluorescent probes being one of the most widely used. Fluorescent probes offer unique advantages, such as high sensitivity, spatiotemporal resolution, and minimal damage to biological samples. They have been widely developed for in situ imaging of living cells and detection of various active molecules.

[0003] Lipid droplets (LDs) are lipid-rich subcellular organelles composed of a neutral lipid core surrounded by a phospholipid monolayer. Research has found that LDs play a key role in cellular lipid storage and supply. However, abnormal levels of cellular LDs are closely associated with many metabolic diseases. For example, nonalcoholic fatty liver disease is often accompanied by LD overexpression, with LDs typically increasing in number and size.

[0004] Prior art discloses benzothiadiazole fluorescent probes for lipid droplet staining and cell tracking. However, these probes suffer from inaccurate staining, low tracking fidelity, and high cytotoxicity. Furthermore, these probes also suffer from low selectivity and poor cell imaging.

[0005] Therefore, there is an urgent need to develop a fluorescent probe that can accurately stain and track intracellular LDs with high fidelity, thereby directly gaining insight into the pathological processes associated with abnormal lipid metabolism, which has important technical significance and research value. Summary of the Invention

[0006] To solve the technical problems existing in the prior art, the present invention provides a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, a preparation method, and an application thereof, which can accurately stain and track intracellular LDs with high fidelity while improving their selectivity and cell imaging effects and reducing their cytotoxicity. In HepG2 cell imaging experiments, the probe has low cytotoxicity and can accurately locate cells.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole has the following molecular structure:

[0009] .

[0010] A method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole comprises the following steps: preparing compound b, preparing compound 1b, and preparing a lipid droplet-sensitive fluorescent probe;

[0011] The method for preparing compound b is to add sodium borohydride to a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole, and react at room temperature to obtain compound b;

[0012] The compound b has the following molecular structural formula:

[0013] ;

[0014] The method for preparing compound 1b is as follows: adding compound b to ethanol, heating to reflux, to obtain a reaction mixture; then adding a selenium dioxide aqueous solution to the reaction mixture, heating to reflux for reaction, to obtain compound 1b;

[0015] The compound 1b has the following molecular structure:

[0016] ;

[0017] The method for preparing the lipid droplet-sensitive fluorescent probe comprises contacting compound 1b with a styrene derivative a in a solvent environment under the protection of an inert gas atmosphere in the presence of a catalyst and an acid-binding agent, and heating the resulting mixture to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole.

[0018] The styrene derivative a has the following molecular structural formula:

[0019] .

[0020] Preferably, in the preparation of compound b, the molar amount of sodium borohydride is at least 18 times that of 4,7-dibromobenzo-[c][1,2,5]thiadiazole.

[0021] Preferably, in the preparation of compound b, the reaction time at room temperature is not less than 12 h.

[0022] Preferably, in the preparation of compound 1b, the molar amount of selenium dioxide is at least 5 times that of compound b.

[0023] Preferably, in the preparation of compound 1b, the heating reflux reaction temperature is not lower than 80° C., and the heating reflux reaction is carried out for not less than 16 hours.

[0024] Preferably, in the preparation of the lipid droplet-sensitive fluorescent probe, the molar amount of the styrene derivative a is at least twice that of compound 1b.

[0025] Preferably, in the preparation of the lipid droplet-sensitive fluorescent probe, the heating reaction temperature is not lower than 100° C., and the heating reaction time is not less than 4 hours.

[0026] Furthermore, in the preparation of the lipid droplet-sensitive fluorescent probe, the catalyst is Pd(OAc)2;

[0027] The molar percentage of the catalyst is at least 20% of the total molar amount of the compound 1b and the styrene derivative a.

[0028] Furthermore, the preparation method of the lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole has the following specific synthetic route:

[0029]

[0030] Furthermore, the method for preparing compound b is as follows: under low temperature conditions, sodium borohydride is added to a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole, mixed evenly, reacted at room temperature, cooled to 0°C, added with distilled water and mixed evenly, desolvated under reduced pressure, and the residue is diluted with ethyl acetate, washed with brine, and the organic layer is dried and desolvated under reduced pressure to obtain compound b.

[0031] Furthermore, the method for preparing compound 1b is as follows: adding compound b to ethanol, heating to reflux, and obtaining a reaction mixture; then adding a selenium dioxide aqueous solution to the reaction mixture, heating to reflux for reaction, cooling to room temperature, and separating to obtain a precipitate; the precipitate is washed with water and then dried to obtain compound 1b;

[0032] Furthermore, the method for preparing a lipid droplet-sensitive fluorescent probe is as follows: in a solvent environment, under the protection of an inert gas atmosphere, in the presence of a catalyst and an acid binder, compound 1b is contacted with a styrene derivative a, after heating reaction, and then purified by silica gel chromatography to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole.

[0033] An application of the aforementioned lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole in lipid droplet detection and / or cell tracking in biological cells.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention regulates the lipophilicity and targeting of the fluorescent probe by chemically modifying the benzothiadiazole skeleton and combining functional groups, thereby improving the specific binding ability to lipid droplets. It can accurately stain and track intracellular LDs with high fidelity while improving its selectivity and cell imaging effect and reducing its cytotoxicity. In the HepG2 cell imaging experiment, the cytotoxicity was low and the cells could be accurately located.

[0036] (2) The preparation method of the lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has a wide range of raw material sources, simple synthesis steps, low production cost, and is conducive to large-scale production.

[0037] (3) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has a good response to lipid droplets, enters cells quickly, and has good stability.

[0038] (4) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has low cytotoxicity in cell imaging experiments and can image lipid droplets in cells.

[0039] (5) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has the characteristics of low cost, high sensitivity, high selectivity, and good lipid droplet targeting effect, and can be effectively applied to lipid droplet detection and / or cell tracking in biological cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The fluorescent probe BDS-LDs prepared in Example 1 of the present invention 1 HNMR spectrum.

[0041] Figure 2 The UV absorption spectra of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention in different solvents.

[0042] Figure 3 The fluorescence spectra of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention with different ratios of 1,4-dioxane and methanol.

[0043] Figure 4 This is a selectivity test diagram of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention.

[0044] Figure 5 This is a graph showing the temporal stability of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention.

[0045] Figure 6 The fluorescence spectra of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention under different ratios of glycerol and methanol.

[0046] Figure 7 This is the MTT analysis chart of the probe BDS-LDs prepared in Example 1 of the present invention.

[0047] Figure 8 This is a lipid droplet colocalization experiment of the probe BDS-LDs prepared in Example 1 of the present invention; in the figure, (a) is the bright field of HepG2 cells; (b) is the green channel (λex=405nm,λem=500-550nm) stained with BODIPY (500nM); (c) is the red channel (λex=561nm,λem=570-620nm) stained with BDS-LDs (10μM); (d) is the merged image of (b) and (c); and (e) is the fluorescence colocalization curve of (b) and (c). DETAILED DESCRIPTION

[0048] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0049] In each example, BDS-LDs represents a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, and the compound numbers in the examples correspond to the compound numbers in the synthesis route of the above invention.

[0050] Example 1

[0051] This embodiment provides a lipid droplet-sensitive fluorescent probe (BDS-LDs) based on selenobenzothiadiazole, which has the following molecular structure:

[0052] .

[0053] This example also provides a method for preparing the aforementioned lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole. The specific synthesis route is as follows:

[0054] .

[0055] The specific steps are as follows:

[0056] 1. Preparation of compound b

[0057] To a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole (6.00 g, 20.4 mmol) in ethanol (250 mL) was added sodium borohydride (14.40 g, 380.6 mmol) in three batches at 0°C with stirring, with a stirring time of 3 min after each addition of sodium borohydride. After the addition of sodium borohydride was completed, stirring was continued at 0°C for 10 min, and the reaction mixture was stirred at room temperature for 12 h. The mixture was then cooled to 0°C and treated with distilled water (100 mL) (specifically, 0°C distilled water was added to the above 0°C reaction mixture and stirred uniformly). After most of the ethanol and distilled water were removed under reduced pressure, the residue (10 mL) was diluted with ethyl acetate (EtOAc) (200 mL), then washed with brine (200 mL), and the organic layer was dried over anhydrous Na2SO4. The solvent was removed under reduced pressure to obtain compound b (5.38 g, yield 99%) as a yellow solid.

[0058] 2. Preparation of compound 1b

[0059] Compound b (0.16 g, 0.71 mmol) was dissolved in ethanol (40 mL) and heated under reflux for 5 h to obtain a reaction mixture. Selenium dioxide (0.39 g, 3.55 mmol, 5 eq.) was mixed thoroughly in boiling water (20 mL) and added to the reaction mixture over 5 min. A yellow precipitate began to form after complete addition. The reaction was maintained at reflux temperature of 80°C for 16 h and then cooled to room temperature. The precipitate was separated by vacuum filtration, washed with ice-cold water (2 × 10 mL), and dried to obtain the desired product, compound 1b (0.19 g, 85% yield), as a mustard yellow solid, which was used without further purification.

[0060] 3. Preparation of lipid droplet-sensitive fluorescent probes

[0061] To a Schlenk tube (25 mL) containing compound 1b (68.2 mg, 0.2 mmol) were added styrene derivative a (70 mg, 0.4 mmol), Pd(OAc)2 (26.9 mg, 0.12 mmol, 20 mol%), K2CO3 (0.11 g, 0.8 mol), and DMF (2.5 mL). The reaction mixture was then stirred at 100°C under a N2 atmosphere for 4 h. The product was purified by silica gel chromatography using a mixture of petroleum ether / ethyl acetate (9:1 by volume) as the eluent to obtain the lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole.

[0062] The lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole 1 HNMR spectrum Figure 1 shown.

[0063] The molecular formula of the lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole is: C30H 34 N4Se.

[0064] This embodiment also provides the application of the aforementioned lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole in lipid droplet detection and / or cell tracking in biological cells.

[0065] Example 2

[0066] Spectral property test of lipid droplet-sensitive fluorescent probe BDS-LDs:

[0067] First, the fluorescent probe BDS-LDs was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mmol / L. 20 μL of the stock solution was added to 2.0 mL of solvents of different polarities to obtain a probe with a concentration of 10 μmol / L. This probe with a concentration of 10 μmol / L was used in all photophysical experiments, and UV absorption tests were performed in different solvents (toluene, 1,4-dioxane, acetone, tetrahydrofuran, dimethyl sulfoxide, and distilled water). Figure 2 As shown, it was found that the absorption peak of the fluorescent probe BDS-LDs was around 550nm, and the maximum absorption wavelength had a slight change in the solvents of the above polarities, which means that the dipole moment of the fluorescent probe BDS-LDs in the ground state was less affected by different solvents.

[0068] The fluorescent probe BDS-LDs was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mmol / L. The stock solution was diluted with a mixture of 1,4-dioxane and methanol at different ratios to form a working solution with a probe concentration of 10 μmol / L for fluorescence testing. The fluorescence emission intensity was tested at an excitation wavelength of 550 nm. Figure 3 As shown, the maximum emission wavelength of the fluorescent probe BDS-LDs red-shifts with increasing microenvironment polarity, and the fluorescence intensity of the probe significantly increases with increasing microenvironmental lipid solubility. These data demonstrate that the fluorescent probe BDS-LDs has a strong response to lipid droplets.

[0069] Example 3

[0070] Anti-interference and stability test of lipid droplet-sensitive fluorescent probe BDS-LDs:

[0071] Each time, 20 mL of the probe stock solution prepared in Example 2 was taken and added to 2 mL of phosphate buffered saline (PBS), and then 20 mL of different interfering solutions with a concentration of 1 mmol / L were added. Figure 4As shown in the figure, when different interfering solutions were added, the fluorescence intensity of the fluorescent probe remained almost unchanged; however, when 1,4-dioxane was added, the fluorescence intensity was significantly enhanced. These results indicate that the fluorescent probe BDS-LDs has good selectivity and also indirectly confirm that the fluorescence intensity of the fluorescent probe BDS-LDs increases with the lipid solubility of the microenvironment.

[0072] Each time, 20 mL of the probe stock solution prepared in Example 2 was taken and added to 2 mL of 1,4-dioxane and methanol respectively. These two solvents were used to detect the stability of the probe, and the stability of the probe in different polar environments of low polarity and high polarity was determined. Figure 5 As shown in the figure, the probe has good stability in both high polarity (methanol) and low polarity (1,4-dioxane), indicating that the probe can maintain good stability in solvent environments with different polarities.

[0073] Example 4

[0074] Viscosity response test of lipid droplet-sensitive fluorescent probe BDS-LDs:

[0075] 20 mL of the probe stock solution prepared in Example 2 was taken and added to 2 mL of glycerol / methanol mixed solvents with different volume ratios to test the sensitivity of the fluorescent probe BDS-LDs to viscosity detection. Figure 6 As shown in the figure, as the viscosity of the mixed solvent increases (from 0% to 100% glycerol by volume), the fluorescence intensity of the fluorescent probe BDS-LDs does not change significantly, indicating that the fluorescent probe BDS-LDs has little response to viscosity. This also shows that the fluorescent probe BDS-LDs can only detect the polarity of the microenvironment, demonstrating the excellent specificity of the probe.

[0076] Example 5

[0077] Biological toxicity detection of lipid droplet-sensitive fluorescent probe BDS-LDs:

[0078] Before cell imaging, it is necessary to conduct a cytotoxicity test on the fluorescent probe BDS-LDs. The standard MTT colorimetric method is used to detect the cytotoxicity of the fluorescent probe BDS-LDs to HepG2 cells in vitro. Specifically, first, 2×10 5 Cells at a concentration of 10 cells / mL were seeded in a 96-well plate and then treated with different concentrations of the fluorescent probe BDS-LDs (0 μM, 2 μM, 5 μM, 10 μM, 15 μM, and 20 μM) for 24 h.

[0079] Subsequently, 10 μL of MTT (5 mg / mL) was added to each well and incubated for another 3 h. Finally, the medium was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The plate was gently shaken for about 10 min and the absorbance was measured using a microplate reader. Figure 7 MTT assay of HepG2 cells using the fluorescent probe BDS-LDs showed a viability of over 85%, indicating that the fluorescent probe BDS-LDs can be used as a practical tool for labeling lipid droplets and lysosomes in complex biological environments.

[0080] Example 6

[0081] Lipid droplet co-localization imaging test of lipid droplet-sensitive fluorescent probe BDS-LDs:

[0082] The lipid droplet-sensitive fluorescent probe BDS-LDs was used to perform cellular co-localization imaging of lipid droplets. Specifically, HepG2 cells, BODIPY (500nM) and fluorescent probe BDS-LDs (10μM) were incubated for 20min, and then the cells were washed three times with sterile PBS buffer before performing cellular co-localization imaging. Figure 8 As shown in c, there is obvious red fluorescence on the cell lipid droplets (λex=561nm, λem=570-620nm), while Figure 8 In b, fluorescence imaging was collected by the commercial organic dye BODIPY (λex=405nm,λem=500-550nm), showing obvious green fluorescence. Figure 8 The merged image of d and Figure 8 The fluorescence colocalization curve in e shows that the green and red images are essentially overlapping, with a Pearson coefficient as high as 98%. All of the above characteristics indicate that the lipid droplet-sensitive fluorescent probe BDS-LDs can be used for cellular lipid droplet labeling.

[0083] Unless otherwise specified, all percentages used in the present invention are by mass.

[0084] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, characterized in that: It has the following molecular structure: 。 2. A method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 1, characterized in that: The method comprises the following steps: preparing compound b, preparing compound 1b, and preparing a lipid droplet-sensitive fluorescent probe; The method for preparing compound b is to add sodium borohydride to a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole, and react at room temperature to obtain compound b; The compound b has the following molecular structural formula: ； The method for preparing compound 1b is as follows: adding compound b to ethanol, heating to reflux, to obtain a reaction mixture; then adding a selenium dioxide aqueous solution to the reaction mixture, heating to reflux for reaction, to obtain compound 1b; The compound 1b has the following molecular structure: ； The method for preparing the lipid droplet-sensitive fluorescent probe comprises contacting compound 1b with a styrene derivative a in a solvent environment under the protection of an inert gas atmosphere in the presence of a catalyst and an acid-binding agent, and heating the resulting mixture to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole. The styrene derivative a has the following molecular structural formula: 。 3. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of compound b, the molar amount of sodium borohydride is at least 18 times that of 4,7-dibromobenzo-[c][1,2,5]thiadiazole.

4. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, wherein: In the preparation of compound b, the reaction time at room temperature is not less than 12 h.

5. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of compound 1b, the molar amount of selenium dioxide is at least 5 times that of compound b.

6. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of compound 1b, the heating reflux reaction temperature is not lower than 80° C., and the heating reflux reaction is carried out for not less than 16 hours.

7. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of the lipid droplet-sensitive fluorescent probe, the molar amount of the styrene derivative a is at least twice that of compound 1b.

8. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of the lipid droplet-sensitive fluorescent probe, the heating reaction temperature is not lower than 100° C., and the heating reaction time is not less than 4 hours.

9. The method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole according to claim 2, characterized in that: In the preparation of the lipid droplet-sensitive fluorescent probe, the catalyst is Pd(OAc)2; The molar percentage of the catalyst is at least 20% of the total molar amount of the compound 1b and the styrene derivative a.

10. An application of a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, characterized in that: Use of the compound of the molecular structure formula as claimed in claim 1 in the preparation of a lipid droplet-sensitive fluorescent probe for intracellular lipid droplet detection and / or cell tracking in organisms.

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