Selenobenzothiadiazole-based lipid droplet sensitive fluorescent probe, preparation method and application

Abstract

The invention provides a lipid droplet sensitive fluorescent probe based on seleno-benzothiadiazole as well as a preparation method and application of the lipid droplet sensitive fluorescent probe, and relates to the field of small organic molecule fluorescent probes. The preparation method of the seleno-benzothiadiazole-based lipid droplet sensitive fluorescent probe comprises the following steps: preparing a compound b, preparing a compound 1b, and preparing the lipid droplet sensitive fluorescent probe. The lipid droplet sensitive fluorescent probe based on selenobenzothiadiazole can perform precise dyeing and high-fidelity tracking on LD in cells, improve the selectivity and the cell imaging effect of the LD and reduce the cytotoxicity of the LD; in HepG2 cell imaging experiments, the cell toxicity is low, and cells can be accurately positioned.

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] Organic functional materials are increasingly being used. Among them, the p-series of polycyclic aromatic or heteroaromatic compounds such as 2,1,3-benzothiadiazole and 2,1,3-benzoselenadiazole are being actively studied as cheap alternatives to traditional inorganic semiconductors in the development of organic light-emitting diodes (OLEDs), organic photovoltaics (OPVs), and bioprobes for bioimaging analysis. There are many different methods for using fluorescence detection, among which fluorescent probes are one of the most widely used methods. Fluorescent probes have unique advantages such as high sensitivity, spatiotemporal resolution, and low damage to biological samples, and 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 and surrounded by a phospholipid monolayer. Studies have found that LDs play a key role in cellular lipid storage and supply. However, abnormal levels of cellular LDs are closely related to many metabolic diseases. For example, non-alcoholic fatty liver disease is often accompanied by LD overexpression, in which LDs typically show an increase in number and size.

[0004] The prior art discloses benzothiadiazole fluorescent probes for lipid droplet staining and cell tracking. However, the existing benzothiadiazole fluorescent probes have the problems of inaccurate staining, low tracking fidelity and high cytotoxicity. At the same time, the existing benzothiadiazole fluorescent probes also have the problems of low selectivity and poor cell imaging effect.

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

[0006] In order 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 its selectivity and cell imaging effect and reducing its cytotoxicity; in the HepG2 cell imaging experiment, the cytotoxicity is low and the cells can be accurately located.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole has the following molecular structure: .

[0008] A method for preparing a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, comprising 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 structure: ; The method for preparing compound 1b is to put compound b into ethanol, heat to reflux, and obtain a reaction mixture; then put selenium dioxide aqueous solution into the reaction mixture, heat to reflux for reaction, and obtain compound 1b; The compound 1b has the following molecular structure: ; The method for preparing a 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 reaction to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole; The styrene derivative a has the following molecular structure: .

[0009] 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.

[0010] Preferably, in the preparation of compound b, the reaction time at room temperature is not less than 12 h.

[0011] Preferably, in the preparation of compound 1b, the molar amount of selenium dioxide is at least 5 times that of compound b.

[0012] Preferably, in the preparation of compound 1b, the heating reflux reaction temperature is not less than 80° C., and the heating reflux reaction is for not less than 16 h.

[0013] 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.

[0014] 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.

[0015] Furthermore, in the preparation of the lipid droplet-sensitive fluorescent probe, the catalyst is Pd(OAc)2; The molar percentage of the catalyst used is at least 20% of the total molar amount of the compound 1b and the styrene derivative a.

[0016] Furthermore, the preparation method of the lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole has a specific synthesis route as follows:

[0017] Furthermore, the method for preparing compound b is, under low temperature conditions, adding sodium borohydride to a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole, mixing evenly, reacting at room temperature, cooling to 0°C, adding distilled water and mixing evenly, desolventizing under reduced pressure, diluting the residue with ethyl acetate, washing with brine, drying the organic layer, and desolventizing under reduced pressure to obtain compound b.

[0018] Furthermore, the method for preparing compound 1b is to add compound b into ethanol, heat to reflux, and obtain a reaction mixture; then add a selenium dioxide aqueous solution into the reaction mixture, heat to reflux for reaction, cool to room temperature, and separate to obtain a precipitate; the precipitate is washed with water and then dried to obtain compound 1b; Furthermore, the method for preparing the lipid droplet-sensitive fluorescent probe is that 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, it is purified by a silica gel column to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole.

[0019] An application of the aforementioned lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole in lipid droplet detection and / or cell tracking in biological cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention adjusts the lipophilicity and targeting of the fluorescent probe by chemically modifying the benzothiadiazole skeleton and combining functional 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 is low and the cells can be accurately located.

[0021] (2) The method for preparing the lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has a wide source of raw materials, simple synthesis steps, low production cost, and is conducive to large-scale production.

[0022] (3) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has a good response effect to lipid droplets, enters cells quickly, and has good stability.

[0023] (4) The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole of the present invention has low cytotoxicity in cell imaging experiments and is capable of imaging lipid droplets in cells.

[0024] (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

[0025] Figure 1 The fluorescent probe BDS-LDs prepared in Example 1 of the present invention 1 HNMR spectrum.

[0026] Figure 2 The UV absorption spectra of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention in different solvents.

[0027] 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.

[0028] Figure 4 This is a selectivity test diagram of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention.

[0029] Figure 5 This is a time stability test chart of the fluorescent probe BDS-LDs prepared in Example 1 of the present invention.

[0030] 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.

[0031] Figure 7 This is the MTT analysis chart of the probe BDS-LDs prepared in Example 1 of the present invention.

[0032] Figure 8 This is a lipid droplet co-localization 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 stained with BODIPY (500nM) (λex=405nm, λem=500-550nm); (c) is the red channel stained with BDS-LDs (10μM) (λex=561nm, λem=570-620nm); (d) is the merged image of (b) and (c); (e) is the fluorescence co-localization curve of (b) and (c). DETAILED DESCRIPTION

[0033] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0034] 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.

[0035] Example 1 This embodiment provides a lipid droplet-sensitive fluorescent probe (BDS-LDs) based on selenobenzothiadiazole, which has the following molecular structure: .

[0036] This example also provides a method for preparing the aforementioned lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole, and the specific synthesis route is as follows: .

[0037] The specific steps are as follows: 1. Preparation of compound b At 0°C, sodium borohydride (14.40 g, 380.6 mmol) was added to a solution of 4,7-dibromobenzo-[c][1,2,5]thiadiazole (6.00 g, 20.4 mmol) in ethanol (250 mL) in three batches under stirring conditions, and the stirring time after each batch of sodium borohydride was 3 min; 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; then cooled to 0°C, and the reaction mixture was treated with distilled water (100 mL) (the specific operation was to add 0°C distilled water to the above 0°C reaction mixture and stir evenly); 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), and then washed with brine (200 mL), and the organic layer was dried over anhydrous Na2SO4; the solvent was removed under reduced pressure to obtain a yellow solid compound b (5.38 g, yield 99%).

[0038] 2. Preparation of compound 1b 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 evenly in boiling water (20 mL) and then added to the reaction mixture within 5 min. After complete addition, a yellow precipitate began to form. The reaction was maintained at a 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 target product, which was a mustard yellow solid compound 1b (0.19 g, yield 85%), which was used without further purification.

[0039] 3. Preparation of lipid droplet-sensitive fluorescent probes 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) were added to a Schlenk tube (25 mL) containing compound 1b (68.2 mg, 0.2 mmol). The reaction mixture was then stirred at 100 °C for 4 h under a N2 atmosphere. A mixture of petroleum ether / ethyl acetate (9:1 by volume) was used as the eluent and purified by silica gel chromatography to obtain the lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole.

[0040] The lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole 1 HNMR spectrum Figure 1 shown.

[0041] The molecular formula of the lipid droplet-sensitive fluorescent probe BDS-LDs based on selenobenzothiadiazole is: C30H 34 N4Se.

[0042] 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.

[0043] Example 2 Spectral property test of lipid droplet-sensitive fluorescent probe BDS-LDs: First, the fluorescent probe BDS-LDs was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mmol / L. By adding 20 μL of the stock solution to 2.0 mL of solvents of different polarities, a probe with a concentration of 10 μmol / L was obtained. 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 above-mentioned polar solvents, which means that the dipole moment of the fluorescent probe BDS-LDs in the ground state was less affected by different solvents.

[0044] 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 in 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 in the figure, the maximum emission wavelength of the fluorescent probe BDS-LDs red-shifts as the polarity of the microenvironment increases; and as the lipid solubility of the microenvironment increases, the fluorescence intensity of the probe increases significantly. The above data show that the fluorescent probe BDS-LDs has a good response effect on lipid droplets.

[0045] Example 3 Anti-interference and stability test of lipid droplet-sensitive fluorescent probe BDS-LDs: 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 4 As shown in the figure, when different interfering solutions were added, the fluorescence intensity of the fluorescent probe remained almost unchanged; when 1,4-dioxane was added, the fluorescence intensity was significantly enhanced. The above results show that the fluorescent probe BDS-LDs has good selectivity; it also verifies from the side that the fluorescence intensity of the fluorescent probe BDS-LDs increases with the increase of lipid solubility in the microenvironment.

[0046] 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.

[0047] Example 4 Viscosity response test of lipid droplet-sensitive fluorescent probe BDS-LDs: 20 mL of the probe stock solution prepared in Example 2 was taken and added to 2 mL of a glycerol / methanol mixed solvent of 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 (the volume percentage of glycerol increases from 0% to 100%), the fluorescence intensity of the fluorescent probe BDS-LDs does not change significantly, indicating that the fluorescent probe BDS-LDs has almost no response to viscosity. This also shows that the fluorescent probe BDS-LDs can only detect the polarity of the microenvironment, indicating that the probe has excellent specificity.

[0048] Example 5 Biological toxicity detection of lipid droplet-sensitive fluorescent probe BDS-LDs: 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 fluorescent probe BDS-LDs (0 μM, 2 μM, 5 μM, 10 μM, 15 μM and 20 μM) for 24 h.

[0049] 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 analysis of fluorescent probe BDS-LDs in HepG2 cells showed a survival rate of more than 85%, indicating that fluorescent probe BDS-LDs can be used as a practical tool for labeling lipid droplets and lysosomes in complex biological environments.

[0050] Example 6 Lipid droplet co-localization imaging test of lipid droplet-sensitive fluorescent probe BDS-LDs: The lipid droplet-sensitive fluorescent probe BDS-LDs was used to perform cell co-localization imaging of lipid droplets. Specifically, HepG2 cells, BODIPY (500 nM) and fluorescent probe BDS-LDs (10 μΜ) were incubated for 20 min, and then the cells were washed three times with sterile PBS buffer, and then cell co-localization imaging was performed. Figure 8 As shown in c, there is obvious red fluorescence on the cell lipid droplets (λex=561nm, λem=570-620nm), while Figure 8In 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 basically overlapped, and the Pearson coefficient is as high as 98%. All the above characteristics indicate that the lipid droplet-sensitive fluorescent probe BDS-LDs can be used for cell lipid droplet labeling.

[0051] Unless otherwise specified, all percentages used in the present invention are by mass.

[0052] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, 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-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, 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 structure: ； The method for preparing compound 1b is to put compound b into ethanol, heat to reflux, and obtain a reaction mixture; then put selenium dioxide aqueous solution into the reaction mixture, heat to reflux for reaction, and obtain compound 1b; The compound 1b has the following molecular structure: ； The method for preparing a 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 reaction to obtain a lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole; The styrene derivative a has the following molecular structure: 。 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, characterized in that: 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 less 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 used is at least 20% of the total molar amount of the compound 1b and the styrene derivative a.

10. A use of the lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole as claimed in claim 1, characterized in that: The lipid droplet-sensitive fluorescent probe based on selenobenzothiadiazole is used in lipid droplet detection and / or cell tracking in biological cells.

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