A cell lipid droplet fluorescent dye and its preparation method and application

Abstract

The present invention discloses a cell lipid droplet fluorescent dye and a preparation method and application thereof, belonging to the field of biochemical analysis technology. The structural formula of the fluorescent dye is shown below. The present invention also discloses the application of the fluorescent dye in specifically labeling lipid droplets in AML-12 cells and super-resolution fluorescence imaging. Experiments have confirmed that the fluorescent dye LD660 of the present invention is a fluorescent dye with superior lipid droplet specificity and photostability, and has great application prospects.

Description

Technical Field

[0001] The invention relates to a cell lipid droplet fluorescent dye and a preparation method and application thereof, belonging to the technical field of biochemical analysis. Background Art

[0002] Lipid droplets are spherical organelles in cells, consisting of a phospholipid monolayer outer membrane and a hydrophobic core composed of neutral lipid substances, and there are many proteins distributed on the surface. Lipid droplets are not a simple fat storage organelle, but a complex and dynamically changing multifunctional organelle. Lipid droplets can interact with other organelles and play an important role in lipid metabolism and storage, membrane transport, protein degradation, and signal transduction. Studies have shown that a variety of metabolic diseases, such as obesity, fatty liver, cardiovascular disease, and diabetes, are accompanied by abnormal lipid metabolism. Therefore, the biological research of lipid droplets has received increasing attention.

[0003] In order to visualize lipid droplets and elucidate their multiple functions, some lipophilic fluorescent dyes have been developed and used to image lipid droplets. Among them, two commercial lipid droplet fluorescent dyes are widely used: Nile Red and BODIPY 493 / 503. However, both dyes have certain shortcomings, for example: the Stokes shift of BODIPY 493 / 503 is relatively small, which will cause cross-overlap of excitation light and emission light; Nile Red staining has low selectivity, and stains lipid droplets while also staining other lipid components in the cell, resulting in a low signal-to-noise ratio. Therefore, it is necessary to develop a fluorescent dye for lipid droplet staining with better targeting and higher signal-to-noise ratio to achieve high-quality lipid droplet imaging. Summary of the invention

[0004] The object of the present invention is to provide a compound represented by formula I.

[0005] The compound of formula I provided by the present invention has the following structural formula:

[0006]

[0007] It is named 6-(4-N,N-dimethylaminostyrene)-2,7-difluoro-3-keto-9-(2-methylphenyl)xanthene, abbreviated as LD660.

[0008] The compound represented by formula I is prepared according to the following synthetic route comprising the following steps:

[0009]

[0010] Compound 1 is mixed with 4-dimethylaminostyrene, and reacted in the presence of a base and a catalyst to obtain a compound represented by formula I.

[0011] In the method, the base may be triethylamine or triethanolamine;

[0012] The catalyst may be at least one of palladium acetate, tetrakistriphenylphosphine palladium, and palladium chloride;

[0013] The molar ratio of compound 1 to 4-dimethylaminostyrene may be 1:1 to 10, specifically 1:1, 1:2 to 8, 1:3 to 9 or 1:5 to 10;

[0014] The molar ratio of compound 1 to the catalyst may be 400:1-10, specifically 400:1, 400:1-2, 400:2-5, 400:3-10, 100:1 or 400:7-9;

[0015] The molar volume ratio of compound 1 to the base may be 1 mmol: 1 to 20 mL, specifically 1 mmol: 1 mL, 1 mmol: 2 mL, 1 mmol: 2 to 15 mL, 1 mmol: 10 to 15 mL or 1 mmol: 15 to 20 mL;

[0016] The reaction temperature of the reaction may be 65 to 130°C, specifically 85°C, 65 to 85°C, 85 to 100°C, 120°C or 95 to 130°C, and the reaction time may be 2 to 12h, specifically 2h, 5h, 5 to 10h, 8 to 12h or 5 to 12h;

[0017] The reaction is carried out in an organic solvent, and the organic solvent may be at least one of dichloromethane, acetonitrile, N,N-dimethylamide and dimethyl sulfoxide;

[0018] The reaction is carried out in an inert atmosphere, which can be provided by nitrogen.

[0019] The use of the compound represented by formula I as a fluorescent dye for cell lipid droplets also falls within the protection scope of the present invention.

[0020] The application may specifically be: the compound shown in formula I is used to specifically label lipid droplets in cells, thereby achieving fluorescence imaging and super-resolution fluorescence imaging of lipid droplets.

[0021] The present invention has the following advantages:

[0022] 1) The dye has a large Stokes shift (141nm), which avoids interference from excitation light during imaging;

[0023] 2) The emission wavelength of the dye is longer (660nm), which can effectively reduce the influence of cell autofluorescence background signal;

[0024] 3) The dye is very sensitive to low-polarity environments and has higher staining selectivity for lipid droplets. The fluorescence quantum yield in polar solvents such as water is less than 1%, while the fluorescence quantum yield in non-polar solvents such as toluene can reach 20%;

[0025] 4) The dye has good photostability and can be used for super-resolution fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The absorption and emission spectra of the fluorescent dye LD660 prepared by the present invention in 1,4-dioxane are shown in Figure 1, where the abscissa is the wavelength, the ordinate is the normalized intensity, and the concentration of the fluorescent dye is 5 μM;

[0027] The solid line on the left is the absorption spectrum, and the dashed line on the right is the emission spectrum.

[0028] Figure 2 This is a co-localization imaging diagram of the fluorescent dye LD660 prepared by the present invention and the lipid droplet fluorescent dye BODIPY 493 / 503 in AML-12 cells.

[0029] Figure 3 This is a super-resolution fluorescence imaging diagram of the fluorescent dye LD660 prepared by the present invention in AML-12 cells. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1. Preparation method of fluorescent dye LD660

[0033] Its synthetic route is as follows:

[0034]

[0035] Compound 1 (134.4 mg, 0.3 mmol) and 4-dimethylaminostyrene (44.1 mg, 0.3 mmol) were dissolved in 30 mL of ultra-dry N, N-dimethylamide, and 0.6 mL of triethanolamine (5.51 mmol) and palladium acetate (0.67 mg, 0.003 mmol) were added to the solution, and the mixture was refluxed at 120° C. for 5 h under nitrogen protection. After the reaction was completed, the solvent was removed by vacuum distillation, and column chromatography was performed with petroleum ether / ethyl acetate as the eluent to obtain a purple solid (32 mg, 23%), which was the fluorescent dye LD660 of the present invention.

[0036] The prepared fluorescent dye LD660 was used in Examples 2, 3 and 4 of the present invention.

[0037] The structure of lipid droplet fluorescent dye LD660 was confirmed as follows:

[0038] 1 H NMR (400MHz, CDCl3): δ7.73–7.70(m,1H),7.54–7.46(m,3H),7.43(d,J=7.0Hz,1H),7.40(d,J=7.5Hz,1H),7.35(d,J=1 6.3Hz,1H),7.17(d,J=7.4Hz,1H),7.06(d,J=16.3Hz,1H),6.73(dd,J=13.0,9.7Hz,3H),6.60(dd,J=13.2,8.8Hz,2H), 3.04(s,6H),2.09(s,3H) . ;

[0039] 13 C NMR (100MHz, CDCl3): δ=176.36,157.46,148.91,136.06,135.66,131.98,130.93,129.99,128.95,128.81,126.46,119.45,119 .37,119.18,119.10,113.00,112.96,112.89,112.63,112.40,109.99,109.77,106.66,106.62,65.57,40.40,30.60,19.63ppm;

[0040] High resolution mass spectrometry: C 30 H 24 F2NO2 + , [M+H] + , the calculated value is 468.1770, and the measured value is 468.1770.

[0041] Example 2: Absorption and emission spectra of LD660 in 1,4-dioxane

[0042] Dissolve LD660 in DMSO solution to prepare a 1mM stock solution. Take 15μL of LD660 stock solution each time and add it to 3mL 1,4-dioxane to prepare a 5μM fluorescent dye LD660 test solution, and perform absorption and fluorescence spectrum tests.

[0043] Figure 1 The absorption and fluorescence spectra of the fluorescent dye LD660 in 1,4-dioxane are shown in Figure 1. The concentration of the fluorescent dye is 5 μM, the absorption wavelength of LD660 in 1,4-dioxane is 519 nm, the emission wavelength is 660 nm, and the Stokes shift is 141 nm.

[0044] Example 3: Co-localization experiment of LD660 and lipid droplet dye BODIPY 493 / 503

[0045] 1) AML-12 cells (purchased from Jiangsu Keygen Biotech Co., Ltd.) were cultured in a glass-bottom culture dish for laser confocal fluorescence microscopy at 37°C and 5% (v / v) CO2 atmosphere for 24 h using a special culture medium for AML-12 cells (purchased from Wuhan Pronocell Life Science Co., Ltd., product catalog number CM-0602) to allow the cells to fully adhere to the bottom of the culture dish.

[0046] 2) After the cell culture in step 1) is completed, the culture medium in the culture dish is aspirated, and then 1 mL of phenol red-free DMEM culture medium (purchased from Wuhan Pronocell Life Science Co., Ltd., product catalog number PM150233A) containing 1 μM LD660 and 500 nM BODIPY 493 / 503 (purchased from Thermo Fisher Scientific, product catalog number D3922) is added, and the culture is continued for 15 minutes.

[0047] 3) After the cultivation in step 2) is completed, the culture medium in the culture dish is aspirated, the cells are washed with 20mM phosphate buffer (pH7.4), and finally 1mL of 20mM phosphate buffer (pH 7.4) is added. The culture dish is placed on a confocal fluorescence microscope for fluorescence imaging. For BODIPY 493 / 503 channel, the excitation light source is a 448nm laser, and the fluorescence signal collection range is 500-550nm; for LD660 channel, the excitation light source is a 448nm laser, and the fluorescence signal collection range is 600-700nm.

[0048] The imaging results are Figure 2As shown, a is the image of BODIPY 493 / 503 in the green channel; b is the image of LD660 in the red channel; c is the superposition of a and b; d is the intensity scatter plot of the two channels; e is the spectral intensity superposition of a and b. We can find that the fluorescent dye LD660 prepared by the present invention can achieve good co-localization with the commercial lipid droplet fluorescent dye BODIPY 493 / 503 in AML-12 cells, indicating that the fluorescent dye LD660 prepared by the present invention has excellent cell lipid droplet targeting.

[0049] Example 4: Three-dimensional super-resolution fluorescence imaging of lipid droplets in cells by LD660

[0050] 1) The steps are the same as step 1) of Example 2

[0051] 2) After the cell culture in step 1) is completed, the culture medium in the culture dish is aspirated, and then 1 mL of phenol red-free DMEM culture medium containing 1 μM LD660 (purchased from Wuhan Pronocell Life Science Technology Co., Ltd., product catalog number PM150233A) is added and culture is continued for 15 minutes.

[0052] 3) After the incubation in step 2) is completed, the culture medium in the culture dish is aspirated, the cells are washed with 20 mM phosphate buffer (pH 7.4), and then 1 mL of 4% paraformaldehyde is added and the incubation is continued for 30 minutes.

[0053] 4) After the incubation in step 3) is completed, the 4% paraformaldehyde in the culture dish is aspirated, the cells are washed with 20mM phosphate buffer (pH 7.4), and finally 1mL of 20mM phosphate buffer (pH 7.4) is added. The culture dish is placed on a confocal fluorescence microscope for fluorescence imaging. The excitation light source is a 448nm laser, and the fluorescence signal collection range is 600-700nm. Leica LAS X software is used to reconstruct the three-dimensional image.

[0054] The imaging results are Figure 3 As shown, A is a confocal imaging image, and B is a stimulated emission depletion (STED) imaging image. The spatial distribution of lipid droplets can be clearly seen from the figure, indicating that the fluorescent dye LD660 of the present invention has excellent photostability and can be used for three-dimensional super-resolution fluorescence imaging of lipid droplets.

[0055] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. The compound shown in formula I has the structural formula shown below: Formula I.

2. A method for preparing the compound of formula I according to claim 1, comprising the steps of: Compound 1 is mixed with 4-dimethylaminostyrene, and reacted in the presence of a base and a catalyst to obtain a compound represented by formula I.

3. The method according to claim 2, characterized in that: In the method, the base is triethylamine or triethanolamine; The catalyst is at least one of palladium acetate, tetrakistriphenylphosphine palladium and palladium chloride.

4. The method according to claim 2 or 3, characterized in that: The molar ratio of compound 1 to 4-dimethylaminostyrene is 1:1-10; The molar ratio of compound 1 to the catalyst is 400:1-10; The molar volume ratio of compound 1 to the base is 1 mmol:1-20 mL.

5. The method according to claim 2, characterized in that: The reaction temperature of the reaction is 65-130° C. and the reaction time is 2-12 h.

6. The method according to claim 2, characterized in that: The reaction is carried out in an organic solvent, and the organic solvent is at least one of dichloromethane, acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide.

7. The method according to claim 2, characterized in that: The reaction is carried out in an inert atmosphere.

8. Use of the compound represented by formula I in claim 1 in the preparation of cell lipid droplet fluorescent dye.

9. The use according to claim 8, characterized in that: The application is: the compound shown in formula I is used to specifically mark lipid droplets in cells to achieve fluorescence imaging and super-resolution fluorescence imaging of lipid droplets.

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