A lysosomal lipid droplet-sensitive fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the technical field of organic small molecule fluorescent probes, and specifically relates to a lysosomal lipid droplet-sensitive fluorescent probe and its preparation method and application. The probe can simultaneously target lipid droplets and lysosomes. In the HeLa cell imaging experiment, the cytotoxicity is low, the cells can be accurately located, and the lipid droplets and lysosomes in the cells can be imaged simultaneously. The present invention is a fluorescent probe with low cost, high sensitivity, small sample dosage, and good targeting effect on lipid droplets and lysosomes.

Description

Technical Field

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

[0002] Lipid droplets (LDs) and lysosomes are important organelles in cells. LDs are the most lipid-soluble subcellular organelles in cells and play important roles in physiological processes such as energy storage, protein degradation, membrane synthesis and transport, lipid metabolism regulation, and signal transduction. However, the imbalance of LDs may lead to a series of diseases, such as diabetes, fatty liver, hyperlipidemia, atherosclerosis, and even Alzheimer's disease. Lysosomes can degrade and recycle exogenous and endogenous biomolecules in living cells and can consume lipid droplets to provide energy for cells. Abnormalities in lysosomes can lead to inflammation and diseases closely related to tumors. Therefore, the ability of a single fluorescent probe to detect LDs and lysosomes in living cells is of great significance for studying the interaction between lipid droplets and lysosomes.

[0003] Currently, there are many methods using fluorescence detection, among which fluorescent probes are one of the most widely used methods. Fluorescent probes have unique advantages, such as high sensitivity, excellent 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. In the past few decades, fluorescent probes for detecting LDs or lysosomes have emerged in an endless stream. Therefore, it is of great significance to be able to image two subcellular compartments in living cells with a single fluorescent probe. Summary of the invention

[0004] The present invention designs and synthesizes a lysosomal lipid droplet-sensitive fluorescent probe, which can simultaneously target lipid droplets and lysosomes. In the HeLa cell imaging experiment, the cytotoxicity is low and the cells can be accurately located.

[0005] The present invention also provides a method for simply and efficiently preparing a lysosomal lipid droplet-sensitive fluorescent probe.

[0006] The present invention also provides an application of the fluorescent probe.

[0007] The present invention adopts the following technical scheme to synthesize an organic small molecule fluorescent probe that can simultaneously target lysosomes and lipid droplets.

[0008] The molecular structure of the fluorescent probe synthesized by the present invention is as follows:

[0009]

[0010] The synthetic route of the probe BTD-Lyso is as follows:

[0011]

[0012] Compound 1 (51.8 mg, 0.2 mmol), compound 2 (75.7 mg, 0.4 mmol), Pd(OAc)2 (9.0 mg, 0.04 mmol), K2CO3 (110.6 mg, 0.8 mmol) were dissolved in THF (2.5 mL) and stirred at 100°C for 4 hours under a nitrogen atmosphere. After the reaction, the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v=10 / 1) to obtain a reddish brown solid BTD-Lyso (53.7 mg, 73%) 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=8.3Hz,1H),8.19(d,J=16.1Hz,1H),8.02(d,J=8.1H z,1H),7.78–7.58(m,3H),7.03(d,J=7.9Hz,2H),3.76(s,4H),3.20(d,J=43.1Hz,4H). 13 C NMR (400MHz, DMSO-d6) δ152.3,138.7,138.5,137.2,136.9,136.7,129.4,127.1,123.6,119.7,115.0,115.0,95.0,66.4,47.8.HRMS(ESI):calcd for C18H16N4O3S[M+H] + 396.1016, found 369.1010.

[0013] The purpose of the lysosomal lipid droplet-sensitive fluorescent probe of the present invention is: the fluorescent probe can be used for the detection of lipid droplets and lysosomes in complex biological cells at the same time; the probe is subjected to fluorescence spectrum testing and cell imaging;

[0014] The present invention adopts the above technical solution to have the following advantages:

[0015] (1) The synthesis steps of the probe are simple and the raw materials are widely available;

[0016] (2) The probe responds well to lipid droplets, enters cells quickly, and has good stability.

[0017] (3) In cell imaging experiments, it has low cytotoxicity and can simultaneously image lipid droplets and lysosomes in cells.

[0018] (4) Therefore, the present invention is a fluorescent probe with low cost, high sensitivity, small sample usage, and good targeting effect on lipid droplets and lysosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 For the probe BTD-Lyso 1 H NMR spectrum;

[0020] Figure 2 For the probe BTD-Lyso 13 C NMR spectrum;

[0021] Figure 3 This is the UV absorption spectrum of the probe BTD-Lyso in different solvents;

[0022] Figure 4 This is the fluorescence emission spectrum of the probe BTD-Lyso in different ratios of Dioxane and MeOH;

[0023] Figure 5 for Figure 4 Normalized spectrum of ;

[0024] Figure 6 This is the MTT result diagram of the probe BTD-Lyso;

[0025] Figure 7 This is the fluorescence dynamics diagram of the probe BTD-Lyso in cells;

[0026] Figure 8 This is a lipid droplet co-localization experiment of the probe BTD-Lyso; a) Bright field of HeLa cells; b) BODIPY (500nM) stained green channel (λ ex =405nm,λ em =470-500nm); c) BTD-Lyso (10μM) staining red channel (λ ex =496nm,λ em =700-800nm); d) is the merged image of (b) and (c); e) is the fluorescence co-localization curve of b) and c);

[0027] Fig. 9 Lysosomal co-localization experiment of probe BTD-Lyso a) Bright field of HeLa cells; b) Lyso-TrackerGreen (50nM) stained green channel (λ ex =405nm,λ em =500-530nm); c) BTD-Lyso (10μM) staining red channel (λ ex =496nm,λ em =700-800nm); d) is the merged image of (b) and (c); e) is the fluorescence co-localization curve of b) and c). DETAILED DESCRIPTION

[0028] The present invention is further described below with reference to the examples and drawings, wherein BTD-Lyso represents a fluorescent probe.

[0029] Example 1

[0030] The synthetic route of the probe BTD-Lyso is as follows:

[0031]

[0032] Compound 1 (51.8 mg, 0.2 mmol), compound 2 (75.7 mg, 0.4 mmol), Pd(OAc)2 (9.0 mg, 0.04 mmol), K2CO3 (110.6 mg, 0.8 mmol) were dissolved in THF (2.5 mL) and stirred at 100°C for 4 hours under a nitrogen atmosphere. After the reaction, the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v=10 / 1) to obtain a reddish brown solid BTD-Lyso (53.7 mg, 73%) 1 H NMR (400MHz, DMSO-d6) δ8.70(d,J=8.3Hz,1H),8.19(d,J=16.1Hz,1H),8.02(d,J=8.1H z,1H),7.78–7.58(m,3H),7.03(d,J=7.9Hz,2H),3.76(s,4H),3.20(d,J=43.1Hz,4H). 13 C NMR (400MHz, DMSO-d6) δ152.3,138.7,138.5,137.2,136.9,136.7,129.4,127.1,123.6,119.7,115.0,115.0,95.0,66.4,47.8.HRMS(ESI):calcd for C 18 H 16 N4O3S[M+H] + 396.1016, found 369.1010.

[0033] Probe 1 H NMR spectrum see Figure 1 , 13 C NMR spectrum see Figure 2 .

[0034] The molecular formula of the probe BTD-Lyso is: C 18 H 16 N4O3S.

[0035] Example 2

[0036] The fluorescent probe BTD-Lyso was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. The stock solution was diluted with common organic solvents and PBS to a 10 μM working solution for UV absorption testing. Figure 3 As shown, it is found that its absorption peak is around 500 nm, and the maximum absorption wavelength has a slight change in the above-mentioned polar solvents, which means that the dipole moment is less affected by different solvents in the ground state.

[0037] Example 3

[0038] The fluorescent probe BTD-Lyso was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. The stock solution was diluted with different proportions of dioxane and methanol to form a 10 μM working solution for fluorescence testing. The fluorescence emission intensity was tested at an excitation wavelength of 500 nm. Figure 4 As shown in Figure 5, the probe shows a certain fluorescence intensity in the wavelength range of 700-800nm. As the proportion of Dioxane in the solvent increases, the polarity of the solvent decreases and the fluorescence intensity of the probe increases. The above data show that the probe has a good response effect on lipid droplets.

[0039] Example 4

[0040] Before cell imaging, we need to conduct a cytotoxicity test on BTD-Lyso. The standard MTT colorimetric method is used to detect the cytotoxicity of the probe BTD-Lyso to HeLa cells in vitro. First, 2×10 5 Cells at 100 cells / mL were seeded in a 96-well plate and then treated with different concentrations of BTD-Lyso (0, 2, 5, 10, 15, and 20 μM) for 24 hours. Subsequently, 10 μL of MTT (5 mg / mL) was added to each well and incubated for another 3 hours. Finally, the culture medium was removed and 100 μL of DMSO was added to dissolve the formazan crystals. The plate was gently shaken for about 10 minutes and the absorbance at 570 nm was measured using a microplate reader. Figure 6 As shown, MTT assay of BTD-Lyso in HeLa cells showed a viability of over 80%, indicating that this probe can serve as a practical tool for lipid droplet and lysosome labeling in complex biological environments.

[0041] Example 5

[0042] The kinetics of BTD-Lyso in cells. The specific operation is as follows: BTD-Lyso (10 μM) is added to a confocal culture dish containing HeLa cells, and the cells are imaged every 5 minutes starting from the addition of BTD-Lyso. Figure 7As shown in the figure, it was found that at 5 minutes, the cells had a more obvious color. At 25 minutes, the fluorescence intensity reached the maximum value, and then the fluorescence intensity remained almost unchanged. This shows that BTD-Lyso can be imaged quickly in cell imaging and has good stability.

[0043] Example 6

[0044] BTD-Lyso performs cell co-localization imaging of lipid droplets. The specific operation is as follows: HeLa cells, BODIPY (500nM) and BTD-Lyso (10μΜ) are incubated for 20 minutes, and then the cells are washed three times with sterile PBS buffer, and then cell co-localization imaging is performed. Figure 8 As shown, Figure 8 As shown in c, there is obvious red fluorescence (λ ex =496nm,λ em =700-800nm), and Figure 8 In b, fluorescence images were collected by the commercial organic dye BODIPY (λ ex =405nm,λ em =470-500nm), showing obvious green fluorescence. In addition, the co-localization map shows that the green and red maps are basically overlapped, and the Pearson coefficient is as high as 86%. All the above characteristics indicate that the probe BTD-Lyso can be used for cell LDs labeling.

[0045] Example 7

[0046] BTD-Lyso performs cell co-localization imaging of lysosomes. The specific operation is as follows: HeLa cells, Lyso-Tracker Green (50nM) and BTD-Lyso (10μΜ) are incubated for 20 minutes, and then the cells are washed three times with sterile PBS buffer, and then cell co-localization imaging is performed. Fig. 9 As shown, Fig. 9 As shown in c, there is obvious red fluorescence (λ ex =496nm,λ em =700-800nm), and Fig. 9 In b, fluorescence imaging was collected by the commercial organic dye Lyso-TrackerGreen, showing obvious green fluorescence (λ ex =405nm,λ em =500-530nm). In addition, the colocalization map shows that the green and red maps are basically overlapped, and the Pearson coefficient is as high as 83%. All the above characteristics indicate that the probe BTD-Lyso can be used for cell lysosome labeling.

[0047] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A lysosomal lipid droplet-sensitive fluorescent probe BTD-Lyso, characterized in that: The fluorescent probe BTD-Lyso has a structure shown in Formula I; , Formula I.

2. The method for preparing the fluorescent probe BTD-Lyso according to claim 1, characterized in that: The method comprises the following steps: dissolving compound 1, compound 2, Pd(OAc)2 and K2CO3 in THF, stirring at 100°C for 4 hours under a nitrogen atmosphere, and after the reaction, purifying the crude product by silica gel column chromatography to obtain the fluorescent probe BTD-Lyso; Compound 1 has the structure shown in Formula II: , Formula II; Compound 2 has the structure shown in Formula III: , Formula III.

3. A sensing element for lipid droplets and lysosomes in a biological cell system, characterized in that: It comprises the fluorescent probe BTD-Lyso as described in claim 1.

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