Probe Cvs-B-NHS based on coumarin derivative as well as preparation method and application of probe Cvs-B-NHS

Abstract

The invention discloses a probe Cvs-B-NHS based on a coumarin derivative as well as a preparation method and application of the probe Cvs-B-NHS. According to the fluorescent probe Cvs-B-NHS of the coumarin derivative, a coumarin mother nucleus and succinimide ester are organically combined, the excellent fluorescence characteristic of coumarin is reserved, efficient protein labeling capacity is achieved, high-sensitivity fluorescence signal output and specific labeling of protein are achieved, and the fluorescent probe Cvs-B-NHS has the advantages of being high in sensitivity and high in sensitivity. And the accuracy and reliability of protein detection and imaging are improved. The invention discloses a TDP-43 protein phase separation labeling method based on a Cvs-B-NHS probe, which provides a new means for researching the action mechanism of a TDP-43 (G335D) mutant in neurodegenerative diseases, and is beneficial to promoting the research progress in the field of neurodegenerative diseases. The application of the Cvs-B-NHS probe in evaluating the polarity change of the microenvironment in the protein liquid-liquid phase separation process in the cytoplasm environment caused by mitochondrial signal disturbance provides a powerful tool for the research of the protein phase separation microenvironment.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent compounds and their preparation and application, and specifically relates to a probe Cvs-B-NHS based on coumarin derivatives, a preparation method thereof and an application. Background Art

[0002] Phase separation of proteins in the cytoplasm is one of the inducements of neurodegenerative diseases. This phase separation process is regulated by the inherent amino acid sequence and affected by the surrounding environment. The phase separation of proteins will form a microenvironment different from the cytoplasmic environment locally, manifested as an increase in microviscosity and a decrease in micro-polarity (inside). The designed fluorescent probe can report the biophysical properties of phase-separated proteins based on the intrinsic optical signal of the molecule. Changes in the cytoplasmic environment may interfere with the protein phase separation process (such as promoting depolymerization or recruiting other molecules), and are accompanied by changes in biophysical properties.

[0003] Coumarin is a class of compounds with excellent optical properties, which are widely used in the fields of fluorescent probes, bioimaging and drug carriers. However, due to the lack of the ability to specifically bind to biomolecules (such as proteins), the application of natural coumarin molecules in biomedicine is limited. Succinimidyl ester is a highly reactive group that can specifically react with amino groups in proteins to form stable amide bonds. Introducing succinimidyl ester into the coumarin nucleus can endow it with the ability to label proteins while retaining the fluorescence characteristics of coumarin.

[0004] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, one of the characteristics of which is the abnormal aggregation and phase separation of TDP-43 protein. The abnormal nucleocytoplasmic distribution of TDP-43 protein is closely related to the pathological process of ALS. Normally, TDP-43 is mainly localized in the nucleus, but in ALS patients, TDP-43 migrates incorrectly to the cytoplasm and forms inclusion bodies. Therefore, studying the intracellular distribution and phase separation characteristics of TDP-43 is of great significance for understanding the pathogenesis of ALS.

[0005] Existing fluorescent probes have problems such as insufficient sensitivity, poor specificity or limited environmental adaptability in labeling proteins and monitoring phase separation behavior. Therefore, developing a new type of fluorescent probe that can efficiently label proteins and monitor their phase separation behavior in real time, while having excellent environmental sensitivity and fluorescence performance, is a current research hotspot and difficulty. Summary of the Invention

[0006] Objective of the Invention: The objective of the present invention is to provide a probe Cvs-B-NHS based on coumarin derivatives to achieve specific labeling of proteins for real-time detection and dynamic imaging of proteins. Meanwhile, a preparation method of the probe and its applications in aspects such as protein phase separation research are provided, so as to provide a powerful tool for protein-related research.

[0007] Technical Solution: The present invention provides a probe Cvs-B-NHS based on coumarin derivatives, and the structural formula of the probe Cvs-B-NHS is shown as follows:

[0008]

[0009] The present invention also provides a preparation method of the above-mentioned probe Cvs-B-NHS based on coumarin derivatives. The preparation method includes: heating 7-diethylamino-4-hydroxy-3-acetylcoumarin and 4-formylbenzoic acid 2,5-dioxopyrrolidin-1-yl ester in an equi-proportion mixed solvent of ethanol and acetonitrile and catalyzing with piperidine. After reflux reaction, column chromatography separation is carried out to obtain compound 4. Compound 4 is dissolved in toluene and heated, boron trifluoride etherate is injected, and after the reaction, it is diluted with petroleum ether to obtain a precipitate, which is purified to obtain Cvs-B-NHS. The structural formula of the compound 4 is:

[0010]

[0011] Further, the 7-diethylamino-4-hydroxy-3-acetylcoumarin is obtained by the following method: reacting diphenyl malonate with 3-hydroxy-N,N-diethylaniline in toluene at 118 °C for 12 h, and after purification, 7-diethylamino-4-hydroxycoumarin is obtained; reacting 7-diethylamino-4-hydroxycoumarin with acetic anhydride in pyridine, after the reaction is completed, pure water is added, and after precipitation, it is purified to obtain 7-diethylamino-4-hydroxy-3-acetylcoumarin.

[0012] Further, the 4-formylbenzoic acid 2,5-dioxopyrrolidin-1-yl ester is obtained by the following method: adding 1-hydroxypyrrolidine-2,5-dione and EDCI to a solution of p-formylbenzoic acid and dichloromethane, stirring overnight in the dark, and after purification, 4-formylbenzoic acid 2,5-dioxopyrrolidin-1-yl ester is obtained.

[0013] The present invention also provides an application of the above-mentioned probe Cvs-B-NHS based on coumarin derivatives in the labeling of TDP-43 protein phase separation.

[0014] Further, the application is to image the TDP-43(G335D)NLS mutant in the cytoplasm.

[0015] The present invention also provides the application of the above-mentioned coumarin derivative-based probe Cvs-B-NHS in polarity detection.

[0016] Furthermore, the application includes detecting the polarity change of the intracellular microenvironment.

[0017] The present invention also provides the application of the above-mentioned coumarin derivative-based probe Cvs-B-NHS in real-time monitoring of the polarity / viscosity change of the protein phase separation microenvironment in the cytoplasmic environment under mitochondrial signal perturbation.

[0018] Furthermore, the concentration of the added probe Cvs-B-NHS is 1-2 μM.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: The fluorescent probe Cvs-B-NHS of the coumarin derivative in the present invention organically combines the coumarin mother nucleus and succinimide ester, not only retaining the excellent fluorescence characteristics of coumarin, but also endowing high-efficiency protein labeling ability, improving the accuracy and reliability of protein detection and imaging. The TDP-43 protein phase separation labeling method based on the Cvs-B-NHS probe provides a new means for studying the mechanism of action of the TDP-43(G335D) mutant in neurodegenerative diseases, and helps to promote the research progress in the field of neurodegenerative diseases. The application of the Cvs-B-NHS probe in evaluating the polarity change of the microenvironment during the protein liquid-liquid phase separation process in the cytoplasmic environment under mitochondrial signal perturbation provides a powerful tool for the research of the protein phase separation microenvironment. Description of the Drawings

[0020] Figure 1 Schematic diagram of the chemical structure and synthesis route of the Cvs-B-NHS probe.

[0021] Figure 2 Intracellular distribution and phase separation characteristics of the TDP-43(G335D) mutant and its NLS deletion mutant.

[0022] Figure 3 Polarity sensitivity of the Cvs-B-NHS probe.

[0023] Figure 4 FLIM imaging results of the polarity change of the protein phase separation microenvironment in the cytoplasmic environment under mitochondrial signal perturbation by the Cvs-B-NHS probe. Detailed Embodiments

[0024] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1 Preparation of the probe molecule Cvs-B-NHS

[0026] 1. Synthesis of Intermediate Compound 1

[0027] First, dissolve diphenyl malonate (6.4 g, 25 mmol) in toluene (25 mL), and gradually add it dropwise to the pre - weighed 3 - hydroxy - N,N - diethylaniline (3.3 g, 20 mmol) while stirring. Keep stirring during the addition, and after thorough mixing, heat the mixture to 118 °C and reflux for 12 h. Stop heating and allow it to cool naturally to room temperature. Filter the reaction mixture by suction. Wash the collected solid with petroleum ether and dichloromethane, and dry it in a vacuum drying oven for 24 h to obtain pure 7 - diethylamino - 4 - hydroxycoumarin, which is Compound 1. (Beige solid powder, yield 94%)

[0028] 2. Synthesis of Intermediate Compound 2

[0029] Weigh 7 - diethylamino - 4 - hydroxycoumarin (2.33 g, 0.01 mo1) and dissolve it thoroughly in pyridine (4 mL). Heat it to 85 °C and maintain the temperature. Then, add acetic anhydride (1.89 mL, 0.02 mol) dropwise and mix evenly. Raise the temperature to 115 °C and react for 2 h. Wait for the reaction solution to cool naturally to room temperature, add pure water (4 mL), and a yellow - black solid will precipitate. Filter the cooled reaction solution by suction, wash the solid with isopropanol, and dry it in a vacuum drying oven for 24 h to obtain pure 7 - diethylamino - 4 - hydroxy - 3 - acetylcoumarin, which is Compound 2. (Light yellow powder, yield 90%)

[0030] 3. Synthesis of Intermediate Compound 3

[0031] Add 1 - hydroxypyrrolidine - 2,5 - dione (760 mg, 6.6 mmol) and EDCI (2.5 g, 13.2 mmol) to a solution of 4 - formylbenzoic acid (500 mg, 3.3 mmol) and CH2Cl2 (20 mL), and stir overnight in the dark. Monitor the completion of the reaction by TLC, and dilute the reaction mixture with distilled water. Extract the reaction mixture with CH2Cl2 (2 x 25 mL), and wash the mixture with brine (1 x 25 mL). Dry the combined organic phases over anhydrous MgSO4, filter to collect the organic phases, and concentrate the organic phases in vacuo. Separate and purify by silica gel gradient column chromatography to obtain 2,5 - dioxopyrrolidin - 1 - yl 4 - formylbenzoate, which is Compound 3. (Yield 93%)

[0032] 4. Synthesis of Intermediate Compound 4

[0033] A mixture of Compound 2 (275 mg, 1 mmol) and Compound 3 (247.21 mg, 1 mmol) was heated to 85 °C in a mixed solvent (ethanol:acetonitrile = 1:1) (6 mL). Then, five drops of piperidine were slowly added to the flask as a catalyst. The reaction mixture was refluxed for 8 h, cooled to room temperature, dried under reduced pressure, and then separated by column chromatography (dichloromethane:petroleum ether = 1:2) to obtain Compound 4, and the product was a reddish-brown solid powder.

[0034] 5. Synthesis of the target compound probe Cvs-B-NHS

[0035] Compound 4 (500 mg, 2 mmol) was dissolved in toluene (6 mL), and then heated to 118 °C. After the temperature stabilized, boron trifluoride etherate (426 mg, 3 mmol) was slowly injected into the flask using an extended syringe, and stirred for 0.5 h. At this time, the reaction mixture was yellow. While it was hot, it was diluted with petroleum ether (12 mL), and a red precipitate quickly precipitated. The precipitated precipitate was filtered by suction and washed with petroleum ether, and then purified by silica gel column and dried in a vacuum drying oven for 24 h to obtain pure Cvs-B-NHS (reddish-brown solid powder, yield 97%). The synthesis route is as Figure 1 shown.

[0036] 6. Analysis and characterization of the probe Cvs-B-NHS:

[0037] 1H NMR (400 MHz, Chloroform-d) δ 8.50 (d, J = 15.6 Hz, 1H), 8.28 (d, J = 15.6 Hz, 1H), 7.97 (d, J = 9.3 Hz, 1H), 7.77 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 6.66 (dd, J = 9.3, 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 3.52 (q, J = 7.1 Hz, 4H), 1.69 (s, 4H), 1.27 (d, J = 7.0 Hz, 6H).

[0038] 13C NMR (101 MHz, Chloroform-d) δ 181.65, 169.17, 159.31, 158.96, 156.08, 139.93, 135.33, 129.96, 129.32, 127.68, 120.98, 110.51, 102.87, 98.42, 96.60, 45.63, 31.44, 29.70, 24.55, 12.53.

[0039] Example 2 Labeling of the phase separation of TDP-43 protein by the probe molecule Cvs-B-NHS

[0040] This experiment aimed to study the distribution and phase separation characteristics of the TDP-43(G335D) mutant and its nuclear localization signal deletion mutant (TDP-43(G335D) NLSmutant ) in cells, and to explore its accessibility using the Cvs-B-NHS labeling probe. In the experiment, TDP-43(G335D)-EGFP and TDP-43(G335D) NLSmutant -EGFP plasmids (constructed by Jiangsu Saisuofei Biotechnology Co., Ltd., and the nucleotide sequences are shown in SEQ ID NO.1) were transfected into HEK293 cells respectively. After culturing for 24 - 48 h, the cells were stained with 2 μM CvS B-NHS probe for 2 h, stained with Hoechst 333342 nuclear dye for 8 - 10 min, and after washing with PBS, a small amount of culture medium was added. It was found by fluorescence microscopy that TDP-43(G335D)-EGFP was mainly distributed in the nucleus and showed obvious phase separation, forming biomolecular condensates; while TDP-43(G335D) NLSmutant -EGFP was mainly distributed in the cytoplasm. The experimental results showed that Cvs-B-NHS could not label TDP-43(G335D)-EGFP in the nucleus, but could effectively label TDP-43(G335D) NLSmutant -EGFP in the cytoplasm, which might be related to the barrier effect of the nuclear membrane and the accessibility of the protein in different regions of the cell( Figure 2 ).

[0041] Example 3 Polarity Sensitivity of Cvs-B-NHS Probe

[0042] Different solvents (TOL (ε = 2.4), THF (ε = 7.6), DCM (ε = 8.9), EtOH (ε = 28.4), MeOH (ε = 33.7) and ACN (ε = 38.8)) were selected to evaluate the polarity sensitivity of the compound. First, the Cvs-B-NHS probe was prepared into a 2 mM stock solution with DMSO, and corresponding amounts of the solution were taken and added to solvents with different polarities to prepare test samples with a final concentration of 10 μM. The fluorescence intensities of each group were recorded using an F-7100 fluorescence and phosphorescence spectrophotometer, and the fluorescence lifetimes were collected using a fluorescence lifetime measurement spectrofluorometer FS5. The polarity-dependent curve was plotted with the logarithm of the solvent dielectric constant as the X-axis and the logarithm of the fluorescence intensity as the Y-axis, and the polarity-dependent parameter (k) was determined according to the slope of the fitted curve. In addition, a polarity-dependent curve was also plotted with the logarithm of the solvent dielectric constant as the X-axis and the logarithm of the average fluorescence lifetime as the Y-axis, and the polarity-dependent parameter (k) was determined according to the slope of the fitted curve( Figure 3)). The results on the left figure show that there are significant differences in fluorescence intensity in different solvents. The fluorescence intensity of TOL (toluene) is the highest, followed by THF (tetrahydrofuran) and DCM (dichloromethane), while the fluorescence intensities of EtOH (ethanol), MeOH (methanol) and ACN (acetonitrile) are lower. This indicates that the polarity of the solvent has an important influence on the fluorescence intensity. Solvents with lower polarity (such as TOL) are more conducive to fluorescence emission, while solvents with higher polarity (such as EtOH and MeOH) may cause fluorescence quenching. The right figure shows the linear relationship between the fluorescence intensity (logarithm) and the solvent dielectric constant (logarithm), and the fitting equation is: y = -1.0098x + 4.0927, (R 2 = 0.9356), indicating a strong correlation between the two. This negative correlation shows that the higher the dielectric constant of the solvent, the lower the fluorescence intensity. This is consistent with the results in the left figure, further supporting the significant influence of solvent polarity on fluorescence behavior.

[0043] Example 4 FLIM imaging of the polarity / viscosity changes in the protein phase separation microenvironment in the cytoplasmic environment under mitochondrial signal perturbation by the Cvs-B-NHS probe

[0044] First, after HEK293 cells were adherently cultured in a culture dish until 70%-80% confluence, the plasmid of TDP-43 (G335D)-NLS mutant (constructed by Jiangsu Saisuofei Biotechnology Co., Ltd.) was transfected into the cells. 24 h after transfection, the cells were treated with drugs respectively: 0.5% H2O2 (1 min), 30 μM CCCP (10 min), 10 μM Rot (10 min), 2 μM Doxycycline (4 h), 200 nM Actinomycin D (6 h), 10 mU / mL Ble (48 h), 100 ng / mL LPS (48 h) and DMSO (solvent control). After the drug treatment was completed, the cells were stained with 2 μM CvS B-NHS probe for 2 h, and then imaged by fluorescence lifetime imaging microscopy (FLIM) to analyze the polarity changes in the protein phase separation microenvironment in the cytoplasmic environment under mitochondrial signal perturbation. The imaging results show ( Figure 4): (A) is the control group; (B) is the mitochondrial function inhibitor group, and the treatments with H2O2, CCCP, and Rot significantly affect mitochondrial function and cytoplasmic polarity; (C) is the transcription inhibitor group, and Doxycycline and Actinomycin D mainly affect the transcription level, with relatively small changes in fluorescence lifetime, indicating that their effects on the polarity of the protein phase separation microenvironment are weak; (D) is the stress factor group, and after treatment with stress factors such as Ble and LPS, the fluorescence lifetime also shows certain changes, further confirming the changes in the polarity of the protein phase separation microenvironment under cellular stress; (E) FLIM imaging reveals that under drug intervention, the cytoplasmic polarity changes are closely related to the expression level and localization of the TDP-43 mutant, indicating that the activation of the cellular stress response and inflammatory signaling pathways has a significant impact on the protein phase separation microenvironment.

Claims

1. A probe Cvs-B-NHS based on coumarin derivatives, characterized in that, The structural formula of the probe Cvs-B-NHS is as follows:

2. The preparation method of the coumarin derivative-based probe Cvs-B-NHS according to claim 1, characterized in that, The preparation method includes: heating 7-diethylamino-4-hydroxy-3-acetylcoumarin and 2,5-dioxopyrrolidin-1-yl 4-formylbenzoate in an equimolar mixed solvent of ethanol and acetonitrile, adding piperidine as a catalyst, refluxing and reacting, and then separating by column chromatography to obtain compound 4. Dissolve compound 4 in toluene and heat it, inject boron trifluoride etherate, dilute with petroleum ether after the reaction to obtain a precipitate, and obtain Cvs-B-NHS after purification; the structural formula of compound 4 is:

3. The preparation method according to claim 2, characterized in that, The 7-diethylamino-4-hydroxy-3-acetylcoumarin is obtained by the following method: refluxing diphenyl malonate and 3-hydroxy-N,N-diethylaniline in toluene at 118 °C for 12 h, and obtaining 7-diethylamino-4-hydroxycoumarin after purification; reacting 7-diethylamino-4-hydroxycoumarin with acetic anhydride in pyridine, adding pure water after the reaction is completed, precipitating, and purifying it to obtain 7-diethylamino-4-hydroxy-3-acetylcoumarin.

4. The preparation method according to claim 2, characterized in that, The 2,5-dioxopyrrolidin-1-yl 4-formylbenzoate is obtained by the following method: adding 1-hydroxypyrrolidine-2,5-dione and EDCI to a solution of p-formylbenzoic acid and dichloromethane, stirring overnight in the dark, and obtaining 2,5-dioxopyrrolidin-1-yl 4-formylbenzoate after purification.

5. Use of the coumarin derivative-based probe Cvs-B-NHS according to claim 1 in the labeling of TDP-43 protein phase separation.

6. The application according to claim 5, wherein The use is to image the TDP-43(G335D)NLS mutant in the cytoplasm.

7. Use of the coumarin derivative-based probe Cvs-B-NHS according to claim 1 in the detection of polarity.

8. The application according to claim 7, characterized in that The use includes detecting the polarity change of the intracellular microenvironment.

9. Use of the coumarin derivative-based probe Cvs-B-NHS according to claim 1 in the real-time monitoring of the polarity / viscosity change of the protein phase separation microenvironment in the cytoplasmic environment under mitochondrial signal perturbation.

10. The application according to any one of claims 5 to 9, characterized in that, The concentration of the probe Cvs-B-NHS added is 1 - 2 μM.

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