A near-infrared fluorescent probe for detecting acetylcholinesterase in cells and preparation method and use thereof

Abstract

The application discloses a near-infrared fluorescent probe for detecting acetylcholinesterase, a preparation method and application thereof, wherein the structure of the near-infrared fluorescent probe is shown in the following formula: The near-infrared fluorescent probe can produce a 'turn on' type fluorescent response to acetylcholinesterase, the emission wavelength 630 nm is located in the near-infrared region, and the detection limit is as low as 4.8 U / L. The Stokes shift of the probe is as high as 160 nm, the response can be completed within 30 min, and the probe has the advantages of good pH stability, high selectivity, good biocompatibility and the like, and can be used for detecting acetylcholinesterase in exogenous and endogenous living cells, and can realize effective differentiation of nerve cells and other cells.

Description

Technical Field

[0001] This invention relates to a novel fluorescent probe for detecting acetylcholinesterase, its preparation method, and its application method. It has the advantages of large Stokes shift, good selectivity, high sensitivity, and fast response speed. It can be used for the detection of acetylcholinesterase in exogenous and endogenous living cells, and can effectively distinguish nerve cells from other cells. Background Technology

[0002] Cholinesterases (ChEs), primarily including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), play crucial roles in various fields such as toxicology, pathology, and neurobiology. Acetylcholine (ACh), a vital component of nervous system metabolism, can be hydrolyzed by ChE into choline and acetic acid. Studies have shown that AChE exhibits superior efficiency and high specificity in the hydrolysis of ACh compared to BChE. Abnormal AChE activity may contribute to neurodegenerative diseases such as fatal familial insomnia, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. It is also closely related to physiological functions such as cell development, differentiation, and apoptosis. Research indicates that AChE is highly overexpressed in aggressive brain tumors such as glioblastoma (GBM) and is associated with cell proliferation and tumorigenesis. Therefore, highly selective and sensitive imaging of AChE activity is of great significance, both for diagnostic purposes and for gaining a deeper understanding of its role in complex cellular processes.

[0003] To date, reported methods for detecting AChE activity include colorimetry, UV-Vis spectrophotometry, and electrochemical techniques, which to some extent meet clinical and research needs. However, these methods typically have limited sensitivity and specificity and are easily affected by complex biological samples. Near-infrared fluorescent probes based on acetylcholinesterase, on the other hand, have attracted significant attention due to their ability to eliminate interference from other signaling molecules and their advantages of high sensitivity, high specificity, non-invasiveness, deep tissue penetration, and real-time detection. Currently reported fluorescent probes mostly emit fluorescence in the UV-Vis region, overlapping with the absorption of body tissues and the fluorescence spectrum generated by the body itself, significantly affecting detection results. Fluorescence emission wavelengths exceeding 600 nm effectively penetrate tissues and eliminate biological background interference; near-infrared fluorescence meets this requirement. Therefore, developing capable near-infrared fluorescent probes for acetylcholinesterase will provide a powerful tool for real-time imaging studies of acetylcholinesterase fluctuations in cells. Summary of the Invention

[0004] This invention aims to provide a near-infrared fluorescent probe for detecting acetylcholinesterase in cells, its preparation method, and its applications. The technical problem to be solved is to obtain a molecular structure with near-infrared fluorescence that can specifically recognize acetylcholinesterase through molecular design, enabling fluorescence imaging and serving as a detection probe for acetylcholinesterase. The near-infrared fluorescent probe of this invention produces a "turnon" type fluorescence response to acetylcholinesterase, with an emission wavelength of 630 nm in the near-infrared region and a detection limit as low as 4.8 U / L. The probe has a Stokes shift as high as 160 nm, the response can be completed within 30 min, and it has advantages such as good pH stability, high selectivity, and good biocompatibility. It can be used for the detection of exogenous and endogenous acetylcholinesterase in living cells and can effectively distinguish nerve cells from other cells. The preparation method of the near-infrared fluorescent probe of this invention is simple, mild, and has a high yield.

[0005] The near-infrared fluorescent probe of this invention, abbreviated as HL, has the following structural formula:

[0006] .

[0007] The preparation method of the near-infrared fluorescent probe HL of the present invention includes the following steps:

[0008] Step 1: In a pressure-resistant bottle, 5 g of 2,7-dihydroxynaphthalene, 11.87 g of sodium metabisulfite, and 7 mL of dimethylamine (40% aqueous solution) were added to 30 mL of water. The mixture was heated at 150 °C for 5 hours. After the reaction was completed, the mixture was cooled to room temperature, dichloromethane was added, and the organic layer was washed with brine. The mixture was dried over anhydrous sodium sulfate and concentrated. Subsequently, it was purified by silica gel column chromatography to obtain white crystalline compound 1.

[0009] Step 2: Under nitrogen protection, 0.7 mL of phosphorus oxychloride was added to 1.5 mL of N,N-dimethylformamide and reacted at 0°C for 10 minutes. Then, 0.5 g of compound 1 was dissolved in an appropriate amount of N,N-dimethylformamide and added to the reaction system. The reaction was then carried out at 0°C for 30 minutes, followed by heating to 70°C and reacting for 2 hours. After the reaction was completed, the mixture was quenched with ice water, and sodium acetate was added to adjust the pH of the system to 8. After the solid was completely precipitated, the mixture was filtered, washed with pure water, and dried to obtain the green solid compound 2.

[0010] Step 3: Dissolve 5 g isophorone and 2.3 g malononitrile in 30 mL of anhydrous ethanol, then add 0.5 mL piperidine, heat and reflux at 80 °C for 8 hours. After the reaction is complete, remove the solvent by vacuum distillation, extract with ethyl acetate, and obtain white crystalline compound 3 by column chromatography.

[0011] Step 4: Dissolve 0.5 g of compound 2 and 0.52 g of compound 3 in 5 mL of anhydrous ethanol, add 2 drops of piperidine, and heat under reflux at 75 °C for 8 hours. After the reaction is complete, remove the solvent by vacuum distillation and obtain the red solid compound 4 (HLN) by column chromatography.

[0012] Step 5: Dissolve 0.5 g of compound 4 in 5 mL of acetonitrile, then add 0.3 g of potassium carbonate and react at 0 °C for 30 minutes. Then add 0.3 g of dimethylcarbamoyl chloride to the reaction and react for 30 minutes. Finally, react at 50 °C for 9 hours. After the reaction is complete, remove the solvent by vacuum distillation and obtain an orange-red solid HL by column chromatography.

[0013] In step 1, the eluent for purifying the crude product by column chromatography is petroleum ether: ethyl acetate = 100:1, v / v.

[0014] In step 3, the eluent for purifying the crude product by column chromatography is petroleum ether: ethyl acetate = 20:1, v / v.

[0015] In step 4, the eluent for purifying the crude product by column chromatography is dichloromethane:methanol = 100:1, v / v.

[0016] In step 5, the eluent for purifying the crude product by column chromatography is dichloromethane:methanol = 100:1, v / v.

[0017] The synthesis route is shown below:

[0018]

[0019]

[0020]

[0021] The present invention relates to the application of the near-infrared fluorescent probe HL in the preparation of acetylcholinesterase detection reagents.

[0022] The near-infrared fluorescent probe HL of this invention emits almost no fluorescence on its own, but after reacting with acetylcholinesterase, it emits fluorescence in the near-infrared region. This probe uses dimethylamino as an electron donor and dicyanoisophorone as an electron acceptor to form a donor-π-acceptor structure, thus creating a red channel. When reacting with acetylcholinesterase, the N,N-dicarboxamide group on the probe is removed, opening the fluorescence and forming the red channel.

[0023] .

[0024] The near-infrared fluorescent probe HL of this invention can be used to detect acetylcholinesterase in exogenous and endogenous living cells, and can effectively distinguish nerve cells from other cells.

[0025] The detection method is as follows:

[0026] A 2 mM stock solution was prepared by dissolving HL in DMSO. 15 μL of this stock solution was then added to 3 mL of PBS containing different concentrations of acetylcholinesterase. Fluorescence and UV spectra of 10 μM acetylcholinesterase in different test solutions were obtained. Without acetylcholinesterase, HL showed no fluorescence emission at 630 nm under 470 nm excitation. However, after adding acetylcholinesterase, the fluorescence intensity at 630 nm increased significantly, and the fluorescence intensity gradually increased with increasing acetylcholinesterase concentration. This indicates that the target compound HL of this invention can be used as a probe for the detection of acetylcholinesterase, and its fluorescence value shows a good linear relationship with the concentration of acetylcholinesterase. These results demonstrate that HL can effectively detect acetylcholinesterase in solution and can be used as an acetylcholinesterase detection probe. Furthermore, the results show that the probe maintains stable fluorescence signals over a long period after the reaction and within the physiological pH range. Attached Figure Description

[0027] Figure 1 These are the UV absorption spectra and 3D fluorescence spectra of the reaction between HL and acetylcholinesterase. (a) UV absorption spectrum of HL (10 μM) in response to acetylcholinesterase (0.5 u / mL), (b) 3D fluorescence spectrum of HL (10 μM) in response to acetylcholinesterase (6 u / mL).

[0028] Figure 2 The fluorescence emission spectra of HL in response to acetylcholinesterase and the Stokes shift of HL are shown. (a) Fluorescence emission spectra of HL (10 μM) in response to acetylcholinesterase (6 u / mL), (b) Stokes shift of HL.

[0029] Figure 3 The fluorescence titration and linearity of the reaction of HL with acetylcholinesterase, as well as the detection limit, are shown. (a) Fluorescence titration of HL (10 μM) in response to acetylcholinesterase (0–1 u / mL), (b) Linearity of HL (10 μM) in response to acetylcholinesterase (0–1 u / mL).

[0030] Figure 4 The test results are the response time and pH stability of HL to acetylcholinesterase. (a) HL (10 μM) response time to acetylcholinesterase (6 u / mL), (b) HL (10 μM) response to acetylcholinesterase (6 u / mL) pH stability test.

[0031] Figure 5 This is a selectivity test for HL. (a) Selectivity test of HL (10 μM) with different ions, (b) Selectivity test of HL (10 μM) with different enzymes.

[0032] Figure 6 This is a liquid chromatography-mass spectrometry validation of HL in response to acetylcholinesterase. Among them, (a) high performance liquid chromatography of HL, HL + AChE and HLN; (b) high resolution mass spectrometry of HLN; (c) high resolution mass spectrometry of HL.

[0033] Figure 7 This is a molecular docking simulation of HL-responsive acetylcholinesterase.

[0034] Figure 8 HL is used for cell imaging to distinguish neural cells (PC-12) from other cells. (a) Confocal imaging of AML-12 cells, HepG2 cells, HeLa cells, 4T1 cells and PC-12 cells using HL (10 μM); (b) The fluorescence intensity of the red channel in (a).

[0035] Figure 9 This is a cell imaging assay using HL to detect exogenous acetylcholinesterase, exploring HL's ability to detect exogenous acetylcholinesterase in cells. (a) shows HL imaging of exogenous acetylcholinesterase in PC-12 cells; (b) shows the fluorescence intensity of the red channel in (a).

[0036] Figure 10 This is an assay for detecting endogenous acetylcholinesterase using HL, exploring the ability of HL to detect endogenous acetylcholinesterase in cells. (a) Imaging of endogenous acetylcholinesterase by HL in PC-12 cells; (b) The fluorescence intensity of the red channel in (a). Detailed Implementation

[0037] The present invention will be further illustrated by the following examples.

[0038] Example 1: Synthesis of HL

[0039] 0.5 g of compound 4 was dissolved in 5 mL of acetonitrile, and then 0.3 g of potassium carbonate was added. The mixture was reacted at 0 °C for 30 minutes. Then, 0.3 g of dimethylcarbamoyl chloride was added to the reaction mixture and reacted for another 30 minutes, followed by reaction at 50 °C for 9 hours. After the reaction was complete, the solvent was removed by vacuum distillation, and the resulting solution was purified by column chromatography to obtain an orange-red solid, HL, in 60% yield.

[0040] 1H NMR (400 MHz, CDCl3, ppm) δ 7.85 (d, J = 2.2 Hz, 1H), 7.81-7.73 (m,2H), 7.56 (d, J = 16.6 Hz, 1H), 7.30 (s, 1H), 7.18 (m, 1H), 7.01 (d, J = 16.6Hz, 1H), 6.84 (s, 1H), 3.18 (s, 3H), 3.04 (s, 3H), 2.83 (s, 6H), 2.63 (d, J =4.3 Hz, 4H), 1.14 (s, 6H). 13 C NMR (101 MHz, CDCl3, ppm) δ 169.74, 155.30,155.17, 151.84, 150.73, 135.08, 133.26, 132.61, 130.10, 129.97, 127.99,125.59, 123.32, 122.76, 120.12, 117.88, 115.12, 113.87, 113.14, 44.82, 43.23,39.26, 36.90, 36.75, 32.18, 28.41, 28.25.

[0041] Example 2: UV absorption and 3D fluorescence spectra of HL in solvent in response to acetylcholinesterase

[0042] To investigate the changes in HL in response to acetylcholinesterase in a solvent, acetylcholinesterase (0.5 u / mL) was used for testing. Figure 1 As shown in (a), after HL reacts with acetylcholinesterase, the UV absorption peak of HL undergoes a blue shift, which is basically consistent with the control structure HLN. Figure 1 (b) It can be seen that after HL reacts with acetylcholinesterase, an emission peak appears at 630 nm. This indicates that the fluorescence channel signal of HL gradually opens after reacting with acetylcholinesterase, showing promise for the identification of acetylcholinesterase.

[0043] Example 3: Fluorescence emission spectrum and Stokes shift of HL in solvent in response to acetylcholinesterase

[0044] To investigate the fluorescence changes of HL in response to acetylcholinesterase in a solvent, acetylcholinesterase (6 u / mL) was used for testing. Figure 2 (a) It can be seen that the fluorescence emission of HL is enhanced after reacting with acetylcholinesterase. From Figure 2(b) It can be seen that the emission wavelength of HL after responding to acetylcholinesterase under 470 nm excitation is 630 nm, and after normalization, we found that its Stokes shift reaches 160 nm. Monitoring the reaction time of HL in solvent in response to acetylcholinesterase shows that the basic reaction between HL and acetylcholinesterase ends at 30 min.

[0045] Example 4: Fluorescent titration and linear relationship of HL in response to acetylcholinesterase in solvent

[0046] To investigate the optical titration of HL in solvent in response to acetylcholinesterase, fluorescent titration was performed using acetylcholinesterase (0-1 u / mL). Figure 3 (a) It can be seen that the fluorescence intensity of HL at 630 nm gradually increases with increasing acetylcholinesterase concentration. From Figure 3 (b) It can be seen that the fluorescence signal of the probe shows a good linear relationship in the detection of acetylcholinesterase (0-1 u / mL), with a linear correlation coefficient R. 2 =0.995, and the detection limit is 4.8 U / L. This indicates that HL has a good monitoring ability for acetylcholinesterase.

[0047] Example 5: Response time and pH stability of HL to acetylcholinesterase in solvent

[0048] To investigate the response time and pH stability of HL in solvents in response to acetylcholinesterase, acetylcholinesterase (6 u / mL) was used for testing. Figure 4 (a) It can be seen that the reaction between HL and acetylcholinesterase basically ends at 30 min. For example... Figure 4 (b) By comparing the changes in fluorescence values ​​of HL and acetylcholinesterase after 10 min in solvents with different pH values, it can be seen that the probe can respond normally to acetylcholinesterase within the pH range of the physiological environment.

[0049] Example 6: Selectivity experiment of HL in response to acetylcholinesterase in solvent

[0050] To investigate the specificity of HL in response to acetylcholinesterase in solvents, we used HL in solvents to detect common ions, reactive oxygen species, amino acids, and enzymes. For example... Figure 5 (a,b) indicates that the probe is unaffected by other ions, amino acids, enzymes, and other reactive oxygen species, and has a good response to acetylcholinesterase only.

[0051] Example 7: Possible response mechanism of probe HL to acetylcholinesterase

[0052] like Figure 6As shown, the retention time of HL is approximately 5.30 min, and the corresponding mass spectrum peak is located at 455.3, which is consistent with [HL]. + (Calculated mass-to-charge ratio m / z: 455.2) consistent. After the addition of acetylcholinesterase, a new peak appeared in the liquid chromatography with a retention time of 4.47 min. The mass spectrometry of this new peak showed a signal at 384.3, corresponding to [HLN]. + The molecular weight (m / z: 384.3) was determined. Therefore, we confirmed that the reaction of HL with acetylcholinesterase removes the N,N-dicarboxamide group, thereby generating the product HLN.

[0053] Example 8: Molecular docking simulation of the response of probe HL to acetylcholinesterase

[0054] like Figure 7 As shown, to determine the interaction forces between the probe HL and acetylcholinesterase, we simulated the binding conformation of HL in the active cavity of acetylcholinesterase (PDB:1B41) using molecular docking technology. Docking results showed that the amino acid residues PHE-297, TRP-236, and THR-238 of the AChE protein formed hydrogen bonds with nitrile groups for lengths of 3.2 am, 2.6 am, and 3.2 am, respectively. The calculated molecular docking binding energy ΔE = -30.2085 kJ / mol indicates that the ligand HL can bind to the large protein AChE receptor in its natural state. In summary, the AChE protein receptor and the small probe HL can effectively form intramolecular hydrogen bonds, which facilitates the efficient binding of the probe to the enzyme's active site.

[0055] Example 9: Cell imaging using probe HL to distinguish nerve cells (PC-12) from other cells.

[0056] Compared to other cells, PC-12 nerve cells contain a higher level of acetylcholinesterase, so the level of acetylcholinesterase can be used as a biological indicator to distinguish nerve cells from other cells. Therefore, we investigated the ability of HL to distinguish nerve cells from other cells. We co-incubated HL with AmL-12, HepG2, HeLa, 4T1, and PC-12, respectively. Figure 8 As shown, only the PC-12 nerve cells exhibited significantly higher fluorescence intensity. ImageJ quantification revealed that the relative fluorescence intensity of nerve cells was more than four times that of other cells, demonstrating high distinguishability. These experiments demonstrate that the probe HL possesses the ability to distinguish nerve cells from other cells with high precision.

[0057] Example 10: Assay of exogenous acetylcholinesterase in cells by probe HL

[0058] Whether HL can be used to detect acetylcholinesterase in cells is very important, as it reflects whether HL can be used to monitor changes in acetylcholinesterase concentration in cells. For example... Figure 9 As shown, during the monitoring of exogenous acetylcholinesterase in PC-12 cells, compared with the control group, the fluorescence intensity of the red channel gradually increased after incubation with acetylcholinesterase in other groups. This indicates that HL can monitor the concentration changes of exogenous acetylcholinesterase in cells.

[0059] Example 11: Assay of endogenous acetylcholinesterase in cells by probe HL

[0060] To investigate the ability of HL to monitor cellular acetylcholinesterase, PC-12 cells were induced with oxidative stress by adding LPS. Figure 10 As shown, compared with the control group, the fluorescence intensity of the red channel gradually increased in other groups after LPS was added to the cells to induce oxidative stress. Cells produced more acetylcholinesterase after LPS-induced oxidative stress, indicating that HL could effectively capture the changes in acetylcholinesterase during this process, which holds promise for monitoring changes in acetylcholinesterase in vivo.

Claims

1. A near-infrared fluorescent probe, abbreviated as HL, characterized in that... Its structural formula is shown below: 。 2. The method for preparing the near-infrared fluorescent probe HL according to claim 1, characterized in that... Includes the following steps: Step 1: 2,7-Dihydroxynaphthalene, sodium metabisulfite, and dimethylamine were added to water in a pressure-resistant bottle, and the mixture was heated to react at 150°C. After the reaction was completed, the mixture was cooled to room temperature, dichloromethane was added, and the organic layer was washed with brine. The mixture was dried over anhydrous sodium sulfate and concentrated. Subsequently, it was purified by silica gel column chromatography to obtain white crystalline compound 1. Step 2: Under nitrogen protection, phosphorus oxychloride was added to N,N-dimethylformamide and reacted at 0°C for 10 minutes. Then, compound 1 was added to the reaction system, and the reaction was continued at 0°C for 30 minutes. The temperature was then increased to 70°C and reacted for 2 hours. After the reaction was completed, the mixture was quenched with ice water, and sodium acetate was added to adjust the pH of the system to 8. After the solid was completely precipitated, the mixture was filtered, washed with pure water, and dried to obtain the green solid compound 2. Step 3: Dissolve isophorone and malononitrile in anhydrous ethanol, then add piperidine, and heat to reflux at 80°C. After the reaction is complete, remove the solvent by vacuum distillation, extract with ethyl acetate, and obtain white crystalline compound 3 by column chromatography. Step 4: Dissolve compound 2 and compound 3 in anhydrous ethanol, then add piperidine, heat to reflux, remove the solvent by vacuum distillation after the reaction is complete, and obtain red solid compound 4 by column chromatography; Step 5: Dissolve compound 4 in acetonitrile, add potassium carbonate, and react at 0°C for 30 minutes. Then add dimethylcarbamoyl chloride to the reaction and react for 30 minutes, followed by reaction at 50°C for 9 hours. After the reaction is complete, remove the solvent by vacuum distillation, and obtain an orange-red solid HL by column chromatography. The synthesis route is shown below: ； ； 。 3. The application of the near-infrared fluorescent probe HL according to claim 1 in the preparation of acetylcholinesterase detection reagent.

4. The application according to claim 3, characterized in that: The detection reagent can detect endogenous and / or intrinsic acetylcholinesterase in living cells.

5. The application according to claim 3, characterized in that: The detection reagent can distinguish nerve cells from other cells.

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