A "on-off" type butyrylcholinesterase near-infrared fluorescent probe and a preparation method and application thereof

Abstract

This invention discloses an "on-off" type butyrylcholinesterase near-infrared fluorescent probe, its preparation method, and its applications. The structural formula of the fluorescent probe QCL-BChE is shown in Formula I. When excited at a wavelength of 720 nm, QCL-BChE shows almost no fluorescence emission at 765 nm. When QCL-BChE coexists with BChE, a significant fluorescence emission peak appears at 765 nm. As the concentration of BChE increases, the fluorescence intensity at 765 nm gradually increases, and other common interfering ions, substances, and enzyme species do not significantly interfere with or affect the detection of BChE. Because the emission wavelength is in the near-infrared region, the interference of system background fluorescence is greatly reduced, improving the sensitivity for detecting BChE activity. In addition, QCL-BChE has been successfully applied to the fluorescence imaging of endogenous BChE in LO2 cells and HepG2 cells.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a biocompatible "off-on" type butyrylcholinesterase near-infrared fluorescent probe QCL-BChE, its preparation method, and its application. Background Technology

[0002] Butyrylcholinesterase (BChE; EC 3.1.1.8) is an important member of the cholinesterase family, widely distributed in plasma, liver, muscle tissue, and brain tissue. As a serine hydrolase synthesized in the hepatic endoplasmic reticulum, it plays a crucial role in cell metabolism, apoptosis, and maintaining normal physiological functions. Studies have shown that abnormal BChE expression in the liver is associated with various liver diseases. For example, patients with non-alcoholic fatty liver disease have elevated BChE expression, while excessively low BChE expression can lead to irreversible liver damage, resulting in chronic liver disease, acute hepatitis, cirrhosis, or cancer. Therefore, BChE is considered one of the biomarkers for the clinical diagnosis of liver diseases. Furthermore, the active site of BChE can function on various substrates, and its abnormal expression is also significantly associated with Alzheimer's disease, multiple sclerosis, lipid metabolism disorders, diabetes, and cardiovascular disease. BChE also has pharmacological functions and can be used to combat cocaine addiction and organophosphate poisoning. Therefore, the detection and research of BChE in vivo, as well as the discovery of related inhibitory drugs, play an extremely important role in clinical practice.

[0003] Currently, there are four main methods used for monitoring and evaluating BChE, including: 1) using 13 C and 3 The methods employed include: 1) radiometric determination using H-labeled substrates such as acetylcholine; 2) isothermal titration calorimetry (ITC) using natural substrates; 3) UV-Vis spectrophotometry to determine cholinesterase or its metabolites; and 4) direct or indirect fluorescence measurement using fluorescent substrates. Because BChE and acetylcholinesterase (AChE) have similar structures and share the same substrates, the above methods are difficult to accurately detect BChE in the presence of AChE and cannot achieve real-time imaging of BChE in vivo. Compared to these methods, molecular fluorescent probe technology, with its advantages of high sensitivity, high selectivity, strong anti-interference ability, simple operation, and non-destructive detection of cells, tissues, and organisms, is increasingly being applied to the in-situ detection and imaging of BChE. Currently, fluorescent probes used for BChE detection generally suffer from drawbacks such as short emission wavelengths, susceptibility to interference from biological background fluorescence, and low sensitivity. Near-infrared fluorescent probes, with their advantages of long-wavelength emission (>700 nm), strong cell penetration, and less background fluorescence interference, are more suitable for the detection and imaging of BChE in vivo.

[0004] Therefore, developing near-infrared fluorescent probes suitable for real-time monitoring and imaging of BChE in biological cells and tissues is of great significance. Summary of the Invention

[0005] One objective of this invention is to provide a near-infrared fluorescent probe molecule—QCL-BChE—that can rapidly identify BChE.

[0006] The QCL-BChE provided by this invention has the structural formula shown in Formula I:

[0007] .

[0008] Another object of the present invention is to provide a method for preparing QCL-BChE as shown in Formula I.

[0009] The preparation method of QCL-BChE provided by this invention is shown in the flowchart below. Figure 1 Specifically, it includes the following steps: 1) Cyclopentanone is reacted with phosphorus tribromide (PBr3) to form compound 1; 2) React compound 1 with 2-hydroxy-4-methoxybenzaldehyde to generate compound 2; 3) Compound 2 is demethylated by boron tribromide (BBr3) to generate compound 3; 4) React compound 3 with N-ethylquinaldine iodide to generate QCL-OH; 5) React compound QCL-OH with cyclopropylformyl chloride to obtain compound QCL-BChE as shown in Formula I;

[0010] In step 1) of the above method, the specific method for generating compound 1 from cyclopentanone under the action of phosphorus tribromide (PBr3) is as follows: at 0°C, phosphorus tribromide (PBr3) is added to a mixed solution of DMF and CHCl3, stirred for 45 min, then cyclopentanone is added, and the reaction is stirred to obtain compound 1. The molar ratio of cyclopentanone to phosphorus tribromide (PBr3) in the reaction is 1:2.3; the reaction temperature is 25°C, and the reaction time is 16 h.

[0011] In step 2) of the above method, the specific method for reacting compound 1 and 2-hydroxy-4-methoxybenzaldehyde to generate compound 2 is as follows: compound 1 and 2-hydroxy-4-methoxybenzaldehyde are dissolved in DMF, cesium carbonate is added and the mixture is stirred to obtain compound 2. The molar ratio of compound 1, 2-hydroxy-4-methoxybenzaldehyde, and cesium carbonate in the reaction is 1:1.2:2.5; the reaction temperature is 25℃, and the reaction time is 16 h.

[0012] In step 3) of the above method, the specific method for removing the methyl group from compound 2 to generate compound 3 under the action of boron tribromide (BBr3) is as follows: Under nitrogen protection, compound 2 is dissolved in anhydrous dichloromethane, boron tribromide (BBr3) is slowly added under an ice-water bath, and after stirring for 1 h, the mixture is slowly raised to room temperature to carry out the reaction, thereby obtaining compound 3. The molar ratio of compound 2 to boron tribromide (BBr3) in the reaction is 1:30; the reaction temperature is 25℃, and the reaction time is 4 h.

[0013] In step 4) of the above method, the specific method for reacting compound 3 and N-ethylquinaldine iodide to generate QCL-OH is as follows: compound 3 and N-ethylquinaldine iodide are dissolved in anhydrous ethanol, then piperidine is added, and the mixture is refluxed under nitrogen protection to obtain compound QCL-OH. The molar ratio of compound 3, N-ethylquinaldine iodide, and piperidine in the reaction is 1:1.2:1.96, respectively; the reaction temperature is 80℃, and the reaction time is 4 h.

[0014] In step 5) of the above method, the specific method for reacting compound QCL-OH with cyclopropylformyl chloride to obtain compound QCL-BChE as shown in Formula I is as follows: Under nitrogen protection, compound QCL-OH is dissolved in dichloromethane, and then triethylamine is added. Then, cyclopropylformyl chloride dissolved in dichloromethane is added dropwise to react and obtain compound QCL-BChE. The molar ratio of compound QCL-OH, cyclopropylformyl chloride, and triethylamine in the reaction is 1:1.2:2; the reaction temperature is 25℃, and the reaction time is 6 h.

[0015] Another object of the present invention is to provide the use of QCL-BChE.

[0016] The uses of QCL-BchE provided by this invention are selected from at least one of the following 1)-8): 1) A fluorescent probe made of QCL-BChE; 2) Applications of QCL-BChE as a fluorescent probe or as a fluorescent probe for detecting BChE; 3) Chemical sensors containing QCL-BChE; 4) Application of QCL-BChE in the preparation of chemical sensors or chemical sensors for detecting BChE; 5) Application of QCL-BChE in BChE detection; 6) Application of the fluorescent probe in 1) above or the chemical sensor in 3) above in the detection of BChE; 7) Application of the fluorescent probe in 1) above or the chemical sensor in 3) above in cell fluorescence imaging; 8) Application of QCL-BChE in the screening and / or evaluation of BChE inhibitors.

[0017] The fluorescent probe and chemical sensor can be used for the detection and / or fluorescence imaging of BChE.

[0018] The BChE can be endogenous or exogenous.

[0019] In some embodiments of the present invention, the fluorescent probe or chemical sensor may be applied to cells (such as cells containing endogenous BChE).

[0020] In some embodiments of the present invention, the fluorescent probe or chemical sensor can be used for fluorescence imaging of endogenous BChE in normal human hepatocytes (such as LO2 cells) and human liver cancer cells (such as HepG2 cells).

[0021] In some embodiments of the present invention, the fluorescent probe or chemical sensor can be used to distinguish between normal human liver cells (such as LO2 cells) and human liver cancer cells (such as HepG2 cells).

[0022] The inventors of this invention experimentally confirmed that QCL-BChE itself does not exhibit significant fluorescence emission at 765 nm. However, when QCL-BChE coexists with BChE, a fluorescence emission peak appears at 765 nm. With increasing BChE concentration (0-0.2 U / mL), the fluorescence at 765 nm gradually increases, and the fluorescence emission intensity F at 765 nm increases. 765 nm The method exhibits a good linear relationship with BChE concentration, and the detection limit is 2.24 × 10⁻⁶. -4 The sensitivity of QCL-BChE is superior to most currently reported BChE fluorescence detection methods, indicating that QCL-BChE is suitable for high-sensitivity detection of BChE, which can be performed using fluorescence spectroscopy.

[0023] QCL-BChE exhibits excellent selectivity for the fluorescence response of BChE, and is effective against common ions and interfering substances (such as Na+). + K + Ca 2+ Mg 2+ Fe 3+ Al 3+ Cl - I - S 2- Ac - SO4 2-The presence of ions (GSH, Gly, Cys, Ala, Arg, Glu, AChE, Trypsin, Tyrosinase, Chymotrypsin, and Lysozyme) in the fluorescence assay of BChE results in almost no response, thus eliminating interference from numerous interfering ions, substances, and enzyme species, leading to high detection specificity. Due to its excellent biocompatibility and low cytotoxicity, QCL-BChE can easily achieve high-resolution bioimaging of BChE in cells. Furthermore, the high sensitivity of QCL-BChE for BChE detection requires only a small amount of sample, broadening the application range of this method. Attached Figure Description

[0024] Figure 1 This is a flowchart of the preparation process of QCL-BChE.

[0025] Figure 2 The 1H NMR spectrum of compound 2.

[0026] Figure 3 This is the carbon NMR spectrum of compound 2.

[0027] Figure 4 This is the high-resolution mass spectrum of compound 2.

[0028] Figure 5 The 1H NMR spectrum of compound 3 is shown.

[0029] Figure 6 The image shows the carbon NMR spectrum of compound 3.

[0030] Figure 7 This is the high-resolution mass spectrum of compound 3.

[0031] Figure 8 The 1H NMR spectrum of QCL-OH is shown.

[0032] Figure 9 The carbon NMR spectrum of QCL-OH is shown.

[0033] Figure 10 This is a high-resolution mass spectrum of QCL-OH.

[0034] Figure 11 The NMR spectrum of QCL-BChE is shown as a 1H NMR spectrum.

[0035] Figure 12 The image shows the carbon NMR spectrum of QCL-BChE.

[0036] Figure 13 This is a high-resolution mass spectrum of QCL-BChE.

[0037] Figure 14The UV absorption spectrum (a) and fluorescence spectrum (b) of the system before and after the reaction of QCL-BChE (10 μM) with BChE (1 U / mL) and QCL-OH (10 μM) are shown; (c) and (d) are the fluorescence intensity changes of the system with QCL-BChE (10 μM) and BChE (1 U / mL) coexisting and the system with QCL-BChE (10 μM) alone at different pH and different temperatures, respectively.

[0038] Figure 15 The graph shows the fluorescence intensity of the coexisting systems of QCL-BChE (10 μM) and different concentrations of BChE (0~2 U / mL) over time (a); the UV absorption spectrum (b) and fluorescence spectrum (c) of the coexisting systems of QCL-BChE (10 μM) and different concentrations of BChE (0~2 U / mL); and the linear relationship between the fluorescence intensity and BChE concentration in the coexisting systems of QCL-BChE (10 μM) and different concentrations of BChE (0~2 U / mL) (d).

[0039] Figure 16 The selectivity and anti-interference effect of QCL-BChE (10 μM) on BChE (a); the relationship between the inhibition efficiency of different concentrations of Tacrine (2~150 μM) on BChE (b); the Michaelis-Menten plot (c) and the corresponding Lineweaver-Burk plot (d) of the enzyme-catalyzed reaction of BChE (1 U / mL) with different concentrations of QCL-BChE (1~30 μM).

[0040] Figure 17 (a) shows confocal fluorescence imaging of LO2 cells and QCL-BChE (10 μM) for different time periods; (b) shows the average fluorescence intensity at each time point in (a).

[0041] Figure 18(a) shows the confocal fluorescence imaging of LO2 and HepG2 cells, from top to bottom: untreated cells; cells incubated with QCL-BChE (10 μM) for 30 min; cells pretreated with Tacrine (100 μM) for 1 h and then co-incubated with QCL-BChE (10 μM) for 30 min; cells pretreated with Donepezil (100 μM) for 1 h and then co-incubated with QCL-BChE (10 μM) for 30 min; cells pretreated with APAP (100 μM) for 12 h and then co-incubated with QCL-BChE (10 μM) for 30 min; (b) shows the cell viability after co-incubation of LO2 and HepG2 cells with different concentrations of QCL-BChE; (c) shows the average fluorescence intensity of each group in Figure (a).

[0042] Figure 19 Fluorescence images of mixed cells (LO2 cells and HepG2 cells). Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Example 1: Preparation of the chemical sensor molecule QCL-BChE The reaction process is as follows Figure 1 As shown, the specific method is as follows: PBr3 (6.2 mL, 65.25 mmol) was added to a mixture of DMF (5.6 mL, 72.5 mmol) and chloroform (25 mL) at 0 °C. After 45 min, cyclopentanone (2.5 mL, 28.25 mmol) was added, and the mixture was stirred at room temperature (25 °C) for 16 h. After the reaction was complete, the pH was adjusted to neutral with saturated sodium bicarbonate (NaHCO3) solution. The mixture was extracted with dichloromethane and washed with saturated sodium chloride solution. The organic phase was collected and dried over anhydrous sodium sulfate. The solid was removed by filtration, and the solvent was removed by rotary evaporation to give a yellow-brown oily compound 1 (yield 70.4%). This substance did not require purification and was used directly in the next step.

[0046] Compound 1 (2.6 g, 15 mmol), 2-hydroxy-4-methoxybenzaldehyde (2.739 g, 18 mmol), and cesium carbonate (12.2 g, 37.5 mmol) were added to DMF (18.75 mL) and stirred at room temperature (25 °C) for 16 h. After the reaction was complete, the cesium carbonate residue was filtered off. The filtrate was concentrated and redissolved in ethyl acetate, washed with saturated sodium chloride (NaCl) solution, and extracted with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, the solid was filtered off, and the solvent was removed by rotary evaporation. Purification was performed by column chromatography, with the eluent being petroleum ether (boiling range 60–90 °C) / ethyl acetate (15:1). v / v ), yielding a yellowish-brown solid compound 2, yield: 42%.

[0047] Compound 2 (228 mg, 1 mmol) was dissolved in anhydrous dichloromethane (30 mL) under nitrogen protection. BBr3 (2.89 mL, 30 mmol) was slowly added to the solution in an ice-water bath. After stirring for 1 h in an ice-water bath, the mixture was slowly brought to room temperature (25 °C) and the reaction continued for 4 h. After the reaction was complete, saturated sodium bicarbonate (NaHCO3) solution was slowly added in an ice-water bath to quench the reaction and adjust the pH to neutral. The mixture was washed with saturated sodium chloride solution and treated with dichloromethane / methanol (10:1). v / v Extraction. The organic phase was collected and dried over anhydrous sodium sulfate. The solid was filtered off, and the solvent was removed by rotary evaporation. Purification was performed by column chromatography, using petroleum ether (boiling range 60-90℃) / ethyl acetate (1:1). v / v The reaction yielded a yellow solid compound 3, with a yield of 94.3%.

[0048] Compound 3 (107.1 mL, 0.5 mmol) and N-ethylquinaldine iodide (224.3 mg, 0.75 mmol) were dissolved in anhydrous ethanol (10 mL), and piperidine (100 μL, 0.98 mmol) was added. The reaction mixture was refluxed at 80 °C with stirring for 4 h under nitrogen protection. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography using dichloromethane / methanol (20:1) as the eluent. v / v The dark green solid compound QCL-OH was obtained with a yield of 92.9%.

[0049] QCL-OH (74.3 mg, 0.15 mmol) was dissolved in anhydrous dichloromethane (20 mL). Triethylamine (41.7 μL, 0.3 mmol) was added under nitrogen protection, and the mixture was cooled to 0 °C. Cyclopropylformyl chloride (14.5 μL, 0.18 mmol) was dissolved in anhydrous dichloromethane (8 mL) and then added dropwise to the aforementioned reaction system. The mixture was stirred at room temperature (25 °C) for 6 h under nitrogen protection. After the reaction was complete, deionized water was added to adjust the pH to neutral. The solution was then mixed with dichloromethane / methanol (10:1). v / v Extraction was performed, the organic phase was collected and dried over anhydrous sodium sulfate, the solid was filtered off, and the solvent was removed by rotary evaporation. Purification was then performed by column chromatography, with the eluent being dichloromethane / methanol (25:1). v / v The dark green solid compound QCL-BChE was obtained with a yield of 97.2%.

[0050] NMR and high-resolution identification results of compound 2: 1 H NMR (CD2Cl2, 500 MHz) = 9.96 (s, 1H); 7.05 (d, J = 8.4 Hz, 1H); 6.68-6.66 (m, 2H); 6.56 (s, 1H); 3.80 (s, 3H); 2.71-2.69 (m, 2H); 2.63-2.61 (m, 2H). 13 C NMR (CD2Cl2, 150 MHz) = 183.8, 163.7, 161.0, 152.9, 137.5, 127.5, 121.9, 116.4, 115.6, 110.9, 101.5, 55.7, 24.3, 23.6. The 1H and 1C NMR spectra are shown below. Figure 2 and Figure 3 Instrument model: Bruker Avance 500 MHz. High-resolution mass spectrometry identification results of compound 2: HR-MS (ESI, m / z) cacld for C 14 H 12 O3 + [M+H] + :229.0865, found 229.0866. See results. Figure 4 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 2.

[0051] NMR and high-resolution identification results of compound 3: 1 H NMR (CDCl3, 800 MHz) = 9.88 (s, 1H), 7.16 (d, J = 8.1 Hz, 1H); 6.82 (s, 1H); 6.63 (s, 1H), 6.62 (dd, J 1 = 8.7 Hz J 2 = 2.2 Hz, 1H); 2.69 (t, J = 6.2 Hz, 2H); 2.53 (d, J = 6.0 Hz, 2H). 13 C NMR (CDCl3, 200 MHz) = 182.7, 163.4, 159.3, 152.2, 135.4, 128.0, 122.9, 115.1, 113.4, 114.4, 102.6, 23.6, 23.3. The 1H and 1C NMR spectra are shown below. Figure 5 and Figure 6 Instrument model: Bruker Avance III-800 MHz. High-resolution mass spectrometry identification results of compound 3: HR-MS (ESI, m / z) cacld forC 13 H 10 O3 + [M+H] + :215.0708, found 215.0708. See results. Figure 7 Instrument model: UPLC-Q / TOF XevoG2-XS. The above results confirm that the obtained compound is indeed the target compound 3.

[0052] NMR and high-resolution identification results of QCL-OH: 1 H NMR (DMSO-d6, 500 MHz) = 1.50 (t, J =7.0 Hz, 3H); 2.85 (s, 4H); 4.87 (q, J = 6.9 Hz, 2H); 6.55 (d, J = 14.5 Hz, 1H); 6.66 (d, J = 8.4 Hz, 1H); 6.72 (s, 1H); 6.95 (s, 1H); 7.23 (d, J = 8.4 Hz, 1H); 7.75 (t,J = 7.6 Hz, 1H); 8.00 (t, J = 7.4 Hz, 1H); 8.17 (d, J = 7.9 Hz, 1H); 8.26(d, J = 14.5 Hz, 1H); 8.31 (d, J = 9.0 Hz, 1H); 8.50 (d, J = 9.3 Hz, 1H); 8.63 (d, J = 9.2 Hz, 1H); 10.32 (s, 1H). 13 C NMR (DMSO-d6, 125 MHz) = 13.1, 23.9, 25.4, 45.1, 102.7, 111.1, 113.1, 114.6, 117.2, 118.0, 120.4, 123.8, 126.7, 127.5, 128.2, 130.0, 131.0, 134.1, 136.1, 138.3, 141.0, 153.1, 153.9, 159.7, 160.9. The 1H and 1C NMR spectra are shown below. Figure 8 and Figure 9 Instrument model: Bruker Avance 500 MHz. High-resolution mass spectrometry identification results of QCL-OH: HR-MS (ESI, m / z) cacld for C 25 H 22 NO2 + [M] + : 368.1645, found 368.1650. See results below. Figure 10 The above results confirm that the obtained compound is indeed the target compound QCL-OH.

[0053] NMR and high-resolution identification results of QCL-BChE: 1 H NMR (DMSO-d6, 500 MHz) = 8.78 (d, J =9.0 Hz, 1H); 8.52 (d, J = 9.5 Hz, 1H); 8.40 (d, J = 9.0 Hz, 1H); 8.26-8.18 (m,2H); 8.06 (td, J 1 = 7.5 Hz J2 = 1.5 Hz, 1H); 7.81 (t, J = 7.5 Hz, 1H); 7.39 (d, J =8.5 Hz, 1H); 7.12 (d, J = 2.0 Hz, 1H); 6.99 (dd, J 1 = 8.0 Hz J 2= ​​2.5 Hz, 1H); 6.92(s, 1H); 6.71 (d, J = 15.0 Hz, 1H); 4.96 (q, J = 7.0 Hz, 2H); 2.88 (s, 4H); 1.96-1.87 (m, 1H); 1.53 (t, J = 7.0 Hz, 3H); 1.12-1.07 (m, 2H); 1.07-1.01 (m, 2H). 13 CNMR (DMSO-d6, 125 MHz) = 172.5, 159.0, 154.2, 151.7, 150.8, 141.9, 139.9, 138.1, 138.0, 134.4, 130.0, 128.0, 127.5, 127.21, 121.1, 120.4, 120.3, 118.23, 118.16, 117.8, 113.2, 109.6, 45.5, 25.3, 24.0, 13.4, 12.6, 9.2. The 1H and 1C NMR spectra are shown below. Figure 11 and Figure 12 Instrument model: Bruker Avance 500 MHz. High-resolution mass spectrometry identification results of QCL-BChE: HR-MS (ESI, m / z) cacld for C 29 H 26 NO3 + [M] + :436.1907, found 436.1910. See results. Figure 13 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound QCL-BChE.

[0054] Example 2: Fluorescence detection of BChE using QCL-BChE as an analytical reagent. 1. Sensitivity of QCL-BChE for fluorescence detection of BChE QCL-BChE was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1.0 mM stock solution, labeled as QCL-BChE stock solution; QCL-OH was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1.0 mM stock solution, labeled as QCL-OH stock solution; 2 mg of BChE was dissolved in a certain amount of phosphate buffer solution (PBS, 10 mM, pH = 7.4) to prepare a 400 U / mL BChE solution, which was then serially diluted to a 50 U / mL BChE solution, labeled as BChE stock solution.

[0055] Add different volumes of BChE stock solution (50 U / mL) to a certain volume of QCL-BChE stock solution (1.0 mM), then add an appropriate amount of PBS buffer / DMSO mixture (7:3). v / v The final concentration of QCL-BChE in each system was 10 μM, prepared using PBS (10.0 mM, pH = 7.4). The concentrations of BChE were 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.5, and 2 U / mL, respectively. After incubation at 37°C for 40 minutes, each system was transferred to a 1 cm quartz cell, and the UV absorption and fluorescence spectra of the reaction system were measured.

[0056] Figure 14 (a) and (b) show the UV absorption and fluorescence spectra of the systems before and after the reaction of QCL-BChE (10 μM) with BChE (1 U / mL), and QCL-OH (10 μM), respectively. The figures show that the maximum UV-Vis absorption wavelength of QCL-BChE is 553 nm, while QCL-OH has two UV absorption peaks at 630 nm and 714 nm. When BChE is added to the QCL-BChE solution, the UV absorption spectrum of this system almost coincides with that of QCL-OH. QCL-BChE shows no fluorescence emission at 765 nm, while QCL-OH shows strong fluorescence emission at 765 nm. When BChE is added to QCL-BChE, the system exhibits strong fluorescence emission at 765 nm, almost coinciding with the fluorescence spectrum of QCL-OH. These results demonstrate that QCL-BChE has excellent UV absorption and fluorescence response to BChE. The spectrum of the coexisting system almost overlaps with that of QCL-OH, proving that QCL-BChE may lose its recognition group and become QCL-OH under the action of BChE. Figure 14(c) and (d) show the fluorescence intensity changes at different pH and temperature for the coexistence of QCL-BChE (10 μM) and BChE (1 U / mL) and the system containing QCL-BChE (10 μM) alone. Figure 14 (c) It can be seen that the fluorescence intensity of QCL-BChE itself is weak when the pH is between 4.0 and 9.0. The fluorescence signal of the QCL-BChE and BChE coexisting system is weak under acidic conditions, but gradually increases with increasing pH, maintaining a strong fluorescence signal at pH = 7.4. Figure 14 (d) It can be seen that QCL-BChE itself has almost no fluorescence signal, while the coexistence system of QCL-BChE and BChE has a strong fluorescence signal in the range of 30~40℃, especially maintaining a strong fluorescence intensity at physiological temperature (37℃), indicating that QCL-BChE is suitable for the detection of BChE in vivo.

[0057] Figure 15 (a) is a graph showing the fluorescence intensity change over time in the coexistence systems of QCL-BChE (10 μM) and different concentrations of BChE (0~2 U / mL). The graph shows that the rate of change in fluorescence intensity with time increases with increasing BChE concentration. When the BChE concentration exceeds 1 U / mL, the fluorescence intensity plateaus around 40 min. Figure 15 (b) and (c) are the UV absorption spectra and fluorescence spectra of QCL-BChE (10 μM) coexisting with different concentrations of BChE (0~2 U / mL), respectively. Figure 15 (b) It can be seen that as the concentration of BChE increases, the absorbance of QCL-BChE at 553 nm gradually decreases, while the absorbance at 630 nm and 714 nm gradually increases, and the color of the reaction system gradually changes from purple to blue. From Figure 15 (c) It can be seen that the fluorescence intensity of QCL-BChE at 765 nm gradually increases with the increase of BChE concentration. Figure 15 (d) shows the linear relationship between the fluorescence intensity of QCL-BChE (10 μM) and different concentrations of BChE (0–2 U / mL) in the coexistence system and the BChE concentration. The figure shows that the fluorescence intensity at 765 nm gradually increases with increasing BChE concentration. When the BChE concentration reaches 0.8 U / mL, the change in fluorescence intensity decreases and tends to plateau with further increases in BChE concentration. Within the range of 0–0.2 U / mL, the fluorescence intensity F at 765 nm is... 765 nm There is a good linear relationship between the concentration of BChE and the concentration of BChE, and the linear equation is F. 765 nm= 1551.1309 × C BChE +13.67595, the method's detection limit, calculated by dividing three times the standard deviation of the blank signal by the slope of the standard curve, is 2.23 × 10⁻⁶. -4 U / mL.

[0058] The above results demonstrate that the analytical reagent QCL-BChE has excellent performance and can achieve highly sensitive fluorescence detection of BChE.

[0059] 2. The selectivity and anti-interference ability of QCL-BChE for fluorescence detection of BChE.

[0060] Simultaneously, take several 5 mL EP tubes and perform similar operations, except that the addition of BChE is replaced with the addition of various potential interfering substances, namely: Blank, Na + Ca 2+ K + Mg 2+ Fe 3+ Al 3+ Cl - I - S 2- Ac - SO4 2- The following interfering ions or substances were added: GSH, Gly, Cys, Ala, Arg, and Glu (concentration of 1 mM) and AChE, Trypsin, Tyrosinase, Chymotrypsin, and Lysozyme (concentration of 2 U / mL). First, the fluorescence intensity of QCL-BChE and each interfering substance was measured. Then, BChE (1 U / mL) was added to the above system, and its fluorescence intensity was measured. The results are as follows: Figure 16 As shown in (a), before the addition of BChE, the system contained only the probe QCL-BChE and various interfering ions, substances, or enzymes. The fluorescence intensity of the system did not change significantly, indicating that the interfering ions, substances, or enzymes did not provide a good fluorescence response or interfere with the probe QCL-BChE. Subsequently, the addition of BChE (1 U / mL) to the system significantly increased the fluorescence intensity at 765 nm, indicating that the probe had a good response to BChE. This experimental phenomenon also demonstrates that the presence of the interfering ions and substances does not significantly interfere with or affect the detection of BChE by QCL-BChE as an analytical reagent, and that QCL-BChE exhibits high selectivity for the fluorescence detection of BChE as a detection reagent.

[0061] 3. Screening for BChE inhibitors and detecting the rate of BChE enzymatic reaction using QCL-BChE. Tacrine (a BChE inhibitor) was selected for inhibitor screening. Several 5mL EP tubes were filled with BChE (1 U / mL) and different concentrations of tacrine (2, 5, 10, 20, 50, 100, and 150 μM), and incubated at 37°C for 40 min. Then, QCL-BChE (10 μM) was added, and incubation continued at 37°C for another 40 min. Fluorescence intensity was measured, and the results are shown below. Figure 16 As shown in (b). Figure 16 As shown in (b): with increasing tacrine concentration, the inhibitory efficiency first increases rapidly and then increases slowly. Based on the linear relationship between inhibitory efficiency and the logarithm of tacrine concentration, the IC50 of tacrine against BChE was calculated. 50 The concentration was 14.71199 μM. These results indicate that QCL-BChE can be used to screen for inhibitors of BChE.

[0062] The enzymatic reaction rate between QCL-BChE and BChE was studied. Several 5 mL EP tubes were used, and BChE (1 U / mL) and different concentrations of QCL-BChE (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 μM) were added. Fluorescence intensity was measured multiple times within 10 minutes, and the initial reaction rate was measured within the linear range. Figure 16 (c) Michaelis-Menten diagram of the reaction catalyzed by BChE with different concentrations of QCL-BChE enzyme. Figure 16 (c) It can be seen that the initial reaction rate V0 gradually increases with the increase of QCL-BChE concentration. To calculate the Michaelis constant K... m and maximum reaction rate V max Lineweaver-Burk plots were created using the initial reaction rate V0 and the reciprocal of the QCL-BChE concentration. The results are shown in [Figure number missing]. Figure 16 (d). From Figure 16 (d) Calculate the maximum reaction rate V from the data. max The flow rate is 0.93 μM / min, and the Michaelis constant K is... m It was 14.67 μM, far lower than that of acetylcholine (K). m = 80 μM), indicating that BChE has a high affinity and catalytic sensitivity to QCL-BChE.

[0063] 4. Performance comparison of QCL-BChE with other BChE fluorescent probes The fluorescence detection performance of QCL-BChE for BChE was summarized and compared with the performance of fluorescent probes for BChE detection in the literature. The results are shown in Table 1. As can be seen from Table 1, the overall performance of QCL-BChE for fluorescence detection of BChE is superior to that of similar reported chemical sensors.

[0064] Table 1. Performance comparison of QCL-BChE and BChE fluorescent probes in the literature.

[0065] References [1]Pei XY, Fang YH, Gu H., Zheng SY, Bin XX, Wang F., HeM. F., Lu S., Chen 2023, 287: 122044. [2]Zhang WD, Zhang JM, Qin CZ, Wang XR, Zhou YB A far-red / near-infrared fluorescence probe with large Stokes shift for monitoring butyrylcholinesterase (BChE) in living cells and in vivo. Analytica ChimicaActa, 2022, 1235: 340540. [3]Ma J. L., Lu X. F., Zhai H. L., Li Q., Qiao L., Guo Y. Rationaldesign of a near-infrared fluorescence probe for highly selective sensingbutyrylcholinesterase (BChE) and its bioimaging applications in living cell.Talanta, 2020, 219: 121278. [4]Liu S. Y., Xiong H., Yang J. Q., Yang S. H., Li Y. F., Yang W. C.,Yang G. F. Discovery of butyrylcholinesterase-activated near-infraredfluorogenic probe for live-cell and in vivo imaging. ACS Sensors, 2018, 3:2118-2128. [5]Tang Z. S., Xu S. L., Zhuang H. R., Qin J. Z., Zou H. R., Liu C.H., Xu Z. H., Zhang H., Hou X.F. A Golgi-targeted near-infrared fluorescentprobe for imaging butyrylcholinesterase in living cells. Spectrochimica ActaPart A: Molecular and Biomolecular Spectroscopy, 2026, 349: 127342. [6]Liu Q., Yu J. Y., Chen L. C., Han J. X., Cai X. Y., Hu S. J., ChuX. F., Zhang W. J., Wang Z. F. A deep-red hemicyanine fluorescent probe forimaging butyrylcholinesterase in living cells and in mice with APAP-inducedliver injury. Talanta, 2025, 286: 127478. [7]Cui J., Wang C. J, Feng T., He Q. M., Nie H. L, Yang W., Peng X.S. An endoplasmic reticulum-targeting NIR fluorescent probe for monitoringthe fluctuations of endogenous butyrylcholinesterase in live cells. Talanta,2025, 295: 128379. [8]Tao C. C., Wang C. J., Zeng C. Y., Li L. S., Bai J., Nie H. L. Ahighly selective ratiometric fluorescent probe for butyrylcholinesterase:Applications in quantification, residue screening, and toxicological studiesof organophosphorus pesticides. Food Chemistry,2025, 482: 144143. [9]Yin X. Y., Zhang G. N., Song G. X., Li X. R., Liu X. M., Wang L.F., Zhang H., Tang Z. X. A novel near-infrared fluorescent probe forbutyrylcholinesterase: Researchfor screening of natural anti-AD inhibitors.Analytica Chimica Acta, 2024, 1331: 343348.

[10] Yu Z. Q., Ma Y. S., Xu S., Yang L., Zhou Y. Q., Yang X. F., KongX. Q., Lv Y. F., Zhang J., Yan M. Bioinspired design of highly specificfluorescent probe for butyrylcholinesterase imaging in living cells andAlzheimer’s disease model. Sensors&Actuators: B. Chemical, 2024, 410: 135662.

[11] Kang W. X., Ma M., Xu L. L., Tang S., Li J. K., Ma P. Y., Song D.Q., Sun Y. Customized fluorescent probe for peering into the expression ofbutyrylcholinesterase in thyroid cancer. Analytica Chimica Acta, 2023, 1282:341932.

[12] Liu Z. X., Shi Y. W., Ding X. F., Zhang Q., Zhang P., Ding C. F.2-(2-Hydroxyphenyl)benzoazole-based ratiometric fluorescence probe for thedetection and imaging of butyrylcholinesterase activity in Alzheimer’sdisease mice brain. Dyes and Pigments, 2026, 246: 113354.

[13] Liu Q., Liu M., Hu S. J., Chu X.F., Zhou J. P., Ma D., Miao M.,Yang X. Y., Wang Z. F. Fast-response NIR “off-on” fluorescent sensor forhepaticinjury visualization through butyrylcholinesterase expressionmonitoring. Analytica Chimica Acta, 2025, 1367: 344332.

[14] Jian Zhang, Xiaojie Tang, Honglan Qi, Zhao Li, Xiaowei He. A newnear-infrared fluorescence probe for highly selective and sensitive detectionand imaging of Butyrylcholinesterase in Alzheimer’s disease mice. Talanta,2025, 285: 127377.

[15] Ji SY, Wang WJ, Ma SJ, Zhang PC, Xiang ZC, Liu GY, Wu J., Wang K., Pan J. A novel enzyme-activated tandem fluorescent probe for dual detection of BChE and Aβ plaques in Alzheimer's disease. Talanta, 2026, 300: 129169. Example 3: Fluorescence imaging of BChE in cells using QCL-BChE as an analytical reagent. Human normal hepatocytes (LO2 cells) and human hepatocellular carcinoma cells (HepG2 cells) were used as research subjects to conduct CCK-8 assays on the cytotoxicity of QCL-BChE. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells were treated with different concentrations of QCL-BChE (0–100 μM) and cultured for 12 hours before CCK-8 assays were performed. The results are shown below. Figure 18 (b). As can be seen from the figure, the cell survival rate in different concentrations of QCL-BChE is greater than 85%, indicating that QCL-BChE has low cytotoxicity to LO2 cells and HepG2 cells, and can be applied to cell imaging studies.

[0066] To determine the optimal incubation time between QCL-BChE and cells, multiple groups of LO2 cells were co-incubated with QCL-BChE (10 μM) for different times, followed by fluorescence imaging. The results are as follows: Figure 17 As shown, the fluorescence intensity of LO2 cells increased with prolonged incubation time with the probe QCL-BChE, eventually plateauing at 30 minutes. Therefore, subsequent imaging experiments used 30 minutes as the optimal incubation time for the probe QCL-BChE with cells.

[0067] Fluorescence imaging of endogenous BChE in normal human hepatocytes (LO2 cells) and human hepatocellular carcinoma cells (HepG2 cells) was performed using QCL-BChE. Both types of cells were treated in the same way and divided into five groups. The specific operation steps are as follows: The first group was the control group, and the cells were directly subjected to fluorescence imaging after incubation; the second group of cells were co-incubated with QCL-BChE (10 μM) for 30 minutes; the third group of cells were co-incubated with 100 μM Tacrine (a co-inhibitor of BChE and AChE) for 1 hour, washed with PBS buffer, and then co-incubated with QCL-BChE (10 μM) for 30 minutes; the fourth group of cells were co-incubated with 100 μM Donepezil (an acetylcholinesterase-specific inhibitor) for 1 hour, washed with PBS buffer, and then co-incubated with QCL-BChE (10 μM) for 30 minutes; the fifth group of cells were co-incubated with 100 μM APAP (4-Acetamidophenol, an acute liver injury inducer) for 12 hours, washed with PBS buffer, and then co-incubated with QCL-BChE (10 μM) for 30 minutes, and fluorescence imaging was performed as follows. Figure 18 As shown in (a). The results of the second group of experiments showed that LO2 cells and HepG2 cells showed obvious fluorescence signals after co-incubation with QCL-BChE, while the control group in the first group did not show obvious fluorescence signals. In addition, the fluorescence intensity of LO2 cells was significantly stronger than that of HepG2 cells, which was due to the significant reduction in BChE activity in HepG2 cells. Comparing the results of the second, third and fourth groups of experiments, we can find that the fluorescence signal of the system treated with tacrine and then co-incubated with QCL-BChE was significantly weakened, while the fluorescence signal of the system treated with donepezil and then co-incubated with QCL-BChE did not change significantly. This is because tacrine can inhibit the activity of BChE and AChE, while donepezil can only inhibit the activity of AChE. These experimental results not only demonstrate the specificity of QCL-BChE in detecting BChE in cells, but also allow for the monitoring of changes in intracellular BChE activity. To verify the actual effect of QCL-BChE in the diagnosis of acute liver injury, we conducted a fifth group of experiments. The results showed that the fluorescence signal of cells was significantly reduced after treatment with APAP (an acute liver injury inducer), which is consistent with the fact that the expression of BChE in cells with acute liver injury is reduced.

[0068] To investigate the difference in BChE expression between LO2 and HepG2 cells, we performed fluorescence imaging after mixing the two cell types. The specific procedure was as follows: First, LO2 and HepG2 cells were thoroughly mixed, and then the mixed cells were co-incubated with QCL-BChE (10 μM). Fluorescence imaging was performed every 3 minutes to observe the fluorescence. The results are shown below. Figure 19 As shown in the figure. The results revealed that the fluorescence images of the mixed cells showed significant differences over time. LO2 cells and HepG2 cells were located under white light, and the fluorescence images at the same locations were observed. The fluorescence intensity of LO2 cells was significantly stronger than that of HepG2 cells. This demonstrates that BChE expression gradually weakens after hepatocellular carcinogenesis.

[0069] The above experimental results show that QCL-BChE can perform fluorescence imaging of endogenous BChE in cells and has the ability to distinguish between normal human hepatocytes (LO2 cells) and human liver cancer cells (HepG2 cells).

[0070] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The compound QCL-BChE shown in Formula I: 。 2. The method for preparing the compound QCL-BChE according to claim 1, comprising the following steps: 1) Cyclopentanone is reacted with phosphorus tribromide (PBr3) to form compound 1; 2) React compound 1 with 2-hydroxy-4-methoxybenzaldehyde to generate compound 2; 3) Compound 2 is demethylated by boron tribromide (BBr3) to generate compound 3; 4) React compound 3 with N-ethylquinaldine iodide to generate QCL-OH; 5) React compound QCL-OH with cyclopropylformyl chloride to obtain compound QCL-BChE as shown in Formula I; 。 3. The preparation method according to claim 2, characterized in that: In step 1), the specific method for generating compound 1 from cyclopentanone under the action of phosphorus tribromide (PBr3) is as follows: at 0°C, phosphorus tribromide (PBr3) is added to a mixed solution of DMF and CHCl3, stirred for 45 min, then cyclopentanone is added, and the reaction is stirred to obtain compound 1; in the reaction, the molar ratio of cyclopentanone to phosphorus tribromide (PBr3) is 1:2.3; the reaction temperature is 25°C, and the reaction time is 16 h; And / or, in step 2), the specific method for reacting compound 1 and 2-hydroxy-4-methoxybenzaldehyde to generate compound 2 is as follows: dissolving compound 1 and 2-hydroxy-4-methoxybenzaldehyde in DMF, adding cesium carbonate and stirring to react, thereby obtaining compound 2; the molar ratio of compound 1, 2-hydroxy-4-methoxybenzaldehyde and cesium carbonate in the reaction is 1:1.2:2.5 respectively; the reaction temperature is 25℃ and the reaction time is 16 h; And / or, in step 3), the specific method for removing the methyl group from compound 2 under the action of boron tribromide (BBr3) to generate compound 3 is as follows: under nitrogen protection, compound 2 is dissolved in anhydrous dichloromethane, boron tribromide (BBr3) is slowly added under an ice-water bath, stirred for 1 h, and then slowly raised to room temperature to react and obtain compound 3; the molar ratio of compound 2 to boron tribromide (BBr3) in the reaction is 1:30; the reaction temperature is 25℃ and the reaction time is 4 h; And / or, in step 4), the specific method for reacting compound 3 and N-ethylquinaldine iodide to generate QCL-OH is as follows: compound 3 and N-ethylquinaldine iodide are dissolved in anhydrous ethanol, then piperidine is added, and the mixture is refluxed under nitrogen protection to obtain compound QCL-OH; the molar ratio of compound 3, N-ethylquinaldine iodide, and piperidine in the reaction is 1:1.2:1.96; the reaction temperature is 80℃, and the reaction time is 4 h; And / or, in step 5), the specific method for reacting compound QCL-OH with cyclopropylformyl chloride to obtain compound QCL-BChE of formula I is as follows: under nitrogen protection, compound QCL-OH is dissolved in dichloromethane, triethylamine is added, and then cyclopropylformyl chloride dissolved in dichloromethane is added dropwise to react and obtain compound QCL-BchE; the molar ratio of compound QCL-OH, cyclopropylformyl chloride and triethylamine in the reaction is 1:1.2:2 respectively; the reaction temperature is 25℃ and the reaction time is 6 h.

4. A fluorescent probe, characterized in that: The fluorescent probe is the compound QCL-BChE as described in claim 1.

5. A chemical sensor, characterized in that: The chemical sensor contains the compound QCL-BChE as described in claim 1.

6. The application of the compound QCL-BChE of claim 1, the fluorescent probe of claim 4, or the chemical sensor of claim 5 in the detection of BChE or in cell fluorescence imaging; wherein the cell is a cell containing endogenous BChE.

7. The application of the compound QCL-BChE according to claim 1, selected from at least one of the following: a) Application of QCL-BChE as a fluorescent probe or as a fluorescent probe for detecting BChE; b) Application of QCL-BChE in the preparation of chemical sensors or in the preparation of chemical sensors for detecting BChE; c) Application of QCL-BChE in the screening and / or evaluation of BChE inhibitors; d) Application of QCL-BChE in BChE detection.

8. The application according to claim 7, characterized in that: The fluorescent probe or chemical sensor is used for the detection and / or fluorescence imaging of BChE.

9. The application according to claim 7 or 8, characterized in that: The BChE is either exogenous or endogenous.

10. The application according to any one of claims 7-9, characterized in that: The fluorescent probe or chemical sensor is applied to cells; Furthermore, the cells are normal human liver cells and human liver cancer cells.

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