A butyrylcholinesterase and viscosity dual-responsive FRET fluorescent probe, and a preparation method and use thereof

Abstract

The application discloses a FRET fluorescent probe with dual response to acetylcholinesterase and viscosity, a preparation method and application thereof, wherein the structure of the FRET fluorescent probe is shown in the following formula: The fluorescent probe is based on a fluorescence resonance energy transfer (FRET) mechanism, responds to acetylcholinesterase (AChE) at a short wave of 520 nm, and the detection limit is as low as 0.104 U / L, and responds to viscosity at a long wave of 680 nm, and the wavelength difference is as high as 160 nm, and the dual-channel response has no crosstalk. Cell toxicity test shows that the probe has good biocompatibility, and confocal fluorescence microscopic imaging experiment shows that the probe has good light stability for cells, has the ability of simultaneously monitoring the concentration change of AChE in cells and the viscosity fluctuation of cells, and is suitable for confocal fluorescence imaging for tracing the concentration change of AChE in cells and the viscosity fluctuation.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and fluorescence imaging technology, specifically relating to a FRET fluorescent probe that is dually responsive to acetylcholinesterase and viscosity, its preparation method, and its applications. Background Technology

[0002] In recent years, the simultaneous detection of key biomolecules and microenvironment parameters within living systems has become a cutting-edge research topic in chemical biology. Acetylcholinesterase (AChE), a core hydrolytic enzyme in nerve signal transduction, is a key marker of neurodegenerative diseases such as Alzheimer's disease and organophosphate poisoning when its activity is abnormal. Meanwhile, intracellular viscosity, as an important physical parameter of the microenvironment, directly affects substance transport and metabolism, and its imbalance is closely related to various disease processes such as diabetes, liver fibrosis, and neuropathy. Traditional detection methods, such as the Elman method for AChE determination or in vitro rheological techniques for viscosity assessment, have inherent limitations, including cumbersome operation and the inability to perform in-situ real-time monitoring.

[0003] With the development of fluorescence sensing technology, probes based on the fluorescence resonance energy transfer (FRET) principle have provided powerful tools for the dynamic visualization of intracellular biological parameters due to their advantages such as high sensitivity, ratiometric signal output, and spatiotemporal resolution imaging. Currently, studies have developed FRET probes targeting AChE activity or viscosity response, achieving precise detection of single parameters. However, complex life activities often involve the synergistic changes of multiple physiological and pathological variables. For example, in the progression of Alzheimer's disease, AChE dysfunction and increased local viscosity caused by amyloid protein aggregation often coexist. Existing single-function probes struggle to simultaneously reveal the intrinsic relationships between these multiple factors, hindering in-depth analysis of disease mechanisms.

[0004] Therefore, developing a FRET fluorescent probe that can simultaneously respond to AChE and viscosity, achieving a "two-in-one" effect, is of urgent significance for advancing research on related diseases. Such probes not only promise crosstalk-free, ratiometric quantitative analysis of enzyme activity and microenvironment physical properties in the same biological sample, but also provide an unprecedented dynamic visualization tool for studying their interaction in physiological and pathological processes, marking a crucial step forward for multifunctional fluorescence sensing technology towards simulating complex biological systems. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a FRET fluorescent probe that is dual-responsive to acetylcholinesterase (AChE) and viscosity, along with its preparation method and applications. The technical problem this invention aims to solve is to obtain a molecular structure that is dual-responsive to AChE and viscosity through molecular design, requiring that this molecular structure exhibit a dual-channel, crosstalk-free fluorescence response to both AChE and viscosity. This invention, based on the fluorescence resonance energy transfer (FRET) mechanism, is a mitochondrial-targeting fluorescent probe capable of detecting acetylcholinesterase concentration and viscosity fluctuations in solution, as well as providing fluorescence tracing of changes in acetylcholinesterase concentration and microenvironment viscosity fluctuations within living cells. It possesses advantages such as selectivity, high detection efficiency, and good biocompatibility.

[0006] This invention designs and synthesizes a dual-response FRET fluorescent probe, YONA, using naphthimide as the fluorophore. Naphthalimide serves as the electron donor, and a quaternized quinoline unit with a strong positive charge acts as the electron acceptor. The donor and acceptor are connected via a piperazine bridge, allowing electrons from the donor to be transferred to the acceptor for FRET. Dimethylformyl chloride is introduced as the recognition group for acetylcholinesterase into the phenolic site, and a benzyl group is used to connect the fluorescent backbone and the recognition group to obtain probe YONA. Upon response to acetylcholinesterase, the benzyl moiety undergoes a "1,6-elimination" process, releasing the fluorophore and enhancing fluorescence emission at 520 nm. Simultaneously, based on the TICT effect, in low-viscosity environments, the intramolecular rotational freedom of YONA leads to an orthogonal conformation of the donor and acceptor, resulting in a TICT effect and fluorescence quenching of the probe. In high-viscosity environments, molecular rotation is restricted, the TICT effect weakens, and non-radiative energy consumption decreases, causing the probe to emit a strong red fluorescent signal at 680 nm. Tests on its optical properties and cytotoxicity demonstrated that YONA exhibits high selectivity, low detection limit, and good biocompatibility. This study evaluated the feasibility of using YONA for specific fluorescence imaging of AChE and viscosity in living cells through analysis of confocal fluorescence imaging, aiming to provide a convenient chemical tool for the visual detection of AChE and viscosity in solution and in living cells, and for research on related diseases.

[0007] The FRET fluorescent probe of this invention, abbreviated as YONA, has the following structure:

[0008] .

[0009] The method for preparing the FRET fluorescent probe of the present invention includes the following steps:

[0010] Step 1: Under nitrogen protection, 2-bromo-1,8-naphthalene anhydride and p-aminobenzoic acid were dissolved in DMF and reacted at 90°C for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and a yellow precipitate was formed. The yellow solid was collected by filtration and recorded as intermediate 1.

[0011] Step 2: Under nitrogen protection, intermediate 1 and tetrahydropyrrole are dissolved in N,N dimethylformamide, trimethylamine is added, and the mixture is heated to reflux. After the reaction is complete, the reaction solution is cooled to room temperature, and then the pH is adjusted until a large amount of orange solid precipitates. The orange solid is collected by filtration and recorded as intermediate 2.

[0012] Step 3: Under nitrogen protection, piperazine was heated to reflux in a mixture of water and 2-methoxyethanol for 20 minutes. Then, a 2-methoxyethanol solution of 4-fluorobenzaldehyde was added dropwise over 30 minutes. The mixture was stirred at 110°C for 5 hours. After the reaction was completed, the mixture was poured into ice water, extracted and dried, and separated by column chromatography to obtain a white solid, which was designated as intermediate 3.

[0013] Step 4: Dissolve p-hydroxybenzyl alcohol and dimethylcarbamoyl chloride in acetone, then add cesium carbonate powder and heat to reflux. After the reaction is complete, filter, extract and dry to obtain a colorless oily compound, which is designated as intermediate 4.

[0014] Step 5: Under nitrogen protection, intermediate 4 and potassium iodide were dissolved in acetonitrile, then TMCS was added and heated to reflux. After the reaction was completed, the mixture was poured into ice water, extracted and dried to obtain a red solid, which was designated as intermediate 5.

[0015] Step 6: Dissolve intermediate 5 in acetonitrile solution, add 4-methylquinoline, stir and react overnight at 80°C, evaporate the solvent, extract and dry, separate by column chromatography, and obtain a yellow solid, which is designated as intermediate 6.

[0016] Step 7: Dissolve intermediates 3 and 6 in ethanol, add piperidine, stir and react overnight at 80°C, evaporate the solvent, extract and dry, separate by column chromatography, and obtain a red solid, which is designated as intermediate 7.

[0017] Step 8: Dissolve intermediates 7 and 2 in dichloromethane, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and react at room temperature for 24 h; evaporate the solvent, extract and dry, and separate by column chromatography to obtain red solid YONA.

[0018] Step 9: Diethylamine and intermediate 2 were dissolved in dichloromethane, then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added, and the mixture was reacted at room temperature for 24 h. The solvent was evaporated, the mixture was extracted and dried, and the residue was separated by column chromatography to obtain a yellow solid, Donor. This solid serves as an electron donor group for subsequent mechanism verification.

[0019] Step 10: Dissolve p-diethylaminobenzaldehyde and intermediate 6 in ethanol, stir overnight at 80°C, evaporate the solvent, extract and dry, separate by column chromatography, and obtain purple solid Acceptor-1. This serves as an electron acceptor group for subsequent mechanism verification.

[0020] The synthesis route is shown below:

[0021]

[0022] The application of the FRET fluorescent probe of this invention in the preparation of AChE and / or viscosity detection reagents.

[0023] The detection reagent is in I 520 nm The fluorescence intensity at point I is linearly related to the concentration of AChE. 680 nm The logarithm of the fluorescence intensity is linearly related to the logarithm of the system viscosity.

[0024] This invention relates to the FRET fluorescent probe YONA, which uses naphthimide as the fluorophore. When only the probe is present, FRET is activated, and the donor electrons are transferred to the acceptor via energy resonance. After receiving the electrons, the acceptor's fluorescence is turned off. When the probe and AChE coexist, the probe's molecular structure changes to YONA, FRET is turned off, and the donor electrons cannot be transferred to the acceptor. The donor receives electrons and emits strong fluorescence at 520 nm, thus activating the fluorescence and enabling a switch-type detection of AChE. Its AChE response is illustrated below:

[0025]

[0026] The detection method is as follows:

[0027] A 2 mM stock solution was prepared by dissolving YONA in DMSO. 15 μL of this stock solution was then added to 3 mL of PBS containing different concentrations of AChE. The fluorescence and UV spectra of 10 μM YONA in different test solutions were obtained. With increasing AChE concentration, the absorbance of YONA increased at 485 nm. A new fluorescence emission appeared at 520 nm, and the fluorescence intensity gradually increased. Furthermore, the fluorescence of YONA increased by more than 35 times before and after the reaction with AChE, demonstrating the ability of on-type fluorescence detection. The fluorescence intensity showed a good linear relationship with the concentration of AChE (R0). 2=0.99), YONA responded normally to AChE within the physiological pH range and showed good response to AChE in cells. 15 μL of the stock solution was added to 3 mL of PBS containing different concentrations of Gly to obtain the fluorescence and UV spectra of 10 μM YONA in different test solutions. With increasing Gly concentration, the absorbance of YONA increased at 480 nm. The fluorescence intensity of YONA gradually increased at 680 nm, and its fluorescence intensity showed a good logarithmic linear relationship with the viscosity of Gly (R0). 2 =0.99).

[0028] This invention provides a FRET fluorescent probe for detecting acetylcholinesterase (AChE) and viscosity. It exhibits a fluorescence-activated response to different concentrations of AChE, with a good linear relationship between fluorescence intensity and AChE concentration, and an effective limit of detection as low as 0.104 U / L. Furthermore, the probe YONA, in a glycerol and water system (40%-100%), responds well to viscosity, with a good linear relationship between the logarithm of the fluorescence signal and the logarithm of the viscosity. Cytotoxicity tests demonstrate the good biocompatibility of YONA, and confocal fluorescence microscopy experiments show its good photostability, enabling it to monitor changes in intracellular and extracellular AChE concentrations and detect intracellular viscosity fluctuations. Attached Figure Description

[0029] Figure 1 This section describes the photophysical properties of the probe YONA in response to AChE. (a) shows the changes in the UV absorption spectrum of YONA (10 μM) reacting with different concentrations of AChE; (b) shows the changes in the fluorescence emission spectrum of YONA (10 μM) reacting with different concentrations of AChE (0–2 U / mL); and (c) shows a good linear relationship between the fluorescence intensity and the concentration of AChE after the reaction of YONA (10 μM) with different concentrations of AChE (0.2–2 U / mL). 2 =0.997), the limit of detection was 0.104 U / L; (d) the reaction time of YONA (10 μM) in response to AChE (2 U / mL).

[0030] Figure 2 This describes the photophysical properties of YONA in response to viscosity. (a) shows the changes in the UV absorption spectra of YONA (10 μM) with different water and glycerol systems; (b) shows the changes in the fluorescence emission spectra of YONA (10 μM) with water and glycerol systems of different viscosities (0-100%); and (c) shows the linear relationship (R0) between the logarithm of fluorescence intensity and the logarithm of viscosity for YONA (10 μM) with water and glycerol systems of different viscosities (40%-100%). 2=0.993); (d) 3D fluorescence of YONA (10 μM) in glycerol.

[0031] Figure 3 (a) The normalized absorption spectrum of the acceptor and the normalized fluorescence spectrum of the donor in PBS; (b) The emission spectra of YONA (10 μM) and the donor Donor (10 μM) in PBS.

[0032] Figure 4 This study investigates the pH stability of YONA. It explores whether the pH stability of YONA (10 μM) reacted with AChE (2 U / mL) for 30 min, and whether the physiological pH range affects YONA during the reaction with AChE.

[0033] Figure 5 The selectivity of YONA for (a) viscosity and (b) AChE was tested. The interaction between YONA (10 μM) and Cu was investigated. 2+ Fe 2+ Ni 2+ Ca 2+ Zn 2+ K + Mg 2+ CO3 2- NO 3- SO4 2- NO 2- CN - S 2- S2O3 2- F - HPO4 2- HCO 3- HSO4 2- Changes in fluorescence intensity after reactions with glycine, cysteine, glutathione (GSH), tyrosine (TYR), carboxylesterase (CES), leucine aminopeptidase (LAP), sulfatase (SULF), nicotinamide adenine dinucleotide (NADH), nitroreductase (NTR), glycerol, and AChE.

[0034] Figure 6 This is a test of YONA on endogenous AChE in live cells. (a) shows confocal imaging after co-incubating PC-12 cells and HepG2 cells with YONA (10 μM) for 30 min, respectively, to investigate the detection ability of YONA on endogenous AChE; (b) shows the fluorescence intensity of the red and green channels in Figure (a).

[0035] Figure 7This study tested YONA's ability to detect exogenous AChE in live cells. PC-12 cells were first co-incubated with AChE for 30 min. Then, YONA (10 μM) was co-incubated with both PC-12 cells treated with AChE and those without AChE (Control group) for 30 min before confocal imaging to investigate YONA's ability to detect exogenous AChE. (b) shows the fluorescence intensity of the red and green channels in Figure (a).

[0036] Figure 8 This study tested YONA's ability to detect changes in intracellular viscosity. Cells were pretreated with nystain for 30 min, followed by incubation with YONA (10 μM) for 30 min in both the pretreated cells and the control group, and then confocal imaging was performed. The aim was to investigate YONA's ability to detect intracellular viscosity fluctuations. (b) shows the fluorescence intensity of the red and green channels in Figure (a).

[0037] Figure 9 This study tested YONA's ability to detect changes in intracellular viscosity and AChE induced by LPS in living cells. First, cells were pretreated with LPS for 30 min. Then, YONA (10 μM) was incubated with both the pretreated cells and the control group for 30 min before confocal imaging. The aim was to investigate YONA's ability to detect intracellular AChE and viscosity. (b) shows the fluorescence intensity of the red and green channels in Figure (a). Detailed Implementation

[0038] The present invention will be further illustrated by the following examples.

[0039] Example 1: Synthesis of YONA

[0040] Compound 2 (309 mg, 0.8 mmol) was dissolved in DMF under a nitrogen atmosphere and at 0 °C. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (153 mg, 0.8 mmol) and 4-dimethylaminopyridine (DMAP) (25 mg, 0.2 mmol) were added and reacted for 0.5 hours. Then, compound 7 (200 mg, 0.4 mmol) was added and reacted for another 0.5 hours. Finally, the reaction was carried out at room temperature for 18 hours. After the reaction was complete, diethyl ether was added to the mixture until a red solid precipitated. The solid was then filtered and dried to obtain the crude product. The crude product was purified by column chromatography (dichloromethane / methanol = 10:1) to obtain the target product YONA as a red powder.

[0041] 1H NMR (400 MHz, DMSO-d6) δ 9.43 (d, J = 6.7 Hz, 1H), 9.11 – 9.06 (m,1H), 8.82 (dd, J = 8.7, 1.1 Hz, 1H), 8.54 – 8.45 (m, 2H), 8.35 (d, J = 8.9Hz, 1H), 8.29 – 8.23 ​​(m, 2H), 8.18 – 8.11 (m, 2H), 7.95 (dt, J = 6.9, 3.3 Hz, 3H), 7.67 (dd, J = 8.6, 7.3 Hz, 1H), 7.63 – 7.58 (m, 2H), 7.45 (d, J = 8.2Hz, 2H), 7.40 – 7.36 (m, 2H), 7.12 (dd, J = 8.8, 6.8 Hz, 4H), 6.94 (d, J =8.9 Hz, 1H), 6.19 (s, 2H), 3.82 (d, J = 6.4 Hz, 4H), 3.51 (s, 8H), 3.00 (s,3H), 2.88 (s, 3H), 2.07 – 2.02 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 169.30,164.53, 163.49, 154.49, 154.30, 152.93, 151.84, 147.65, 145.35, 138.44,138.21, 135.73, 135.49, 133.53, 131.85, 131.81, 131.63, 131.29, 130.18,130.01, 129.25, 128.64, 128.16, 127.02, 125.97, 123.76, 123.05, 122.37, 119.79, 115.46, 114.91, 109.37, 109.00, 58.72, 55.49, 53.47, 36.81, 36.61, 31.82, 29.57, 29.11, 27.08, 26.12, 22.63, 14.49.

[0042] Example 2: Optical titration, linearity, and response time of YONA in solvent in response to AChE

[0043] To investigate the optical titration of YONA in solvent in response to AChE, UV absorption and fluorescence titration were performed using AChE (0-2 U / mL). Figure 1 (a) It can be seen that the UV absorption of YONA at 485 nm gradually increases with increasing AChE concentration. From Figure 1 (b) It can be seen that with the addition of AChE, YONA reached the titration endpoint at 2 (U / mL). From Figure 1 (c) It can be seen that the fluorescence signal of YONA shows a good linear relationship in the detection of AChE (0.2-2 U / mL), with a linear correlation coefficient R0. 2 =0.997, the limit of detection is 0.104 U / L. From Figure 1 (d) shows the reaction time of PEG-gal in response to AChE (2 U / mL) in the solvent. It can be seen that the reaction between YONA and AChE basically ends at 30 min. This indicates that YONA and AChE can emit a good fluorescence signal after reaction, and AChE can be accurately monitored in the solvent.

[0044] Example 3: Optical titration, linearity, and 3D fluorescence of YONA in a glycerol and water system

[0045] To explore the optical titration of YONA in a glycerol and water system in response to viscosity, UV absorption and fluorescence titration were performed using glycerol (0-100%). Figure 2 (a) It can be seen that the UV absorption at 480 nm gradually increases with increasing glycerol concentration. From Figure 2 (b) It can be seen that the emission peak at 680 nm gradually increases with increasing glycerol concentration. From Figure 2 (c) It can be seen that the fluorescence signal of YONA shows a good linear relationship in the detection of glycerol (40%-100%), with a linear correlation coefficient R. 2 =0.993. This indicates that YONA can detect changes in viscosity within a solvent. From Figure 2 (d) It can be seen that the optimal excitation wavelength of the probe YONA in response to viscosity is 480 nm.

[0046] Example 4: FRET mechanism of probe YONA in response to AChE in solvent

[0047] To investigate the FRET mechanism of YONA in response to AChE (2 U / mL) in solvent. Figure 3(a) The normalized absorption spectrum of the acceptor and the normalized fluorescence spectrum of the donor in PBS show a significant overlap, indicating that the donor-to-acceptor FRET process is significantly effective. Figure (b) shows the emission spectra of YONA (10 µM) and Donor (10 µM) in PBS, obtained using the formula E=1-F. DA / F D The energy transfer efficiency of the YONA molecule can be calculated to be 85.2%. This demonstrates that the probe YONA responds to AChE in solvent via a FRET mechanism.

[0048] Example 5: pH stability of YONA in solvent in response to AChE

[0049] To investigate the pH stability of YONA in response to AChE (2 U / mL). Figure 4 By comparing the fluorescence changes of YONA and AChE in solvents with different pH values ​​after 30 minutes, it was found that YONA can respond to AChE within the pH range of the physiological environment.

[0050] Example 6: Selective Response Experiment of YONA to AChE in Solvent

[0051] To investigate the specificity of YONA in response to AChE (2 U / mL) in solvent, YONA was tested against common enzymes, metal ions, and reactive oxygen species in solvent. Figure 5 (a) This indicates that YONA is unaffected by other metal ions, enzymes, and other reactive oxygen species at 520 nm, exhibiting only a good response to AChE, and the fluorescence intensity after the response is much higher than that of other detectable substances. Figure 5 (b) This indicates that YONA is not affected by other metal ions, enzymes and other reactive oxygen species at 680 nm, and has a good response to viscosity only, and the fluorescence intensity after the response is much higher than that of other detectable substances.

[0052] Example 7: Detection of endogenous AChE in PC-12 and G2 cells using YONA

[0053] To investigate the imaging ability of YONA on endogenous AChE in cells, the probe YONA (10 µM) was incubated with normal PC-12 and G2 cells for 30 min, respectively. Confocal imaging was then performed. Figure 6 Because PC-12 cells contain endogenous AChE, the fluorescence intensity of the channel at 520 nm in PC-12 cells was significantly enhanced compared to G2 cells, while the channel at 680 nm showed no significant change. These results indicate that YONA has the ability to detect endogenous AChE within cells.

[0054] Example 8: YONA detection of exogenous AChE

[0055] To investigate the imaging ability of YONA against exogenous AChE in cells, the probe YONA (10 µM) was incubated with PC-12 cells and AChE for 30 min. Confocal imaging was then performed. Figure 7 In the 520 nm channel, cells with added AChE showed stronger fluorescence enhancement than the normal group, indicating that YONA has the ability to detect endogenous and exogenous AChE in cells.

[0056] Example 9: YONA's test on viscosity change

[0057] To explore the ability of YONA to detect viscosity changes in cells, PC-12 cells were induced with 10 μM nystain, followed by incubation with 10 μM YONA for 30 min, and then confocal imaging was performed. Figure 8 As shown, after viscosity induction, the fluorescence intensity of the 680 nm channel in PC-12 cells was significantly enhanced compared to normal cells, while the green channel remained almost unchanged. These results indicate that YONA has the ability to detect changes in intracellular viscosity.

[0058] Example 10: YONA's effect on LPS-induced changes in intracellular AChE and viscosity

[0059] To investigate the effect of YONA on LPS-induced changes in intracellular AChE and viscosity, PC-12 cells were induced with 10 μM LPS, followed by incubation with 10 μM YONA for 30 min, and then confocal imaging was performed. Figure 9 As shown, after viscosity induction, the fluorescence intensity at 520 nm and 680 nm in PC-12 cells was significantly enhanced, indicating that YONA has the ability to detect LPS-induced changes in intracellular AChE and viscosity.

Claims

1. A FRET fluorescent probe, abbreviated as YONA, characterized in that... Its structure is as follows: 。 2. The method for preparing the FRET fluorescent probe according to claim 1, characterized in that... Includes the following steps: Step 1: Under nitrogen protection, 2-bromo-1,8-naphthalene anhydride and p-aminobenzoic acid were dissolved in DMF and reacted at 90°C. After the reaction was completed, the reaction solution was cooled to room temperature, and a yellow precipitate was formed. The yellow solid was collected by filtration and recorded as intermediate 1. Step 2: Under nitrogen protection, intermediate 1 and tetrahydropyrrole were dissolved in N,N dimethylformamide, trimethylamine was added, and the mixture was heated to reflux. After the reaction was completed, the reaction solution was cooled to room temperature, and then the pH was adjusted until a large amount of orange solid precipitated. The orange solid was collected by filtration and recorded as intermediate 2. Step 3: Under nitrogen protection, piperazine was heated to reflux in a mixture of water and 2-methoxyethanol for 20 minutes. Then, a 2-methoxyethanol solution of 4-fluorobenzaldehyde was added dropwise over 30 minutes. The mixture was stirred at 110°C. After the reaction was completed, the mixture was poured into ice water, extracted and dried, and separated by column chromatography to obtain a white solid, which was designated as intermediate 3. Step 4: Dissolve p-hydroxybenzyl alcohol and dimethylcarbamoyl chloride in acetone, then add cesium carbonate powder and heat to reflux. After the reaction is complete, filter, extract and dry to obtain a colorless oily compound, denoted as intermediate 4. Step 5: Under nitrogen protection, intermediate 4 and potassium iodide were dissolved in acetonitrile, then TMCS was added and heated to reflux. After the reaction was completed, the mixture was poured into ice water, extracted and dried to obtain a red solid, which was designated as intermediate 5. Step 6: Dissolve intermediate 5 in acetonitrile solution, add 4-methylquinoline, stir the reaction at 80°C, evaporate the solvent after the reaction is complete, extract and dry, separate by column chromatography to obtain a yellow solid, which is denoted as intermediate 6; Step 7: Dissolve intermediates 3 and 6 in ethanol, add piperidine, stir the reaction at 80°C, evaporate the solvent after the reaction is complete, extract and dry, separate by column chromatography, and obtain a red solid, which is denoted as intermediate 7. Step 8: Dissolve intermediates 7 and 2 in dichloromethane, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and react at room temperature; after the reaction is complete, evaporate the solvent, extract and dry, and separate by column chromatography to obtain red solid YONA; The synthesis route is shown below: 。 3. The use of the FRET fluorescent probe of claim 1 in the preparation of AChE and / or viscosity detection reagents.

4. The application according to claim 3, characterized in that: The detection reagent is in I 520 nm The fluorescence intensity at the point is linearly related to the concentration of AChE.

5. The application according to claim 3, characterized in that: The detection reagent is in I 680 nm The logarithm of the fluorescence intensity is linearly related to the logarithm of the system viscosity.

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