Fluorescent probe for detecting monoamine oxidase as well as preparation method and application of fluorescent probe

Abstract

The invention provides a fluorescent probe for detecting monoamine oxidase as well as a preparation method and application of the fluorescent probe. The fluorescent probe disclosed by the invention is high in sensitivity and high in speed when being used for detecting the monoamine oxidase.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe for detecting monoamine oxidase, its preparation method and application, belonging to the technical field of fluorescence detection. Background Art

[0002] Monoamine oxidase (MAO) belongs to the flavin-dependent enzyme family and is produced in most cell types. Its main function is to catalyze the oxidation of biogenic amines to produce corresponding aldehydes. Measuring MAO is helpful for the diagnosis of liver diseases, especially for the early diagnosis of liver cirrhosis. For this important enzyme in the living system, further study of its biological distribution and physiological functions is beneficial to effective intervention to prevent the occurrence and development of related major diseases. With the improvement of organic synthesis technology, high-performance fluorescent probes based on basic dyes have been prepared, which can be used as effective visual fluorescence tracking tools for monoamine oxidase in the living system. The existing fluorescent probes for detecting MAO have low detection sensitivity or require dual detection with other enzymes, making it difficult to meet the actual application needs. Summary of the Invention

[0003] The present invention provides a fluorescent probe for detecting monoamine oxidase, its preparation method and application, which can effectively solve the above problems.

[0004] A fluorescent probe for detecting monoamine oxidase has the following structural formula:

[0005]

[0006] A method for detecting monoamine oxidase is to add the above-mentioned fluorescent probe into a sample to be tested, react for a period of time, detect the fluorescence spectrum, and qualitatively or quantitatively analyze monoamine oxidase according to the spectrum.

[0007] In some embodiments, the wavelength of the excitation light for detecting the fluorescence spectrum is 610 - 630 nm.

[0008] In some embodiments, the temperature for the reaction for a period of time is 36 - 38 °C, the time is 3.5 - 4.5 h, and the pH is 7.2 - 7.6.

[0009] In some embodiments, the use concentration of the fluorescent probe is 0.01 - 0.05 mM.

[0010] A preparation method for the above-mentioned fluorescent probe for detecting monoamine oxidase includes the following steps:

[0011] (1) Stir and heat the mixture of basic blue 3, sodium carbonate and dichloromethane to 40 - 50 °C under the protection of inert gas, and slowly add an aqueous solution of sodium dithionite at this temperature. React the mixture at 40 - 50 °C until the solution changes from blue to red. After the reaction is completed, stop heating and keep stirring for 8 - 12 min, then let it stand until the system is stratified. When the system cools to room temperature, slowly add triphosgene dissolved in dichloromethane under ice bath conditions. Stop the reaction after 50 - 70 min of reaction. Extract, collect the organic phase, and purify the crude product to obtain the light blue solid target product, which is product 1.

[0012] (2) Dissolve p-hydroxybenzaldehyde, tert-butyl (3-bromopropyl)carbamate and anhydrous potassium carbonate in acetonitrile. Stir and react at 80 - 90 °C for 8 - 12 h, then end the reaction. Filter to remove the solid, remove the solvent, and obtain a white oily substance, which is product 2.

[0013] (3) Dissolve product 2 in dichloromethane, add trifluoroacetic acid dropwise, stir at room temperature for 25 - 35 min, rotary evaporate to remove the solvent and trifluoroacetic acid. Then add dichloromethane, add triethylamine dropwise to adjust the pH to 7, and then add allyl chloroformate. React at room temperature for 7 - 9 h and then end the reaction. Remove the solvent and purify the crude product to obtain product 3.

[0014] (4) Dissolve product 3 in methanol, add sodium borohydride under ice bath conditions. After reacting for 1.5 - 2.5 h, rotary evaporate to remove the solvent, add an appropriate amount of water to the system, and extract three times with ethyl acetate. Remove the solvent to obtain the light yellow oily target product, which is product 4.

[0015] (5) Dissolve product 1, product 4, 4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate in dichloromethane. React at room temperature under the protection of inert gas for 7.5 - 8.5 h and then end the reaction. Rotary evaporate to remove the solvent and purify the crude product to obtain the light blue solid target product, which is product 5.

[0016] (6) Dissolve product 5 in tetrahydrofuran, add tetrakis(triphenylphosphine)palladium, and then add formic acid dropwise. React at 25 - 35 °C for 3.5 - 4.5 h and then end the reaction. Rotary evaporate to remove the solvent and purify the crude product to obtain the light blue solid target product, which is the described fluorescent probe.

[0017] In some embodiments, in step (1), the molar ratio of basic blue 3:sodium carbonate:sodium dithionite:triphosgene is 1:3.5 - 4.5:3.5 - 4.5:1.5 - 2.5; in step (2), the molar ratio of p-hydroxybenzaldehyde:tert-butyl (3-bromopropyl)carbamate:anhydrous potassium carbonate is 3 - 5:4 - 6:18 - 22.

[0018] In some embodiments, in step (3), the molar ratio of product 2: trifluoroacetic acid: allyl chloroformate is 1: 1.5 - 2.5: 1.0 - 1.5; in step (4), the molar ratio of product 3: sodium borohydride is 1: 1 - 2.

[0019] In some embodiments, in step (5), the molar ratio of product 1: product 4: DMAP: anhydrous sodium carbonate is 1: 0.8 - 1.2: 1.0 - 2.0: 2.5 - 3.5.

[0020] In some embodiments, in step (6), the molar ratio of product 5: tetrakis(triphenylphosphine)palladium: formic acid is 1: 0.08 - 0.12: 2.5 - 3.5.

[0021] The beneficial effects of the present invention are as follows:

[0022] The fluorescent probe of the present invention has a unique activation mechanism, that is, it can be effectively activated and release a significant fluorescent signal in a specific environment where only monoamine oxidase (MAO) exists. This characteristic makes the fluorescent probe have broad application prospects in the medical field, especially in the process of monitoring diseases caused by overexpression of monoamine oxidase, such as Parkinson's disease, epilepsy and other diseases, and can play an important role.

[0023] The fluorescent probe of the present invention shows extremely high sensitivity in detecting monoamine oxidase, can quickly respond in the presence of extremely low concentration of MAO, and the detection speed is also significantly better than the prior art. This characteristic greatly improves the detection efficiency and accuracy, and provides a powerful tool for clinical diagnosis.

[0024] The method for detecting monoamine oxidase of the present invention is designed to be relatively simple and easy to operate, and the detection process is very convenient. Users only need to use a conventional fluorescence detector to complete the entire detection process, without complex instruments and cumbersome operation steps, which greatly reduces the detection threshold and cost.

[0025] The detection signal generated by the method for detecting monoamine oxidase of the present invention is very obvious, and the color change of the reaction solution can be directly observed by the naked eye. This intuitive detection result is not only convenient for quick judgment, but also improves the credibility and reliability of the detection, providing strong support for the early detection and timely treatment of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0027] Figure 1 1H NMR spectrum of the fluorescent probe prepared in Example 1.

[0028] Figure 2 1H-13C NMR spectrum of the fluorescent probe prepared in Example 1.

[0029] Figure 3 High-resolution mass spectrum of the fluorescent probe prepared in Example 1.

[0030] Figure 4 Fluorescence spectrum of the fluorescent probe in response to monoamine oxidase in Example 2.

[0031] Figure 5 Cell imaging of the fluorescent probe incubated with cells in Example 3.

[0032] Figure 6 Synthetic route diagram of the fluorescent probe of the embodiment of the present invention. Detailed implementation manners

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention.

[0034] The embodiments of the present invention provide a fluorescent probe for detecting monoamine oxidase, and its structural formula is as follows:

[0035]

[0036] The present invention provides a near-infrared fluorescent probe based on basic dyes, with the Chinese name: 4-(3-aminopropoxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate, and its English name: 4-(3-aminopropoxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate. It is used to detect monoamine oxidase (MAO), with basic blue 3 as the fluorophore and propylamino as the recognition site for MAO. When the probe acts on MAO, the propylamino group is hydrolyzed, and the basic blue 3 fluorophore is released through the self-immolative group, emitting red fluorescence. This compound can be used for the detection of monoamine oxidase, and the detection signal is obvious, and the color change of the reaction solution is visible to the naked eye, providing an effective visual fluorescence tracking tool for biological detection.

[0037] An embodiment of the present invention provides a method for detecting monoamine oxidase. The fluorescent probe is added to a sample to be tested, and after reacting for a period of time, the fluorescence spectrum is detected, and monoamine oxidase is qualitatively or quantitatively analyzed according to the spectrum. When the fluorescent probe acts on monoamine oxidase, a chemical reaction will occur, resulting in the release or structural change of the fluorophore, thereby generating a detectable fluorescent signal. There is a certain corresponding relationship between the change of the fluorescence spectrum and the content of monoamine oxidase. By analyzing the spectral characteristics, such as parameters such as fluorescence intensity and wavelength shift, it is possible to qualitatively determine the presence of monoamine oxidase and quantitatively analyze its specific concentration, providing comprehensive detection information for research and application. This process is equivalent to amplifying the signal of the presence of monoamine oxidase, so that even if the content of the enzyme in the sample is low, it can be detected through the change of the fluorescent signal. The fluorescent probe is designed with a specific recognition site for monoamine oxidase. During the reaction process, the probe specifically binds or reacts with monoamine oxidase, and this specificity ensures the accuracy of the detection and reduces the interference of other substances. This step is simple to operate. Just adding the fluorescent probe to the sample to be tested can initiate the reaction, without complex pretreatment or special equipment assistance, reducing the operation threshold and being applicable to various laboratory environments.

[0038] In some embodiments, the wavelength of the excitation light for detecting the fluorescence spectrum is 610 - 630 nm. The use of excitation light with a wavelength of 610 - 630 nm is carefully selected according to the optical properties of the fluorescent probe. Within this wavelength range, the fluorescent probe can be effectively excited and emit high-intensity fluorescence. By detecting the fluorescence spectrum, the change of the fluorescent signal can be accurately captured, and even very weak signals can be captured by the detection instrument, thereby improving the detection sensitivity.

[0039] In some embodiments, the temperature for the reaction for a period of time is 36 - 38 °C, the time is 3.5 - 4.5 h, and the pH is 7.2 - 7.6. Setting the reaction temperature at 36 - 38 °C, this temperature range is close to the normal human body temperature, which can simulate the enzymatic activity environment under physiological conditions, ensure that monoamine oxidase has a high activity at this temperature, thereby promoting its reaction with the fluorescent probe, and ensuring the accuracy and reliability of the detection results. The reaction time is set to 3.5 - 4.5 h, and this time period is determined based on the kinetic characteristics of the reaction between the fluorescent probe and monoamine oxidase. Within this time, the reaction can proceed fully, making the fluorescence signal reach a stable and easily detectable state, avoiding unclear signals caused by too short reaction time or unnecessary resource waste and side reactions caused by too long reaction time. The pH value is maintained between 7.2 - 7.6, also to simulate the physiological environment, because most enzymes have the best activity under neutral or near-neutral pH conditions. This pH range is conducive to monoamine oxidase maintaining its natural structure and activity, ensuring the efficient progress of its reaction with the fluorescent probe, and at the same time preventing the denaturation and inactivation of the probe or enzyme due to too high or too low pH.

[0040] In some embodiments, the concentration of the fluorescent probe used is 0.01 - 0.05 mM. Controlling the concentration of the fluorescent probe used within the range of 0.01 - 0.05 mM is to ensure sufficient fluorescence signal intensity while avoiding background interference and non-specific binding caused by too high a concentration. The lower concentration range helps to reduce the detection cost, reduce the potential impact on the sample, and at the same time ensure that the fluorescence signal mainly comes from the specific reaction with monoamine oxidase, improving the signal-to-noise ratio and accuracy of the detection.

[0041] An embodiment of the present invention provides a preparation method for detecting monoamine oxidase as described above, comprising the following steps:

[0042] (1) Stir and heat a mixture of basic blue 3, sodium carbonate, and dichloromethane to 40 - 50 °C under the protection of an inert gas, and slowly add an aqueous solution of sodium dithionite at this temperature. React the mixture at 40 - 50 °C until the solution changes from blue to red. After the reaction is completed, stop heating and keep stirring for 8 - 12 min, and let it stand until the system is stratified; when the system cools to room temperature, slowly add triphosgene dissolved in dichloromethane under ice bath conditions, and stop the reaction after reacting for 50 - 70 min; extract, collect the organic phase, purify the crude product, and obtain a light blue solid target product, which is product 1;

[0043] (2) Dissolve p-hydroxybenzaldehyde, tert-butyl (3-bromopropyl)carbamate, and anhydrous potassium carbonate in acetonitrile; stir and react at 80 - 90 °C for 8 - 12 h and then end the reaction; filter to remove the solid, remove the solvent, and obtain a white oily substance, which is product 2;

[0044] (3) Dissolve product 2 in dichloromethane, add trifluoroacetic acid dropwise, stir at room temperature for 25 - 35 min, rotary evaporate to remove the solvent and trifluoroacetic acid; then add dichloromethane, add triethylamine dropwise to adjust the pH to 7, add allyl chloroformate, react at room temperature for 7 - 9 h until the reaction is completed, remove the solvent and purify the crude product to obtain product 3;

[0045] (4) Dissolve product 3 in methanol, add sodium borohydride under ice bath, after reacting for 1.5 - 2.5 h, rotary evaporate to remove the solvent, add appropriate amount of water to the system, extract three times with ethyl acetate, remove the solvent to obtain a pale yellow oily target product, which is product 4;

[0046] (5) Dissolve product 1, product 4, 4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate in dichloromethane, react under inert gas protection at room temperature for 7.5 - 8.5 h until the reaction is completed; rotary evaporate to remove the solvent and purify the crude product to obtain a pale blue solid target product, which is product 5;

[0047] (6) Dissolve product 5 in tetrahydrofuran, add tetrakis(triphenylphosphine)palladium, then add formic acid dropwise, react at 25 - 35 °C for 3.5 - 4.5 h until the reaction is completed, rotary evaporate to remove the solvent and purify the crude product to obtain a pale blue solid target product, which is the described fluorescent probe.

[0048] In some embodiments, in step (1), the molar ratio of basic blue 3: sodium carbonate: sodium dithionite: triphosgene is 1: 3.5 - 4.5: 3.5 - 4.5: 1.5 - 2.5; in step (2), the molar ratio of p-hydroxybenzaldehyde: tert-butyl (3-bromopropyl)carbamate: anhydrous potassium carbonate is 3 - 5: 4 - 6: 18 - 22.

[0049] Basic blue 3, as the starting material of the reaction, is mixed with sodium carbonate and sodium dithionite in a certain molar ratio, which can ensure that there is an adequate alkaline environment and reducing ability at the initial stage of the reaction, promoting the structural change and reaction of basic blue 3. The appropriate excess of sodium dithionite helps to fully reduce basic blue 3, causing its structure to transform and preparing for the subsequent reaction with triphosgene. Controlling the amount of triphosgene at an appropriate ratio can ensure a full reaction with the reduced basic blue 3 to generate target product 1, improving the conversion rate and yield of the reaction. A reasonable molar ratio helps to reduce the occurrence of side reactions. For example, if the amount of triphosgene is excessive, it may lead to overreaction or the formation of unnecessary by-products; while if the amount of sodium dithionite is insufficient, basic blue 3 may not be fully reduced, affecting the purity and quality of the product. By precisely controlling the molar ratio, high-purity product 1 can be obtained, which is beneficial for the subsequent steps.

[0050] p-Hydroxybenzaldehyde reacts with tert-butyl (3-bromopropyl)carbamate in the presence of anhydrous potassium carbonate. The appropriate control of the amount of p-hydroxybenzaldehyde can ensure its reaction with a sufficient amount of tert-butyl (3-bromopropyl)carbamate to form product 2. As a basic substance, the reasonable control of the amount of anhydrous potassium carbonate can provide suitable reaction conditions, promote the smooth progress of the reaction, improve the reaction efficiency and the yield of the product. This molar ratio range helps to avoid side reactions caused by the excess of a certain reactant. For example, if the amount of tert-butyl (3-bromopropyl)carbamate is excessively large, it may react with p-hydroxybenzaldehyde in various side reactions to form a complex mixture with a complicated structure, increasing the difficulty of separation and purification and reducing the purity and quality of product 2.

[0051] In some embodiments, in step (3), the molar ratio of product 2: trifluoroacetic acid: allyl chloroformate is 1: 1.5 - 2.5: 1.0 - 1.5; in step (4), the molar ratio of product 3: sodium borohydride is 1: 1 - 2.

[0052] When product 2 reacts with trifluoroacetic acid, the appropriate control of the amount of trifluoroacetic acid can ensure the effective removal of the protecting group of product 2 while avoiding the destruction of the product structure under overly acidic conditions. The subsequent addition of allyl chloroformate, with its reasonable amount control, can ensure a full reaction with the treated product 2 to form product 3, optimize the reaction conditions, and improve the selectivity and yield of the reaction. The appropriate molar ratio helps to obtain product 3 with a stable structure and suitable activity. If the amount of allyl chloroformate is insufficient, the reaction may be incomplete and the structure of product 3 may be unstable; while an excess may introduce unnecessary chemical activity and affect the progress of subsequent reaction steps.

[0053] Product 3 undergoes a reduction reaction under the action of sodium borohydride. The control of the amount of sodium borohydride in an appropriate ratio can ensure the full reduction of specific functional groups in product 3 to form the target product 4. An appropriate amount of sodium borohydride can not only provide sufficient reducing ability but also avoid side reactions caused by excess, such as structural changes or impurity generation due to over-reduction. This molar ratio range helps to achieve a mild and controllable reaction. By controlling the amount of sodium borohydride, the reaction rate and degree can be adjusted, avoiding potential safety hazards and product losses caused by violent reactions, ensuring the smooth progress of the reaction under suitable conditions, and obtaining high-quality product 4.

[0054] In some embodiments, in step (5), the molar ratio of product 1: product 4: DMAP: anhydrous sodium carbonate is 1: 0.8 - 1.2: 1.0 - 2.0: 2.5 - 3.5. Product 1 and product 4 undergo a coupling reaction in the presence of DMAP and anhydrous sodium carbonate. Controlling the molar ratio of product 1 to product 4 within a certain range can ensure that the two participate in the reaction in a suitable proportion to produce the target product 5. As a catalyst, the appropriate addition of DMAP can significantly improve the reaction efficiency and selectivity, and promote the smooth progress of the coupling reaction between product 1 and product 4. Controlling the amount of anhydrous sodium carbonate can provide a suitable alkaline environment, further optimize the reaction conditions, and improve the reaction yield and quality. A reasonable molar ratio helps to obtain product 5 with high purity and excellent properties. If product 1 or product 4 is in excess, it may cause unreacted materials to mix into the product, affecting its purity and performance. While the appropriate control of DMAP and anhydrous sodium carbonate can reduce the formation of by-products and ensure the structural integrity and functional characteristics of product 5.

[0055] In some embodiments, in step (6), the molar ratio of product 5: tetrakis(triphenylphosphine)palladium: formic acid is 1: 0.08 - 0.12: 2.5 - 3.5. Product 5 reacts with formic acid under the catalysis of tetrakis(triphenylphosphine)palladium. As a catalyst, the reasonable control of the amount of tetrakis(triphenylphosphine)palladium can ensure the efficient progress of the catalytic reaction, while avoiding side reactions or product structure changes caused by excessive catalyst. Controlling the amount of formic acid in an appropriate proportion can ensure sufficient reaction with product 5 to produce the final fluorescent probe. This molar ratio range helps to accurately control the reaction end point. By controlling the amount of formic acid, overreaction or underreaction can be avoided, ensuring that the reaction stops at the appropriate time point, thereby obtaining a fluorescent probe with stable structure and excellent performance. At the same time, a reasonable molar ratio also helps to reduce the difficulty of separation and purification after the reaction and improve the purity and quality of the product.

[0056] Example 1

[0057] Synthesis and characterization of the fluorescent probe:

[0058] (1) Stir and heat a mixture of 1795 mg of Basic Blue 3 (5 mmol), 2120 mg of sodium carbonate (20 mmol) and dichloromethane under nitrogen protection to 45 °C, and slowly add 3282 mg of sodium dithionite (20 mmol) dissolved in water at this temperature. React the mixture at 45 °C until the solution changes from blue to red (about 30 min to 1 h). After the reaction is completed, stop heating and keep stirring for 10 min, and let it stand until the system is stratified; wait for the system to cool to room temperature, and slowly add 2960 mg of triphosgene (10 mmol) dissolved in dichloromethane under ice bath conditions. Stop the reaction after 1 hour. Extract three times with dichloromethane, collect the organic phase, rotary evaporate to remove the solvent, and purify the crude product by silica gel column chromatography to obtain the light blue solid target product. That is Product 1. The molar ratio of the feed is Basic Blue 3:sodium carbonate:sodium dithionite:triphosgene = 1:4:4:2.

[0059] (2) Dissolve 488 mg of p-hydroxybenzaldehyde (4 mmol), 1190 mg of tert-butyl (3-bromopropyl)carbamate (5 mmol) and 2760 mg of anhydrous potassium carbonate (20 mmol) in 32 mL of acetonitrile. Stir at 85 °C. After reacting for 10 hours, the reaction ends. Filter to remove the solid. Rotary evaporate to remove the solvent to obtain a white oil. That is Product 2. The molar ratio of the feed is p-hydroxybenzaldehyde:tert-butyl (3-bromopropyl)carbamate:anhydrous potassium carbonate = 4:5:20.

[0060] (3) Dissolve 720 mg of Product 2 (4 mmol) in 10 mL of dichloromethane, and add 8 mL of trifluoroacetic acid dropwise. Stir at room temperature for 30 minutes. Rotary evaporate to remove trifluoroacetic acid. Add dichloromethane again, and add triethylamine dropwise to adjust the pH to 7. Then add 576 mg of allyl chloroformate (4.8 mmol), and stop the reaction after reacting at room temperature for 8 hours. Rotary evaporate to remove the solvent, and purify the crude product by silica gel column chromatography. That is Product 3. The molar ratio of the feed is Product 2:trifluoroacetic acid:allyl chloroformate = 1:2:1.2.

[0061] (4) Dissolve 263 mg of Product 3 (1 mmol) in 10 mL of methanol, and add 55.5 mg of sodium borohydride (1.5 mmol) under ice bath. Stop the reaction after 2 hours. Rotary evaporate to remove the solvent, add an appropriate amount of water to the system, and extract three times with ethyl acetate. Rotary evaporate to remove the solvent to obtain the light yellow oily target product. That is Product 4. The molar ratio of the feed is Product 3:sodium borohydride = 1:1.5.

[0062] (5) Dissolve 389 mg of product 1 (1 mmol), 265 mg of product 4 (1 mmol), 183 mg of 4-(dimethylamino)pyridine (DMAP) (1.5 mmol) and 3.18 mg of anhydrous sodium carbonate (3 mmol) in dichloromethane. After reacting for 8 hours under nitrogen protection at room temperature, the reaction is completed. Rotate evaporate to remove the solvent and purify the crude product by silica gel column chromatography to obtain the light blue solid target product, which is product 5. The feeding molar ratio is product 1: product 4: DMAP: anhydrous sodium carbonate = 1:1:1.5:3.

[0063] (6) Dissolve 616 mg of product 5 (1 mmol) in 10 mL of tetrahydrofuran, add 111.5 mg of tetrakis(triphenylphosphine)palladium(0) (0.1 mmol), and then dropwise add 138 mg of formic acid (3 mmol). After reacting at 30 °C for 4 h, the reaction is completed. Rotate evaporate to remove the solvent and purify the crude product by silica gel column chromatography to obtain the light blue solid target product, which is the fluorescent probe described in claim 1. The feeding molar ratio is product 5: tetrakis(triphenylphosphine)palladium(0): formic acid = 1:0.1:3.

[0064] Figure 1 The 1H NMR spectrum of the fluorescent probe is shown.

[0065] Fluorescent probe: 1H NMR of H19. 1H NMR (600 MHz, CDCl3) δ 7.32 (t, J = 8.4 Hz, 4H), 6.87 (d, J = 8.5 Hz, 2H), 6.34 (d, J = 8.9 Hz, 4H), 5.17 (s, 2H), 4.04 (t, J = 5.9 Hz, 2H), 3.31 (q, J = 7.0 Hz, 8H), 2.97 (t, J = 6.7 Hz, 2H), 2.04–1.95 (m, 2H), 1.14 (t, J = 7.0 Hz, 12H).

[0066] Figure 1 The 1H NMR spectrum of [the fluorescent probe] shows that the fluorescent probe has a complex structure, containing multiple aromatic rings, methylene groups, ethyl groups, etc., and there are specific chemical bondings and spatial arrangements among these groups.

[0067] Figure 2 The 13C NMR spectrum of the fluorescent probe is shown.

[0068] Fluorescent probe: 13C NMR (151 MHz, CDCl3) δ 158.70, 153.95, 151.23, 146.38, 129.97, 128.64, 125.13, 117.25, 114.40, 106.43, 99.50, 67.50, 65.70, 44.56, 38.79, 31.46, 12.56.

[0069] Figure 2 The carbon-13 nuclear magnetic resonance spectrum further confirmed the complex structure of the fluorescent probe, which contains multiple aromatic rings, carbonyl groups, methylene groups, ethyl groups, etc., and there are specific chemical bondings and spatial arrangements among these groups.

[0070] Figure 3 The high-resolution mass spectrum of the fluorescent probe is shown.

[0071] Fluorescent probe: Calc. for C31H42N4O4 2+ [M+2H]2+ 267.1603, found 267.1598.

[0072] The theoretical calculated value (Calc.) is very close to the actual detected value (found), which indicates that the molecular structure of the fluorescent probe conforms to the expected molecular formula C31H42N4O4, further verifying the accuracy of its structure. The exact match of the high-resolution mass spectrum shows that the synthesized compound has the expected molecular composition and structure.

[0073] Example 2

[0074] (1) Prepare a 10 mM phosphate buffer solution with pH 7.4; dissolve the fluorescent probe in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the freeze-dried monoamine oxidase in water to prepare a 30 U / mL monoamine oxidase stock solution.

[0075] (3) Fluorescence spectrum: Take 478.3 μL of the phosphate buffer solution, 5 μL of the fluorescent probe stock solution, and 16.7 μL of the monoamine oxidase stock solution and add them to a cuvette. After reacting at 37 °C for 4 h, perform fluorescence spectrum scanning with an excitation light of 620 nm at 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, and 240 min.

[0076] The results are as Figure 4As shown. The spectrum shows that after adding monoamine oxidase, the fluorescence intensity of the fluorescent probe at 676 nm gradually increases with time. The fluorescence intensity at the initial time point (0 min) is relatively low, and as the reaction time extends, the fluorescence intensity gradually increases, indicating that a specific reaction has occurred between the fluorescent probe and monoamine oxidase. The fluorescent probe is designed with an MAO recognition site. When MAO is present, it will specifically interact with the probe, resulting in a change in the probe structure. This structural change usually involves the release of the fluorophore or a change in the environment around the fluorophore, thus enhancing the fluorescence intensity. As the reaction time extends, more probes react with MAO, leading to a gradual increase in the fluorescence signal. The continuous increase in fluorescence intensity over time indicates that the probe has high sensitivity to MAO and can detect the presence of MAO even at a low concentration. Since the change in the fluorescence signal is the result of a specific reaction, the probe has high specificity to MAO and is not easily interfered by other substances. This fluorescent probe exhibits good performance in detecting MAO and can be used for the rapid and sensitive detection of MAO in biological samples. In disease diagnosis and research, the level of MAO is related to various diseases such as Parkinson's disease and epilepsy, so this probe has potential clinical application value.

[0077] Example 3

[0078] (1) Prepare a 10 mM phosphate buffered saline solution with a pH of 7.4; dissolve the fluorescent probe in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the MAO inhibitor pargyline in water to prepare a 2 mM stock solution.

[0079] (2) Cell test: Take 10 μL of the fluorescent probe stock solution and add it to 2 mL of the phosphate buffered saline solution to make its concentration 10 μM; incubate human hepatocarcinoma cells Hepa1-6 with the above solution at 37 °C for 4 h. After incubation, wash the cells 3 times with 2 mL of the phosphate buffered saline solution and image under a laser scanning confocal microscope; For the second group, take 20 μL of the pargyline stock solution and add it to 2 mL of the phosphate buffered saline solution to make its concentration 20 μM; incubate human hepatocarcinoma cells Hepa1-6 with the above solution at 37 °C for 0.5 h. After incubation, add 10 μL of the fluorescent probe stock solution to make its concentration 10 μM; incubate Hepa1-6 with the above solution at 37 °C for 4 h. After incubation, wash the cells 3 times with 2 mL of the phosphate buffered saline solution and image under a laser scanning confocal microscope.

[0080] The results are as Figure 5As shown, compared with the group added with the MAO inhibitor pargyline, the cells in the group without the inhibitor emitted a strong fluorescence turn-on signal, which proved at the cellular level that the probe was activated by MAO in the cells. In the absence of the MAO inhibitor, MAO in the cells could freely interact with the fluorescent probe. MAO catalyzed the hydrolysis or other chemical reactions of specific groups in the probe, resulting in the release or structural change of the fluorophore, thus enhancing the fluorescence signal. Pargyline, as an inhibitor of MAO, could inhibit the activity of MAO and reduce its interaction with the fluorescent probe. Therefore, the fluorescent probe could not be effectively activated and the fluorescence signal was weak. The fact that the fluorescent probe could enter the cells and function inside the cells indicated its good biocompatibility. The specific response of the probe to MAO was verified by cell experiments, further proving its feasibility and effectiveness in detecting MAO in biological systems, providing strong support for subsequent biomedical research and clinical applications.

[0081] The above description is only the preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting monoamine oxidase, characterized in that: Its structural formula is as follows:

2. A method for detecting monoamine oxidase, characterized in that: The fluorescent probe described in claim 1 is added to the sample to be tested, reacted for a period of time, and the fluorescence spectrum is detected, and the monoamine oxidase is qualitatively or quantitatively determined according to the spectrum.

3. The method according to claim 2, characterized in that The wavelength of the excitation light for detecting the fluorescence spectrum is 610-630nm.

4. The method according to claim 2, characterized in that: The temperature of the reaction for a period of time is 36-38°C, the time is 3.5-4.5h, and the pH is 7.2-7.

6.

5. The method according to claim 2, characterized in that: The fluorescent probe is used at a concentration of 0.01-0.05 mM.

6. A preparation method for detecting monoamine oxidase according to claim 1, characterized in that: The following steps are involved: (1) A mixture of basic blue 3, sodium carbonate and dichloromethane is stirred and heated to 40-50° C. under the protection of an inert gas, and an aqueous sodium dithionite solution is slowly added at this temperature, and the mixture is reacted at 40-50° C. until the solution changes from blue to red. After the reaction is completed, heating is stopped and stirring is maintained for 8-12 minutes, and the system is allowed to stand until the system is separated; after the system is cooled to room temperature, triphosgene dissolved in dichloromethane is slowly added under ice bath conditions, and the reaction is stopped after reacting for 50-70 minutes; extraction, the organic phase is collected, and the crude product is purified to obtain a light blue solid target product, namely, product 1; (2) dissolving p-hydroxybenzaldehyde, tert-butyl (3-bromopropyl)carbamate and anhydrous potassium carbonate in acetonitrile; stirring the reaction at 80-90° C. for 8-12 hours and then terminating the reaction; filtering out the solid and removing the solvent to obtain a white oil, which is the product 2; (3) Product 2 is dissolved in dichloromethane, trifluoroacetic acid is added dropwise, and the mixture is stirred at room temperature for 25-35 min. The solvent is removed by rotary evaporation to remove the trifluoroacetic acid. Dichloromethane is then added, and triethylamine is added dropwise to adjust the pH to 7. Allyl chloroformate is then added. The reaction is allowed to complete at room temperature for 7-9 h. The solvent is removed and the crude product is purified to obtain product 3. (4) Product 3 was dissolved in methanol, sodium borohydride was added under ice bath, and the reaction was continued for 1.5-2.5 hours. The solvent was removed by rotary evaporation, an appropriate amount of water was added to the system, and the system was extracted with ethyl acetate three times. The solvent was removed to obtain the target product as a light yellow oil, which was product 4; (5) Product 1, product 4,4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate were dissolved in dichloromethane, and the reaction was completed after 7.5-8.5 hours at room temperature under inert gas protection; the solvent was removed by rotary evaporation and the crude product was purified to obtain a light blue solid target product, namely product 5; (6) The product 5 is dissolved in tetrahydrofuran, tetrakis(triphenylphosphine)palladium is added, and then formic acid is added dropwise. The reaction is carried out at 25-35° C. for 3.5-4.5 hours. After the reaction is completed, the solvent is removed by rotary evaporation and the crude product is purified to obtain a light blue solid target product, which is the fluorescent probe.

7. The preparation method for detecting monoamine oxidase according to claim 6, characterized in that: In step (1), the molar ratio of basic blue 3: sodium carbonate: sodium dithionite: triphosgene is 1:3.5-4.5:3.5-4.5:1.5-2.5; in step (2), the molar ratio of p-hydroxybenzaldehyde: tert-butyl (3-bromopropyl)carbamate: anhydrous potassium carbonate is 3-5:4-6:18-22.

8. The method for detecting monoamine oxidase according to claim 6, characterized in that: In step (3), the molar ratio of product 2: trifluoroacetic acid: allyl chloroformate is 1:1.5-2.5:1.0-1.5; in step (4), the molar ratio of product 3: sodium borohydride is 1:1-2.

9. The preparation method for detecting monoamine oxidase according to claim 6, characterized in that: In step (5), the molar ratio of product 1: product 4: DMAP: anhydrous sodium carbonate is 1: 0.8-1.2: 1.0-2.0: 2.5-3.

5.

10. The preparation method for detecting monoamine oxidase according to claim 6, characterized in that: In step (6), the molar ratio of product 5: tetrakis(triphenylphosphine)palladium:formic acid is 1:0.08-0.12:2.5-3.5.

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