Near-infrared fluorescent probe based on alkaline dye double-enzyme activation and preparation and application thereof

Abstract

The application provides a near-infrared fluorescent probe based on double-enzyme activation of basic dyes, a preparation method and application of the probe, and the Chinese name of the probe compound is 4-(3-(2-amino-4-methylpentanoylamino)propoxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate. The probe can simultaneously detect leucine aminopeptidase (LAP) and monoamine oxidase (MAO), under the action of leucine aminopeptidase, the terminal leucine is hydrolyzed to expose propylamino as a recognition site of MAO, when the probe reacts with MAO, the propylamino is hydrolyzed to release a basic blue 3 fluorophore through a self-immolative group, and red fluorescence is emitted. The compound completes the detection of leucine aminopeptidase and monoamine oxidase through a cascade reaction, the detection signal is obvious, and the color change of the reaction solution can be observed by naked eyes. The application provides an effective visual fluorescent tracking tool for double-enzyme detection in biological detection.

Description

TECHNICAL FIELD

[0001] The application relates to basic dyes and fluorescent detection, and particularly belongs to a near-infrared fluorescent probe based on double-enzyme activation of basic dyes and a preparation method and application thereof. BACKGROUND

[0002] Leucine aminopeptidase (LAP) is an important metallopeptidase that selectively catalyzes the hydrolytic removal of leucine residues at the N-terminus of proteins and polypeptides. Importantly, researchers have reported that diseased hepatocytes have higher LAP activity than normal hepatocytes. Therefore, LAP can be used as a specific marker for distinguishing diseased hepatocytes from normal hepatocytes. Monoamine oxidase (MAO) belongs to the flavin-dependent enzyme family and can be produced in most cell types, and its main function is to catalyze the oxidation of biological amines to produce the corresponding aldehydes. Determining MAO is helpful for the diagnosis of liver diseases, especially the early diagnosis of liver cirrhosis. For these two important enzymes in the life system, although some enzyme-activated molecular probes activated only by LAP or MAO have been developed, their non-specificity leads to less expression in tissues, but the activation of the ubiquitous protease can lead to false positive signals and limit their clinical application. Therefore, we synthesized a fluorescent probe activated by both LAP and MAO. The fluorescent probe can be used for distinguishing detection of normal hepatocytes and drug-induced hepatotoxic cells. SUMMARY

[0003] The application aims to provide a near-infrared fluorescent probe based on basic dyes and a preparation method and application thereof, and use the fluorescent probe for simultaneous detection of monoamine oxidase and leucine aminopeptidase.

[0004] The application provides a fluorescent probe based on double-enzyme activation of basic dyes for simultaneous detection of monoamine oxidase and leucine aminopeptidase, which is 4-(3-(2-amino-4-methylpentanamido)propoxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate in Chinese and 4-(3-(2-amino-4-methylpentanamido)propoxy)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate in English.

[0005] The structural formula is as follows:

[0006]

[0007] The application provides a preparation method of the near-infrared fluorescent probe based on double-enzyme activation of basic dyes, which comprises the following steps:

[0008] (1) Dissolve basic blue 3 and sodium carbonate in a mixture of dichloromethane and water, stir and heat to 45℃ under nitrogen protection, and slowly add sodium hydrosulfite dissolved in water at this temperature, react the mixture at 45℃ until the solution changes from blue to purple red, stop heating after the reaction is completed and keep stirring for 10 min, stand until the system is layered; after the system is cooled to room temperature, slowly add triphosgene dissolved in dichloromethane under ice bath conditions, stop the reaction after 1 hour of reaction; extract with dichloromethane for three times, collect the organic phase, remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain the target product as a light blue solid, which is product 1; the molar ratio of the feed is basic blue 3: sodium carbonate: sodium hydrosulfite: triphosgene = 1:4:4:2;

[0009] (2) Dissolve p-hydroxybenzaldehyde, (3-bromopropyl) tert-butyl carbamate and anhydrous potassium carbonate in acetonitrile, stir at 85℃, and stop the reaction after 10 hours of reaction; filter off the solids, remove the solvent by rotary evaporation to obtain white oil, which is product 2; the molar ratio of the feed is p-hydroxybenzaldehyde: (3-bromopropyl) tert-butyl carbamate: anhydrous potassium carbonate = 4:5:20;

[0010] (3) Dissolve product 2 in dichloromethane, add trifluoroacetic acid dropwise; stir at room temperature for 30 min; remove the solvent by rotary evaporation to remove trifluoroacetic acid; add dichloromethane again, and add triethylamine dropwise to adjust pH = 7 to obtain reaction liquid 1; take another round-bottom flask, dissolve 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and alloc-L-leucine in dichloromethane, stir at room temperature for 30 min to obtain reaction liquid 2; add reaction liquid 2 to reaction liquid 1, and stop the reaction after 8 hours of reaction at room temperature; remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain product 3; the molar ratio of the feed is product 2: trifluoroacetic acid: EEDQ: alloc-L-leucine = 1:2:1.5:1;

[0011] (4) Dissolve product 3 in methanol, and add sodium borohydride under ice bath; stop the reaction after 2 hours of reaction; add an appropriate amount of water to the system, and extract with ethyl acetate for three times; remove the solvent by rotary evaporation to obtain the target product as a light yellow oil, which is product 4; the molar ratio of the feed is product 3: sodium borohydride = 1:1.5;

[0012] (5) Dissolve product 1, product 4, 4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate in dichloromethane, and stop the reaction after 8 hours of reaction at room temperature under nitrogen protection; remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain the target product as a light blue solid, which is product 5; the molar ratio of the feed is product 1: product 4: DMAP: anhydrous sodium carbonate = 1:1:1.5:3;

[0013] (6) The product 5 is dissolved in tetrahydrofuran, tetra(triphenylphosphine)palladium is added, and formic acid is added dropwise, and the reaction is completed after 4h of reaction at 30℃; the solvent is removed by rotary evaporation, and the crude product is purified by silica gel column chromatography to obtain the target product, i.e. the fluorescent probe, as a light blue solid; the molar ratio of the raw materials is product 5: tetra(triphenylphosphine)palladium: formic acid = 1: 0.1: 3.

[0014] The prepared fluorescent probe can be applied in the preparation of a reagent for simultaneously detecting leucine aminopeptidase and monoamine oxidase.

[0015] The application provides a method for simultaneously detecting leucine aminopeptidase and monoamine oxidase, and the steps are as follows:

[0016] (1) A 10mM phosphate buffer solution with pH 7.4 is prepared; the fluorescent probe as described above is dissolved in dimethyl sulfoxide to prepare a 2mM stock solution; monoamine oxidase freeze-dried powder is dissolved in water to prepare a 30U / mL monoamine oxidase stock solution; leucine aminopeptidase freeze-dried powder is dissolved in water to prepare a 30U / mL monoamine oxidase stock solution;

[0017] (2) Fluorescence spectrum: 461.6μL of the phosphate buffer solution, 5μL of the fluorescent probe stock solution, 16.7μL of the monoamine oxidase stock solution and 16.7μL of the leucine aminopeptidase stock solution are added to a cuvette, and fluorescence spectrum scanning is immediately performed with 620nm excitation light, and the time is 0min, 15min, 30min, 45min, 60min, 120min, 180min and 240min;

[0018] (3) Fluorescence spectrum: ① 478.3μL of the phosphate buffer solution, 5μL of the fluorescent probe stock solution and 16.7μL of the monoamine oxidase stock solution are added to a cuvette, and fluorescence spectrum scanning is performed with 620nm excitation light after 4h of reaction at 37℃, ② 478.3μL of the phosphate buffer solution, 5μL of the fluorescent probe stock solution and 16.7μL of the leucine aminopeptidase stock solution are added to a cuvette, and fluorescence spectrum scanning is performed with 620nm excitation light after 4h of reaction at 37℃, ③ 461.6μL of the phosphate buffer solution, 5μL of the fluorescent probe stock solution, 16.7μL of the monoamine oxidase stock solution and 16.7μL of the leucine aminopeptidase stock solution are added to a cuvette, and fluorescence spectrum scanning is performed with 620nm excitation light after 4h of reaction at 37℃, and ④ 495μL of the phosphate buffer solution and 5μL of the fluorescent probe stock solution are added to a cuvette, and fluorescence spectrum scanning is performed with 620nm excitation light after 4h of reaction at 37℃.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1、The present application has simple operation, simple detection, and only needs a fluorescence detector.

[0021] 2、The present application has obvious detection signal, and the color change of the reaction solution can be observed by naked eyes.

[0022] 3、The present application needs two enzyme activations, and has stronger specificity. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 NMR hydrogen spectrum of the fluorescent probe prepared in Example 1

[0024] Figure 2 NMR hydrogen-carbon spectrum of the fluorescent probe prepared in Example 1

[0025] Figure 3 High-resolution mass spectrum of the fluorescent probe prepared in Example 1

[0026] Figure 4 Fluorescence spectrum of the fluorescent probe responding to monoamine oxidase and leucine aminopeptidase in Example 2

[0027] Figure 5 Fluorescence spectrum of the fluorescent probe responding to monoamine oxidase, responding to leucine aminopeptidase, and responding to monoamine oxidase and leucine aminopeptidase in Example 3

[0028] Figure 6 Cell imaging diagram of the fluorescent probe incubating cells in Example 4

[0029] Figure 7 Cell imaging diagram of the fluorescent probe incubating cells in Example 5 DETAILED DESCRIPTION

[0030] The present application will be further described below in combination with examples and drawings, but the present application is not limited by the following examples.

[0031] Example 1

[0032] Synthesis and characterization of the fluorescent probe:

[0033] (1) 1795 mg of basic blue 3 (5 mmol), 2120 mg of sodium carbonate (20 mmol) were dissolved in a mixture of dichloromethane (15 mL) and water (10 mL) under nitrogen protection, and heated to 45°C, and 3282 mg of sodium hydrosulfite (20 mmol) dissolved in water was slowly added at this temperature, and the mixture was reacted at 45°C until the solution changed from blue to purple red, and after the reaction was completed, the heating was stopped and stirred for 10 min, and the system was allowed to stand until the layers were separated; after the system was cooled to room temperature, 2960 mg of triphosgene (10 mmol) dissolved in dichloromethane was slowly added under ice bath conditions. After 1 hour of reaction, the reaction was stopped, extracted with dichloromethane three times, the organic phase was collected, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain the target product as a light blue solid, which was product 1;

[0034] (2) 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) were dissolved in 32 mL of acetonitrile and stirred at 85°C. After 10 hours of reaction, the reaction was completed, the solid was filtered off, and the solvent was removed by rotary evaporation to obtain a white oil, which was product 2;

[0035] (3) 720 mg of product 2 (4 mmol) was dissolved in 10 mL of dichloromethane, and 8 mL of trifluoroacetic acid was added dropwise. Stir at room temperature for 30 minutes, remove the solvent by rotary evaporation, remove the trifluoroacetic acid, add dichloromethane, and add triethylamine to adjust the pH to 7 to obtain reaction solution 1. Take another round bottom flask, dissolve 1488 mg of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (6 mmol) and 860 mg of alloc-L-leucine (4 mmol) in dichloromethane, and stir at room temperature for 30 min to obtain reaction solution 2. Add reaction solution 2 to reaction solution 1, and react at room temperature for 8 hours after the reaction is completed. Remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain product 3;

[0036] (4) 752 mg of product 3 (2 mmol) was dissolved in methanol, and 111 mg of sodium borohydride (3 mmol) was added under ice bath. After 2 hours, the reaction was completed; remove the solvent by rotary evaporation, add an appropriate amount of water to the system, extract with ethyl acetate three times, remove the solvent by rotary evaporation, and obtain the target product as a light yellow oil; which is product 4;

[0037] (5) 778 mg of product 1 (2 mmol), 756 mg of product 4 (2 mmol), 366 mg of 4- (dimethylamino)pyridine (DMAP) (3 mmol) and 648 mg of anhydrous sodium carbonate (6 mmol) were dissolved in 15 mL of dichloromethane, and the reaction was completed after 8 hours of reaction at room temperature under nitrogen protection; the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain the target product as a light blue solid; that is, product 5;

[0038] (6) 364.5 mg of product 5 (0.5 mmol) was dissolved in 10 mL of tetrahydrofuran, 57.75 mg of tetrakis (triphenylphosphine) palladium (0.05 mmol) was added, and then 69 mg of formic acid (1.5 mmol) was added dropwise. The reaction was completed after 4 hours of reaction at 30°C; the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain the target product as a light blue solid, that is, the fluorescent probe.

[0039] Figure 1 The figure shows the nuclear magnetic resonance hydrogen spectrum (H NMR) of the fluorescent probe. 1

[0040] The fluorescent probe: 1 H NMR (500 MHz, DMSO) δ 8.05 (t, J = 5.5 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 6.37 (dd, J = 9.1, 2.7 Hz, 2H), 6.31 (d, J = 2.7 Hz, 2H), 5.12 (s, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.29 (q, J = 6.9 Hz, 8H), 3.25 - 3.17 (m, 3H), 1.84 (dd, J = 12.9, 6.5 Hz, 2H), 1.68 - 1.60 (m, 1H), 1.42 - 1.35 (m, 1H), 1.26 - 1.23 (m, 1H), 1.08 - 1.03 (m, 12H), 0.84 (dd, J = 17.8, 6.6 Hz, 6H).

[0041] Figure 2 The figure shows the nuclear magnetic resonance hydrogen spectrum (H NMR) of the fluorescent probe. 1 C NMR) of the fluorescent probe.

[0042] The fluorescent probe: 13 ​C NMR (126 MHz, DMSO) δ 174.91, 158.57, 153.17, 150.83, 146.09, 129.98, 128.19, 125.25, 116.56, 114.44, 106.31, 98.77, 67.22, 65.41, 52.98, 48.69, 43.90, 35.55, 24.21, 23.16, 22.03, 12.44.

[0043] Figure 3 High resolution mass spectrum of the fluorescent probe is shown.

[0044] Fluorescent probe: Calc. for C 37 H 52 N5O5 + [M+H] + 646.3968, found 646.3963

[0045] Example 2

[0046] (1) Prepare 10 mM phosphate buffer solution at pH 7.4; dissolve the fluorescent probe prepared in Example 1 in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the monoamine oxidase freeze-dried powder in water to prepare a 30 U / mL monoamine oxidase stock solution; dissolve the leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL monoamine oxidase stock solution;

[0047] (2) Fluorescence spectrum: Take 461.6 μL of phosphate buffer solution, 5 μL of fluorescent probe stock solution, 16.7 μL of monoamine oxidase stock solution, and 16.7 μL of leucine aminopeptidase stock solution into a cuvette, and perform fluorescence spectrum scanning at 620 nm excitation light after 4 h of reaction at 37°C. The time is 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, and 240 min. The spectrum shows that the additional addition of leucine aminopeptidase and monoamine oxidase increases the fluorescence intensity of the fluorescent probe at 676 nm over time. The results are shown in Figure 4

[0048] Example 3

[0049] (1) Prepare 10 mM phosphate buffer solution at pH 7.4; dissolve the fluorescent probe prepared in Example 1 in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the monoamine oxidase freeze-dried powder in water to prepare a 30 U / mL monoamine oxidase stock solution; dissolve the leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL monoamine oxidase stock solution;

[0050] ​(2) Fluorescence spectrum: ① 478.3 μL of phosphate buffered saline solution, 5 μL of fluorescent probe stock solution, 16.7 μL of monoamine oxidase stock solution were added into a cuvette, and after reaction at 37 °C for 4 h, fluorescence spectrum scanning was performed with 620 nm excitation light; ② 478.3 μL of phosphate buffered saline solution, 5 μL of fluorescent probe stock solution, 16.7 μL of leucine aminopeptidase stock solution were added into a cuvette, and after reaction at 37 °C for 4 h, fluorescence spectrum scanning was performed with 620 nm excitation light; ③ 461.6 μL of phosphate buffered saline solution, 5 μL of fluorescent probe stock solution, 16.7 μL of monoamine oxidase stock solution, 16.7 μL of leucine aminopeptidase stock solution were added into a cuvette, and after reaction at 37 °C for 4 h, fluorescence spectrum scanning was performed with 620 nm excitation light; ④ 495 μL of phosphate buffered saline solution, 5 μL of fluorescent probe stock solution were added into a cuvette, and after reaction at 37 °C for 4 h, fluorescence spectrum scanning was performed with 620 nm excitation light; The spectrum showed that: with 620 nm as the excitation, the fluorescence intensity of the fluorescent probe at 676 nm did not change significantly when leucine aminopeptidase and monoamine oxidase were added separately, but the fluorescence intensity of the fluorescent probe at 676 nm was enhanced when leucine aminopeptidase and monoamine oxidase were added together; the results are shown in Figure 5

[0051] Example 4

[0052] (1) Prepare 10 mM phosphate buffered saline solution at pH 7.4; dissolve the fluorescent probe prepared in Example 1 in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the LAP inhibitor ubenimex in water to prepare a 2 mM stock solution; dissolve the MAO inhibitor nialamide in water to prepare a 2 mM stock solution.

[0053] ​(2) Cell test: 10 μL of the fluorescent probe stock solution was added to 2 mL of phosphate buffered saline solution to make the concentration 10 μM; the human liver cancer cells Hepa1-6 were incubated with the above solution at 37 °C for 4 h, and then washed with 2 mL of phosphate buffered saline solution for 3 times, and imaged under the laser scanning confocal microscope; the second group took 20 μL of ubenimex stock solution to add to 2 mL of phosphate buffered saline solution to make the concentration 20 μM; the human liver cancer cells Hepa1-6 were incubated with the above solution at 37 °C for 0.5 h, and then 10 μL of the fluorescent probe stock solution was added to make the concentration 10 μM; the above solution was used to incubate Hepa1-6 at 37 °C for 4 h, and then washed with 2 mL of phosphate buffered saline solution for 3 times, and imaged under the laser scanning confocal microscope; the third group took 20 μL of dipyridamole stock solution to add to 2 mL of phosphate buffered saline solution to make the concentration 20 μM; the human liver cancer cells Hepa1-6 were incubated with the above solution at 37 °C for 0.5 h, and then 10 μL of the fluorescent probe stock solution was added to make the concentration 10 μM; the above solution was used to incubate Hepa1-6 at 37 °C for 4 h, and then washed with 2 mL of phosphate buffered saline solution for 3 times, and imaged under the laser scanning confocal microscope; the results are shown in Figure 6 .

[0054] Example 5

[0055] (1) 10 mM phosphate buffered saline solution with pH 7.4 was prepared; the fluorescent probe prepared in Example 1 was dissolved in dimethyl sulfoxide to prepare a 2 mM stock solution; paracetamol (APAP) was dissolved in water to prepare a 20 mM stock solution.

[0056] (2) Cell test: 10 μL of the fluorescent probe stock solution was added to 2 mL of phosphate buffered saline solution to make the concentration 10 μM; the normal liver cells HL7702 were incubated with the above solution at 37 °C for 4 h, and then washed with 2 mL of phosphate buffered saline solution for 3 times, and imaged under the laser scanning confocal microscope; the second group took 20 μL of paracetamol stock solution to add to 2 mL of phosphate buffered saline solution to make the concentration 200 μM; the normal liver cells HL7702 were incubated with the above solution at 37 °C for 12 h, and then 10 μL of the fluorescent probe stock solution was added to make the concentration 10 μM; after incubation, the cells were washed with 2 mL of phosphate buffered saline solution for 3 times, and imaged under the laser scanning confocal microscope; the results are shown in Figure 7 .

Claims

1. A near-infrared fluorescent probe based on dual-enzyme activation by a basic dye, characterized in that, The structure is as follows: 。 2. The method for preparing the fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve Basic Blue 3 and sodium carbonate in a mixture of dichloromethane and water, stir and heat to 45°C under nitrogen protection, and slowly add sodium dithionite dissolved in water at this temperature. React the mixture at 45°C until the solution changes from blue to purple-red. After the reaction is complete, stop heating and keep stirring for 10 min. Let the system stand until the layers are separated. After the system cools to room temperature, slowly add triphosgene dissolved in dichloromethane under ice bath conditions. Stop the reaction after 1 hour. Extract three times with dichloromethane, collect the organic phase, remove the solvent by rotary evaporation, and purify the crude product by silica gel column chromatography to obtain the light blue solid target product, which is product 1. The molar ratio of feed is Basic Blue 3: sodium carbonate: sodium dithionite: triphosgene = 1:4:4:

2. (2) Dissolve p-hydroxybenzaldehyde, tert-butyl (3-bromopropyl)carbamate and anhydrous potassium carbonate in acetonitrile, stir at 85°C, and the reaction ends after 10 hours; filter to remove the solid, remove the solvent by rotary evaporation, and obtain a white oily substance, which is product 2; the molar ratio of the feed is p-hydroxybenzaldehyde: tert-butyl (3-bromopropyl)carbamate: anhydrous potassium carbonate = 4: 5: 20; (3) Dissolve product 2 in dichloromethane and add trifluoroacetic acid dropwise; stir at room temperature for 30 minutes; remove the solvent by rotary evaporation and remove trifluoroacetic acid; add dichloromethane again and add triethylamine dropwise to adjust pH = 7 to obtain reaction solution 1; then take a round bottom flask and dissolve 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and alloc-L-leucine in dichloromethane, stir at room temperature for 30 minutes to obtain reaction solution 2; add reaction solution 2 to reaction solution 1, react at room temperature for 8 hours to end the reaction, remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain product 3; the molar ratio of the reactants is product 2: trifluoroacetic acid: EEDQ: alloc-L-leucine = 1:2:1.5:1; (4) Dissolve product 3 in methanol, add sodium borohydride under ice bath, and the reaction ends after 2 hours; remove the solvent by rotary evaporation, add an appropriate amount of water to the system, and extract three times with ethyl acetate; remove the solvent by rotary evaporation to obtain the pale yellow oily target product, which is product 4; the molar ratio of the feed is product 3: sodium borohydride = 1:1.5; (5) Products 1, 4, 4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate were dissolved in dichloromethane and reacted under nitrogen protection at room temperature for 8 hours until the reaction ended. The solvent was removed by rotary evaporation and the crude product was purified by silica gel column chromatography to obtain the light blue solid target product, which is product 5. The molar ratio of the reactants was product 1: product 4: DMAP: anhydrous sodium carbonate = 1: 1: 1.5:

3. (6) Dissolve product 5 in tetrahydrofuran, add tetra(triphenylphosphine)palladium, then add formic acid dropwise, and react at 30°C for 4 h to finish the reaction; remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain a light blue solid target product, which is the fluorescent probe; the molar ratio of the feed is product 5: tetra(triphenylphosphine)palladium: formic acid = 1:0.1:

3.

3. The use of the fluorescent probe as described in claim 1 in the preparation of reagents for the simultaneous detection of leucine aminopeptidase and monoamine oxidase.

4. The application as described in claim 3, characterized in that, The steps for detecting leucine aminopeptidase and monoamine oxidase using the reagents are as follows: (1) Prepare a 10 mM phosphate buffer solution with pH 7.4; dissolve the fluorescent probe of claim 1 in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the monoamine oxidase lyophilized powder in water to prepare a 30 U / mL monoamine oxidase stock solution; dissolve the leucine aminopeptidase lyophilized powder in water to prepare a 30 U / mL monoamine oxidase stock solution. (2) Fluorescence spectrum: 461.6 µL of phosphate buffer solution, 5 µL of fluorescent probe stock solution, 16.7 µL of monoamine oxidase stock solution and 16.7 µL of leucine aminopeptidase stock solution were added to a cuvette and immediately subjected to fluorescence spectrum scanning with 620 nm excitation light at 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min and 240 min. (3) Fluorescence spectrum: 461.6 µL of phosphate buffer solution, 5 µL of fluorescent probe stock solution, 16.7 µL of monoamine oxidase stock solution and 16.7 µL of leucine aminopeptidase stock solution were added to a cuvette and reacted at 37 °C for 4 h. The fluorescence spectrum was then scanned with 620 nm excitation light.

5. The application of the fluorescent probe as described in claim 1 in the preparation of cell imaging reagents.

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