A fluorescent probe based on basic dye and a preparation method and application thereof

Abstract

The application provides a near-infrared fluorescent probe based on basic dye and a preparation method and application thereof, and the probe is named 4-(2-amino-4-methylpentanoylamino)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-methylate in Chinese. The probe is used for detecting leucine aminopeptidase (LAP), basic blue 3 is used as a fluorophore, and L-leucine is used as a recognition site of the LAP. When the probe is reacted with the LAP, the leucine is hydrolyzed, the basic blue 3 fluorophore is released through a self-immolative group, red fluorescence is emitted, the probe can be used for detecting the leucine aminopeptidase, the detection signal is obvious, the color change of a reaction solution is visible to the naked eye, and the probe provides an effective visual fluorescent tracking tool for the leucine aminopeptidase for biological detection.

Description

TECHNICAL FIELD

[0001] The application relates to basic dyes and fluorescence detection, in particular to a fluorescence probe based on a basic dye and a preparation method of the fluorescence probe, and application of the fluorescence probe in detection of leucine aminopeptidase. BACKGROUND

[0002] Leucine aminopeptidase (LAP) is an important metallopeptidase which 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. As an important hydrolytic enzyme in the life system, further research on the biological distribution and physiological role of leucine aminopeptidase is conducive to effective intervention to prevent the occurrence and development of related major diseases. With the improvement of organic synthesis technology, a high-performance fluorescence probe based on a basic dye is prepared, which can be used as an effective visual fluorescence tracking tool for leucine aminopeptidase in the life system. SUMMARY

[0003] The application aims to provide a near-infrared fluorescence probe based on a basic dye and a preparation method of the fluorescence probe, and use of the fluorescence probe for specific detection of leucine aminopeptidase.

[0004] The application provides a fluorescence probe based on a basic dye for detecting leucine aminopeptidase, which is 4-(2-amino-4-methylpentanamido)benzyl 3,7-bis(diethylamino)-10H-phenoxazine-10-carboxylate in Chinese and in English.

[0005] The structural formula is as follows:

[0006]

[0007] The application provides a preparation method of the fluorescence probe based on the basic dye for detecting leucine aminopeptidase, which comprises the following steps:

[0008] 1) Dissolve basic blue 3 and sodium carbonate in dichloromethane and water mixture, 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 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 of 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-aminobenzyl alcohol, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and Fmoc-L-leucine in dichloromethane, stir at room temperature, and end the reaction after 24 hours of reaction; remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain the target product of light yellow solid, which is product 2; the molar ratio of the feed is p-aminobenzyl alcohol: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline: Fmoc-L-leucine = 1.1:1.1:1;

[0010] (3) Dissolve product 1, product 2, 4-(dimethylamino)pyridine (DMAP) and anhydrous sodium carbonate in dichloromethane, end 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 of light blue solid, which is product 3; the molar ratio of the feed is product 1: product 2: 4-(dimethylamino)pyridine: anhydrous sodium carbonate = 1:1.2:1:3;

[0011] (4) Dissolve product 3 in N,N-dimethylformamide (DMF), add piperidine dropwise at room temperature, end the reaction after 1 hour, add a large amount of water to the system, extract with ethyl acetate three times; remove the solvent by rotary evaporation and purify the crude product by silica gel column chromatography to obtain the target product of light blue solid, which is the fluorescent probe of claim 1.

[0012] The application provides a method for detecting leucine aminopeptidase, and the steps are as follows:

[0013] (1) Prepare a 10 mM phosphate buffer solution with a pH of 7.4; dissolve the fluorescent probe as described above in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL leucine aminopeptidase stock solution;

[0014] (2) Fluorescence spectrum: 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 4 h of reaction at 37 DEG C, fluorescence spectrum scanning was carried out with 620 nm excitation light, time was 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, 240 min;

[0015] (3) Kinetic curve: 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 4 h of reaction at 37 DEG C, kinetic scanning was carried out with 620 nm excitation wavelength, time length was 4800 s;

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. The fluorescent probe of the present application can sensitively and rapidly detect leucine aminopeptidase;

[0018] 2. The present application is relatively simple in operation, and detection is simple, and only a fluorescence detector is needed;

[0019] 3. The present application has obvious detection signal, and color change of the reaction solution can be observed by naked eye. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 NMR hydrogen spectrum of the fluorescent probe prepared in Example 1

[0021] Figure 2 NMR hydrogen-carbon spectrum of the fluorescent probe prepared in Example 1

[0022] Figure 3 High-resolution mass spectrum of the fluorescent probe prepared in Example 1

[0023] Figure 4 Fluorescence spectrum of the fluorescent probe and leucine aminopeptidase response in Example 2

[0024] Figure 5 Fluorescence intensity value at 676 nm of the fluorescent probe and leucine aminopeptidase response changes with time in Example 3

[0025] Figure 6 Cell imaging diagram of the fluorescent probe incubated cells in Example 4 DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] Synthesis and characterization of fluorescent probes:

[0029] (1) Dissolve 1795 mg of Basic Blue 3 (5 mmol) and 2120 mg of sodium carbonate (20 mmol) in a mixture of dichloromethane (15 mL) and water (10 mL). Stir and heat to 45 °C under nitrogen protection. At this temperature, slowly add 3282 mg of sodium dithionite (20 mmol) dissolved in water. 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 separate. After the system cools to room temperature, 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, 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.

[0030] (2) 1550 mg of p-aminobenzyl alcohol (2.59 mmol), 3150 mg of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (2.59 mmol) and 4150 mg of Fmoc-L-leucine (2.35 mmol) were dissolved in dichloromethane and stirred at room temperature. The reaction was stopped after 24 hours. The solvent was removed by rotary evaporation and the crude product was purified by silica gel column chromatography to obtain the pale yellow solid target product, which is product 2.

[0031] (3) Dissolve 387 mg of product 1 (1 mmol), 458 mg of product 2 (1.2 mmol), 122 mg of 4-(dimethylamino)pyridine (DMAP) (1 mmol) and 318 mg of anhydrous sodium carbonate (3 mmol) in dichloromethane. After reacting under nitrogen protection at room temperature for 8 hours, the reaction was terminated. 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 3.

[0032] (4) Dissolve 16 mg of product 3 (0.02 mmol) in N,N-dimethylformamide (DMF), and add 0.25 mL of piperidine dropwise at room temperature. Stop the reaction after 1 hour; add a large amount of water to the system, extract three times with ethyl acetate, 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 the fluorescent probe described in claim 1.

[0033] Figure 1 This represents the proton NMR spectrum of the fluorescent probe. 1 H NMR spectrum.

[0034] Fluorescent probe: 1H NMR (500 MHz, DMSO) δ 7.65 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 6.39 (dd, J = 9.1, 2.8 Hz, 2H), 6.32 (d, J = 2.8 Hz, 2H), 5.14 (s, 2H), 3.38 (dd, J = 8.7, 5.6 Hz, 1H), 3.30 (q, J = 6.9 Hz, 8H), 1.74 (tt, J = 13.2, 6.6 Hz, 1H), 1.49 (ddd, J = 13.6, 8.2, 5.7 Hz, 1H), 1.39 - 1.32 (m, 1H), 1.06 (t, J = 7.0 Hz, 12H), 0.90 (dd, J = 12.4, 6.6 Hz, 6H).

[0035] Figure 2 The proton nuclear magnetic resonance spectrum of the fluorescent probe is shown. 1 C NMR) spectrum.

[0036] Fluorescent probe: 13 C NMR (126 MHz, DMSO) δ 174.75, 153.51, 151.21, 146.44, 139.25, 131.24, 129.21, 125.64, 119.46, 116.86, 106.65, 99.10, 67.59, 54.16, 44.26, 24.64, 23.60, 22.35, 12.78.

[0037] Figure 3 The high resolution mass spectrum of the fluorescent probe is shown.

[0038] Fluorescent probe: Calc. for C 34 H 46 N5O4 + [M+H] + 588.3550, found 588.3560

[0039] Example 2

[0040] (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 leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL leucine aminopeptidase stock solution;

[0041] (4) Fluorescence spectrum: 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 4 h of reaction at 37 °C, fluorescence spectrum scanning was performed with 620 nm excitation light, time was 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, 240 min; results are shown in Figure 4 The spectrum shows that with 620 nm excitation, the additional addition of leucine aminopeptidase enhances the fluorescence intensity of the fluorescent probe at 676 nm with the increase of time.

[0042] Example 3

[0043] (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 leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL leucine aminopeptidase stock solution;

[0044] (2) Take 830 μL of phosphate buffered saline solution, 2.5 μL of fluorescent probe stock solution, 167 μL of leucine aminopeptidase stock solution and add them into a cuvette, and after 4 h of reaction at 37 °C, kinetic scanning was performed with 620 nm excitation wavelength, scanning time was 4800 s. Results are shown in Figure 5 The spectrum shows that with 620 nm excitation, the additional addition of leucine aminopeptidase enhances the fluorescence intensity of the fluorescent probe at 676 nm with the increase of time.

[0045] Example 4

[0046] (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.

[0047] (2) Cell test: take 10 μL of fluorescent probe stock solution and add it into 2 mL of phosphate buffered saline solution to make its concentration 10 μM; incubate human hepatoma cells Hepa1-6 with the above solution at 37 °C for 4 h, after incubation, wash the cells with 2 mL of phosphate buffered saline solution for 3 times, and image under laser scanning confocal microscope; the second group takes 20 μL of ubenimex stock solution and adds it into 2 mL of phosphate buffered saline solution to make its concentration 20 μM; incubate human hepatoma cells Hepa1-6 with the above solution at 37 °C for 0.5 h, after incubation, add 10 μL of 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 with 2 mL of phosphate buffered saline solution for 3 times, and image under laser scanning confocal microscope; results are shown in Figure 6as shown.

Claims

1. Application of a basic dye-based near-infrared fluorescent probe in the preparation of a leucine aminopeptidase detection reagent, the structural formula of the fluorescent probe is as follows: 。 2. A method for detecting leucine aminopeptidase using a basic dye-based near-infrared fluorescent probe, characterized by, The steps are: (1) Prepare a 10 mM phosphate buffer solution at pH 7.4; dissolve the fluorescent probe in dimethyl sulfoxide to prepare a 2 mM stock solution; dissolve the leucine aminopeptidase freeze-dried powder in water to prepare a 30 U / mL leucine aminopeptidase stock solution; (2) 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 leucine aminopeptidase stock solution and add them to a cuvette, then perform fluorescence spectrum scanning at an excitation wavelength of 620 nm after 4 h of reaction at 37℃, with time points of 0 min, 15 min, 30 min, 45 min, 60 min, 120 min, 180 min, and 240 min; (3) Kinetic curve: take 478.3 μL of the phosphate buffer solution, 5 μL of the fluorescent probe stock solution, and 16.7 μL of the leucine aminopeptidase stock solution and add them to a cuvette, then perform kinetic scanning at an excitation wavelength of 620 nm after 4 h of reaction at 37℃, with a time length of 4800 s; The structural formula of the fluorescent probe is as follows: 。 3. Application of a basic dye-based near-infrared fluorescent probe in the preparation of a cell imaging reagent, the structural formula of the fluorescent probe is as follows: 。

Images