CN110746410B - Leucine aminopeptidase and monoamine oxidase activated near-infrared fluorescent probe, synthetic method and biological application - Google Patents
Leucine aminopeptidase and monoamine oxidase activated near-infrared fluorescent probe, synthetic method and biological application Download PDFInfo
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Abstract
The invention discloses a near-infrared fluorescent probe activated by leucine aminopeptidase and monoamine oxidase, a synthetic method and biological application, wherein the adopted synthetic method comprises the following steps: (1) synthesizing a novel leucine aminopeptidase and monoamine oxidase activated near infrared fluorescent probe (NML); (2) the probe is applied to biological imaging and serum detection. The fluorescent probe responding to leucine aminopeptidase and monoamine oxidase is synthesized for the first time, and the problem of low specificity of the traditional single-enzyme fluorescent probe is solved; the fluorescent probe has near-infrared fluorescence property, so that background fluorescence signals can be effectively reduced, and the sensitivity of the probe is improved; the fluorescent probe has good dyeing effect on living cells and high dyeing efficiency, and can detect endogenous leucine aminopeptidase and monoamine oxidase of the cells.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and relates to a near-infrared fluorescent probe activated by leucine aminopeptidase and monoamine oxidase, a synthetic method and biological application.
Background
Liver diseases such as hepatitis, liver cirrhosis and liver cancer are major diseases worldwide. Currently, millions of people die from liver disease every year. The best treatment for liver disease today is to obtain accurate diagnostic information and perform treatment before it deteriorates. However, similar symptoms of different liver diseases at an early stage limit accurate diagnosis of liver diseases. Standard methods for diagnosis of liver disease, such as histopathological examination and molecular medical imaging, are often ineffective in the middle and late stages of the disease, often resulting in delayed diagnosis. Therefore, there is an urgent need to develop a simple and accurate method for diagnosing and identifying various liver diseases and achieving the purpose of early detection and treatment of the diseases.
The molecular fluorescence imaging method can detect the change of physiological process by an activated fluorescent probe, has high selectivity, high resolution and excellent space-time sampling capability, and can provide powerful assistance for the diagnosis and imaging of liver diseases. In addition, compared with the traditional liver disease diagnosis method, the fluorescent probe can avoid the radioactive hazards generated by the current invasive imaging technology, such as Positron Emission Tomography (PET), Computed Tomography (CT), Single Photon Emission Computed Tomography (SPECT) and the like. Enzymes are common targets in molecular bioimaging and disease diagnosis. At present, the development of non-invasive imaging probes for detecting enzymatic activity in vivo remains an urgent need for cancer diagnosis and related disease detection. Over the past few years, many activated small molecule imaging probes have been widely used in the design of targeted diagnostic or therapeutic compounds. These probe molecules can be selectively activated by disease markers (e.g., enzymes) that are up-regulated in diseased tissue, thus revealing a completely different fluorescent signal from normal tissue. However, the levels of certain disease markers in many liver disease cells and tissues are not significantly increased. Therefore, detection and imaging with only a single activation type of probe may result in false positive signals, failing to achieve accurate diagnosis of a particular liver disease. The double-factor activated probe can effectively overcome the problem and has good application prospect in accurate diagnosis and differentiation of liver diseases.
Leucine Aminopeptidase (LAP) is one of important metallopeptidases, and is involved in various physiological processes ranging from tumor cell invasion, proliferation, drug resistance and angiogenesis to liver injury, and diseased hepatocytes have higher LAP enzyme activity than normal hepatocytes. Monoamine oxidase (MAO) is another flavoprotein enzyme that is widely present in most cell types in vivo. In medical diagnosis, serum MAO is commonly used as one of the indicators for evaluating the degree of cirrhosis. Although many fluorescent probes activated by proteases associated with liver diseases have been developed, most of these probes are activated by a single enzyme, resulting in non-specific activation, thereby limiting their clinical applications. Therefore, we tried to design a dual-factor activated fluorescent probe to achieve accurate imaging and diagnosis of liver disease by using high activity of LAP and MAO in serum as dual criteria for liver disease differentiation.
Disclosure of Invention
The invention aims to provide a near-infrared fluorescent probe activated by leucine aminopeptidase and monoamine oxidase, a synthetic method and biological application, so as to solve the technical problems of low sensitivity, poor specificity and the like of the current fluorescent probe responded by a single enzyme and improve the contrast ratio of fluorescence imaging.
The invention provides a near-infrared fluorescent probe, which has the following structural formula:
the invention provides a synthesis method of a near-infrared fluorescent probe activated by leucine aminopeptidase and monoamine oxidase, which comprises the following steps:
(1) dissolving p-hydroxybenzaldehyde and N-Boc-3-bromopropylamine in acetonitrile, adding potassium carbonate, heating and refluxing for 2-3 h, cooling, filtering, and evaporating under reduced pressure to remove the solvent to obtain a light yellow oily liquid, wherein the light yellow oily liquid is marked as a compound 1;
(2) adding the compound 1 into a dichloromethane solution of trifluoroacetic acid, stirring at normal temperature for 0.5-1 h, and removing the solvent by reduced pressure evaporation to obtain brown oily liquid, which is marked as a compound 2;
(3) adding a compound 2, N-Boc-leucine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 1-hydroxybenzotriazole and N, N-diisopropylethylamine into a dichloromethane solution, stirring at room temperature for 10-12 h, pouring a reaction mixture into water, washing twice, separating an organic phase, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove a solvent, and performing column chromatography separation with petroleum ether and ethyl acetate to obtain a white solid which is marked as a compound 3;
(4) slowly adding sodium borohydride into a methanol suspension of the compound 3, stirring for 2-3 hours at room temperature, diluting the mixture with dichloromethane, washing with water and brine, separating an organic layer, drying with anhydrous sodium sulfate, removing the solvent by reduced pressure evaporation, performing column chromatography, and using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 4;
(5) dropwise adding phosphorus tribromide into a dichloromethane solution of a compound 4, stirring at room temperature for 3-5 h, diluting the mixture with dichloromethane, washing with water and brine, separating an organic layer, drying with anhydrous sodium sulfate, and removing the solvent by reduced pressure evaporation to obtain a crude product of a compound 5;
(6) dissolving 4-bromo-resorcinol and triethylamine in N, N-dimethylformamide, heating and stirring under nitrogen atmosphere, introducing the N, N-dimethylformamide solution of the compound 1 into the mixture through an injector, heating and stirring for 2-3 h, pouring the reaction solution into ice water, extracting with dichloromethane, separating an organic layer, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove the solvent, and purifying to obtain a compound 6 which is a blue-green solid;
(7) mixing the crude product of the compound 5, the compound 6, sodium bicarbonate, 18-crown ether-6 and potassium iodide in acetone, stirring the obtained mixture for 20-24 hours under the protection of nitrogen, then adding dichloromethane, washing the obtained solution with water and brine, drying with anhydrous sodium sulfate, removing the solvent by reduced pressure evaporation, and separating by column chromatography to obtain a compound 7;
(8) and (3) dropwise adding a dioxane hydrochloride solution into a dichloromethane solution of the compound 7, stirring for 10-20 min under the protection of nitrogen, and performing fast separation by column chromatography to obtain a bluish purple solid, which is recorded as NML.
The reaction formula is as follows:
the mass ratio of the p-hydroxybenzaldehyde, the N-Boc-3-bromopropylamine and the potassium carbonate in the step (1) is 1: 1.25: 5.
the volume ratio of the dichloromethane to the trifluoroacetic acid in the step (2) is 1: 1.
the mass ratio of the compound 2, N-Boc-leucine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 1-hydroxybenzotriazole and N, N-diisopropylethylamine in the step (3) is 1: 1: 1.2: 1: 1.2: 3.
the eluent in the step (3) is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the mixture is 2: 1.
the mass ratio of the compound 3 to the sodium borohydride in the step (4) is 1: 2.
the amount ratio of the compound 4 to the phosphorus tribromide in the step (5) is 1: 2.
the mass ratio of the compound 1, 4-bromo-resorcinol and triethylamine in the step (6) is 1: 2.5: 5.
the ratio of the amounts of the compound 5, the compound 6, sodium bicarbonate, 18-crown-6 and potassium iodide in the step (7) is 2: 1: 2: 1: 5.
and (3) eluent adopted by the column chromatography in the step (7) is a mixture of dichloromethane and methanol, and the volume ratio of the mixture is 200: 1-50: 1.
the mass ratio of the compound 7 to HCl in dioxane hydrochloride in the step (8) is 1: 30 to 50.
Dioxane hydrochloride solution is a commercially available, colorless and transparent liquid, and serves to provide excess HCl, which ultimately leads to NML formation of compound 7.
And (3) the eluent adopted by the column chromatography in the step (8) is a mixture of dichloromethane and methanol, and the volume ratio of the mixture is 200: 1-50: 1.
the invention provides application of a near-infrared fluorescent probe activated by leucine aminopeptidase and monoamine oxidase in detecting the activities of leucine aminopeptidase and monoamine oxidase in an in vitro buffer solution.
The invention also provides application of the near-infrared fluorescent probe activated by the leucine aminopeptidase and the monoamine oxidase in detecting the activities of the leucine aminopeptidase and the monoamine oxidase in living cells.
The invention adopts a near-infrared fluorescent dye which is based on an intramolecular charge transfer mechanism, and the fluorescence property of the near-infrared fluorescent dye can be regulated and controlled by protecting and deprotecting the hydroxyl group of the near-infrared fluorescent dye. The dye is utilized to synthesize the leucine aminopeptidase and monoamine oxidase activated near-infrared fluorescent probe NML, the fluorescent probe has better water solubility, and after the fluorescent probe reacts under the catalysis of leucine aminopeptidase and monoamine oxidase, a fluorophore NF with near-infrared luminescence is released, so that high-resolution and high-contrast fluorescent imaging of the leucine aminopeptidase and monoamine oxidase can be realized.
Compared with the prior art, the invention has the beneficial technical effects that:
1) the fluorescent probe has near-infrared luminescence property, can effectively reduce background fluorescence signals and improve the sensitivity of the probe;
2) the fluorescent probe has higher selectivity on leucine aminopeptidase and monoamine oxidase;
4) the fluorescent probe has good dyeing effect on living cells and high dyeing efficiency;
5) the fluorescent probe can detect and image cell endogenous leucine aminopeptidase and monoamine oxidase;
6) the fluorescent probe has good light stability and stable fluorescent signal in living cell imaging.
Drawings
FIG. 1 is a graph showing the change of fluorescence emission spectra of the fluorescent probe with increasing activities of leucine aminopeptidase and monoamine oxidase, with the abscissa being wavelength (nm) and the ordinate being fluorescence emission intensity.
FIG. 2 is a reaction kinetic curve of the fluorescent probe with leucine aminopeptidase and monoamine oxidase, with the abscissa representing reaction time (min) and the ordinate representing fluorescence emission intensity.
FIG. 3 is a confocal imaging test chart of the fluorescence probe for liver cancer cells and normal liver cells (the staining solution is PBS buffer solution, pH 7.4).
FIG. 4 is a confocal image of the fluorescent probe for detection of endogenous leucine aminopeptidase and monoamine oxidase in living cells (the staining solution is PBS buffer, pH 7.4).
FIG. 5 is a confocal imaging detection of the fluorescence probe for live cell mitochondria (staining solution PBS buffer, pH 7.4).
FIG. 6 is the serum of the fluorescent probe imaging and differentiating different liver disease model mice and humans.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The process is conventional unless otherwise specified, and the starting materials are commercially available from open sources.
Example 1 Synthesis of near Infrared leucine aminopeptidase and monoamine oxidase fluorescent Probe NML
(1) Synthesis of Compound 1: p-hydroxybenzaldehyde (2.44g, 20mmol), (3-bromo-propyl) carbamic acid tert-butyl ester (5.95g, 25mmol) and anhydrous potassium carbonate (13.8g, 100mmol) were placed in a flask containing acetonitrile (80 mL). Stirring the mixture at 85 ℃ for 2-3 hours; the solution was cooled and separated by filtration. The filtrate was removed under reduced pressure to give the product as a pale yellow oily liquid (4.81g, yield 86%) without purification.1H NMR(400MHz,CDCl3)δ9.79(s,1H),7.74(d,J＝8.0Hz,2H),6.92(d,J＝8.1Hz,2H),5.07(s,1H),4.04(t,J＝5.5Hz,2H),3.27(s,2H),1.95(d,J＝7.1Hz,2H),1.37(s,9H).13C NMR(101MHz,CDCl3)δ190.73,163.85,156.06,131.90,129.85,114.70,79.07,66.00,37.60,29.46,28.34；
(2) Synthesis of Compound 2: dissolving compound 1(2.79g, 10mmol) in trifluoroacetic acid/dichloromethane (20mL/20mL) at room temperature for 0.5-1 h; the solvent was removed by evaporation under reduced pressure and the crude product 2 obtained was used in the next reaction without further purification;
(3) synthesis of Compound 3: compound 2(1.79g, 10mmol), N-Boc-leucine (2.31g, 10mmol), EDCI (1.55g, 10mmol), DMAP (1.22g, 10mmol), HOBt (1.35g, 10mmol), and DIPEA (3.87g, 30mmol) were dissolved in dichloromethane (50mL) at room temperature, and stirred at room temperature for 10 to 12 hours. The solution was then washed with water and brine, and dried over anhydrous sodium sulfate. Removing the solvent under reduced pressureThe residue was purified by flash chromatography using petroleum ether/ethyl acetate (2/1) as eluent to give compound 3 as a white solid (2.45g, 62.5% yield);1H NMR(400MHz,CDCl3)δ9.87(s,1H),7.82(d,J＝8.1Hz,2H),7.00(d,J＝8.1Hz,2H),6.73(s,1H),5.05(d,J＝6.4Hz,1H),4.10(t,J＝5.4Hz,3H),3.46(d,J＝3.6Hz,2H),2.08-2.00(m,2H),1.66(d,J＝5.7Hz,2H),1.54-1.46(m,1H),1.43(d,J＝7.8Hz,9H),0.93(s,6H).13C NMR(101MHz,CDCl3)δ190.80,172.89,163.74,155.84,131.99,130.02,114.74,80.09,66.21,53.22,41.23,36.74,28.98,28.29,24.77,22.93,21.96；
(4) synthesis of Compound 4: to a suspension of compound 3(1.18g, 3.0mmol) in methanol (80mL) at 0 deg.C was slowly added sodium borohydride (228mg, 6mmol), and the resulting suspension was stirred at room temperature for 2-3 hours. The mixture was then diluted with dichloromethane (100mL) and washed with water and brine. The organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure and the residue was chromatographed on silica gel using petroleum ether/ethyl acetate (1/1) as eluent to give compound 4 as a white solid (1.00g, yield 85%).1H NMR(400MHz,DMSO)δ7.89(s,1H),7.21(d,J＝7.5Hz,2H),6.86(d,J＝7.4Hz,3H),5.05(s,1H),4.41(d,J＝4.4Hz,2H),3.94(s,3H),3.36(s,2H),3.20(s,2H),1.83(s,2H),1.56(s,1H),1.37(s,9H),0.89-0.81(m,6H).13C NMR(101MHz,DMSO)δ173.00,157.89,155.77,134.96,128.32,114.47,78.35,65.65,63.01,53.31,41.35,35.98,29.32,28.64,24.76,23.36,22.09；
(5) Synthesis of Compound 5: to a solution of compound 4(985mg, 2.5mmol) in dichloromethane (50mL) was added tribromophosphine (0.474mL, 5.0mmol) dropwise at 0 ℃. And stirring the reaction solution at room temperature for 3-5 hours. The mixture was then diluted with dichloromethane (20mL) and washed with water and brine; the organic layer was separated and dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure and the crude product 5 obtained was used in the next reaction without further purification;
(6) synthesis of Compound 6: 4-bromo-resorcinol (1.88g, 10mmol) and triethylamine (2mL) were placed in a flask containing N, N-dimethylformamide (10mL) and the mixture was brought to 50 deg.CAnd stirred under nitrogen for 10 minutes. A solution of Compound 1(3.0g, 5mmol) in N, N-dimethylformamide (10.0mL) was introduced into the mixture via syringe and the reaction mixture was heated at 55 ℃ for 2-3 hours. Then, the reaction solution was poured into ice water and extracted with dichloromethane. The organic layer was separated and dried over anhydrous sodium sulfate, the solvent was removed by evaporation under reduced pressure, and after purification by silica gel chromatography (dichloromethane/ethanol, 10: 1, v/v), compound 6 was obtained as a blue-green solid (2.24g, 38%).1H NMR(CDCl3,400MHz)δ8.14(d,J＝8.11Hz,1H),7.59(s,1H),7.32-7.29(m,J＝7.30Hz,3H),7.10-7.06(t,J＝7.08Hz,1H),6.87(d,J＝6.86Hz,1H),6.70(s,1H),5.62(d,J＝5.61Hz,1H),3.37(s,3H),2.70-2.67(t,J＝2.69Hz,2H),2.64-2.61(t,J＝2.62Hz,2H),1.86(t,J＝1.90Hz,2H),1.63(s,6H)；
(7) Synthesis of compound 7: compound 6(462mg, 1mmol), compound 5(912mg, 2mmol), sodium bicarbonate (168mg, 2mmol), 18-crown-6 (264mg, 1mmol) and potassium iodide (1.66) g, 10mmol) were combined in 40mL of acetone and the resulting mixture was stirred at 40 ℃ under nitrogen for 20-24 hours. Then, dichloromethane (50mL) was added, and the resulting solution was washed with water and brine, and dried over anhydrous sodium sulfate. After removal of the solvent, the residue was purified by flash chromatography on silica gel using dichloromethane/methanol 200/1-50/1 as eluent to give compound 7 as a blue-violet solid (246.7mg, 29% yield).1H NMR(400MHz,CDCl3)δ8.60(d,J＝14.9Hz,1H),7.56(d,J＝9.8Hz,2H),7.47(d,J＝7.6Hz,3H),7.45-7.40(m,2H),7.11(s,1H),7.03(s,1H),6.92(d,J＝7.7Hz,2H),6.86(d,J＝8.0Hz,1H),6.78(d,J＝11.8Hz,1H),6.50(d,J＝14.9Hz,1H),5.31(s,2H),5.06(d,J＝7.0Hz,1H),4.07(s,3H),4.02(d,J＝5.6Hz,2H),3.44(s,2H),2.73(d,J＝18.7Hz,4H),2.28(d,J＝7.0Hz,2H),2.00(d,J＝6.1Hz,2H),1.90(s,2H),1.82(s,6H),1.65(d,J＝4.0Hz,2H),1.45(s,3H),1.42(s,9H),0.92(d,J＝4.4Hz,6H).13C NMR(101MHz,CDCl3)δ178.11,172.82,160.71,158.73,157.66,153.35,145.82,142.13,141.59,131.53,130.83,129.33,129.12,128.56,127.64(s,4H),122.60,116.48,115.25,114.53,112.86,109.45,105.15,101.57,77.32,71.78,66.18,53.51,50.90,37.09,34.87,29.27,29.02,28.06,24.77,23.02,21.93,20.16。
(8) Synthesis of NML: to a solution of compound 7(100mg, 0.12mmol) in dichloromethane (2mL) was added dropwise a dioxane hydrochloride solution (1mL, 4mol/L) at 0 deg.C, and the resulting mixture was stirred at 0 deg.C for 10-20 minutes. The residue was then flash purified by silica gel flash chromatography using dichloromethane/methanol (100/1) as eluent to give a blue-violet solid (30mg, 41% yield) named NML.
Nuclear magnetic and mass spectral characterization of NML
1H NMR(400MHz,MeOD)δ8.71(d,J＝14.9Hz,1H),7.70(d,J＝6.8Hz,2H),7.59(dd,J＝16.3,7.5Hz,2H),7.48(dd,J＝13.8,7.5Hz,3H),7.22(d,J＝9.9Hz,2H),6.98(d,J＝7.6Hz,2H),6.56(d,J＝15.0Hz,1H),5.28(s,2H),4.04(d,J＝5.2Hz,2H),3.91(s,3H),3.82(s,1H),3.67(s,1H),2.82-2.67(m,4H),2.05-1.99(m,2H),1.92(s,2H),1.84(s,6H),1.65(d,J＝6.0Hz,3H),1.28(s,2H),0.98-0.92(m,6H).13C NMR(101MHz,MeOD)δ178.08,172.79,160.68,158.70,157.63,153.32,145.80,142.10,141.56,131.51,130.80,129.30,129.10,128.53,127.62,122.57,116.46,115.23,114.50,112.84,109.42,105.13,101.55,71.75,66.16,53.49,50.87,37.06,34.85,29.24,28.99,28.03,24.74,22.99,21.91,20.13.ESI-MS:m/z calcd for NML(C42H49BrN3O4 +,[M]),740.29；found,740.2.
EXAMPLE 2NML Probe detection of leucine aminopeptidase and monoamine oxidase in vitro
NML probe was prepared as a 1mM DMSO stock solution and stored at-20 ℃. The assay was PBS buffer (10mM, pH 7.4, 10% DMSO). The reaction system of the NML probe and the enzyme was shaken at 37 ℃ for 2 hours, and then its fluorescence emission spectrum was measured. The excitation wavelength of the fluorescence instrument is 670nm, and the receiving range of the emission wavelength is 700-900 nm. The results are shown in fig. 1, and it can be seen from fig. 1 that the NML probe responded well to leucine aminopeptidase and monoamine oxidase.
Example 3 reaction kinetics study of NML probes with enzymes
The reaction system was PBS buffer (10mM, pH 7.4, 10% DMSO). The fluorescence emission spectra were measured in real time at 37 ℃ with NML probe and leucine aminopeptidase and monoamine oxidase enzymes of different activities until the fluorescence intensity did not change any more. The intensity of the maximum emission peak was then plotted on the ordinate and the reaction time on the abscissa to obtain the reaction kinetics curve shown in fig. 2, from which it can be seen that the NML probe responds rapidly to both enzymes. The excitation wavelength of the fluorometer was set to 670nm and the emission wavelength was set to 720 nm.
Example 4NML Probe imaging experiments on staining of viable cells
a) L02 and HepG2 cells were seeded in optical culture dishes in advance, 4 ten thousand cells were seeded per dish and incubated for 24 h. After incubating the NML probe with the cells for 2h, the original medium (DMEM, containing 5% FBS and 10% double antibody) was aspirated, PBS buffer (10mM, pH 7.4) was added, and the cells were washed 3 times with PBS and the fluorescence signal was detected with confocal laser microscopy. The results are shown in fig. 3, and it can be seen from fig. 3 that the NML probe can distinguish between LO2 and HepG2 cells;
b) HepG2 cells were seeded in optical culture dishes in advance, 4 ten thousand cells were seeded per dish and incubated for 24 h. Leucine aminopeptidase inhibitor (ubenimex) and monoamine oxidase inhibitor (clorgoline) were added separately and incubated for half an hour. The probe NML was added to the cells, then the original medium (DMEM, containing 5% FBS and 10% double antibody) was aspirated, PBS buffer (10mM, pH 7.4) was added, and the fluorescence signal was detected with a confocal laser microscope. As shown in fig. 4, it can be seen from fig. 4 that the NML probe can detect leucine aminopeptidase and monoamine oxidase in cells well, and when the two enzymes are inhibited, the probe cannot give a fluorescent signal;
c) HepG2 cells were seeded in optical culture dishes in advance, 4 ten thousand cells were seeded per dish and incubated for 24 h. After the NML probe and the cells are incubated for 2h, a mitochondrial positioning agent is added for incubation for 0.5h, then the original culture medium (DMEM, containing 5% FBS and 10% double antibody) is aspirated, PBS buffer (10mM, pH 7.4) is added, the cells are washed for 3 times by PBS, and the fluorescence signals of the cells are detected by a laser confocal microscope. As a result, as shown in fig. 5, it can be seen from fig. 5 that the NML probe is mainly distributed in mitochondria in the cell.
Example 5 serum with NML probes for differentiating different models of liver disease
a) NML probe was added to PBS buffer (10mM, pH 7.4) containing different liver disease model sera, incubated at 37 ℃ for 2 hours and its fluorescence signal was detected with a small animal imager. The results are shown in fig. 6, and it can be seen from fig. 6 that the NML probe can well distinguish the sera of normal mice, acetaminophen-induced liver injury mice and carbon tetrachloride-induced cirrhosis mice;
b) NML probe was added to PBS buffer (10mM, pH 7.4) containing different liver disease model sera, incubated at 37 ℃ for 2 hours and its fluorescence signal was detected with a small animal imager. The results are shown in FIG. 6, and it can be seen from FIG. 6 that the NML probe can well discriminate the sera of normal persons, hepatitis patients and liver cirrhosis patients.
Claims (10)
1. A near-infrared leucine aminopeptidase and monoamine oxidase responsive fluorescent probe, the structural formula of the fluorescent probe is as follows:
2. a method of synthesizing the near infrared leucine aminopeptidase and monoamine oxidase-responsive fluorescent probe of claim 1, comprising the steps of:
(1) dissolving p-hydroxybenzaldehyde and N-Boc-3-bromopropylamine in acetonitrile, adding potassium carbonate, heating and refluxing for 2-3 h, cooling, filtering, and evaporating under reduced pressure to remove the solvent to obtain a light yellow oily liquid, wherein the light yellow oily liquid is marked as a compound 1;
(2) adding the compound 1 into a dichloromethane solution of trifluoroacetic acid, stirring at normal temperature for 0.5-1 h, and removing the solvent by reduced pressure evaporation to obtain brown oily liquid, which is marked as a compound 2;
(3) adding a compound 2, N-Boc-leucine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 1-hydroxybenzotriazole and N, N-diisopropylethylamine into a dichloromethane solution, stirring at room temperature for 10-12 h, pouring a reaction mixture into water, washing twice, separating an organic phase, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove a solvent, and performing column chromatography separation with petroleum ether and ethyl acetate to obtain a white solid which is marked as a compound 3;
(4) slowly adding sodium borohydride into a methanol suspension of the compound 3, stirring for 2-3 hours at room temperature, diluting the mixture with dichloromethane, washing with water and brine, separating an organic layer, drying with anhydrous sodium sulfate, removing the solvent by reduced pressure evaporation, performing column chromatography, and using a mixed solution of petroleum ether and ethyl acetate as an eluent to obtain a compound 4;
(5) dropwise adding phosphorus tribromide into a dichloromethane solution of a compound 4, stirring at room temperature for 3-5 h, diluting the mixture with dichloromethane, washing with water and brine, separating an organic layer, drying with anhydrous sodium sulfate, and removing the solvent by reduced pressure evaporation to obtain a crude product of a compound 5;
(6) dissolving 4-bromo-resorcinol and triethylamine in N, N-dimethylformamide, heating and stirring under a nitrogen atmosphere, introducing the N, N-dimethylformamide solution of the compound 1 into the mixture through an injector, heating and stirring for 2-3 hours, pouring the reaction solution into ice water, extracting with dichloromethane, separating an organic layer, drying with anhydrous sodium sulfate, evaporating under reduced pressure to remove the solvent, and purifying to obtain a blue-green solid which is marked as a compound 6;
(7) mixing the crude product of the compound 5, the compound 6, sodium bicarbonate, 18-crown ether-6 and potassium iodide in acetone, stirring the obtained mixture for 20-24 hours under the protection of nitrogen, then adding dichloromethane, washing the obtained solution with water and brine, drying with anhydrous sodium sulfate, removing the solvent by reduced pressure evaporation, and separating by column chromatography to obtain a compound 7;
(8) dropwise adding a dioxane hydrochloride solution into a dichloromethane solution of the compound 7, stirring for 10-20 min under the protection of nitrogen, and performing fast separation by column chromatography to obtain a bluish purple solid, which is recorded as NML;
the synthetic route is as follows:
3. the method for synthesizing a near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe of claim 2, wherein the ratio of the amounts of p-hydroxybenzaldehyde, N-Boc-3-bromopropylamine and potassium carbonate in step (1) is 1: 1.25: 5.
4. the method for synthesizing the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the volume ratio of dichloromethane to trifluoroacetic acid in the step (2) is 1: 1.
5. the method for synthesizing the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the ratio of the amounts of the compounds 2, N-Boc-leucine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, 1-hydroxybenzotriazole and N, N-diisopropylethylamine in the step (3) is 1: 1: 1.2: 1: 1.2: 3; the eluent in the step (3) is a mixture of petroleum ether and ethyl acetate, and the volume ratio of the mixture is 2: 1.
6. the method for synthesizing the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the ratio of the amounts of the compound 3 and the sodium borohydride in the step (4) is 1: 2.
7. the method for synthesizing the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the ratio of the amounts of the compound 4 and the phosphorus tribromide in step (5) is 1: 2.
8. the method for synthesizing the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the ratio of the amounts of the compounds 1, 4-bromo-resorcinol and triethylamine in the step (6) is 1: 2.5: 5.
9. the method for synthesizing a near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probe according to claim 2, wherein the ratio of the amounts of the compound 5, the compound 6, sodium bicarbonate, 18-crown-6 and potassium iodide in the step (7) is 2: 1: 2: 1: 5; and (3) eluent adopted by the column chromatography in the step (7) is a mixture of dichloromethane and methanol, and the volume ratio of the mixture is 200: 1-50: 1.
10. use of the near-infrared leucine aminopeptidase and monoamine oxidase fluorescent probes according to claim 1 or 2 for the preparation of buffered solutions in vitro or of detection reagents for the detection of the activities of leucine aminopeptidase and monoamine oxidase in living cells.
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