A fluorescent probe for detecting methionine aminopeptidase 1 and its preparation method and application

Abstract

The present invention discloses a fluorescent probe for detecting methionine aminopeptidase 1, and its preparation method and application. The fluorescent probe structural formula is shown in formula (1). The present invention also discloses its preparation method and application, and discloses a method for quantitatively detecting the activity of methionine aminopeptidase 1 using the fluorescent probe. The specific fluorescent probe provided by the present invention has high specificity during application and can be detected by a fluorescence detector. At the same time, the DDAN-MT provided by the present invention has a long-wavelength fluorescence emission wavelength, which can better reduce the interference of biological background fluorescence and has high sensitivity. In addition, the DDAN-MT provided by the present invention can be obtained by simple chemical synthesis, the synthesis process is simple and easy, and the fluorescence method detection cost is low.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a fluorescent probe for detecting methionine aminopeptidase 1, and a preparation method and application thereof. Background Art

[0002] Tuberculosis (TB) is a fatal disease caused by mycobacteria. Mycobacterium tuberculosis is the primary pathogen of human tuberculosis and poses a serious threat to public health. In recent years, the rapid emergence of multidrug-resistant and extensively drug-resistant strains has made drug-resistant TB a treatment challenge. Prior art has developed first- and second-line anti-TB drugs, such as isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin, for the treatment of susceptible Mycobacterium tuberculosis. However, these drugs have little clinical therapeutic effect on TB caused by drug-resistant Mycobacterium tuberculosis.

[0003] Methionine aminopeptidase 1 (MtMET-AP1) is a binuclear metalloproteinase responsible for cleaving the initiating methionine at the N-terminus of nascent peptide chains. The gene encoding this enzyme is essential for cell survival and is conserved across all life forms, from bacteria to humans. MtMET-AP1 has been found to be a key factor in inducing virulence expression in Mycobacterium tuberculosis (MtTB) within host cells. By providing the methionine required for initiation of protein expression, MtMET-AP1 influences the pathogenicity of M. tuberculosis and represents a novel drug target. This discovery is of great significance for the development of new drugs and treatments for tuberculosis.

[0004] Therefore, how to provide a specific fluorescent probe substrate with high sensitivity and strong resistance to environmental interference suitable for in vivo and in vitro detection of methionine aminopeptidase 1, and establish a fluorescent detection method for high-throughput screening of MtMET-AP1 inhibitors are technical problems that technicians in this field urgently need to solve. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a fluorescent probe for detecting methionine aminopeptidase 1 that is highly specific, inexpensive, readily available and sensitive, as well as a preparation method and application thereof.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] A fluorescent probe for detecting methionine aminopeptidase 1, wherein the fluorescent probe has a structural formula as shown in formula (1).

[0008]

[0009] Beneficial Effects: The present invention uses the methionine group as the metabolic recognition site of methionine aminopeptidase 1 and 1,3-dichloro-7-amino-9,9-dimethyl-2(9H)-acridone as a fluorescent group. It can be used for the visual detection of Mycobacterium tuberculosis methionine aminopeptidase.

[0010] A method for preparing a fluorescent probe for detecting methionine aminopeptidase 1 comprises the following steps:

[0011] The fluorescent probe for detecting methionine aminopeptidase 1 is prepared by acylation reaction using methionine and 1,3-dichloro-7-amino-9,9-dimethyl-2(9H)-acridone as raw materials.

[0012] A fluorescent probe for detecting methionine aminopeptidase 1 is used in detecting the activity of recombinantly expressed methionine aminopeptidase 1 or endogenous methionine aminopeptidase 1 of Mycobacterium tuberculosis.

[0013] Beneficial effects: The hydrolysis product of the specific fluorescent probe in the present invention is a long-emission wavelength fluorescent probe, which is not easily interfered by the biological system matrix and impurities during the methionine aminopeptidase 1 activity detection process and can be used for the quantitative determination of methionine aminopeptidase 1 activity in various recombinant methionine aminopeptidase 1 or Mycobacterium tuberculosis.

[0014] A method for detecting methionine aminopeptidase 1 comprises using the fluorescent probe as a specific substrate of methionine aminopeptidase 1 to carry out a hydrolysis reaction, and then using fluorescence detection to quantitatively detect the generation rate of 7-amino-1,3-dichloro-9,9-dimethylacridin-2(9-hydrogen)-one per unit time to quantitatively determine the activity of methionine aminopeptidase 1.

[0015] More preferably, the methionine aminopeptidase 1 is recombinantly expressed methionine aminopeptidase 1 or methionine aminopeptidase 1 from Mycobacterium tuberculosis.

[0016] Detection of methionine aminopeptidase 1 can provide technical support for the discovery of active molecules that inhibit Mycobacterium tuberculosis.

[0017] Preferably, before the hydrolysis reaction, the reaction system of methionine aminopeptidase 1 is pre-incubated;

[0018] The reaction system includes a buffer solution and methionine aminopeptidase 1, and the concentration of methionine aminopeptidase 1 is 0-17.5 μg / mL;

[0019] The pre-incubation temperature is 37° C., the pre-incubation time is 3 minutes, and the pre-incubation pH is 7-8.

[0020] The detection of enzyme activity in a nearly neutral system is conducive to the application of the probe in the visual detection of enzyme activity in biological systems.

[0021] Preferably, in the hydrolysis reaction, the concentration of the fluorescent probe is 2.5 μmol / L;

[0022] The hydrolysis reaction temperature is 37°C and the time is 30 minutes;

[0023] The present invention only requires a catalytic reaction time of 30 minutes, which reflects a faster reaction rate of the probe.

[0024] Preferably, the excitation wavelength in the fluorescence detection is 600 nm and the maximum emission wavelength is 668 nm.

[0025] Beneficial effects: The fluorescent probe and its hydrolysis product provided by the present invention have different optical properties, and a fluorescence detector can be used to achieve rapid and sensitive detection of the product.

[0026] Application of a fluorescent probe for detecting methionine aminopeptidase 1 in the rapid screening of methionine aminopeptidase 1 inhibitors and the quantitative evaluation of their inhibitory ability.

[0027] This system can be used to quickly and efficiently discover inhibitors of methionine aminopeptidase 1, providing a rich resource of candidate substances for anti-tuberculosis drug screening.

[0028] Application of a fluorescent probe for detecting methionine aminopeptidase 1 in the evaluation of methionine aminopeptidase 1 activity in Mycobacterium tuberculosis.

[0029] Visual evaluation of methionine aminopeptidase activity in Mycobacterium tuberculosis is crucial for assessing the virulence of pathogens and also provides guidance for the discovery of specific antibacterial drugs.

[0030] A fluorescent probe for detecting methionine aminopeptidase 1 is used in high-throughput screening of methionine aminopeptidase 1 inhibitors. Inhibitors have the potential to inhibit Mycobacterium tuberculosis and are an important resource for anti-tuberculosis drug discovery.

[0031] The present invention discloses a fluorescent probe for detecting methionine aminopeptidase 1, as well as its preparation method and application. The specific fluorescent probe substrate provided by the present invention can be selectively hydrolyzed by methionine aminopeptidase 1 to generate a hydrolysis product with significantly altered fluorescence properties. This probe has high specificity and can be detected by a fluorescence detector. Furthermore, the probe reaction can be used to quantitatively evaluate the activity and function of methionine aminopeptidase 1 in Mycobacterium tuberculosis and can be used to screen for methionine aminopeptidase 1 inhibitors. Furthermore, the DDAN-MT provided by the present invention has a long-wavelength fluorescence emission wavelength, which can effectively reduce the interference of biological background fluorescence and has high sensitivity. Furthermore, the DDAN-MT provided by the present invention can be obtained through simple chemical synthesis, with a simple and easy synthesis process and low-cost fluorescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0033] Figure 1 The DDAN-MT H NMR spectrum in Example 1 ( 1 H-NMR);

[0034] Figure 2 The DDAN-MT carbon NMR spectrum in Example 1 ( 13 C-NMR);

[0035] Figure 3 This is the high-resolution mass spectrum of DDAN-MT in Example 1;

[0036] Figure 4 The UV absorption and fluorescence emission spectra of DDAN-MT and its hydrolysis products;

[0037] Figure 5 The results of the screening experiment of different hydrolases on DDAN-MT in Example 2 are as follows;

[0038] Figure 6 The results of the screening experiment of different ions and amino acids on DDAN-MT in Example 3 are shown;

[0039] Figure 7 The linear change of the enzyme catalyzing the DDAN-MT probe reaction by methionine aminopeptidase 1 in Example 4;

[0040] Among them, a is the fluorescence spectrum under the catalytic action of different enzyme activities, and b is the correlation analysis between enzyme activity and fluorescence intensity. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] The raw materials in the examples of the present invention were all purchased from commercial sources.

[0044] Example 1

[0045] A fluorescent probe for detecting methionine aminopeptidase 1, the structural formula of which is shown in formula (1):

[0046]

[0047] The fluorescent probe is named 2-amino-N-(6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl)-4-(methylthio)butanamide (DDAN-MT).

[0048] Its synthetic route is:

[0049]

[0050] The preparation method specifically comprises the following steps:

[0051] (1) Compound 1 (1.52 g) and compound 2 (2.1 g) were placed in a 100 mL single-necked bottle, THF (5 mL) and H2O (5 mL) were added to dissolve (orange-red), and stirred in an ice bath for 10 min. Then, a NaOH aqueous solution (10.6 mL, 2 M) was slowly added dropwise to the reaction solution. After the reaction solution changed from orange-red to blue, it was stirred in an ice bath for 2 h. The reaction solution was then poured into a mixture of ethyl acetate (300 mL) and saturated NH4Cl aqueous solution (350 mL), stirred, and separated. The organic layer was then washed with saturated NH4Cl aqueous solution (350 mL) to obtain a blue-purple organic layer (Compound 3).

[0052] (2) Na2S2O4 (50 g, sodium hydroxide powder) was dissolved in H2O (500 mL) to obtain a Na2S2O4 aqueous solution. The above blue-purple organic layer was added to the Na2S2O4 aqueous solution (250 mL) and stirred for 25 min. The reaction solution changed from blue-purple to orange-yellow. The layers were separated, and the Na2S2O4 aqueous solution (250 mL) was added to the organic layer and stirred for 25 min. The layers were separated again, and the organic layer was washed with saturated NaCl water and dried to obtain a brown-black solid (Compound 4).

[0053] (3) Place HCl aqueous solution (200 ml, 2N) in a 250 mL two-necked flask and pass N2 for 15 min. Dissolve compound 4 in MeOH (12 ml) and slowly drip into the HCl solution. Then, reflux at 105°C for 1.5 h under N2. Cool the reaction solution to room temperature, then add ethyl acrylate (200 mL) and stir to separate the layers. The aqueous layer is basically colorless, while the ethyl acrylate layer is brown. Separate the layers. Extract the aqueous phase again with ethyl acrylate (150 mL). Combine the organic layers, wash with saturated NaCl water, and separate the layers to obtain a brown organic layer.

[0054] (4) 3.2 g of NaIO4 was dissolved in 110 mL of H2O, and the above brown organic layer was added. The mixture was stirred at room temperature for 1 h, and the reaction solution changed from brown to red. The liquid was separated, and the organic phase was washed with saturated NaCl water, and the black-red solid was dried. The crude product was ultrasonically beaten with EtOH (8 ml), filtered, and dried to obtain compound 6. Compounds 6 and 7 were dissolved in CH2Cl2 and stirred at room temperature for 1 h for deprotection. After removing the solvent, the residue was purified on a SiO2 column with CH2Cl2 / MeOH (16 / 1, v / v) to obtain DDAN-MT as an orange solid (20 mg, yield: 32.4%).

[0055] The spectral data for the structural identification of DDAN-MT are as follows:

[0056] H NMR spectroscopy (e.g. Figure 1 ): 1 H-NMR (600MHz, DMSO-d6) δ7.85(s,1H),7.74(d,J=2.1Hz,1H),7.73(d,J=2.1Hz,1H),7.67(s, 1H),4.08–4.05(m,1H),2.61–2.56(m,2H),2.19–2.13(m,1H),2.13–2.07(m,4H),1.83(s,6H).

[0057] C NMR spectroscopy (e.g. Figure 2 ): 13C-NMR(150MHz,DMSO-d6)δ172.42,167.77,148.51,141.62,140.52,139.32,139.11,136 .98,135.70,133.68,132.55,118.95,117.32,52.65,38.52,30.59,28.22,26.31,14.41.

[0058] High-resolution mass spectrometry (eg Figure 3 ): HRMS(+)m / z 438.0810[M+H] + , calculated value m / z 438.0732.

[0059] Example 2

[0060] In vitro determination of the selectivity of different hydrolases:

[0061] (1) Prepare 199 μL of 9 different in vitro metabolic reaction systems containing various hydrolases. The reaction system consists of pH 7.4 buffer (100 mM Hepes, 50 mM NaCl, and 200 μM CoCl2) and hydrolases (11 μg / mL), and pre-incubate at 37°C with shaking for 3 minutes.

[0062] Among them, the nine different types of hydrolases were human serum albumin, bovine serum albumin, dipeptidyl peptidase IV, aminopeptidase N, α-glucosidase, β-glucuronidase, human lipase, porcine lipase, and methionine aminopeptidase 1;

[0063] (2) Add 1 μL of 500 μM DDAN-MT (final concentration 2.5 μM) to each of the nine in vitro metabolic reaction systems pre-incubated in step (1), react at 37°C for 30 min, then add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0064] (3) The mixture was centrifuged at 4°C and 20,000 × g for 20 min in a high-speed refrigerated centrifuge. The supernatant was collected and the yield of 7-amino-1,3-dichloro-9,9-dimethylacridin-2(9-hydro)-one was determined by fluorescence detection (fluorescence detection conditions: Ex = 600 nm, Em = 668 nm).

[0065] like Figure 4 DDAN-MT and its enzymatic metabolite DDAN showed different UV absorption spectra, with DDAN-MT showing a significant red shift. DDAN exhibited a strong fluorescence spectrum under 600 nm excitation light.

[0066] like Figure 5The fluorescence detection results showed that the reaction rate of the reaction catalyzed by methionine aminopeptidase 1 (MtMET-AP1) was much higher than that of other hydrolases, indicating that the reaction of DDAN-MT catalyzed by methionine aminopeptidase 1 had good selectivity. The DDAN-MT provided by the present invention can be used for the activity determination of methionine aminopeptidase 1.

[0067] Example 3

[0068] In vitro determination of the effects of different metal ions and amino acids on the fluorescence intensity of DDAN-MT:

[0069] (1) Different types of metal ions (final concentration 200 μM) or amino acids (final concentration 10 μM) were added to pH 7.4 buffer (100 mM Hepes, 50 mM NaCl, and 200 μM CoCl2) and pre-incubated at 37°C with shaking for 3 minutes.

[0070] The metal ion includes one of sodium ion, zinc ion, sulfate ion, carbonate ion, manganese ion, calcium ion, iodide ion, chromium ion, barium ion, nickel ion, potassium ion, magnesium ion, copper ion, divalent iron ion, trivalent iron ion and cobalt ion;

[0071] The amino acids include one of lysine, serine, histidine, glycine, arginine, glutamine, glutamic acid, tryptophan, glutathione, and tyrosine;

[0072] (2) Add 1 μL of 500 μM DDAN-MT (final concentration 2.5 μM) to the pre-incubated in vitro metabolic reaction system obtained in step (1), react at 37°C for 30 min, then add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0073] (3) Centrifuge the mixture at 4°C and 20,000×g for 20 minutes using a high-speed refrigerated centrifuge. Take the supernatant and determine the yield of 7-amino-1,3-dichloro-9,9-dimethylacridin-2(9-hydro)-one by fluorescence detection (fluorescence detection conditions: Ex = 600 nm, Em = 668 nm).

[0074] like Figure 6 The fluorescence test results showed that various metal ions or amino acids had no effect on the fluorescence intensity of the probe itself, indicating that the probe has high anti-interference ability.

[0075] Example 4

[0076] Linearity study of the DDAN-MT probe reaction catalyzed by methionine aminopeptidase 1:

[0077] (1) Prepare 199 μL of in vitro metabolic reaction system, which consists of pH 7.4 buffer (100 mM Hepes, 50 mM NaCl, and 200 μM CoCl2) and methionine aminopeptidase 1 (0-17.5 μg / mL), and pre-incubate at 37°C with shaking for 3 min.

[0078] (2) Add 1 μL of 500 μM DDAN-MT (final concentration 2.5 μM) to the pre-incubated reaction system obtained in step (1), react at 37°C for 30 min, then add 100 μL of glacial acetonitrile, shake vigorously, and terminate the reaction;

[0079] (3) Centrifuge the mixture at 4°C and 20,000×g for 20 minutes in a high-speed refrigerated centrifuge. Take the supernatant and determine the yield of 7-amino-1,3-dichloro-9,9-dimethylacridin-2(9-hydro)-one by fluorescence detection (fluorescence detection conditions: Ex = 600 nm, Em = 668 nm).

[0080] like Figure 7 The fluorescence detection results showed that the probe reaction of DDAN-MT catalyzed by methionine aminopeptidase 1 (MtMET-AP1) showed a good enzyme linear relationship in the range of 0-11 μg / mL, r 2 The value is 0.9983, indicating that DDAN-MT in the present invention can be applied to the determination of methionine aminopeptidase 1 activity and expression level in complex samples.

[0081] The above are merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A fluorescent probe for detecting methionine aminopeptidase 1, characterized in that: The structural formula of the fluorescent probe is shown in formula (1):

2. The method for preparing a fluorescent probe for detecting methionine aminopeptidase 1 according to claim 1, wherein: The following steps are involved: The fluorescent probe for detecting methionine aminopeptidase 1 is prepared by acylation reaction using methionine and 1,3-dichloro-7-amino-9,9-dimethyl-2(9H)-acridone as raw materials.

3. Use of the fluorescent probe for detecting methionine aminopeptidase 1 according to claim 1 in the preparation of a drug for detecting the activity of recombinantly expressed methionine aminopeptidase 1 or endogenous methionine aminopeptidase 1 of Mycobacterium tuberculosis.

4. Use of the fluorescent probe for detecting methionine aminopeptidase 1 according to claim 1 in the rapid screening of methionine aminopeptidase 1 inhibitors and the quantitative evaluation of their inhibitory ability.

5. Use of the fluorescent probe for detecting methionine aminopeptidase 1 according to claim 1 in the preparation of a drug for evaluating the activity of methionine aminopeptidase 1 in Mycobacterium tuberculosis. 6 . Use of the fluorescent probe for detecting methionine aminopeptidase 1 according to claim 1 in high-throughput screening of methionine aminopeptidase 1 inhibitors.

Images