Fluorescent probe for detecting myeloperoxidase, preparation method and application

Abstract

The present invention relates to the field of in vitro detection technology. Specifically, a fluorescent probe for detecting myeloperoxidase, a preparation method, and an application thereof are provided. The fluorescent probe has the following general structural formula: wherein R1 and R2 are each independently selected from hydrogen, a substituted or unsubstituted aliphatic hydrocarbon group, a substituted or unsubstituted alicyclic hydrocarbon group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; and X ‑ The fluorescent probe of the present invention can directly and quickly measure the MPO content in biological samples.

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection technology, and in particular to a fluorescent probe for detecting myeloperoxidase, a preparation method and an application thereof. Background Art

[0002] Myeloperoxidase (MPO) is a peroxidase primarily found in neutrophils. It catalyzes the reaction between hydrogen peroxide and halide anions in the body, generating reactive oxygen species such as hypochlorous acid and hypobromous acid. During inflammation, polymorphonuclear cells (PMNs), a type of white blood cell, accumulate in inflamed tissues and release large amounts of MPO. Consequently, MPO is associated with a variety of diseases, particularly acute and chronic inflammatory conditions, including coronary artery disease, atherosclerosis, rheumatoid arthritis, chronic obstructive pulmonary disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and leukemia. As a biomarker, MPO levels in the blood are closely associated with the onset and progression of these diseases (Pharmacol. Ther., 2021, 221, 107711). Therefore, measuring blood MPO levels can provide a key physiological indicator for clinical diagnosis and intervention.

[0003] Methods for detecting MPO levels include enzyme-linked immunosorbent assay (ELISA) and flow cytometry, which often require complex sample preparation or relatively high instrument costs. Fluorescence analysis offers the advantages of rapidity, high sensitivity, high selectivity, and low cost, and is widely used in various in vitro assays. Fluorescence analysis, which measures the concentration of its catalytic product, hypochlorous acid, is an indirect method for measuring MPO levels and cannot rule out interference from hypochlorous acid produced by other peroxides in the sample.

[0004] The current in vitro detection field lacks analytical methods to directly and rapidly determine the MPO content in biological samples, which limits the early detection and intervention treatment of various diseases in clinical practice.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a fluorescent probe for detecting myeloperoxidase, a preparation method and an application thereof, so as to solve the problem that the prior art cannot directly and quickly determine the MPO content in a biological sample.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0008] In a first aspect, the present invention provides a fluorescent probe for detecting myeloperoxidase, wherein the fluorescent probe has the following general structural formula:

[0009] ;

[0010] wherein R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, and X - It is a halogen anion or an oxoacid anion.

[0011] As a further preferred technical solution, the aliphatic hydrocarbon group includes an aliphatic hydrocarbon group having 1 to 4 carbon atoms.

[0012] As a further preferred technical solution, after the fluorescent probe is combined with MPO, it can generate fluorescence at 582 nm.

[0013] In a second aspect, the present invention provides a method for preparing the fluorescent probe for detecting myeloperoxidase, wherein the synthetic route of the method comprises:

[0014] S1. According to Reaction Formula 1, pyronine derivatives were synthesized from 3-(1-piperazinyl)phenol:

[0015]

[0016] Reaction formula 1;

[0017] S2. According to reaction formula 2, prepare an N-tert-butyloxycarbonyl protected fluorescent probe from a pyronine derivative:

[0018]

[0019] Reaction formula 2;

[0020] S3. According to Reaction Formula 3, the protecting group is removed to prepare a fluorescent probe for detecting myeloperoxidase:

[0021]

[0022] Reaction formula 3;

[0023] Wherein, R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, and substituted or unsubstituted heteroaryl group.

[0024] As a further preferred technical solution, R1 and R2 are both methyl, and X is Cl.

[0025] As a further preferred technical solution, S1 includes the following steps:

[0026] Under inert gas protection, 3-(1-piperazinyl)phenol and 4-(dimethylamino)-2-hydroxybenzaldehyde are dissolved in 85% concentrated phosphoric acid, and the reaction system is refluxed at 130-150°C. After the reaction, the reaction system is cooled to room temperature, and a methanol solution containing 10% NH4PF6 is added. After stirring for 30-40 minutes, the system is allowed to stand for 4-5 hours to crystallize. The crystals are washed and dried to obtain a pyronine derivative.

[0027] As a further preferred technical solution, S2 includes the following steps:

[0028] Under the protection of inert gas, α-acetyl-Boc-L-Lys-L-Tyr is dissolved in dry dichloromethane, and EDC, DMAP and N,N-dimethylformamide are added in sequence; after cooling in an ice-water bath for 10-15 minutes, the pyronine derivative is added, and after removing the ice bath, the mixture is stirred at room temperature in the dark; after the reaction is completed, the insoluble matter is filtered off, and the filtrate is diluted and washed to obtain an organic phase; after purifying the organic phase, an N-tert-butyloxycarbonyl-protected fluorescent probe is obtained.

[0029] As a further preferred technical solution, S3 includes the following steps:

[0030] Under the protection of inert gas, the N-tert-butyloxycarbonyl-protected fluorescent probe is dissolved in a pre-cooled HCl-MeOH solution; stirring is continued in an ice-water bath. After the reaction is completed, volatile components are removed by vacuum concentration, and the residue is washed and dried in sequence to obtain a fluorescent probe for detecting myeloperoxidase.

[0031] In a third aspect, the present invention provides a use of the fluorescent probe for detecting myeloperoxidase or the fluorescent probe for detecting myeloperoxidase prepared by the above-mentioned preparation method in detecting myeloperoxidase content, wherein the use comprises:

[0032] obtaining a detection solution containing a fluorescent probe for detecting myeloperoxidase and a standard curve comprising a linear relationship between myeloperoxidase concentration and fluorescence emission intensity;

[0033] Adding the biological sample to the detection solution of the fluorescent probe for detecting myeloperoxidase, maintaining it in a 35-39°C water bath for 8-12 minutes to obtain a test solution;

[0034] The test solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and the concentration of myeloperoxidase is obtained by comparing it with the standard curve.

[0035] As a further preferred technical solution, the standard curve is constructed in the following manner:

[0036] Add a solution containing a determined concentration of catalase to a detection solution containing a fluorescent probe for detecting catalase and keep it in a 35-39°C water bath for 8-12 minutes to obtain a standard solution.

[0037] The standard solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and a standard curve is obtained based on the linear relationship between the myeloperoxidase concentration and the fluorescence emission intensity.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The fluorescent probe for detecting myeloperoxidase and its application of the present invention can be used in (1.6~50.0)×10 -6 This method can detect MPO levels within a 10-mol / L concentration range, enabling simple and rapid determination of MPO concentrations in biological samples. Compared to currently commercially available colorimetric MPO kits, this method offers comparable accuracy with lower cost, shorter assay times, and simpler operation. Furthermore, this method is also suitable for microplate-based assays and can process multiple batches of biological samples simultaneously, including traditional matrix, serum, and cell nuclear tissue samples. It is widely applicable for the early detection and therapeutic intervention of diseases such as inflammation, cardiovascular and cerebrovascular diseases, and neurodegenerative disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 For the fluorescent probe PMF 1 Schematic diagram of H NMR spectrum.

[0041] Figure 2 For the fluorescent probe PMF 13 Schematic diagram of C NMR spectrum.

[0042] Figure 3 Schematic diagram of the fluorescence response spectrum of the fluorescent probe PMF to various endogenous substances in cells;

[0043] Figure 3 (a) Schematic diagram of the effects of reactive oxygen, reactive nitrogen, reactive sulfur species, and some metal cations on the fluorescence emission intensity of the fluorescent probe PMF at 582 nm.

[0044] Figure 3 (b) Schematic diagram of the fluorescence response spectrum of the fluorescent probe PMF to important peroxidases in the body;

[0045] Figure 3 (c) Schematic diagram of the effect of various amino acids on the fluorescence emission intensity of the fluorescent probe PMF at 582 nm;

[0046] Figure 3 (d) Schematic diagram of the effect of important peroxidases in the body on the fluorescence emission intensity of the fluorescent probe PMF at 582 nm.

[0047] Figure 4 is the fluorescence spectrum of the fluorescent probe PMF at different pH;

[0048] Figure 4 (a) Schematic diagram of the fluorescence spectra of the fluorescent probe PMF at different pH values;

[0049] Figure 4 (b) Schematic diagram of the effect of different pH on the fluorescence emission intensity of the fluorescent probe PMF at 582 nm.

[0050] Figure 5 This is the fluorescence titration graph of MPO using the fluorescent probe PMF;

[0051] Figure 5 (a) Schematic diagram of the fluorescence titration of MPO to the fluorescent probe PMF;

[0052] Figure 5 (b) Schematic diagram of the linear relationship between the fluorescence emission intensity of the fluorescent probe PMF at 582 nm and the MPO concentration.

[0053] Figure 6 Schematic diagram of MPO content in serum samples of BALB / c nude mice pulmonary fibrosis model induced by different concentrations of lipopolysaccharide (LPS);

[0054] Figure 6 (a) Schematic diagram of the fluorescence response spectra of the fluorescent probe PMF to normal serum and pulmonary fibrosis serum samples;

[0055] Figure 6 (b) Schematic diagram of the effects of normal serum and pulmonary fibrosis serum samples on the fluorescence emission intensity of the fluorescent probe PMF at 582 nm. DETAILED DESCRIPTION

[0056] The embodiments of the present invention will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are intended only to illustrate the present invention and are not to be construed as limiting the scope of the present invention. Where specific conditions are not specified in the examples, the procedures were carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0057] In one aspect, the present invention provides a fluorescent probe for detecting myeloperoxidase, wherein the fluorescent probe has the following general structural formula:

[0058] ;

[0059] wherein R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, and X -It is a halogen anion or an oxoacid anion.

[0060] In this fluorescent probe, the MPO targeting unit is an N-acetyl-L-lysine-L-tyrosine peptide, the linker unit is 1,4-piperazine, and the fluorescent unit is pyronine. The aliphatic hydrocarbon group includes aliphatic hydrocarbon groups with 1 to 20 carbon atoms.

[0061] In an optional embodiment, the aliphatic hydrocarbon group includes an aliphatic hydrocarbon group having 1 to 4 carbon atoms. Further, the aliphatic hydrocarbon group includes an alkane group, an alkene group, or an alkyne group having 1 to 4 carbon atoms.

[0062] In an optional embodiment, the fluorescent probe can generate fluorescence at 582 nm after binding to MPO.

[0063] In another aspect, the present invention provides a method for preparing the fluorescent probe for detecting myeloperoxidase, wherein the synthetic route of the method comprises:

[0064]

[0065] The above synthetic route can also be written in the following form:

[0066] S1. According to Reaction Formula 1, pyronine derivatives were synthesized from 3-(1-piperazinyl)phenol:

[0067]

[0068] Reaction formula 1;

[0069] S2. According to reaction formula 2, prepare an N-tert-butyloxycarbonyl protected fluorescent probe from a pyronine derivative:

[0070]

[0071] Reaction formula 2;

[0072] S3. According to Reaction Formula 3, the protecting group is removed to prepare a fluorescent probe for detecting myeloperoxidase:

[0073]

[0074] Reaction formula 3;

[0075] Wherein, R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, and substituted or unsubstituted heteroaryl group.

[0076] In an optional embodiment, R1 and R2 are both methyl, and X is Cl.

[0077] In the present invention, in the fluorescent probe prepared according to Reaction Formula 3, X is Cl. If other anions are needed, they can be replaced with corresponding salts to obtain other anions such as hexafluorophosphate.

[0078] In an optional embodiment, the method further comprises adjusting the structure of the anion unit of the purified probe by an anion replacement method to obtain the fluorescent probe containing different anions X. The water solubility of the probe can be improved by selecting the anion unit.

[0079] In an optional embodiment, the S1 includes the following steps:

[0080] Under inert gas protection, 3-(1-piperazinyl)phenol and 4-(dimethylamino)-2-hydroxybenzaldehyde are dissolved in 85% concentrated phosphoric acid, and the reaction system is refluxed at 130-150°C. After the reaction, the reaction system is cooled to room temperature, and a methanol solution containing 10% NH4PF6 is added. After stirring for 30-40 minutes, the system is allowed to stand for 4-5 hours to crystallize. The crystals are washed and dried to obtain a pyronine derivative.

[0081] The above-mentioned inert gas includes but is not limited to helium, neon or argon. 10% of the 10% NH4PF6 methanol solution is the mass percentage.

[0082] Optionally, the mass of 3-(1-piperazinyl)phenol is 210-230 mg (further optionally 220 mg), the amount of substance is 1.2-1.3 mmol (further optionally 1.24 mmol), and the molar multiple is 1.0 eq.

[0083] Optionally, the mass of 4-(dimethylamino)-2-hydroxybenzaldehyde is 200-210 mg (further optionally 204 mg), the amount of substance is 1.2-1.3 mmol (further optionally 1.24 mmol), and the molar multiple is 1.0 eq.

[0084] Optionally, the volume of concentrated phosphoric acid is 15-20 mL.

[0085] During the reaction, the progress of the reaction can be monitored by TLC (thin-layer chromatography). The crystals can be separated by filtration, and the filter cake can then be washed with icy methanol and ultrapure water. After drying, the resulting purple-red crystals are the desired pyronine derivative.

[0086] In an optional embodiment, the S2 includes the following steps:

[0087] Under the protection of inert gas, α-acetyl-Boc-L-Lys-L-Tyr is dissolved in dry dichloromethane, and EDC, DMAP and N,N-dimethylformamide are added in sequence; after cooling in an ice-water bath for 10-15 minutes, the pyronine derivative is added, and after removing the ice bath, the mixture is stirred at room temperature in the dark; after the reaction is completed, the insoluble matter is filtered off, and the filtrate is diluted and washed to obtain an organic phase; after purifying the organic phase, an N-tert-butyloxycarbonyl-protected fluorescent probe is obtained.

[0088] The above-mentioned inert gas includes but is not limited to helium, neon or argon.

[0089] Optionally, the mass of α-acetyl-Boc-L-Lys-L-Tyr is 70-80 mg (further optionally 74 mg), the amount of substance is 0.1-0.2 mmol (further optionally 0.16 mmol), and the molar multiple is 1-1.5 eq (further optionally 1.2 eq).

[0090] Optionally, the volume of dichloromethane is 3-5 mL (further optionally 4 mL).

[0091] Optionally, the mass of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) is 25-35 mg (further optionally 31 mg), the amount of substance is 0.1-0.2 mmol (further optionally 0.16 mmol), and the molar multiple is 1-1.5 eq (further optionally 1.2 eq).

[0092] Optionally, the mass of DMAP (4-dimethylaminopyridine) is 15-20 mg (17 mg), the mass per mole is 0.1-0.2 mmol (further optionally 0.14 mmol), and the molar multiple is 0.5-1.5 eq (1.0 eq).

[0093] Optionally, the volume of N,N-dimethylformamide is 0.3-0.9 mL (further optionally 0.6 mL).

[0094] Optionally, the amount of the pyronine derivative added is 60-65 mg (further optionally 62 mg), the amount of the substance is 0.1-0.2 mmol (further optionally 0.14 mmol), and the molar multiple is 0.5-1.5 eq (1.0 eq).

[0095] During the reaction, the progress of the reaction can be monitored by TLC.

[0096] Alternatively, dilute with 10 mL of dichloromethane.

[0097] Optionally, washing is performed with a 10% by mass aqueous solution of citric acid, and the washing is performed 3 times in total, with 5 mL of the aqueous solution of citric acid used each time.

[0098] Optionally, the purification comprises: drying, concentration under reduced pressure and silica gel column chromatography in sequence.

[0099] Optionally, anhydrous sodium sulfate is used for drying, and the molar ratio of dichloromethane to methanol in the silica gel column is 15:1.

[0100] The obtained N-tert-butyloxycarbonyl protected fluorescent probe product is a dark red solid.

[0101] In an optional embodiment, the S3 includes the following steps:

[0102] Under the protection of inert gas, the N-tert-butyloxycarbonyl-protected fluorescent probe is dissolved in a pre-cooled HCl-MeOH solution; stirring is continued in an ice-water bath. After the reaction is completed, volatile components are removed by vacuum concentration, and the residue is washed and dried in sequence to obtain a fluorescent probe for detecting myeloperoxidase.

[0103] Optionally, the mass of the N-tert-butyloxycarbonyl protected fluorescent probe is 70-90 mg (further optionally 80 mg), the amount of substance is 0.08-0.1 mmol (further optionally 0.09 mmol), and the molar multiple is 0.5-1.5 eq (1.0 eq).

[0104] Optionally, the amount of the HCl-MeOH solution is 2-4 M (further optionally 3 M), and the volume is 3-8 mL (further optionally 5 mL).

[0105] During the reaction, the progress of the reaction can be monitored by TLC.

[0106] Optionally, dichloromethane is used for washing, and the washing is performed 3 times in total, with 5 mL of citric acid aqueous solution used each time.

[0107] The obtained fluorescent probe for detecting myeloperoxidase is a deep red solid.

[0108] In another aspect, the present invention provides a use of the fluorescent probe for detecting myeloperoxidase in detecting the content of myeloperoxidase.

[0109] In an optional embodiment, the application includes:

[0110] obtaining a detection solution containing a fluorescent probe for detecting myeloperoxidase and a standard curve comprising a linear relationship between myeloperoxidase concentration and fluorescence emission intensity;

[0111] Adding the biological sample to the detection solution of the fluorescent probe for detecting myeloperoxidase, maintaining it in a 35-39°C water bath for 8-12 minutes to obtain a test solution;

[0112] The test solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and the concentration of myeloperoxidase is obtained by comparing it with the standard curve.

[0113] Optionally, the water bath temperature is 25, 36, 37, 38, or 39°C and the water bath time is 8, 9, 10, 11, or 12 minutes.

[0114] It can be understood that the detection solution is a solution compatible with the fluorescent probe.

[0115] In an optional embodiment, the detection solution is prepared in the following manner:

[0116] Prepare deoxygenated double distilled water;

[0117] Mixing the deoxygenated double distilled water and dimethyl sulfoxide in a preset ratio to obtain a detection solution compatible with the fluorescent probe;

[0118] The preset ratio is that the volume ratio of double distilled water to dimethyl sulfoxide is 99:1 to 999:1.

[0119] Optionally, in the detection solution containing the fluorescent probe, the concentration of the fluorescent probe is 1×10 -7 mol / L.

[0120] In an optional embodiment, the standard curve is constructed in the following manner:

[0121] Add a solution containing a determined concentration of catalase to a detection solution containing a fluorescent probe for detecting catalase and keep it in a 35-39°C water bath for 8-12 minutes to obtain a standard solution.

[0122] The standard solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and a standard curve is obtained based on the linear relationship between the myeloperoxidase concentration and the fluorescence emission intensity.

[0123] Optionally, the standard curve is constructed in the following manner:

[0124] 0, 10, 20, 30, 40, and 50 nM MPO were added to the detection solution containing the fluorescent probe, and the fluorescence emission intensity at 582 nm was measured; each concentration value was measured in parallel three times, and the average value was taken to establish a standard curve of fluorescence intensity-MPO content.

[0125] Optionally, after the biological samples are diluted in multiples, the emission intensity of each sample at 582 nm is determined and compared with the standard curve to obtain the concentration of MPO in the sample to be tested.

[0126] The present invention will be further described in detail below with reference to the embodiments.

[0127] Example 1

[0128] A fluorescent probe for detecting myeloperoxidase, the preparation method of which comprises:

[0129] S1. Under argon, dissolve 3-(1-piperazinyl)phenol (220 mg, 1.24 mmol, 1.0 eq) and 4-(dimethylamino)-2-hydroxybenzaldehyde (204 mg, 1.24 mmol, 1.0 eq) in 15 mL of 85% concentrated phosphoric acid. Reflux the reaction system at 140°C, and monitor the reaction progress by TLC. After completion of the reaction, cool the system to room temperature, and add a 10% methanol solution containing NH₄PF₆ (10 mL). Stir thoroughly for 30 minutes, then allow the mixture to crystallize for 4 hours. Filter the precipitate, and wash the filter cake with ice-cold methanol and then ultrapure water. After drying, the desired pyronine derivative is obtained as purple-red crystals. Yield: 77.8%.

[0130] Its H NMR spectrum ( 1 H NMR) and carbon nuclear magnetic resonance spectroscopy ( 13 The specific data of C NMR are as follows:

[0131] 1 H NMR: (300 MHz, DMSO-d6, ppm) δ: 8.86 (s, 2H), 7.97 (d, 1H, J = 9.2 Hz), 7.95 (d, 1H, J = 9.2 Hz), 7.41 (d, 1H, J = 9.2 Hz), 7.32 (d, 1H, J = 9.2 Hz),7.24 (s, 1H), 6.93 (s, 1H), 3.93 (t, 4H, J = 11.6 Hz), 3.35 (s, 6H), 3.30 (t,4H, J = 11.6 Hz). 13 C NMR: (75 MHz, DMSO-d6, ppm) δ: 158.57, 158.20, 157.80, 156.83,147.10, 134.16, 133.80, 116.10, 115.64, 115.02, 114.43, 98.45, 96.52, 44.29, 42.92, 41.32.

[0132] S2. Under argon, dissolve α-acetyl-Boc-L-Lys-L-Tyr (74 mg, 0.16 mmol, 1.2 eq) in 4 mL of dry dichloromethane. Add EDC (31 mg, 0.16 mmol, 1.2 eq), DMAP (17 mg, 0.14 mmol, 1.0 eq), and 0.6 mL of N,N-dimethylformamide in sequence. After cooling in an ice-water bath for 10-15 minutes, add 62 mg of the above-mentioned pyronine derivative (0.14 mmol, 1.0 eq). Remove the ice bath, stir at room temperature in the dark, and monitor the reaction progress by TLC. After completion of the reaction, remove the insoluble material by filtration, dilute the filtrate with 10 mL of dichloromethane, and wash with 10% aqueous citric acid (5 mL x 3). Dry the organic phase over anhydrous sodium sulfate and concentrate under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 15:1). The product was a dark red solid. Yield: 42.6%.

[0133] That 1 H NMR and 3 The specific data of C NMR are as follows:

[0134] 1 H NMR: (500 MHz, DMSO-d6, ppm) δ: 9.18 (s, 1H), 8.81 (s, 1H), 8.21 (d, 1H,J = 7.4 Hz), 7.91 (d, 2H, J = 8.8 Hz), 7.32 (d, 1H, J = 9.2 Hz), 7.26 (d, 1H,J = 9.2 Hz), 7.05 (s, 1H), 7.01 (d, 2H, J = 8.0 Hz), 6.90 (s, 1H), 6.76 (t,1H, J = 11.6 Hz), 6.63 (d, 2H, J = 8.0 Hz), 4.82-4.80 (m, 1H), 4.21-4.19 (m,1H), 3.71 (t, 4H, J = 12.8 Hz), 3.61 (br, 2H), 3.48-3.50 (m, 1H), 3.43-3.46(m, 1H), 3.34 (s, 6H), 2.85 (t, 4H, J = 12.8 Hz), 1.83 (s, 3H), 1.53-1.54 (m,1H), 1.41-1.43 (m, 1H), 1.35 (s, 9H), 1.31-1.32 (m, 2H), 1.19-1.23 (m, 2H).

[0135] 13 C NMR: (125 MHz, DMSO-d6, ppm) δ: 171.95, 170.32, 169.64, 158.22, 157.99,157.94, 157.04, 156.47, 156.03, 146.89, 133.93, 133.84, 130.80, 127.56,115.56, 115.40, 114.99, 114.94, 111.46, 97.47, 96.56, 87.45, 77.81, 52.84,50.51, 46.50, 44.30, 41.21, 40.22, 37.23, 32.15, 29.72, 28.74, 23.24, 23.00.

[0136] S3. Under argon, dissolve the N-tert-butyloxycarbonyl-protected fluorescent probe (80 mg, 0.09 mmol, 1.0 eq) in pre-chilled 3 M HCl-MeOH solution (5 mL). Stir continuously in an ice-water bath, and monitor the reaction progress by TLC. After completion of the reaction, remove volatile components by concentration under reduced pressure. The residue is washed thoroughly with dichloromethane (5 mL × 3). After drying, the resulting dark red solid is PMF, a fluorescent probe for detecting myeloperoxidase. Yield: 88.4%.

[0137] That 1 H NMR spectrum Figure 1 The specific data are as follows:

[0138] 1H NMR: (500 MHz, DMSO-d6, ppm) δ: 9.19 (s, 1H), 8.82 (s, 1H), 8.18 (d, 2H,J = 7.4 Hz), 7.95 (d, 1H, J = 7.4 Hz), 7.93 (s, 1H), 7.91 (s, 1H), 7.61 (s,2H), 7.32 (d, 1H, J = 9.4 Hz), 7.28 (d, 1H, J = 9.4 Hz), 7.06 (s, 1H), 7.02(d, 2H, J = 8.2 Hz), 6.91 (s, 1H), 6.64 (d, 2H, J = 8.2 Hz), 4.83-4.82 (m,1H), 4.24-4.23 (m, 1H), 3.71 (t, 2H, J = 10.0 Hz), 3.60 (t, 2H, J = 6.2 Hz), 3.46-3.44 (m, 2H), 3.32 (s, 6H), 2.87-2.83 (m, 1H), 2.79-2.74 (m, 3H), 1.85 (s, 3H), 1.59-1.57 (m, 1H), 1.53-1.44 (m, 3H), 1.27 (t, 4H, J = 16.2 Hz).

[0139] That 13 C NMR spectrum Figure 2 The specific data are as follows:

[0140] 13 C NMR: (125 MHz, DMSO-d6, ppm) δ: 171.82, 170.40, 169.75, 158.26, 158.02, 157.94, 157.02, 156.50, 146.93, 133.96, 133.85, 130.82, 127.42, 115.60,115.42, 115.03, 114.90, 114.46, 97.47, 96.55, 52.52, 50.54, 46.45, 44.31,41.22, 39.23, 37.23, 31.82, 27.06, 23.04, 22.80.

[0141] Example 2

[0142] Detection of MPO content in serum samples:

[0143] The assay system consisted of 100 mM phosphate buffered saline (PBS), pH 7.4, containing 1% by volume of dimethyl sulfoxide (DMSO). PMF concentration was 0.1 μM; reactive oxygen species (ROS) concentration was 50 μM; reactive nitrogen species (RNS) concentration was 50 μM; reactive sulfur species (RSS) concentration (excluding glutathione (GSH)) concentration was 100 μM; GSH concentration was 1.0 mM; amino acid concentration was 100 μM; inorganic salt concentration was 200 μM; vascular peroxidase-1 (VPO1) concentration was 1.0 μM; dual oxidase (DOUX1 / 2) concentration was 1.0 μM. Serum samples from a nude mouse pulmonary fibrosis model were diluted at multiples of 0, 2, 4, 8, 16, and 32. Excitation wavelength was 500 nm. Testing temperature was 23 ± 0.5°C. Both the excitation and emission slit widths were 5 nm.

[0144] Figure 3 (a) ~ Figure 3 (d) is a schematic diagram of the fluorescence response spectrum of PMF to various endogenous cellular substances such as ROS, RNS, RSS (including GSH), amino acids, and peroxidase. It can be seen from the figure that in PBS buffer containing 1% DMSO (volume ratio), the fluorescence intensity of PMF at 582 nm is relatively low. Among all the endogenous cellular species detected, such as ROS, RNS, RSS, amino acids, and other peroxidases, only MPO can significantly enhance the fluorescence emission intensity of the system at 582 nm.

[0145] Figure 4 (a) ~ Figure 4 (b) Schematic diagram of the fluorescence response spectrum of the fluorescent probe PMF at different pH values. Figure 5 (a) ~ Figure 5 (b) Schematic diagram of fluorescence titration of MPO using the fluorescent probe PMF. Figure 6 (a) ~ Figure 6 (b) Schematic diagram of MPO levels in serum samples from a BALB / c nude mouse model of pulmonary fibrosis induced by different concentrations of lipopolysaccharide (LPS). The experimental results show that PMF can effectively measure MPO levels in serum samples from a nude mouse model of pulmonary fibrosis.

[0146] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. It is therefore intended that the appended claims include all such changes and modifications that fall within the scope of the present invention.

Claims

1. A fluorescent probe for detecting myeloperoxidase, characterized in that: The fluorescent probe has the following general structural formula: ； wherein R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, and X - It is a halogen anion or an oxoacid anion.

2. The fluorescent probe for detecting myeloperoxidase according to claim 1, wherein The aliphatic hydrocarbon group includes an aliphatic hydrocarbon group having 1 to 4 carbon atoms.

3. The fluorescent probe for detecting myeloperoxidase according to claim 1 or 2, characterized in that After the fluorescent probe is combined with MPO, it can generate fluorescence at 582 nm.

4. A method for preparing a fluorescent probe for detecting myeloperoxidase according to any one of claims 1 to 3, characterized in that: The synthetic route of the method comprises: S1. According to Reaction Formula 1, pyronine derivatives were synthesized from 3-(1-piperazinyl)phenol: Reaction formula 1; S2. According to reaction formula 2, prepare an N-tert-butyloxycarbonyl protected fluorescent probe from a pyronine derivative: Reaction formula 2; S3. According to Reaction Formula 3, the protecting group is removed to prepare a fluorescent probe for detecting myeloperoxidase: Reaction formula 3; Wherein, R1 and R2 are each independently selected from one of hydrogen, substituted or unsubstituted aliphatic hydrocarbon group, substituted or unsubstituted alicyclic hydrocarbon group, substituted or unsubstituted aryl group, and substituted or unsubstituted heteroaryl group.

5. The method for preparing a fluorescent probe for detecting myeloperoxidase according to claim 4, wherein: R1 and R2 are both methyl groups, and X is Cl.

6. The method for preparing a fluorescent probe for detecting myeloperoxidase according to claim 5, wherein: Said S1 comprises the following steps: Under inert gas protection, 3-(1-piperazinyl)phenol and 4-(dimethylamino)-2-hydroxybenzaldehyde are dissolved in 85% concentrated phosphoric acid, and the reaction system is refluxed at 130-150°C. After the reaction, the reaction system is cooled to room temperature, and a methanol solution containing 10% NH4PF6 is added. After stirring for 30-40 minutes, the system is allowed to stand for 4-5 hours to crystallize. The crystals are washed and dried to obtain a pyronine derivative.

7. The method for preparing a fluorescent probe for detecting myeloperoxidase according to claim 5, wherein: The S2 comprises the following steps: Under the protection of inert gas, α-acetyl-Boc-L-Lys-L-Tyr is dissolved in dry dichloromethane, and EDC, DMAP and N,N-dimethylformamide are added in sequence; after cooling in an ice-water bath for 10-15 minutes, the pyronine derivative is added, and after removing the ice bath, the mixture is stirred at room temperature in the dark; after the reaction is completed, the insoluble matter is filtered off, and the filtrate is diluted and washed to obtain an organic phase; after purifying the organic phase, an N-tert-butyloxycarbonyl-protected fluorescent probe is obtained.

8. The method for preparing a fluorescent probe for detecting myeloperoxidase according to claim 5, wherein: The S3 includes the following steps: Under the protection of inert gas, the N-tert-butyloxycarbonyl-protected fluorescent probe is dissolved in a pre-cooled HCl-MeOH solution; stirring is continued in an ice-water bath. After the reaction is completed, volatile components are removed by vacuum concentration, and the residue is washed and dried in sequence to obtain a fluorescent probe for detecting myeloperoxidase.

9. Use of the fluorescent probe for detecting myeloperoxidase according to any one of claims 1 to 3 or the fluorescent probe for detecting myeloperoxidase prepared by the method according to any one of claims 4 to 8 in detecting myeloperoxidase content, characterized in that: The applications include: obtaining a detection solution containing a fluorescent probe for detecting myeloperoxidase and a standard curve comprising a linear relationship between myeloperoxidase concentration and fluorescence emission intensity; Adding the biological sample to the detection solution of the fluorescent probe for detecting myeloperoxidase, maintaining the solution in a water bath at 35-39°C for 8-12 minutes to obtain a test solution; The test solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and the concentration of myeloperoxidase is obtained by comparing it with the standard curve.

10. The use according to claim 9, characterized in that The standard curve was constructed in the following manner: Add a solution containing a determined concentration of catalase to a detection solution containing a fluorescent probe for detecting catalase and keep it in a 35-39°C water bath for 8-12 minutes to obtain a standard solution. The standard solution is irradiated with light of a preset wavelength, the fluorescence emission intensity is monitored, and a standard curve is obtained based on the linear relationship between the myeloperoxidase concentration and the fluorescence emission intensity.

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