Chemiluminescent probe of N-acetyl-beta-D-glucosaminase, kit and preparation method and application of chemiluminescent probe

Abstract

The invention provides a chemiluminescent probe of N-acetyl-beta-D-glucosaminase, a kit and a preparation method and application of the chemiluminescent probe and the kit, develops the chemiluminescent detection kit of the N-acetyl-beta-D-glucosaminase based on the chemiluminescent probe, and belongs to the technical field of in-vitro detection.The chemiluminescent probe is named as CL-NAG, the structural formula of the CL-NAG is as shown in formula 1. After the chemiluminescence probe reacts with NAG, remarkable chemiluminescence can be generated, and the chemiluminescence probe has the advantages of being high in sensitivity and easy to operate when applied to NAG detection.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro detection, and particularly relates to a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme, a kit, and a preparation method and application thereof. Background Art

[0002] N-acetyl-β-D-glucosaminidase (NAG) is a high molecular weight glycoprotein acid hydrolase with a relative molecular weight of (13-14) × 10 4 , widely present in the lysosomes of various tissues, organs, body fluids and blood cells of the human body, and the highest content is in the lysosomes of the prostate and renal proximal tubular epithelial cells. The half-life of NAG enzyme in plasma is only 5 minutes, and because of its large relative molecular weight, NAG enzyme in plasma cannot be filtered by the glomerulus under normal circumstances. Lysosomes are easily invaded by various attack factors (such as biological toxins, chemical toxins, free radicals, and immune activation factors). When the proximal tubules of the kidney are damaged, the level of NAG in urine increases significantly. Studies have shown that NAG is very sensitive to renal proximal tubular damage and can quickly and directly reflect the degree of active tubular damage and changes in the disease. In clinical practice, serum creatinine, urea nitrogen and endogenous creatinine clearance are often used as indicators for judging glomerular filtration, tubular reabsorption and secretion function, but these indicators cannot indicate early kidney damage and abnormal renal proximal tubular function. Although renal biopsy has high sensitivity, it is expensive and causes puncture damage to patients, which is not suitable for large-scale screening of kidney diseases. Therefore, the non-invasive test of urine NAG has important clinical value for the early diagnosis of acute kidney injury (AKI), drug and heavy metal nephrotoxicity, urinary tract infection (pyelonephritis), and diabetes-related kidney injury.

[0003] At present, the methods for detecting NAG enzyme activity mainly include immunoassay, fluorescence analysis and UV-visible spectrophotometry. Among them, the spectrophotometric method uses the colorimetric endpoint method as the mainstream methodology, including the PNP-NAG method, the CNP-NAG method and the MPT-NAG method. The determination principle of these methods is that under the action of NAG, the substrate undergoes enzymatic hydrolysis to generate new products, and the activity of NAG is calculated by colorimetry or absorbance measurement. The PNP-NAG method requires the setting of a sample blank, and the operation time is long, which is not suitable for large-scale automated analysis; the CNP-NAG method does not require the setting of a sample blank, but the substrate solubility is poor, and the pH value of the buffer solution needs to be strictly controlled during the determination; the MPT-NAG method can avoid the interference of urine chromogen, and does not require the setting of a sample blank, but the substrate cost is relatively high. The above three methods all have problems such as low sensitivity and detection limit and complex operation. Therefore, it is urgent to develop a new method for NAG enzyme detection with high sensitivity and strong anti-interference ability. Summary of the invention

[0004] In order to solve the problems of low sensitivity and complex operation in the existing NAG detection method, the present invention provides a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme. After the chemiluminescent probe reacts with NAG, it can produce significant chemiluminescence. When applied to NAG detection, it has the advantages of high sensitivity and simple operation.

[0005] The present invention also provides a preparation method and application of a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme.

[0006] The present invention is achieved through the following technical solutions: The present invention provides a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme, the chemiluminescent probe is named CL-NAG, and the structural formula of CL-NAG is shown in Formula 1: .

[0007] Based on the same inventive concept, the present invention provides a method for preparing a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme, wherein the chemiluminescent probe comprises CL-NAG, and the preparation method of CL-NAG comprises: S1. Compound 1 was dissolved in methanol, trimethyl orthoformate and tetrabutylammonium tribromide were added under an inert atmosphere for reaction, and after the reaction was completed, the mixture was diluted with ethyl acetate and extracted with sodium bicarbonate. The obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 2; S2. The compound 2 and imidazole were dissolved in dichloromethane, tert-butyldimethylsilyl chloride was added under an inert atmosphere, and after the reaction was completed, the mixture was separated and purified by a silica gel column to obtain a compound 3; S3. The compound 3 is dissolved in dichloromethane, trimethyl phosphite is added under an inert atmosphere, the reaction is carried out at 0±2°C for 15±5 min, titanium tetrachloride is added and the reaction is continued for 30±5 min, and after the reaction is completed, a saturated aqueous solution of sodium bicarbonate is added at 0±2°C, and the mixture is extracted with dichloromethane. The obtained organic phase is dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4; S4. Dissolve the compound 4 in tetrahydrofuran under an inert atmosphere, stir at -78±2°C for 30±5 min, then add lithium diisopropylamide and stir for 20±5 min, dissolve 2-adamantanone in tetrahydrofuran and add to the reaction system, stir for 15±5 min and heat to room temperature for reaction, add tetrabutylammonium fluoride after the reaction is completed, dilute with ethyl acetate, wash with 1.0 M hydrochloric acid, dry the obtained organic phase over anhydrous sodium sulfate, and purify by column chromatography to obtain compound 5; S5. The compound 5 is dissolved in tetrahydrofuran, magnesium chloride and triethylamine are added, and then paraformaldehyde is added, and the mixture is heated to 80±2°C under reflux conditions, and then cooled to room temperature and diluted with dichloromethane, washed with 0.5M hydrochloric acid, and the organic layer is separated and then washed with saturated brine. The obtained organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 6; S6. The compound 6 was dissolved in dichloromethane, methoxyformylmethylenetriphenylphosphine was added, stirred for 15±5 min, then diluted with dichloromethane, washed with 1.0 M hydrochloric acid, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 7; S7. 2-acetylamino-3,4,6-tri-O-acetyl-aD-pyranose, p-hydroxybenzaldehyde, 15-crown ether-5 and potassium carbonate were dissolved in acetonitrile, stirred for reaction, and then diluted with dichloromethane, washed with saturated sodium bicarbonate and brine. The obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain product A. The product A was dissolved in methanol, cooled to 0±2°C, and sodium borohydride was added. After stirring for 2±0.5 h, ammonium chloride was added to quench, and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain product B. The product B was dissolved in dichloromethane, cooled to 0±2°C, phosphorus tribromide was added, and ammonium chloride was added to quench, extracted with dichloromethane, and the obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 8; S8.0±2 ℃, the compound 7 was dissolved in N,N-dimethylformamide under an inert atmosphere, potassium carbonate was added and stirred for 10±5 min, and then the compound 8 was added. After stirring for reaction, the mixture was moved to room temperature and stirred for 30±5 min. After the reaction was completed, the mixture was washed with ammonium chloride, diluted with ethyl acetate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 9; S9. The compound 9 is dissolved in methanol, potassium carbonate is added under an inert atmosphere for reaction, and then diluted with ethyl acetate, washed with saturated brine, and the obtained organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude product and methylene blue are co-dissolved in dichloromethane, oxygen is bubbled through the solution at 0±2°C, and yellow light is irradiated at the same time, and then concentrated under reduced pressure and purified by column chromatography to obtain compound CL-NAG; Wherein, the structural formulas of the compounds 1 to 9 and the CL-NAG are as follows: .

[0008] Furthermore, in step S1, the molar ratio of the compound 1, trimethyl orthoformate and tetrabutylammonium tribromide is 1.0:1.6:0.1; In step S2, the molar ratio of compound 2, tert-butyldimethylsilyl chloride and imidazole is 1.0:1.2:3.0; In step S3, the molar ratio of compound 3, trimethyl phosphite and titanium tetrachloride is 1.0:1.2:3.0.

[0009] Furthermore, in step S4, the molar ratio of compound 4, 2-adamantanone, lithium diisopropylamide and tetrabutylammonium fluoride is 1.0:1.2:1.5:1.1; In step S5, the molar ratio of compound 5, magnesium chloride, triethylamine and paraformaldehyde is 1.0:2.2:2.2:8.0; In step S6, the molar ratio of compound 6 to methoxyformylmethylenetriphenylphosphine is 1.0:1.0.

[0010] Further, in step S7, the molar ratio of p-hydroxybenzaldehyde, the compound 2-acetylamino-3,4,6-tri-O-acetyl-aD-pyranoglucopyranosyl, 15-crown ether-5 and potassium carbonate is 1.0:2.0:0.2:4.5, the molar ratio of the product A to sodium borohydride is 1.0:2.0, and the molar ratio of the product B to phosphorus tribromide is 1.0:2.0; In step S8, the molar ratio of compound 7, compound 8 and potassium carbonate is 1.0:1.0:1.3; In step S9, the molar ratio of compound 9 to potassium carbonate is 1.0:4.2.

[0011] Based on the same inventive concept, the present invention provides an application of a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme in the preparation of a NAG detection reagent.

[0012] Based on the same inventive concept, the present invention provides an application of a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme in the preparation of a kidney injury detection reagent.

[0013] Based on the same inventive concept, the present invention provides a kidney injury detection reagent, which comprises the above-mentioned chemiluminescent probe of N-acetyl-β-D-glucosaminase.

[0014] Based on the same inventive concept, the present invention provides a NAG detection reagent, wherein the NAG detection reagent comprises the above-mentioned chemiluminescent probe of N-acetyl-β-D-glucosaminase.

[0015] Based on the same inventive concept, the present invention also provides a NAG detection kit, which comprises the above-mentioned chemiluminescent probe of N-acetyl-β-D-glucosaminase.

[0016] Based on the same inventive concept, the present invention also provides a chemiluminescent kit for N-acetyl-β-D-glucosamine enzyme, the kit comprising reagent 1, reagent 2 and reagent 3; The reagent 1 includes a CL-NAG probe and DMSO; the reagent 2 includes NAG, copper sulfate and water; the reagent 3 includes potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate and water; The CL-NAG probe is a chemiluminescent probe of the above-mentioned N-acetyl-β-D-glucosamine enzyme.

[0017] Preferably, in the reagent 1, the concentration of CL-NAG probe is 5 mM; In the reagent 2, the concentration of each component is: NAG: 200U / L, copper sulfate: 0.5 mM; In the reagent 3, the concentration of each component is: Potassium dihydrogen phosphate: 1.0588236 mM, sodium chloride: 155.17241 mM, sodium hydrogen phosphate: 2.966418 mM. Potassium dihydrogen phosphate, sodium chloride, sodium hydrogen phosphate heptahydrate and distilled water are prepared into PBS buffer (10mM, pH=7.4) One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. The present invention discloses a chemiluminescent probe for N-acetyl-β-D-glucosamine enzyme, named CL-NAG. The CL-NAG is composed of a NAG-cleavable substrate, an acryl-substituted phenoxydioxetane chemiluminescent body and a self-eliminating linker, p-aminobenzyl alcohol. After the reaction between CL-NAG and NAG, significant chemiluminescence can be generated. The probe is applied to NAG detection, and has significant advantages such as high sensitivity, non-invasiveness and simple operation. It can specifically detect NAG in urine, has good stability and is not interfered by other biological molecules and enzymes.

[0018] 2. The present invention provides a NAG detection kit, which is a NAG chemiluminescence kit with the probe CL-NAG as the main component, has the advantages of strong selectivity, low cytotoxicity, and high signal-to-noise ratio, can be used for the detection of NAG at the living cell level, and has good imaging effect. The kit components are relatively simple and the price is low, and it can be used for the diagnosis and monitoring of early acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 This is the nuclear magnetic resonance hydrogen spectrum of the chemiluminescent probe CL-NAG in DMSO.

[0021] Figure 2 This is the carbon NMR spectrum of the chemiluminescent probe CL-NAG in DMSO.

[0022] Figure 3 High-resolution mass spectrometry of the chemiluminescent probe CL-NAG.

[0023] Figure 4 Figure 2 shows the photophysical properties of the chemiluminescent probe CL-NAG: (A) is the absorption spectrum of the chemiluminescent probe CL-NAG before and after incubation with NAG in a buffer solution; (B) is the chemiluminescence spectrum of the chemiluminescent probe CL-NAG before and after incubation with NAG in a buffer solution; (C) is the fluorescence emission spectrum of the chemiluminescent probe CL-NAG before and after incubation with NAG in a buffer solution; (D) is the chemiluminescence kinetic curve of the chemiluminescent probe CL-NAG in response to different concentrations of NAG (0, 50, 100, 200, 300, 400 U / L); (E) is the fluorescence emission curve of the chemiluminescent probe CL-NAG in response to different concentrations of NAG (0, 50, 100, 200, 300, 400 U / L); -NAG (10μM) changes in chemiluminescence intensity of different concentrations of NAG (0, 2.5, 5, 10, 25, 50, 100, 150, 200, 300, 400 and 500U / L) after incubation at 37℃ for 30min; (F) is the selectivity test of the chemiluminescent probe CL-NAG; (G) is the change in chemiluminescent intensity of the chemiluminescent probe CL-NAG in different pH buffers; (H) is the change in chemiluminescent intensity of the chemiluminescent probe CL-NAG and the main components in urine (Na+, K+, Mg2+, Ca2+, Cl-, C2O42-, SO42-, uric acid, urea, glucose, creatinine) at different times (0, 0.5, 1, 2, 3h); (I) is the change in chemiluminescent intensity of the chemiluminescent probe CL-NAG after incubation with the main components in urine for 3h and then NAG was added to each group and incubated for another 30min.

[0024] Figure 5 Cytotoxicity test diagram: (A) is the cytotoxicity test of chemiluminescent probe CL-NAG on HK-2 (human renal tubular epithelial cells) and HUEVC (human umbilical vein endothelial cells) cells; (B) is the cytotoxicity test of NAG inhibitor NGT on HK-2 cells; (C) is the cytotoxicity test of cisplatin on HK-2 cells.

[0025] Figure 6In the figure, (A) shows the change of chemiluminescent intensity of different counted cells (0, 5000, 10000, 20000, 40000, 60000) after HK-2 cells were incubated with chemiluminescent probe CL-NAG for 30 minutes; (B) shows the linear fitting curve of chemiluminescent probe CL-NAG and different cell numbers of HK-2 cells; (C) explores the inhibition of NAG activity by different concentrations of NGT (1, 2, 3, 4, 5 mM) in buffer; (D) studies the ability of chemiluminescent probe CL-NAG to detect endogenous NAG in living cells; (E) explores the change of chemiluminescent intensity of chemiluminescent probe CL-NAG after cisplatin stimulation of HK-2 cells; (F) compares the change of chemiluminescent intensity of endogenous NAG detected by chemiluminescent probe CL-NAG in different cells (HK-2, HUEVC cells).

[0026] Figure 7 Figure 2 is the standard curve of CL-NAG and different concentrations of NAG (0, 25, 50, 75 and 100 U / L) in urine. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0029] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] The following is a detailed description of a chemiluminescent probe, a kit, a preparation method and an application of N-acetyl-β-D-glucosamine enzyme of the present invention in combination with examples and experimental data.

[0032] Example 1 This example provides a method for preparing a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme. The specific synthesis route is as follows: .

[0033] 1. Synthesis of Compound 2 Compound 1 (1 g, 6.38 mmol) was dissolved in 100 mL of methanol solution and stirred. Trimethyl orthoformate (1.2 ml, 10.22 mmol) and tetrabutylammonium tribromide (300 mg, 0.64 mmol) were added under nitrogen and reacted overnight at room temperature. The reaction was monitored by TLC. After completion, the mixture was diluted with ethyl acetate and extracted with sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 1.17 g of colorless oily liquid compound 2 with a yield of 90.3%.

[0034] 2. Synthesis of Compound 3 Compound 2 (500 mg, 2.48 mmol) and imidazole (500 mg, 7.43 mmol) were dissolved in 60 mL of dichloromethane and stirred. Tert-butyldimethylsilyl chloride (450 mg, 2.97 mmol) was added under nitrogen. The reaction was allowed to react at room temperature for 1 h. The reaction was monitored by TLC and separated and purified by silica gel column to obtain 730 mg of compound 3 with a yield of 93.4%.

[0035] 3. Synthesis of Compound 4 Compound 3 (400 mg, 1.27 mmol) was dissolved in 40 mL of dichloromethane and stirred. Trimethyl phosphite (0.19 mL, 1.65 mmol) was added under nitrogen. The reaction was continued at 0°C for 15 min. Titanium tetrachloride (0.18 mL, 1.65 mmol) was added dropwise. The reaction was continued for 30 min and monitored by TLC. After the reaction, a saturated aqueous solution of sodium bicarbonate was added at 0°C and stirred for 15 min. The mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 430 mg of colorless oily liquid compound 4 with a yield of 86.8%.

[0036] 4. Synthesis of Compound 5 Compound 4 (200 mg, 0.5 mmol) was dissolved in 20 mL of tetrahydrofuran under nitrogen atmosphere at room temperature, moved to -78 °C and cooled with stirring for 30 min, lithium diisopropylamide (0.4 mL, 0.76 mmol) was added dropwise, stirred for 20 min, 2-adamantanone (92 mg, 0.6 mmol) was dissolved in 10 mL of tetrahydrofuran and added dropwise to the reaction system, stirred for 15 min and heated to room temperature for reaction, the reaction was continued, monitored by TLC, and tetrabutylammonium fluoride (0.6 mL, 0.55 mmol) was added after completion, monitored by TLC, and diluted with ethyl acetate after completion of the reaction, washed with 1.0 M hydrochloric acid, and the organic phase was dried over anhydrous sodium sulfate and purified by column chromatography to obtain 140 mg of white solid compound 5 with a yield of 91%.

[0037] 5. Synthesis of Compound 6 Compound 5 (450 mg, 1.5 mmol) was dissolved in 10 mL of tetrahydrofuran, stirred for 10 min, magnesium chloride (310 mg, 3.3 mmol) and triethylamine (0.3 ml, 3.3 mmol) were added, stirred for 10 min, paraformaldehyde (360 mg, 11.8 mmol) was added, heated at 80 °C overnight under reflux conditions, cooled to room temperature, monitored by TLC, diluted with dichloromethane after completion, washed with 0.5 M hydrochloric acid, separated the organic layer, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 130 mg of compound 6 with a yield of 24.9%.

[0038] 6. Synthesis of Compound 7 Compound 6 (100 mg, 3.9 mmol) was dissolved in 5 mL of dichloromethane, and methoxycarbonylmethylenetriphenylphosphine (130 mg, 3.9 mmol) was added. The mixture was stirred for 15 min and monitored by TLC. After completion, the mixture was diluted with dichloromethane, washed with 1.0 M hydrochloric acid and saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 140 mg of white solid compound 7 with a yield of 95%.

[0039] 7. Synthesis of Compound 8 Dissolve p-Hydroxybenzaldehyde (0.75 g, 4.12 mmol), 2-acetylamino-3,4,6-tri-O-acetyl-aD-pyranoglucopyranosyl (3 g, 8.2 mmol), 15-crown-5 (0.3 ml, 0.82 mmol) and potassium carbonate (2.55 g, 18.45 mmol) in 75 mL of acetonitrile and stir at room temperature overnight. Monitor by TLC. After completion, dilute with dichloromethane, wash with saturated sodium bicarbonate and brine. Dry the organic phase over anhydrous sodium sulfate and concentrate under reduced pressure to obtain 4 g of product A with a yield of 95%. Product A (1g, 2.21mmol) was dissolved in 35mL methanol, cooled to 0℃, sodium borohydride (170mg, 4.43mmol) was added, stirred for 2h, ammonium chloride (10mL) was added to quench, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and 0.7g of white solid, i.e., product B, was obtained with a yield of 90%. Product B (500mg, 1.1mmol) was dissolved in 10mL dichloromethane, cooled to 0℃, phosphorus tribromide (0.2ml, 2.2mmol) was added, monitored by TLC, and ammonium chloride (10ml) was added to quench after completion, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and 700mg of white powder was obtained with a yield of 88%.

[0040] The synthetic route of compound 8 is as follows: .

[0041] 8. Synthesis of Compound 9 Compound 7 (110 mg, 0.3 mmol) was dissolved in 3 ml of N,N-dimethylformamide under nitrogen at 0°C and stirred for 15 min. Potassium carbonate (50 mg, 0.4 mmol) was added and stirred for 10 min. Compound 8 (150 mg, 0.3 mmol) was added and stirred for 15 min. The mixture was moved to room temperature and stirred for 30 min. After the reaction was completed, the mixture was washed with ammonium chloride, diluted with ethyl acetate, concentrated under reduced pressure, and purified by column chromatography to obtain 170 mg of light yellow solid compound 9 with a yield of 70.9%.

[0042] 9. Synthesis of CL-NAG Compound 9 (150 mg, 0.18 mmol) was dissolved in 10 mL methanol, potassium carbonate (104 mg, 0.76 mmol) was added under nitrogen, and the reaction was carried out at room temperature for 30 min. After completion, the mixture was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain 200 mg of pale yellow solid crude product. The crude product and methylene blue (1-2 mg) were dissolved in 40 mL of dichloromethane, oxygen was bubbled through the solution at 0°C, and yellow light was irradiated for 4 h, concentrated under reduced pressure, and purified by column chromatography to obtain 120 mg of CL-NAG with a yield of 91%. Its hydrogen nuclear magnetic resonance spectrum, carbon spectrum, and high-resolution mass spectrum are shown as follows: Figure 1-3 shown.

[0043] Example 2 In this example, the photophysical properties of the obtained CL-NAG probe were measured.

[0044] (1) A 1.0 mM CL-NAG stock solution was prepared in DMSO. UV absorption experiments, chemiluminescence experiments, and bioluminescence experiments were performed in PBS buffer (10 mM, pH = 7.4, containing 5% DMSO).

[0045] Photophysical properties of target probe CL-NAG: The target probe CL-NAG stock solution prepared in Example 1 was incubated with a buffer solution containing NAG (200 U / L) at 37°C for 30 min (the final concentration of CL-NAG was 10 μM), and the UV absorption spectrum was measured using a UV-visible spectrophotometer. The results are as follows: Figure 4 A, shows that the absorption peak of the pure probe CL-NAG is low, and the absorption at 290 nm is significantly increased after incubation with NAG. Figure 4B and C are the chemiluminescence and fluorescence emission spectra of the probe. The chemiluminescence and fluorescence emission spectra of the probe were measured at 37°C using the multifunctional microplate reader SpectraMax iD3 from 450nm to 650nm in the presence of NAG, and the spectra without NAG were also measured. The results showed that at 540nm, there was a significant chemiluminescence enhancement (~200 times) after incubation with NAG compared to the absence of NAG, while the enhancement of the fluorescence spectrum was weaker than that of the chemiluminescence spectrum (~13.5 times), indicating that chemiluminescence has a high signal-to-noise ratio compared to fluorescence. Figure 4 D is the probe (10 mM). The chemiluminescence kinetic curves in response to different concentrations of NAG (0, 50, 100, 200, 300, 400 U / L) were measured using a multifunctional microplate reader SpectraMaxi iD3 (chemiluminescence mode) at 37°C for 3 hours. As the concentration of NAG increased, the chemiluminescence intensity also increased. Figure 4 E is the change in chemiluminescence intensity of CL-NAG (10 μM) after incubation with different concentrations of NAG (0, 2.5, 5, 10, 25, 50, 100, 150, 200, 300, 400 and 500 U / L) at 37°C for 30 min. The chemiluminescence intensity of CL-NAG increased with the increase of NAG concentration. Figure 4 F is the selectivity test of CL-NAG. Potential interferents, including ROS, metal ions, biothiols and other enzymes, were used to measure the selectivity of the probe for NAG. The results showed that the probe would not respond to other compounds and had a single selectivity for NAG. Figure 4 G tested the effect of buffer solutions with different pH values ​​on the chemiluminescence intensity and confirmed that the probe could exert its effect on NAG within the pH range of 3 to 11 (CL-NAG: 10 μM, NAG: 200 U / L). Figure 4 H is the probe and the main component in urine (Na + , K + Mg 2+ , Ca 2+ , Cl - , C 2 O 4 2- 、SO 4 2- , uric acid, urea, glucose, creatinine) at different times (0, 0.5, 1, 2, 3h) (CL-NAG: 10μM, the concentration of each component in urine is 0.5mM). Figure 4I is a graph showing the change in chemiluminescence intensity after incubation with the main components in urine for 3 h and then adding NAG (200 U / L) to each group and continuing incubation for 30 min, indicating that the components in urine will not cause significant interference to the probe and the probe is suitable for the detection of urine specimens.

[0046] Example 3 This example conducts experiments on the target probe CL-NAG and HK-2 and HUEVC cells.

[0047] At 37°C and 5% CO 2 HK-2 cells were cultured in DMEM / F-12 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics), and HUEVC cells were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics) in a humidified incubator. Figure 5 A is the cytotoxicity test of the probe on HK-2 and HUEVC cells. HK-2 and HUEVC cells were seeded in 96-well plates (8×10 3 ) for 24h, and then different concentrations of CL-NAG (0, 2.5, 5, 10, 25 and 50mM) were cultured with cells for 24h. Then 10% CCK-8 reagent was added to each well, and the absorbance value at 450nm was measured using the multi-function microplate reader SpectraMax iD3. The results showed that the probe had good biocompatibility. Figure 5 B shows the effects of different concentrations of NAG inhibitor NGT (NAG-thiazoline) (0, 1, 2, 3, 4 and 5 mM) on HK-2 cells (8×10 3 ) cytotoxicity test, HK-2 cells were seeded in 96-well plates (8×10 3 ) for 24 h, and then co-cultured with different concentrations of NGT (0, 1, 2, 3, 4 and 5 mM) for 24 h to measure the cell viability. As shown in the figure, high concentrations of NGT had no effect on cell activity. Figure 5 C is to study the cytotoxicity of cisplatin on HK-2 cells. Similarly, HK-2 cells were seeded in 96-well plates (8×10 3 ) for 24 h and then co-cultured with different concentrations of cisplatin (0, 1, 2, 5, 10 and 20 mM) for 24 h to measure the cell survival rate. In order to reduce the influence of dead cells on subsequent experimental results, the cisplatin concentration (i.e., 5 mM) when the cell survival rate was greater than 80% was selected for subsequent experiments. Figure 6 A shows the change in chemiluminescence intensity of HK-2 cells at different counts (0, 5000, 10000, 20000, 40000, 60000) after the probe (10 mM) was incubated for 30 min. When the number of cells gradually increased, the chemiluminescence intensity also increased. Figure 6B is the linear fitting curve of the probe and different cell numbers of HK-2 cells, showing a good linear relationship between chemiluminescence intensity and cell number. Figure 6 C Inhibition of NAG activity by different concentrations of NGT (1, 2, 3, 4, 5 mM) in buffer. NAG (200 U / L) was pre-added to the experimental wells, and then NGT (final concentrations of 1, 2, 3, 4, 5 mM) and probe (10 mM) were added. The blank group was the probe group without NAG and NGT, and the control group was the probe plus NAG. After incubation at 37°C for 30 minutes, the chemiluminescence intensity of each group was detected. The results showed that compared with the control group without NGT, the presence of NGT reduced the chemiluminescence intensity of CL-NAG, further verifying the property of CL-NAG activated by NAG. Figure 6 D. To investigate the ability of CL-NAG to detect endogenous NAG in living cells. HK-2 cells were treated with NGT and then incubated with the probe as the experimental group. The cells were seeded in a 96-well black plate (6 × 10 3 ) for 24h, treated with NGT (5mM) for 2h, and then incubated with CL-NAG (10μM) for 30min (CL-NAG+NGT in the figure). The results showed that compared with the blank group (simple cells, without probe and NGT), the chemiluminescent signal of the control group incubated with CL-NAG was significantly enhanced, and compared with the results of the experimental group (i.e., CL-NAG+NGT), it was shown that the enhanced chemiluminescent signal originated from CL-NAG activated by NAG. Figure 6 E To investigate whether CL-NAG can detect the upregulation of NAG in HK-2 cells stimulated by cisplatin. HK-2 cells were treated with 5mM cisplatin for 24h, and other groups were not treated with additional treatment. After 24h, they were incubated with 5mM NGT for 2h, and then CL-NAG (10μM) was added and incubated for 30min. The blank group was a simple HK-2 cell group without further treatment. The results showed that the chemiluminescence intensity of HK-2 cells treated with cisplatin was stronger than that of HK-2 cells not treated with cisplatin, indicating that CL-NAG is sensitive enough to detect the upregulation of NAG in HK-2 cells induced by cisplatin. Figure 6 F Comparison of the ability of CL-NAG to detect endogenous NAG in different cells (HK-2 and HUEVC cells). HK-2 cells (6×10 3 ) and HUEVC cells (6×10 3 ) were inoculated in a black 96-well plate. After 24 hours, the probe (10 mM) was added and incubated for 30 minutes before detection. The results showed that the content of endogenous NAG in HK-2 cells was higher than that in HUEVC cells.

[0048] Example 4 This embodiment provides a target NAG chemiluminescence kit.

[0049] A chemiluminescence kit for N-acetyl-β-D-glucosaminase comprises reagent 1, reagent 2, reagent 3 and a white 96-well plate, wherein reagent 1 comprises a CL-NAG probe and DMSO; reagent 2 comprises NAG, copper sulfate and distilled water; and reagent 3 comprises potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate heptahydrate and distilled water.

[0050] Among them, in the reagent 1, the concentration of CL-NAG probe is 5 mM: CL-NAG probe is dissolved in DMSO to prepare a 5 mM probe stock solution.

[0051] In the reagent 2, the concentration of each component is: NAG: 200U / L, copper sulfate: 0.5 mM, distilled water: 5ml.

[0052] In the reagent 3, the concentration of each component is: Potassium dihydrogen phosphate: 1.0588236 mM, sodium chloride: 155.17241 mM, sodium dihydrogen phosphate heptahydrate: 2.966418 mM. Potassium dihydrogen phosphate, sodium chloride, sodium dihydrogen phosphate heptahydrate and distilled water are prepared into PBS buffer (10 mM, pH = 7.4).

[0053] The kit operation steps are as follows:

[0054] Perform performance validation of the kit.

[0055] Establishment of standard curve: Take 1 mg of CL-NAG, weigh accurately, dissolve in 274 mL DMSO in a 2 mL EP tube, and obtain a probe stock solution with a concentration of 5 mM (reagent 1). Add reagent 2 (NAG: 200U / L) to blank urine to prepare a series of solutions of 0, 25, 50, 75, and 100U / L. Follow the kit operating procedures to obtain the standard curve of CL-NAG in urine. The results are as follows: Figure 7 As shown, it can be seen that when the NAG concentration range is 0-100U / L, the linearity is good.

[0056] Precision determination: Standard solutions of NAG solution with concentrations of 5.00, 10.00, 50.00, and 100.00 U / L were measured using CL-NAG (50mM) respectively. The specific determination method is as follows: each concentration was measured once every 2 hours for 5 consecutive times to investigate the intra-day precision of the method; each concentration was measured continuously for 5 days, once a day, to investigate the inter-day precision of the method. As shown in Table 1, both the intra-day and inter-day precisions met the requirements.

[0057] Recovery experiment: NAG solution with concentrations of 5.00, 10.00, 50.00, and 100.00 U / L was measured using CL-NAG (50mM) for three times in parallel. Recovery = measured concentration / configuration concentration × 100%. The results are shown in Table 2. The average recoveries were 100.87%, 102.87%, 98.73%, and 100.26%, respectively, which were within the range of 90% to 110%, and the RSD (%) was less than 5%, meeting the requirements.

[0058] Table 1: Intra-day and inter-day precision of the CL-NAG assay. (n=5)

[0059] Table 2: Recovery test results of CL-NAG. (n=3)

[0060] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0061] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0062] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A chemiluminescent probe for N-acetyl-β-D-glucosamine enzyme, characterized in that: The chemiluminescent probe is named CL-NAG, and the structural formula of CL-NAG is shown in Formula 1: 。 2. The method for preparing a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme according to claim 1, characterized in that: The chemiluminescent probe includes CL-NAG, and the preparation method of CL-NAG includes: S1. Compound 1 was dissolved in methanol, trimethyl orthoformate and tetrabutylammonium tribromide were added under an inert atmosphere for reaction, and after the reaction was completed, the mixture was diluted with ethyl acetate and extracted with sodium bicarbonate. The obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 2; S2. The compound 2 and imidazole were dissolved in dichloromethane, tert-butyldimethylsilyl chloride was added under an inert atmosphere, and after the reaction was completed, the mixture was separated and purified by a silica gel column to obtain a compound 3; S3. The compound 3 is dissolved in dichloromethane, trimethyl phosphite is added under an inert atmosphere, the reaction is carried out at 0±2°C for 15±5 min, titanium tetrachloride is added and the reaction is continued for 30±5 min, and after the reaction is completed, a saturated aqueous solution of sodium bicarbonate is added at 0±2°C, and the mixture is extracted with dichloromethane. The obtained organic phase is dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4; S4. Dissolve the compound 4 in tetrahydrofuran under an inert atmosphere, stir at -78±2°C for 30±5 min, then add lithium diisopropylamide and stir for 20±5 min, dissolve 2-adamantanone in tetrahydrofuran and add to the reaction system, stir for 15±5 min and heat to room temperature for reaction, add tetrabutylammonium fluoride after the reaction is completed, dilute with ethyl acetate, wash with 1.0M hydrochloric acid, dry the obtained organic phase over anhydrous sodium sulfate, and purify by column chromatography to obtain compound 5; S5. The compound 5 is dissolved in tetrahydrofuran, magnesium chloride and triethylamine are added, and then paraformaldehyde is added, and the mixture is heated to 80±2°C under reflux conditions, and then cooled to room temperature and diluted with dichloromethane, washed with 0.5M hydrochloric acid, and the organic layer is separated and then washed with saturated brine. The obtained organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 6; S6. The compound 6 was dissolved in dichloromethane, methoxyformylmethylenetriphenylphosphine was added, and the mixture was stirred for 15±5 min, and then diluted with dichloromethane, washed with 1.0 M hydrochloric acid, and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 7; S7. 2-acetylamino-3,4,6-tri-O-acetyl-aD-pyranose, p-hydroxybenzaldehyde, 15-crown ether-5 and potassium carbonate were dissolved in acetonitrile, stirred for reaction, and then diluted with dichloromethane, washed with saturated sodium bicarbonate and brine. The obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain product A. The product A was dissolved in methanol, cooled to 0±2°C, and sodium borohydride was added. After stirring for 2±0.5 h, ammonium chloride was added to quench, and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain product B. The product B was dissolved in dichloromethane, cooled to 0±2°C, phosphorus tribromide was added, and ammonium chloride was added to quench, extracted with dichloromethane, and the obtained organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 8; S8.0±2 ℃, the compound 7 was dissolved in N,N-dimethylformamide under an inert atmosphere, potassium carbonate was added and stirred for 10±5 min, and then the compound 8 was added. After stirring for reaction, the mixture was moved to room temperature and stirred for 30±5 min. After the reaction was completed, the mixture was washed with ammonium chloride, diluted with ethyl acetate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 9; S9. The compound 9 is dissolved in methanol, potassium carbonate is added under an inert atmosphere for reaction, and then diluted with ethyl acetate, washed with saturated brine, and the obtained organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude product and methylene blue are co-dissolved in dichloromethane, oxygen is bubbled through the solution at 0±2°C, and yellow light is irradiated at the same time, and then concentrated under reduced pressure and purified by column chromatography to obtain compound CL-NAG; Wherein, the structural formulas of the compounds 1 to 9 and the CL-NAG are as follows: 。 3. The method for preparing a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme according to claim 2, characterized in that: In step S1, the molar ratio of compound 1, trimethyl orthoformate and tetrabutylammonium tribromide is 1.0:1.6:0.1; In step S2, the molar ratio of compound 2, tert-butyldimethylsilyl chloride and imidazole is 1.0:1.2:3.0; In step S3, the molar ratio of compound 3, trimethyl phosphite and titanium tetrachloride is 1.0:1.2:3.0; In step S4, the molar ratio of compound 4, 2-adamantanone, lithium diisopropylamide and tetrabutylammonium fluoride is 1.0:1.2:1.5:1.1; In step S5, the molar ratio of compound 5, magnesium chloride, triethylamine and paraformaldehyde is 1.0:2.2:2.2:8.0; In step S6, the molar ratio of the compound 6 to methoxyformylmethylenetriphenylphosphine is 1.0:1.0; In step S7, the molar ratio of p-hydroxybenzaldehyde, the compound 2-acetylamino-3,4,6-tri-O-acetyl-aD-pyranoglucopyranosyl, 15-crown ether-5 and potassium carbonate is 1.0:2.0:0.2:4.5, the molar ratio of the product A to sodium borohydride is 1.0:2.0, and the molar ratio of the product B to phosphorus tribromide is 1.0:2.0; In step S8, the molar ratio of compound 7, compound 8 and potassium carbonate is 1.0:1.0:1.3; In step S9, the molar ratio of compound 9 to potassium carbonate is 1.0:4.

2.

4. Use of a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme as claimed in claim 1 in the preparation of a NAG detection reagent.

5. Use of a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme as claimed in claim 1 in the preparation of a renal injury detection reagent.

6. A kidney injury detection reagent, characterized in that: The kidney injury detection reagent comprises the chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme according to claim 1.

7. A NAG detection reagent, characterized in that: The NAG detection reagent comprises the chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme according to claim 1.

8. A NAG detection kit, characterized in that: The NAG detection kit comprises the chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme according to claim 1.

9. A chemiluminescent kit for N-acetyl-β-D-glucosamine enzyme, characterized in that: The kit comprises reagent 1, reagent 2 and reagent 3; The reagent 1 includes CL-NAG probe and DMSO; the reagent 2 includes NAG, copper sulfate and distilled water; the reagent 3 includes potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate and water; The CL-NAG probe is a chemiluminescent probe of N-acetyl-β-D-glucosamine enzyme as described in claim 1.

10. A chemiluminescent kit for N-acetyl-β-D-glucosamine enzyme according to claim 9, characterized in that: In the reagent 1, the concentration of CL-NAG probe is 5 mM; In the reagent 2, the concentration of each component is: NAG: 200U / L, copper sulfate: 0.5 mM; In the reagent 3, the concentration of each component is: Potassium dihydrogen phosphate: 1.0588236 mM, sodium chloride: 155.17241 mM, disodium hydrogen phosphate: 2.966418 mM.

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