Fluorescent probe for sequentially responding gamma-glutamyltranspeptidase and monoamine oxidase as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of biological medicine analysis, and particularly relates to a fluorescent probe sequentially responding to gamma-glutamyltranspeptidase and monoamine oxidase and application of the fluorescent probe. The structure of the fluorescent probe is as shown in a formula 1, the fluorescent probe in the formula 1 is constructed by sequentially covalently connecting gamma-glutamyltranspeptidase and specific recognition groups of monoamine oxidase and integrating the gamma-glutamyltranspeptidase and the specific recognition groups of monoamine oxidase into a benzothiazole-xanthene fluorescent parent structure, and the fluorescent probe has near-infrared fluorescence luminescence performance. The probe adopts a double enzyme response activation strategy, and can be activated and generate a fluorescence signal only when gamma-glutamyltranspeptidase and monoamine oxidase exist at the same time, so that the fluorescence probe is high in specificity and has excellent accuracy and specificity in the aspects of pathological imaging, drug safety evaluation and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fluorescent probe that responds sequentially to γ-glutamyl transpeptidase and monoamine oxidase, its preparation method, and its application. Background Technology

[0002] Herb-induced liver injury (HILI) refers to drug-induced liver injury (DILI) induced by prescription or over-the-counter products and their metabolites of traditional Chinese medicine (TCM) preparations, decoction pieces, granules, extracts, and dietary supplements containing TCM. It has become a common cause of abnormal liver biochemical indicators in clinical practice. In recent years, with the widespread use of TCM globally and the increasingly sophisticated drug adverse reaction monitoring system, clinical reports of TCM-related liver injury have been on the rise, posing a serious challenge to the safe clinical application and healthy development of the TCM industry. Currently, the detection of TCM hepatotoxicity and related liver injury mainly relies on retrospective detection methods based on an exclusionary strategy, i.e., causal assessment through comprehensive evaluation of patient history, physical signs, serum biochemical indicators, and histopathological examination results. This strategy is insufficient to provide effective information in the early stages of hepatotoxicity, resulting in inadequate accuracy and timeliness in assessment. Therefore, developing a detection strategy capable of real-time and sensitive monitoring of liver injury progression has become an urgent need for early screening of TCM-related liver injury.

[0003] Fluorescent detection technology, with its advantages of high sensitivity, high selectivity, real-time response, and visualization, has shown significant potential in early disease assessment and drug toxicity monitoring. However, most reported fluorescent probes related to drug-induced liver injury still employ a single biomarker response strategy. Since the selected biomarkers are also widely present in blood or other tissues under normal physiological conditions, these probes are easily non-specifically activated during blood / tissue circulation before reaching the liver, generating false-positive signals. This severely limits their practical application in the accurate assessment of liver-specific damage. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent probe that responds sequentially to γ-glutamyl transferase and monoamine oxidase, as well as its preparation method and application.

[0005] The first aspect of this invention provides a fluorescent probe that responds sequentially to γ-glutamyl transpeptidase and monoamine oxidase, the structure of which is shown in formula (1): Equation (1).

[0006] In another preferred embodiment, the fluorescent probe is constructed by covalently linking the γ-glutamyl amino group, the recognition group of γ-glutamyl transpeptidase, and the propylamino group, the recognition group of monoamine oxidase, together on a benzothiazole-oxanthracene fluorescent matrix.

[0007] A second aspect of the present invention provides a method for preparing a fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase, comprising the following steps: (1) Phosphorus tribromide was added dropwise to solvent one, and cyclohexanone was added dropwise under stirring. The reaction was carried out for 8h~12h. After the reaction was completed, the reaction solution was purified to obtain compound 1. (2) 2,4-dihydroxybenzaldehyde, cesium carbonate and compound 1 were suspended in solvent 2 and stirred for 18h~24h. After the reaction was completed, the reaction solution was purified to obtain compound 2. (3) Compound 2, N -(3-bromopropyl)carbamate tert-butyl ester, cesium carbonate and sodium iodide were suspended in solvent three and stirred for 12 h to 24 h. After the reaction was completed, the reaction solution was purified to obtain compound 3. (4) 2-Methylbenzothiazole and iodomethane were dissolved in solvent tri, and the mixture was heated under reflux for 6-8 hours. After the reaction was completed, the reaction solution was filtered to obtain compound 4. (5) Compound 3 and Compound 4 were suspended in solvent 4 and heated for 6-8 hours. After the reaction was completed, the reaction solution was treated to obtain compound 5. (6) Compound 5 was suspended in solvent 5, and trifluoroacetic acid was added and stirred for 1-2 hours. After the reaction was completed, the reaction solution was purified to obtain compound 6. (7) Boc- L - 1-tert-butyl glutamate, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were dissolved in solvent five. After stirring at 0°C for 0.25 h to 0.5 h, an anhydrous dichloromethane solution containing compound 6 was added, and the reaction was stirred at room temperature for 1 h to 3 h. After the reaction was completed, the reaction solution was purified to obtain compound 7. (8) Trifluoroacetic acid was added to solvent 5 in which compound 7 was dissolved, and the reaction was stirred for 1 h to 2 h. The reaction solution was then purified to obtain the fluorescent probe. In another preferred embodiment, the solvent is a mixture of N,N-dimethylformamide and anhydrous dichloromethane in a volume ratio of 2:5. The second solvent is a mixed solvent of N,N-dimethylformamide and anhydrous acetonitrile with a volume ratio of 1:1. The solvent three is anhydrous acetonitrile; The solvent four is a mixed solvent of n-butanol and toluene with a volume ratio of 4:1; Solvent 5 is anhydrous dichloromethane.

[0008] In another preferred embodiment, the molar ratio of phosphorus tribromide to cyclohexanone in step (1) is 3~6:1; In step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, cesium carbonate and compound 1 is 1:1~2:1~2; In step (3), compound 2, N The molar ratio of tert-butyl 3-(3-bromopropyl)carbamate, cesium carbonate, and sodium iodide is 2:2~4:2~4:1; In step (4), the molar ratio of 2-methylbenzothiazole to iodomethane is 1:1~2; In step (5), the molar ratio of compound 3 and compound 4 is 1:1~2; In step (6), the molar ratio of compound 5 to trifluoroacetic acid is 1:10~20; In step (7), Boc- L - 1-tert-butyl glutamate, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, the molar ratio of compound 6 is 3~4:3:3:2; In step (8), the molar ratio of compound 7 to trifluoroacetic acid is 1:10~20.

[0009] In another preferred embodiment, the preparation process of compound 1 in step (1) is as follows: the reaction solution is placed in an ice-water mixture, sodium carbonate powder is added until no bubbles are generated, dichloromethane is used for extraction, and the organic solvent is removed by vacuum concentration. The purification process of compound 2 in step (2) is as follows: the reaction solution is filtered to remove cesium carbonate, concentrated under reduced pressure to remove organic solvent, extracted with dichloromethane, washed with distilled water, concentrated to remove dichloromethane, and the crude product is purified by silica gel column chromatography. The elution system is petroleum ether and ethyl acetate in a volume ratio of 3:1. The purification process of compound 3 in step (3) is as follows: after removing insoluble matter by filtration, the reaction solution is concentrated under reduced pressure to remove organic solvent. The crude product is purified by silica gel column chromatography. The elution system is petroleum ether and ethyl acetate in a volume ratio of 4:1. The purification process of compound 4 in step (4) is as follows: the reaction solution is filtered to obtain a precipitate, which is then washed with acetonitrile and dried to obtain a solid compound; The purification process of compound 5 in step (5) is as follows: the reaction solution is concentrated under reduced pressure to remove the organic solvent, and the crude product is purified by silica gel column chromatography. The elution system is dichloromethane and methanol with a volume ratio of 40:1. The purification process of compound 6 in step (6) is as follows: the reaction solution is concentrated under reduced pressure to remove the solvent, and dichloromethane is continuously added to dissolve the crude product and continue to evaporate to remove residual trifluoroacetic acid. The crude product is purified by silica gel column chromatography, and the elution system is dichloromethane and methanol with a volume ratio of 30:1. The purification process of compound 7 in step (7) is as follows: after removing the solvent by vacuum concentration, it is extracted with dichloromethane, washed with distilled water, and after removing the solvent by concentration, the crude product is purified by silica gel column chromatography. The elution system is dichloromethane and methanol with a volume ratio of 40:1. The purification process of the fluorescent probe compound in step (8) is as follows: After removing the solvent by vacuum concentration, dichloromethane is continuously added to dissolve the crude product and evaporated to remove residual trifluoroacetic acid. The crude product is purified by silica gel column chromatography, and the elution system is dichloromethane and methanol in a volume ratio of 5:1.

[0010] A third aspect of the present invention provides the application of the fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase in the preparation of a diagnostic reagent for visually detecting drug-induced liver injury, characterized in that the drug-induced liver injury includes traditional Chinese medicine-related liver injury.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention is based on the dynamic characteristics of biomarkers in the pathological microenvironment of drug-induced liver injury. Through rational molecular design, the γ-glutamylamino group, the recognition group of γ-glutamyl transferase, and the propylamino group, the recognition group of monoamine oxidase, are directly tandemly linked via an acylation reaction and covalently integrated into a near-infrared fluorescent matrix via ether bonds, successfully constructing a novel fluorescent probe with a sequential activation mechanism. The fluorescent probe of this invention can only be activated under pathological conditions where γ-glutamyl transferase and monoamine oxidase coexist and act sequentially, generating a near-infrared fluorescent signal. This effectively avoids false positives caused by physiological fluctuations of a single biomarker, significantly improving the specificity, particularity, and accuracy of the detection.

[0012] The fluorescent probes provided by this invention can serve as imaging reagents, enabling precise imaging and assessment of the pathological degree of liver injury induced by traditional Chinese medicine (TCM) through dynamic responses to changes in the activity of endogenous γ-glutamyl transpeptidase and monoamine oxidase. Tripterygium wilfordii is a representative example of this invention. Furthermore, these fluorescent probes can intuitively and visually assess the hepatotoxic adverse reactions of TCM components through fluorescence signals. Psoralen A, psoralen B, emodin, matrine, oxymatrine, and saikosaponin A are representative examples of TCM components, thus providing a powerful detection tool for the safety evaluation of TCM. Attached Figure Description

[0013] Figure 1 This is a synthetic route diagram for the fluorescent probe YMG.

[0014] Figure 2 This is the proton NMR spectrum of the fluorescent probe YMG.

[0015] Figure 3 This is the carbon NMR spectrum of the fluorescent probe YMG.

[0016] Figure 4 This is a high-resolution mass spectrum of the fluorescent probe YMG.

[0017] Figure 5 The fluorescence emission spectra of the fluorescent probe YMG (10 μM) after reacting with GGT (90 U / L), MAO (90 U / L), or GGT (90 U / L) + MAO (90 U / L) are shown.

[0018] Figure 6 The figure shows the fluorescence response of the fluorescent probe YMG to GGT and MAO over time. Figure A shows the fluorescence emission spectra of YMG (10 μM) in response to GGT (90 U / L) and MAO (90 U / L) at different times (0-150 min), and Figure B shows the relationship between fluorescence intensity at 728 nm and reaction time.

[0019] Figure 7 The figures show the fluorescence response of the fluorescent probe YMG to GGT and MAO as a function of concentration. Figure A shows the fluorescence emission spectra of YMG (10 μM) reacting with MAO (90 U / L) and different concentrations of GGT (0–90 U / L) for 100 min. Figure B shows the relationship between fluorescence intensity (728 nm) and MAO (90 U / L) and different concentrations of GGT (0–90 U / L). The experiment was repeated three times. Figure C shows the fluorescence emission spectra of YMG (10 μM) reacting with GGT (90 U / L) and different concentrations of MAO (0–90 U / L) for 100 min. Figure D shows the relationship between fluorescence intensity (728 nm) and GGT (90 U / L) and different concentrations of MAO (0–90 U / L). The experiment was repeated three times.

[0020] Figure 8 The response specificity of the fluorescent probe YMG was investigated (fluorescence intensity at 728 nm after YMG was co-incubated with GGT + MAO or other analytes for 100 min). 1. Blank; 2-16. K + Ca 2+ Mg 2+ Zn 2+ I - HCO3 - H2PO4 - NO3 - H2O2, Cys, Hcy, GSH, Arg, Tyr, Lys: 50 μM; 17-20. LAP, ALP, tyrosinase, APN: 100 U / L; 21-22. nitroreductase, β-galactosidase: 5 μg / mL; 23. MAO (90 U / L) + GGT (90 U / L). GSH is reduced glutathione, Cys is cysteine, Hcy is homocysteine, Arg is arginine, Tyr is tyrosine, Lys is lysine, LAP is leucine aminopeptidase, ALP is alkaline phosphatase, tyrosinase is tyrosinase, APN is aminopeptidase N, nitroreductase is nitroreductase, and β-galactosidase is β-galactosidase. The experiment was repeated 3 times.

[0021] Figure 9 Fluorescence imaging of the degree of hepatocyte damage induced by the traditional Chinese medicine component triptolide, detected by the fluorescent probe YMG. Cells were first co-incubated with different concentrations of triptolide (0 nM, 25 nM, 50 nM, 100 nM) for 12 h, and then co-incubated with the probe YMG (10 μM) for 1.5 h. Cell locations were marked using nuclear imaging (DAPI), scale bar: 40 μm.

[0022] Figure 10 Fluorescent imaging of hepatocyte damage induced by different traditional Chinese medicine components, detected by the fluorescent probe YMG. Cells were first co-incubated with 0.03 mM of different traditional Chinese medicine components (psoralen, psoralen B, emodin, matrine, oxymatrine, and saikosaponin A) for 12 h, and then co-incubated with probe YMG (10 μM) for 1.5 h. Cell location was marked using nuclear imaging (DAPI), scale bar: 40 μm. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0025] This invention constructs a fluorescent probe capable of sequentially responding to two liver injury-related biomarkers, which is expected to significantly improve the predictive accuracy of drug-induced liver injury, especially traditional Chinese medicine-related liver injury. Gamma-glutamyl transpeptidase (GGT) is a key enzyme catalyzing the transfer of gamma-glutamyl groups, mainly involved in glutathione metabolism. In normal human serum, it primarily originates from the liver, and its activity level is significantly correlated with the degree of liver tissue damage, making it an important indicator of hepatocyte damage. Monoamine oxidase (MAO), as an oxidoreductase, is significantly upregulated in pathological states such as liver injury, acute hepatitis, and liver fibrosis, and can also be considered one of the biomarkers of drug-induced liver injury. Based on this, a novel near-infrared fluorescent probe capable of sequentially identifying GGT and MAO can be constructed. This probe can only be activated and generate a fluorescent signal when GGT and MAO are abnormally expressed in the pathological microenvironment, thereby significantly improving detection specificity, effectively avoiding false alarms caused by physiological fluctuations of a single indicator, enhancing the accuracy of judging liver damage related to traditional Chinese medicine and the ability to evaluate the safety of traditional Chinese medicine, and providing reliable technical support for early warning and adverse reaction screening of hepatotoxicity of traditional Chinese medicine.

[0026] Example 1: Synthesis of the fluorescent probe YMG Synthetic routes such as Figure 1 As shown, structural identification is as follows Figures 2-4 As shown.

[0027] 1. Synthesis of Compound 1 0 o At temperature C, phosphorus tribromide (8.46 mL, 90 mmol) was slowly added dropwise to a mixture of N,N-dimethylformamide (DMF, 8 mL) and dichloromethane (CH2Cl2, 20 mL). After stirring continuously for 0.5 h, cyclohexanone (3.11 mL, 30 mmol) was added dropwise, and stirring was continued at room temperature (25 °C) for 12 h. The reaction mixture was then slowly placed in an ice-water mixture, and sodium carbonate powder was continuously added until no more bubbles were generated. Extraction was then performed using dichloromethane, and the organic phase was removed by vacuum concentration to obtain 4.51 g of oily compound 1, with a yield of 79.5%, which was directly used in the next reaction.

[0028] 2. Synthesis of Compound 2 2,4-Dihydroxybenzaldehyde (2.76 g, 20 mmol) and cesium carbonate (6.52 g, 20 mmol) were added to a 30 mL DMF / acetonitrile (1:1 v / v) mixture containing compound 1 (3.78 g, 20 mmol). The reaction mixture was stirred continuously at room temperature (25 °C) for 18 h. Cesium carbonate was removed by filtration, and the organic solvent was removed by concentration under reduced pressure. The mixture was then extracted with dichloromethane, washed with distilled water, and concentrated to remove dichloromethane. The crude product was then purified by silica gel column chromatography using petroleum ether and ethyl acetate (3:1 v / v) as eluents to obtain 3.47 g of solid compound 2, with a yield of 76.1%.

[0029] 3. Synthesis of Compound 3 Compound 2 (1.83 g, 8 mmol) and N 2.86 g (12 mmol) of tert-butyl 3-(3-bromopropyl)carbamate was suspended in anhydrous acetonitrile (25 mL). Cesium carbonate (2.61 g, 8 mmol) and sodium iodide (0.60 g, 4 mmol) were added under stirring. The mixture was stirred for 12 h at room temperature (25 °C). After removing insoluble matter by filtration, the reaction solution was concentrated. The crude product was purified by silica gel column chromatography using petroleum ether and ethyl acetate in a volume ratio of 4:1 as eluents to obtain 2.38 g of solid compound 3, with a yield of 77.2%.

[0030] 4. Synthesis of Compound 4 2-Methylbenzothiazole (1.27 mL, 10 mmol) and iodomethane (0.62 mL, 10 mmol) were dissolved in 30 mL of anhydrous acetonitrile. o The mixture was heated under reflux at C for 8 hours. After the reaction was completed, it was cooled to room temperature, filtered to obtain a precipitate, washed with acetonitrile, and dried to give 2.46 g of solid compound 4, with a yield of 84.4%.

[0031] 5. Synthesis of Compound 5 Compound 3 (2.31 g, 6 mmol) and compound 4 (1.75 g, 6 mmol) were added to a mixed solvent of n-butanol / toluene (24 mL / 6 mL), and the reaction mixture was heated to 85°C. o Stirring at C for 6 hours. After the reaction was complete, the organic solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 40:1 as the eluent, yielding 2.38 g of solid compound 5, with a yield of 60.3%.

[0032] 6. Synthesis of Compound 6 Trifluoroacetic acid (2.47 mL, 32.2 mmol) was added to 10 mL of dry dichloromethane containing compound 5 (2.12 g, 3.22 mmol). After stirring for 1 h, the solvent was removed by concentration under reduced pressure. Dichloromethane was continuously added to dissolve the crude product, and the mixture was further evaporated to remove residual trifluoroacetic acid. Finally, the product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 30:1 as the eluent to obtain 1.38 g of solid compound 6, with a yield of 76.7%.

[0033] 7. Synthesis of Compound 7 Boc-L-glutamic acid-1-tert-butyl ester (Boc-Glu-OtBu, 1.21 g, 4 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (HATU, 1.14 g, 3 mmol), and N,N-diisopropylethylamine (DIPEA, 0.52 mL) were dissolved in anhydrous dichloromethane (20 mL). o After stirring at C for 0.5 h, 10 mL of anhydrous dichloromethane containing 1.12 g (2 mmol) of compound 6 was added to the reaction system, and the mixture was stirred at room temperature for 1 h. After removing the solvent by concentration under reduced pressure, the mixture was extracted with dichloromethane and washed with plenty of distilled water to remove the condensing agent. After further concentration to remove the solvent, the crude product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 40:1 to obtain 1.33 g of solid compound 7, with a yield of 79.1%.

[0034] 8. Synthesis of the fluorescent probe YMG Trifluoroacetic acid (1.74 mL) was added to anhydrous dichloromethane (10 mL) containing compound 7 (1.17 g, 1.39 mmol). After stirring for 1 h, the solvent was removed by concentration under reduced pressure. Anhydrous dichloromethane was continuously added to dissolve the crude product, and the mixture was further evaporated to remove residual trifluoroacetic acid. Finally, the product was purified by silica gel column chromatography using dichloromethane and methanol in a volume ratio of 5:1 as the eluent to obtain 0.43 g of solid fluorescent probe compound YMG, with a yield of 45.5%.

[0035] Fluorescent probe YMG structure characterization spectral data: 1 H NMR (600MHz, Methanol- d 4) δ H 8.60–8.45 (m, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.94 (d, J= 10.2 Hz, 1H), 7.80-7.71 (m, 1H),7.64 (dd, J = 16.8, 8.9 Hz, 1H), 7.34 (q, J = 8.8 Hz, 1H), 7.14 (dd, J = 10.6, 4.7Hz, 1H), 7.07-6.99 (m, 1H), 6.92-6.73 (m, 2H), 4.17-4.09 (m, 4H), 3.78 (t, J =6.3 Hz, 1H), 3.42 (dd, J = 9.9, 4.1 Hz, 2H), 2.70 (dt, J = 21.7, 6.5 Hz, 4H), 2.48 (t, J = 7.3 Hz, 2H), 2.15 (t, J = 6.7 Hz, 2H), 2.06-2.01 (m, 2H), 1.92 (t, J =6.1 Hz, 2H), 1.30 (s, 2H). 13 C NMR (151 MHz, Methanol- d 4) δ C 174.95, 163.44,160.26, 155.71, 144.50, 132.31, 130.38, 129.68, 128.65, 124.49, 117.09,116.32, 114.17, 113.90, 102.22, 67.51, 37.64, 35.38, 32.97, 30.36, 30.18,27.83, 25.75, 21.88. HR-ESI-MS: m / z [M] + Calculated value C 31 H 34 N3O5S + 560.2214, found value 560.2994.

[0036] The above synthesis method and characterization results demonstrate that the fluorescent probe YMG was successfully synthesized.

[0037] Example 2: Detection effect of fluorescent probe YMG on GGT / MAO Weigh an appropriate amount of the fluorescent probe YMG and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The buffer solutions prepared for the spectral tests were all mixtures of PBS (10 mM, pH = 7.4) and DMSO at a volume ratio of 19:1. The concentration of the probe YMG in the test solution was 10 μM, the fluorescence excitation wavelength was 680 nm, and the test environment temperature was 37 °C.

[0038] First, the spectral characteristics of the fluorescent probe YMG in response to the enzymes GGT and MAO were investigated. Fluorescence spectra were tested under simulated physiological conditions for four different scenarios: enzyme-free system, GGT only, MAO only, and simultaneous presence of GGT and MAO. The results are as follows: Figure 5 As shown, under excitation at 680 nm, the probe YMG (10 μM) itself exhibits a weak fluorescence signal. However, after co-incubation with either enzyme GGT (90 U / L) or MAO (90 U / L), the fluorescence signal shows no significant change, indicating that the probe cannot react with either GGT or MAO alone to produce a fluorescence signal. But when GGT (90 U / L) and MAO (90 U / L) are simultaneously co-incubated with the probe YMG, the fluorescence intensity of the test system (728 nm) increases significantly, indicating that the probe YMG can only be effectively activated and release a near-infrared fluorescence signal when both GGT and MAO are present.

[0039] When performing response time spectroscopy, a stock solution of the fluorescent probe YMG and appropriate volumes of GGT and MAO stock solutions were added to PBS buffer (pH = 7.4, 5% DMSO, 10mM) to achieve a final probe concentration of 10μM and a final GGT and MAO concentration of 90U / L. The test solutions were incubated for different times (0–150 min) before spectral analysis. The results are as follows: Figure 6 As shown, the fluorescence intensity at 728 nm is time-dependent, gradually increasing with the extension of the reaction time. After about 100 min, the change in fluorescence intensity is not obvious, which also indicates that the probe can be effectively activated and release fluorescence signals when GGT and MAO are present at the same time.

[0040] For concentration-dependent detection, in the same testing environment, the probe YMG (10 μM) was reacted with MAO (90 U / L) and different concentrations of GGT (0~90 U / L) for 100 min, or YMG (10 μM) was reacted with GGT (90 U / L) and different concentrations of MAO (0~90 U / L) for 100 min, followed by spectral analysis. The results are as follows: Figure 7As shown, the fluorescence intensity of the reaction system gradually increased with the increase of GGT and MAO concentrations, indicating that different concentrations of GGT and MAO can induce different fluorescence signal enhancements. The experimental results suggest that the probe has the potential to assess the pathological degree of the microenvironment by responding to fluorescence changes generated by different GGT and MAO activity levels simultaneously.

[0041] Example 3: Environmental Specificity Investigation of Fluorescent Probe YMG To investigate the recognition specificity of the probe YMG, its selectivity was evaluated. YMG (10 μM) was compared with GGT (90 U / L) + MAO (90 U / L) or other analytes (K). + Ca 2+ Mg 2+ Zn 2+ I - HCO3 - H2PO4 - NO3 - The concentrations of H2O2, Cys, Hcy, GSH, Arg, Tyr, and Lys were all 50 μM; the concentrations of LAP, ALP, tyrosinase, and APN were all 100 U / L; and the concentrations of nitroreductase and β-galactosidase were all 5 μg / mL. GSH is reduced glutathione, Cys is cysteine, Hcy is homocysteine, Arg is arginine, Tyr is tyrosine, Lys is lysine, LAP is leucine aminopeptidase, ALP is alkaline phosphatase, tyrosinase is tyrosinase, APN is aminopeptidase N, nitroreductase is nitroreductase, and β-galactosidase is β-galactosidase. The fluorescence emission spectra of the incubated samples after 100 min were analyzed. The results are as follows: Figure 8 As shown, the probe only responds to a significant fluorescence enhancement signal when GGT and MAO are present simultaneously. The addition of other analytes did not cause a significant change in the fluorescence intensity of the test system, indicating that the probe only releases a noticeable fluorescence signal after reacting sequentially with GGT and MAO, demonstrating good selectivity. These results demonstrate that the YMG probe is highly specific, exclusively responding to the enzymes GGT and MAO sequentially, giving it an advantage in detection applications within complex biological microenvironments.

[0042] Example 4: Application of the fluorescent probe YMG in detecting hepatocellular damage related to traditional Chinese medicine and assessing the hepatotoxicity of traditional Chinese medicine components This embodiment utilizes the probe YMG to detect changes in endogenous GGT / MAO activity and assesses the degree of hepatocellular damage related to traditional Chinese medicine (TCM) through cell imaging. Triptolide was used as a representative hepatotoxic component of TCM. AML12 liver cells were co-incubated with different concentrations (0 nM, 25 nM, 50 nM, 100 nM) of triptolide for 12 h, followed by co-incubation with probe YMG at a final concentration of 10 μM for 1.5 h. Cell fluorescence imaging analysis was then performed using the Image Xpress Micro Confocal system. Results are as follows: Figure 9 As shown, the cellular fluorescence intensity increases with increasing triptolide concentration, indicating that the upregulated GGT / MAO expression induced by triptolide-induced hepatocyte injury can synergistically respond to the probe YMG to release a visible fluorescent signal. This probe can conveniently and intuitively reflect the pathological degree of traditional Chinese medicine-related liver injury through fluorescence signals.

[0043] Next, using GGT / MAO-mediated fluorescence signal changes as a detection index, the hepatotoxic adverse reactions of various active ingredients in traditional Chinese medicine (TCM) were assessed using the YMG probe. This example used Bavachin, Isobavachalcone, Emodin, Matrine, Oxymatrine, and Saikosaponin A as representatives. AML12 cells were co-incubated with each TCM ingredient at a concentration of 0.03 mM for 12 h, followed by co-incubation with 10 μM YMG probe for 1.5 h, and then cell imaging analysis was performed. The results are as follows... Figure 10 As shown, after treatment with various traditional Chinese medicine (TCM) components, the cell fluorescence intensity significantly increased, indicating that hepatotoxic TCM components can cause hepatocyte damage, thereby upregulating the expression activities of GGT and MAO, enhancing the synergistic response to the probe YMG, and generating a stronger fluorescence signal. Furthermore, the relative hepatotoxicity of each TCM component can be further assessed by observing the differences in the fluorescence response signal of the probe YMG. In summary, this embodiment demonstrates that the probe YMG can achieve a direct and rapid assessment of the hepatotoxicity of TCM by responding to the visualized fluorescence signal generated by changes in GGT / MAO activity in the liver injury microenvironment, showing promising prospects for practical application.

[0044] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase, characterized in that, The chemical structural formula is shown in formula (1): Equation (1).

2. The fluorescent probe that responds sequentially to γ-glutamyl transpeptidase and monoamine oxidase according to claim 1, characterized in that, This fluorescent probe is constructed by covalently linking the γ-glutamyl amino group, the recognition group of γ-glutamyl transpeptidase, and the propylamino group, the recognition group of monoamine oxidase, and then modifying them together on a benzothiazole-oxanthracene fluorescent matrix.

3. The fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase according to claim 2, characterized in that, The preparation method of the fluorescent probe includes the following steps: (1) Phosphorus tribromide was added dropwise to solvent one, and cyclohexanone was added dropwise under stirring. The reaction was carried out for 8h~12h. After the reaction was completed, the reaction solution was treated to obtain compound 1. (2) 2,4-dihydroxybenzaldehyde, cesium carbonate and compound 1 were suspended in solvent 2 and stirred for 18h~24h. After the reaction was completed, the reaction solution was purified to obtain compound 2. (3) Compound 2, N -(3-bromopropyl)carbamate tert-butyl ester, cesium carbonate and sodium iodide were suspended in solvent three and stirred for 12 h to 24 h. After the reaction was completed, the reaction solution was purified to obtain compound 3. (4) 2-Methylbenzothiazole and iodomethane were dissolved in solvent tri, and the mixture was heated under reflux for 6-8 hours. After the reaction was completed, the reaction solution was filtered to obtain compound 4. (5) Compound 3 and Compound 4 were suspended in solvent 4 and heated for 6-8 hours. After the reaction was completed, the reaction solution was purified to obtain compound 5. (6) Compound 5 was suspended in solvent 5, and trifluoroacetic acid was added and stirred for 1-2 hours. After the reaction was completed, the reaction solution was purified to obtain compound 6. (7) Boc- L - 1-tert-butyl glutamate, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were dissolved in solvent five. After stirring at 0°C for 0.25 h to 0.5 h, an anhydrous dichloromethane solution containing compound 6 was added, and the reaction was stirred at room temperature for 1 h to 3 h. After the reaction was completed, the reaction solution was purified to obtain compound 7. (8) Trifluoroacetic acid was added to solvent 5 in which compound 7 was dissolved, and the reaction was stirred for 1-2 hours. The reaction solution was then purified to obtain the fluorescent probe.

4. The method for preparing the fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase according to claim 3, characterized in that: The solvent is a mixed solvent of N,N-dimethylformamide and anhydrous dichloromethane with a volume ratio of 2:

5. The second solvent is a mixed solvent of N,N-dimethylformamide and anhydrous acetonitrile with a volume ratio of 1:

1. The solvent three is anhydrous acetonitrile; The solvent four is a mixed solvent of n-butanol and toluene with a volume ratio of 4:1; Solvent 5 is anhydrous dichloromethane.

5. The method for preparing the fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase according to claim 3, characterized in that: In step (1), the molar ratio of phosphorus tribromide to cyclohexanone is 3~6:1; In step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, cesium carbonate and compound 1 is 1:1~2:1~2; In step (3), compound 2, N The molar ratio of tert-butyl 3-(3-bromopropyl)carbamate, cesium carbonate, and sodium iodide is 2:2~4:2~4:1; In step (4), the molar ratio of 2-methylbenzothiazole to iodomethane is 1:1~2; In step (5), the molar ratio of compound 3 and compound 4 is 1:1~2; In step (6), the molar ratio of compound 5 to trifluoroacetic acid is 1:10~20; In step (7), Boc- L - 1-tert-butyl glutamate, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, the molar ratio of compound 6 is 3~4:3:3:2; In step (8), the molar ratio of compound 7 to trifluoroacetic acid is 1:10~20.

6. The method for preparing the fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase according to claim 3, characterized in that: The preparation process of compound 1 in step (1) is as follows: the reaction solution is placed in an ice-water mixture, sodium carbonate powder is added until no bubbles are generated, dichloromethane is used for extraction, and the organic solvent is removed by vacuum concentration. The purification process of compound 2 in step (2) is as follows: the reaction solution is filtered to remove cesium carbonate, concentrated under reduced pressure to remove organic solvent, extracted with dichloromethane, washed with distilled water, concentrated to remove dichloromethane, and the crude product is purified by silica gel column chromatography. The elution system is petroleum ether and ethyl acetate in a volume ratio of 3:

1. The purification process of compound 3 in step (3) is as follows: after removing insoluble matter by filtration, the reaction solution is concentrated under reduced pressure to remove organic solvent. The crude product is purified by silica gel column chromatography. The elution system is petroleum ether and ethyl acetate in a volume ratio of 4:

1. The purification process of compound 4 in step (4) is as follows: the reaction solution is filtered to obtain a precipitate, which is then washed with acetonitrile and dried to obtain a solid compound; The purification process of compound 5 in step (5) is as follows: the reaction solution is concentrated under reduced pressure to remove the organic solvent, and the crude product is purified by silica gel column chromatography. The elution system is dichloromethane and methanol with a volume ratio of 40:

1. The purification process of compound 6 in step (6) is as follows: the reaction solution is concentrated under reduced pressure to remove the solvent, and dichloromethane is continuously added to dissolve the crude product and continue to evaporate to remove residual trifluoroacetic acid. The crude product is purified by silica gel column chromatography, and the elution system is dichloromethane and methanol with a volume ratio of 30:

1. The purification process of compound 7 in step (7) is as follows: after removing the solvent by vacuum concentration, it is extracted with dichloromethane, washed with distilled water, and after removing the solvent by concentration, the crude product is purified by silica gel column chromatography. The elution system is dichloromethane and methanol with a volume ratio of 40:

1. The purification process of the fluorescent probe compound in step (8) is as follows: After removing the solvent by vacuum concentration, dichloromethane is continuously added to dissolve the crude product and evaporated to remove residual trifluoroacetic acid. The crude product is purified by silica gel column chromatography, and the elution system is dichloromethane and methanol in a volume ratio of 5:

1.

7. The application of the fluorescent probe that sequentially responds to γ-glutamyl transpeptidase and monoamine oxidase according to claim 1 in the preparation of a diagnostic reagent for visually detecting drug-induced liver injury, characterized in that, Drug-induced liver injury includes liver injury related to traditional Chinese medicine.

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