A probe for monitoring the activity of glutamyl transpeptidase, a preparation method thereof, and applications

Abstract

The present invention provides a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and applications, comprising the following steps: (S10) preparing a hemicyanine photoacoustic group containing two hydrophilic sulfonic acid groups and a hemicyanine molecular solution; (S20) dissolving N-Boc-Glu-OtBu and HATU in an organic solvent, adding DMAP, activating at 0-4 °C, and adding the hemicyanine molecular solution prepared in the step (S10) and continuously stirring for reaction; (S30) removing unreacted raw materials and by-products through washing with water and drying operations; (S40) obtaining the target product GGT-probe probe through an acidic deprotection step and silica gel column chromatography. The probe has strong hydrophilicity. By utilizing the photoacoustic properties derived from hemicyanine, it can non-invasively monitor GGT activity at the in vivo level and is suitable for applications in monitoring liver function and systemic lipid metabolism diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method thereof, and an application thereof. Background Art

[0002] Obesity and atherosclerosis are major health challenges globally. They not only seriously affect the quality of life of patients but also significantly increase the risks of cardiovascular diseases, diabetes, and cancer. Both of these diseases are closely related to lipid metabolism disorders, and glutamyl transpeptidase (GGT), as a key biomarker, plays a crucial role in the monitoring and diagnosis of these diseases.

[0003] Obesity and lipid metabolism disorders: Obesity is a chronic disease caused by excessive accumulation of body fat. It is closely related to various metabolic morbidities, such as type 2 diabetes, hypertension, and cardiovascular diseases. Studies have shown that the GGT level in obese patients often abnormally increases, which is related to liver fat accumulation and increased oxidative stress.

[0004] Pathogenesis of atherosclerosis: Atherosclerosis is a disease in which the inner wall of the artery gradually hardens and narrows, mainly caused by the accumulation of fat and other substances on the blood vessel wall. This accumulation leads to a decrease in blood vessel elasticity and obstruction of blood flow, and may ultimately cause heart disease or stroke. The expression level of GGT in this process is positively correlated with the level of inflammatory markers (such as C-reactive protein) and can be used as a biomarker for early diagnosis and monitoring.

[0005] Biological function of GGT and its association with diseases: GGT is an enzyme widely present in organs such as the liver, pancreas, and kidney. Its main function is to participate in the metabolism of glutathione, regulate the intracellular antioxidant status and signal transduction. Clinically, the measurement of GGT is often used to evaluate liver damage and functional abnormalities. Recent studies have shown that GGT activity not only reflects liver diseases but is also related to the overall lipid metabolism status, especially in obese and non-alcoholic fatty liver disease (NAFLD) patients, the increase in GGT level is closely related to excessive liver fat accumulation and increased oxidative stress.

[0006] [[ID=(20)]]Limitations of diagnostic techniques: Although GGT is an important biomarker, traditional detection techniques such as serological tests often have difficulty meeting the requirements of early diagnosis and disease monitoring due to insufficient sensitivity and specificity. In addition, these methods usually cannot provide dynamic information about disease progression. Therefore, it is necessary to develop a highly specific GGT detection method for the activity of glutamyl transpeptidase. Summary of the Invention

[0007] One advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof, which can solve the problems of insufficient sensitivity and specificity of traditional GGT detection methods, and is suitable for early diagnosis and disease condition monitoring of diseases related to GGT activity such as liver function and systemic lipid metabolism diseases. This will promote the early diagnosis and treatment of related diseases and improve the prognosis of patients.

[0008] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof. The water solubility of the probe for monitoring the activity of glutamyl transpeptidase provided by the present invention has been greatly improved, making it more suitable for in vivo applications. It can not only improve the accuracy of detection but also achieve real-time monitoring of disease progression.

[0009] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof. The novel probe contains two hydrophilic sulfonic acid groups. Compared with the currently reported probes, the hydrophilicity of the novel GGT-probe provided by the present invention is greatly increased, which can significantly improve biocompatibility.

[0010] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof. The GGT-probe provided by the present invention can non-invasively monitor GGT activity at the in vivo level by utilizing the photoacoustic properties derived from hemicyanine, providing a new tool for clinical diagnosis and biomedical research.

[0011] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof. When the GGT-probe is not acting on GGT, its photoacoustic signal is in the off state. When GGT is present, the probe specifically acts on GGT, triggering the cleavage of the protective peptide and activating the signal, thereby detecting the GGT activity in real time and with high sensitivity.

[0012] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application thereof. The main structure of the probe includes a hemicyanine molecule with two hydrophilic groups (sulfonic acid groups), belonging to a photoacoustic material, or including a photoacoustic group. The GGTprobe probe is obtained by condensation of an amino group with a peptide chain containing a GGT recognition site. Under the catalytic action of GGT, the peptide chain is specifically cleaved, thereby releasing the photoacoustic group and achieving the "turning on" of the signal.

[0013] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application. The GGT-probe has high sensitivity and good selectivity for the response to GGT. Experiments show that the probe can respond rapidly at a GGT concentration as low as 57.5 ng / mL, and has no obvious response to other common biomolecules such as proteinase K and trypsin, showing excellent specificity. The photoacoustic signal of the probe rapidly increases under the action of GGT, and can be used to monitor the change of GGT activity in real time.

[0014] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application. The GGT-probe has good biosafety and biocompatibility, and does not show obvious toxicity and side effects in vitro and preliminary in vivo experiments.

[0015] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application. By comparing the fluorescence intensities of the obese group and the non-obese group, the sensitivity and specificity of the GGT-probe under different body weight states can be evaluated. The GGT activity in the serum of obese patients increases, which is reflected in a significant increase in fluorescence intensity, which is consistent with the clinical observation that the risk of metabolic syndrome increases in the obese state, indicating that the GGT-probe can be used to detect GGT in the serum of obese patients.

[0016] Another advantage of the present invention is to provide a novel probe for monitoring the activity of glutamyl transpeptidase, a preparation method and an application. The preparation method is simple, highly operable, the prepared probe has a low cost, and is suitable for clinical popularization and application.

[0017] According to one aspect of the present invention, the present invention provides a preparation method of a novel probe for monitoring the activity of glutamyl transpeptidase, comprising the following steps: (S10) preparing a hemicyanine photoacoustic group containing two hydrophilic sulfonic acid groups and a hemicyanine molecular solution;

[0018] (S20) dissolving N-Boc-Glu-OtBu and HATU in an organic solvent, adding DMAP, activating at 0-4 °C, and adding the hemicyanine molecular solution and continuing to stir and react;

[0019] (S30) removing unreacted raw materials and by-products through washing with water and drying operations;

[0020] (S40) obtaining the target product GGT-probe through an acidic deprotection step and silica gel column chromatography.

[0021] The step (S10) includes the following steps: (S101) reacting 2,2'-(2-methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) and acetonitrile between 60-80 °C to prepare Compound 2; (S102) reacting Compound 2 with an equivalent amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide to prepare Compound 4, wherein the prepared Compound 4 contains two hydrophilic sulfonic acid groups; (S103) deacetylating Compound 4 to obtain the hemicyanine photosensitizer; and (S104) dissolving the hemicyanine photosensitizer in 10 mL of dichloromethane to obtain a hemicyanine molecular solution.

[0022] In the step (S103), Compound 4 is dissolved in a solution of sodium hydroxide, a methanol solution of sodium hydroxide or an acetonitrile solution of sodium hydroxide. The reaction system is refluxed and stirred at 60 °C and reacts within the range of 50 °C to 70 °C for 3 to 5 hours. After the reaction is completed, the excess basic substance in the reaction mixture is neutralized by using dilute hydrochloric acid. The reaction solution is separated by extraction or filtration, the organic solvent phase is collected, and the organic phase is washed repeatedly with saturated brine to remove unreacted reagents and impurities. Then, the collected organic phase is dried to obtain the hemicyanine photosensitizer by deacetylation.

[0023] In the step (S20), the organic solvent used is dichloromethane, carbon tetrachloride or dimethylformamide; the conditions for the stirring reaction are to react at 20-30 °C for 10-15 hours.

[0024] In the step (S20), N-(tert-butoxycarbonyl)-L-glutamic acid 1-tert-butyl ester and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in dichloromethane, and 4-dimethylaminopyridine is added as a catalyst. The reaction is stirred at 0-4 °C for 0.5-1 hour. Then, the hemicyanine molecular solution in the step (S10) is added to the solution prepared in the step (S20), and the mixture is stirred at 25 °C for 12 hours.

[0025] In the step (S30), first, the reaction solution is diluted with dichloromethane, and then washed with deionized water (5 mL each time, washed twice) to remove polar impurities in the solvent. Then, the organic layer is dried over anhydrous sodium sulfate to remove water.

[0026] The step (S40) includes the following steps: (S401) deprotection reaction; and (S402) purification by silica gel column chromatography.

[0027] Step (S40) includes the following steps: (S401) Dissolve the residue obtained in step (S30) in 10 mL of dichloromethane, and then add 5 mL of trifluoroacetic acid (TFA) for an acid deprotection reaction to remove the N-Boc protecting group. Stir the mixture at room temperature for 0.5 hours to complete the deprotection reaction. (S402) Evaporate and remove the solvent, and purify the residue by silica gel column chromatography (using dichloromethane / MeOH as the eluent, with a volume ratio of 95:5) to obtain the target product, the GGT-probe probe.

[0028] According to another aspect of the present invention, the present invention also provides a novel probe for monitoring the activity of glutamyl transpeptidase, wherein the probe is the GGT-probe probe. The GGT-probe probe includes a photoacoustic group, and the photoacoustic group is a hemicyanine molecule with two hydrophilic sulfonic acid groups, which is obtained by condensation connection with an amino group through a peptide chain containing a GGT recognition site. The structure of the GGT-probe probe is: In addition, the GGT-probe probe includes a hemicyanine photoacoustic group with two hydrophilic sulfonic acid groups. By using the hemicyanine derivative with both fluorescence and photoacoustic properties, the GGT activity can be specifically monitored under physiological conditions. When the GGT-probe probe does not interact with GGT, its photoacoustic signal is in the off state. When GGT is present, the probe specifically interacts with GGT, triggering the cleavage of the protective peptide and activating the signal.

[0029] The present invention also provides an application of a novel probe for monitoring the activity of glutamyl transpeptidase. The probe is a strongly hydrophilic GGT-probe probe, which is suitable for applications in monitoring liver function and systemic lipid metabolism diseases, including but not limited to atherosclerosis and obesity.

[0030] The probe is the GGT-probe probe. The GGT-probe probe includes a hemicyanine photoacoustic group with two hydrophilic sulfonic acid groups. By using the acoustic properties of the hemicyanine derivative, the GGT activity can be specifically monitored under physiological conditions. When the GGT-probe probe does not interact with GGT, its photoacoustic signal is in the off state. When GGT is present, the probe specifically interacts with GGT, triggering the cleavage of the protective peptide and activating the signal.

[0031] In the GGT monitoring experiment, the method for detecting the GGT activity in the solution includes the following steps:

[0032] (A) Prepare the GGT stock solution;

[0033] (B) Mix different volumes of the GGT stock solution with the GGT-probe probe;

[0034] (C) Dissolve the mixture in phosphate buffer and adjust the total volume to 100 μL;

[0035] (D) Incubate the mixed solution under a constant temperature condition of 37 °C to simulate the environment in vivo;

[0036] (E) After the incubation process, use absorption spectrometry to observe and record the absorption characteristics of the solution;

[0037] (F) Analyze the functionality and application potential of the GGT-probe.

[0038] Among them, in the step (B), the concentration of the GGT probe is maintained within the range of 5 - 10 μM, and the concentration of the GGT enzyme is maintained within the range of 10 - 500 ng / mL.

[0039] Among them, in the step (B), the mixing ratio of the GGT stock solution to the GGT-probe is: 1:1, 1:2 or 1:5. Brief Description of the Drawings

[0040] Figure 1 It is a synthetic route diagram of a GGT-probe according to an embodiment of the present invention.

[0041] Figure 2 It is a response schematic diagram of the GGT-probe according to the above embodiment of the present invention.

[0042] Figure 3 It is a schematic diagram of the signal change before and after the response of the GGT-probe according to the above embodiment of the present invention.

[0043] Figure 4 It is an absorption spectrum schematic diagram of the response of the GGT-probe to GGT according to the above embodiment of the present invention.

[0044] Figure 5 It is a fluorescence spectrum schematic diagram of the response of the GGT-probe to GGT according to the above embodiment of the present invention.

[0045] Figure 6 It is a photoacoustic spectrum schematic diagram of the response of the GGT-probe to GGT according to the above embodiment of the present invention.

[0046] Figure 7 It is a schematic diagram of the selectivity test of the GGT-probe according to the above embodiment of the present invention.

[0047] Figure 8 It is a schematic diagram of the photoacoustic images of the liver regions of the control mice and atherosclerotic mice after injecting the GGT-probe.

[0048] Figure 9 Schematic diagram of photoacoustic images of the liver regions of control mice and obese mice after injection of GGT-probe.

[0049] Figure 10 Schematic diagram of GGT-probe for detecting GGT in human serum.

[0050] Figure 11 Schematic diagram of the fluorescence intensity values after incubation of GGT-probe with human serum samples. Detailed implementation manners

[0051] The following description is used to introduce the present invention in detail so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other implementation schemes, variant schemes, improvement schemes, equivalent schemes, and other technical schemes that do not depart from the spirit and scope of the present invention.

[0052] In view of the above background, the present invention provides a highly specific GGT detection method, which utilizes the fluorescence and photoacoustic properties derived from hemicyanine, and can non-invasively monitor GGT activity at the in vivo level. This method can not only improve the accuracy of detection, but also achieve real-time monitoring of disease progression, so as to be applied to the early diagnosis and treatment of diseases.

[0053] The GGT-probe of the present invention is a probe based on hemicyanine derivatives. The hemicyanine molecule has both fluorescence and photoacoustic properties and can be used as a fluorescent material (or photoacoustic material). Its design concept is through a specific peptide chain link, which remains inactive when not reacting with GGT enzyme, thus inhibiting the emission of fluorescence signals. The main structure of the probe includes a photoacoustic group, and the two are connected by a peptide chain at the GGT recognition site. Under the catalytic action of GGT, the peptide chain is specifically cleaved, thereby releasing the photoacoustic group and realizing the "turning on" of the signal.

[0054] In addition, two hydrophilic sulfonic acid groups are specifically added to the GGT-probe of the present invention, significantly improving the biocompatibility and water solubility of the probe. The sulfonic acid groups can increase the solubility of the probe in the aqueous phase, improve its stability and dispersion in the biological environment, and further enhance its ability to detect GGT activity. This design enables the probe to interact more efficiently with biological fluids, enhancing the sensitivity and accuracy of the signal. Especially in complex biological systems, it can ensure higher detection accuracy.

[0055] That is to say, when the probe does not interact with GGT, its photoacoustic signal is in the off state. When GGT is present, the probe specifically interacts with GGT, triggering the cleavage of the protective peptide and activating the signal, thereby detecting the activity of GGT in real time and with high sensitivity. By designing with an increased sulfonic acid group, the biocompatibility of the probe is improved, thus achieving more reliable and stable detection, greatly expanding its application potential in clinical diagnosis and biomedical research. The detection limit of this probe for GGT is 57.5 ng / mL, and it has a good linear response.

[0056] The synthesis process involves multiple-step organic synthesis techniques, including the synthesis of peptide chains, the labeling of photoacoustic groups, and the final purification and characterization. High-performance liquid chromatography (HPLC) is used to ensure the purity of the synthesized products.

[0057] Specifically, as Figure 1 shown, the preparation method of the GGT-probe probe of the present invention is as follows:

[0058] The preparation method of the GGT-probe probe includes the following steps:

[0059] (S10) Prepare a hemicyanine photoacoustic group containing two hydrophilic sulfonic acid groups and a hemicyanine molecular solution;

[0060] (S20) Dissolve N-Boc-Glu-OtBu and HATU in an organic solvent, add DMAP, activate at 0-4 °C, and add the hemicyanine molecular solution and continue stirring for reaction;

[0061] (S30) Remove unreacted raw materials and by-products through washing with water and drying operations; and

[0062] (S40) Obtain the target product GGT-probe probe through an acidic deprotection step and silica gel column chromatography.

[0063] The step (S10) includes the following steps: (S101) React 2,2'-(2-methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) with acetonitrile, anhydrous dichloromethane or anhydrous tetrahydrofuran at 60-80 °C to prepare compound 2; (S102) React compound 2 with an equivalent amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide to prepare compound 4, where compound 4 contains two hydrophilic sulfonic acid groups; (S103) Deacetylate compound 4 to obtain the hemicyanine photoacoustic group; and (S104) Dissolve the hemicyanine photoacoustic group in 10 mL of dichloromethane to obtain a hemicyanine molecular solution.

[0064] In the step (S20), HATU is used as a coupling reagent to ensure the mildness of the reaction, reduce the occurrence of side reactions, and avoid degradation or by-products that may occur at high temperatures. In the step (S40), the acidic deprotection reagent used is trifluoroacetic acid (TFA).

[0065] Example 1

[0066] (S101)2,2'-(2-Methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) (Compound 1, 8.0 g, 50 mmol) and iodoethane (7.1 g, 50 mmol) were added to a round-bottom flask, and 30 mL of anhydrous acetonitrile was added as a solvent and mixed. The reaction system was heated to the reflux temperature of 80 °C and maintained at reflux for 12 hours. After the reaction was completed, heating was stopped and the mixture was cooled to room temperature. After the reaction, filtration was carried out through a Buchner funnel, the precipitate was collected, and the solid product was washed with ether. Finally, Compound 2 (7.8 g, yield 90%) was obtained.

[0067] (S102)Compound 2 and an equimolar amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide (Compound 3) were mixed with 30 mL of anhydrous acetonitrile in a round-bottom flask. The reaction system was heated to the reflux temperature of 80 °C and maintained at reflux for 12 hours. After the reaction was completed, heating was stopped and the mixture was cooled to room temperature. After the reaction, filtration was carried out through a Buchner funnel, the precipitate was collected, and extraction was carried out with dichloromethane and saturated brine. The organic phase was collected, dried, and finally Compound 4 was obtained.

[0068] (S103)Synthesize the deacetylated product (hemicyanine photosensitizer). The specific synthesis steps are as follows:

[0069] Compound 4 was dissolved in an ethanol solution of sodium hydroxide, and the reaction system was refluxed and stirred at 60 °C for 4 hours. During the reaction, ensure that the reaction proceeds completely.

[0070] After the reaction was completed, the excess basic substance in the reaction mixture was neutralized by using dilute hydrochloric acid (HCl). Then, the reaction solution was separated by extraction or filtration, and the organic solvent phase was collected. Next, the organic phase was washed several times with saturated brine to remove unreacted reagents and impurities. Finally, the collected organic phase was dried to obtain the desired deacetylated product - hemicyanine photosensitizer.

[0071] (S104)Dissolve the hemicyanine photosensitizer (0.5 mmol) in 10 mL of dichloromethane to obtain a hemicyanine molecular solution.

[0072] (S20) Dissolve N-(tert-butoxycarbonyl)-L-glutamic acid 1-tert-butyl ester (N-Boc-Glu-OtBu) (0.5 mmol, 92 mg) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1 mmol, 379 mg) in 10 mL of dichloromethane, and add 4-dimethylaminopyridine (DMAP) (1 mmol, 122 mg) as a catalyst. Stir the reaction mixture at 0 °C for 1 hour.

[0073] Slowly add the semi-cyanine molecule solution in the step (S104) to the reaction mixture in the step (S20). Then, stir the mixture at 25 °C for 12 hours to ensure complete and gentle reaction. This step helps to form the target coupling product.

[0074] (S30) After the reaction is completed, remove the unreacted raw materials and by-products by washing with water. First, dilute the reaction solution with dichloromethane, and then wash it with deionized water (5 mL each time, wash twice) to remove the polar impurities in the solvent. Then, dry the organic layer with anhydrous sodium sulfate to remove water.

[0075] (S40) It includes the following steps: (S401) Deprotection reaction. Dissolve the residue obtained in the step (S30) in 10 mL of dichloromethane, and then add 5 mL of trifluoroacetic acid (TFA) for acidic deprotection reaction to remove the N-Boc protecting group. Stir the mixture at room temperature for 0.5 hour to complete the deprotection reaction. (S402) Purification by silica gel column chromatography. Evaporate the solvent, and purify the residue by silica gel column chromatography (using dichloromethane / MeOH as the eluent, volume ratio 95:5) to obtain the target product GGT-probe probe (solid, about 210 mg, yield 80%).

[0076] It is worth mentioning that trifluoroacetic acid (TFA) is a commonly used deprotection reagent, which can effectively remove the N-Boc protecting group under mild conditions to generate an activated probe molecule.

[0077] Example 2

[0078] (S101) 2,2'-(2-Methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) (Compound 1, 8.0 g, 50 mmol) and iodoethane (7.1 g, 50 mmol) were added to a round-bottom flask, and 30 mL of anhydrous dichloromethane was added as a solvent and mixed. The reaction system was heated to the reflux temperature of 60 °C and maintained at reflux for 15 hours. After the reaction was completed, heating was stopped and the mixture was cooled to room temperature. After the reaction, filtration was carried out through a Buchner funnel, the precipitate was collected, and the solid product was washed with ether to finally obtain Compound 2 (7.7 g, yield 88.9%).

[0079] (S102) Compound 2 and an equimolar amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide (Compound 3) were mixed with 30 mL of anhydrous dichloromethane in a round-bottom flask. The reaction system was heated to the reflux temperature of 60 °C and maintained at reflux for 14 hours. After the reaction was completed, heating was stopped and the mixture was cooled to room temperature. After the reaction, filtration was carried out through a Buchner funnel, the precipitate was collected, and extraction was carried out with dichloromethane and saturated brine. The organic phase was collected, dried, and finally Compound 4 was obtained.

[0080] (S103) Synthesis of the deacetylated product (hemicyanine photosensitizer), and the specific synthesis steps are as follows:

[0081] Compound 4 was dissolved in a methanol solution of sodium hydroxide, and the reaction system was refluxed and stirred at 70 °C for 3.5 hours. During the reaction, ensure that the reaction proceeds completely.

[0082] After the reaction was completed, the excess basic substance in the reaction mixture was neutralized by using dilute hydrochloric acid (HCl). Then, the reaction solution was separated by extraction or filtration, and the organic solvent phase was collected. Next, the organic phase was washed repeatedly with saturated brine to remove unreacted reagents and impurities. Finally, the collected organic phase was dried to obtain the desired deacetylated product - hemicyanine photosensitizer.

[0083] (S104) The hemicyanine photosensitizer (0.5 mmol) was dissolved in 10 mL of dichloromethane to obtain a hemicyanine molecular solution.

[0084] (S20) Dissolve N-(tert-butoxycarbonyl)-L-glutamic acid 1-tert-butyl ester (N-Boc-Glu-OtBu) (0.5 mmol, 92 mg) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1 mmol, 379 mg) in 10 mL of carbon tetrachloride, and add 4-dimethylaminopyridine (DMAP) (1 mmol, 122 mg) as a catalyst. Stir the reaction mixture at 4 °C for 0.8 hours.

[0085] (S20) Slowly add the semi-cyanine molecular solution in the step (S104) to the mixture obtained from the reaction in the step (S20). Then, stir the mixture at 30 °C for 15 hours to ensure complete reaction. This step helps to form the target coupling product.

[0086] (S30) After the reaction is completed, remove the unreacted raw materials and by-products by washing with water. First, dilute the reaction solution with carbon tetrachloride, and then wash it with deionized water (5 mL each time, wash twice) to remove the polar impurities in the solvent. Then, dry the organic layer with anhydrous sodium sulfate to remove water.

[0087] (S40) It includes the following steps: (S401) Deprotection reaction. Dissolve the residue obtained in the step (S30) in 10 mL of carbon tetrachloride, and then add 5 mL of trifluoroacetic acid (TFA) for acid deprotection reaction to remove the N-Boc protecting group. Stir the mixture at room temperature for 0.5 hours to complete the deprotection reaction. (S402) Purification by silica gel column chromatography. Evaporate the solvent, and purify the residue by silica gel column chromatography (using dichloromethane / MeOH as the eluent, volume ratio 95:5) to obtain the target product GGT-probe probe (solid, about 196.8 mg, yield 75%).

[0088] Example 3

[0089] (S101) Add 2,2'-(2-methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) (Compound 1, 9.0 g, 56.3 mmol) and iodoethane (7.1 g, 50 mmol) to a round-bottom flask, and add 30 mL of anhydrous tetrahydrofuran as a solvent for mixing. Heat the reaction system to the reflux temperature of 70 °C, maintain the reflux reaction for 10 hours, stop heating after the reaction is completed, and cool to room temperature. After the reaction, filter through a Buchner funnel, collect the precipitate, and wash the solid product with ether to finally obtain Compound 2 (7.62 g, yield 87.9%).

[0090] (S102) Compound 2 was mixed with an equivalent amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide (Compound 3) in a round-bottom flask with 30 mL of anhydrous tetrahydrofuran. The reaction system was heated to the reflux temperature of 75 °C and maintained at reflux for 18 hours. After the reaction was completed, heating was stopped and the system was cooled to room temperature. After the reaction, filtration was carried out through a Buchner funnel, the precipitate was collected, and extraction was performed with dichloromethane and saturated brine. The organic phase was collected, dried, and finally Compound 4 was obtained.

[0091] (S103) Synthesis of the deacetylated product (hemicyanine photosensitizer), the specific synthesis steps are as follows:

[0092] Compound 4 was dissolved in an acetonitrile solution of sodium hydroxide. The reaction system was refluxed and stirred at 50 °C for 5 hours. During the reaction, ensure that the reaction proceeds completely.

[0093] After the reaction was completed, the excess basic substance in the reaction mixture was neutralized by using dilute hydrochloric acid (HCl). Then, the reaction solution was separated by extraction or filtration, and the organic solvent phase was collected. Next, the organic phase was washed several times with saturated brine to remove unreacted reagents and impurities. Finally, the collected organic phase was dried to obtain the desired deacetylated product - hemicyanine photosensitizer.

[0094] (S104) The hemicyanine photosensitizer (0.5 mmol) was dissolved in 10 mL of dichloromethane to obtain a hemicyanine molecular solution.

[0095] It should be noted that in the step (S101), the reaction solvent can be replaced with a similar solvent as needed, such as anhydrous dichloromethane, anhydrous tetrahydrofuran, etc. The reaction temperature can be controlled between 60 - 80 °C, and the reaction time can be adjusted within the range of 8 to 16 hours to ensure the complete progress of the reaction.

[0096] In the step (S102), the reaction solvent can be replaced with a similar solvent as needed, such as anhydrous dichloromethane, anhydrous tetrahydrofuran, etc. The reaction temperature can be controlled between 60 - 80 °C, and the reaction time can be adjusted within the range of 8 to 16 hours to ensure the complete progress of the reaction.

[0097] In the reaction for synthesizing the deacetylated product (hemicyanine photosensitizer) in (S103), the reaction solvent can be replaced with a similar solvent as needed, such as anhydrous methanol, acetonitrile, etc. The reaction temperature can be controlled within the range of 50 °C to 70 °C, and the reaction time can be adjusted between 3 and 5 hours to ensure sufficient reaction.

[0098] (S20) Dissolve N-(tert-butoxycarbonyl)-L-glutamic acid 1-tert-butyl ester (N-Boc-Glu-OtBu) (0.5 mmol, 92 mg) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1 mmol, 379 mg) in 10 mL of N,N-dimethylformamide (DMF), and add 4-dimethylaminopyridine (DMAP) (1 mmol, 122 mg) as a catalyst, and stir the reaction at 2 °C for 0.5 - 1 hour.

[0099] Slowly add half of the cyanine molecule solution in the step (S104) to the mixture obtained in the reaction of the step (S20). Then, stir the mixture at 30 °C (room temperature) for 12 hours to ensure that the reaction proceeds completely. This step helps to form the target coupling product.

[0100] (S30) After the reaction is completed, remove the unreacted raw materials and by-products through a water washing operation. First, dilute the reaction solution with N,N-dimethylformamide, and then wash it with deionized water (5 mL each time, wash twice) to remove the polar impurities in the solvent. Then, dry the organic layer over anhydrous sodium sulfate to remove water.

[0101] (S40) It includes the following steps: (S401) Deprotection reaction: Dissolve the residue obtained in the step (S30) in 10 mL of N,N-dimethylformamide, and then add 5 mL of trifluoroacetic acid (TFA) for an acidic deprotection reaction to remove the N-Boc protecting group. Stir the mixture at room temperature for 0.5 hour to complete the deprotection reaction. (S402) Purification by silica gel column chromatography: Evaporate the solvent, and purify the residue by silica gel column chromatography (using dichloromethane / methanol (MeOH) as the eluent, volume ratio 90:5) to obtain the target product GGT-probe probe (solid, about 183 mg, yield 70%).

[0102] The present invention provides a hemicyanine-based probe named GGT-probe, which is designed for highly sensitive and real-time monitoring of liver glutamyltransferase (GGT). Hemicyanine molecules are an ideal choice for biomedical imaging due to their excellent absorption and emission characteristics in the near-infrared region. In order to further improve the biocompatibility and detection performance of the probe, in the present invention, two hydrophilic sulfonic acid groups are added to the molecular structure of the GGT-probe probe.

[0103] The introduction of the sulfonic acid group significantly improves the probe's hydrophilicity and solubility, ensuring better dispersion and effective interaction with target molecules within the body. Furthermore, the sulfonic acid group significantly enhances the probe's compatibility with biomacromolecules by enhancing its hydration, further optimizing the probe's biocompatibility and reducing background signals caused by nonspecific binding.

[0104] GGT-probe can accurately reflect GGT activity by combining hemicyanine molecules with the specific recognition sequence of GGT. Figure 2 As shown in FIG, in the absence of GGT, the amino terminus of the GGT-probe is blocked by a protective peptide, which prevents charge transfer within the molecule, thereby keeping the photoacoustic signal in an off state. The advantage of this design of the present invention is that it can reduce the interference of background signals and improve the selectivity and sensitivity of the probe. Figure 2 In the figure, position 1 indicates that the intramolecular charge transfer is "off", and position 2 indicates that the intramolecular charge transfer is "on".

[0105] When GGT is present in the sample, it will specifically recognize and cleave the linker sequence in the probe, resulting in the removal of the protective peptide and the release of the amino terminus. This change restores the electron supply and promotes charge transfer, activating the photoacoustic signal, allowing direct monitoring of GGT activity, such as Figure 3 As shown, in Figure 3 In the figure, position 3 indicates that the fluorescence / photoacoustic signal of GGT-probe is “off”, and position 4 indicates that the fluorescence / photoacoustic signal of hemicyanine photoacoustic group is “on”.

[0106] By adding a sulfonic acid group to the probe structure, the present invention further optimizes the performance of the GGT-probe, enabling it to have higher sensitivity and accuracy in biological detection while ensuring its excellent biocompatibility, providing a more reliable tool for early diagnosis and real-time monitoring in clinical applications.

[0107] In the in vitro GGT monitoring experiment, the method for detecting γ-glutamyl transpeptidase (GGT) activity in solution includes the following steps:

[0108] (A) Preparation of GGT stock solution;

[0109] (B) Different volumes of GGT stock solution were mixed with GGT-probe;

[0110] (C) Dissolve the mixture in phosphate buffer and adjust the total volume to 100 μL;

[0111] (D) The mixed solution was incubated at a constant temperature of 37°C to simulate the in vivo environment;

[0112] (E) After the incubation process, absorption spectrometry was used to observe and record the absorption characteristics of the solution; and

[0113] (F) Analyze the functionality and application potential of the GGT-probe probe.

[0114] In the above step (B), to ensure the detection sensitivity, the concentration of the GGT stock solution should be within a certain range (e.g., 0.1 - 10 μg / mL), and the concentration of the GGT-probe probe generally remains at a certain excess level to ensure its reaction with GGT. The specific mixing ratios of the GGT stock solution and the GGT-probe probe are as follows: GGT stock solution: GGT-probe probe = 1:1; GGT stock solution: GGT-probe probe = 1:2; GGT stock solution: GGT-probe probe = 1:5, and finally the GGT probe concentration is maintained within the range of 5 - 10 μM, and the GGT enzyme concentration is maintained within the range of 10 - 500 ng / mL).

[0115] In the above step (E), by measuring the light absorption intensity of the solution at a specific wavelength, the molecular structure changes caused by GGT activity were analyzed. Subsequently, a fluorescence spectrometer was used to further record the fluorescence spectra of these solutions. The recording of the fluorescence spectra was to capture the changes in the emitted light after the reaction of the probe with GGT, and these changes reflected the dynamic process of the interaction between the probe and GGT.

[0116] In addition, the present invention also collected photoacoustic (PA) images of the solution at 720 nm. Photoacoustic imaging technology combines the advantages of optics and ultrasound and can provide more in-depth information about the distribution and reaction state of the probe in the organism. Through this imaging technology, the behavior of the probe in biological tissues can be observed non-invasively, which is crucial for understanding the biomedical applications of the probe.

[0117] To evaluate the selectivity of the GGT-probe probe, the present invention incubated it with different biological species. This selectivity experiment aimed to verify the specificity of the probe for GGT and ensure that the probe could specifically recognize and respond to GGT when other biomolecules were present. Through these experiments, not only the functionality and application potential of the probe could be verified, but also its behavior pattern in complex biological systems could be deeply understood.

[0118] The results of the above in vitro experiments were analyzed as follows:

[0119] Absorption spectrum analysis: To comprehensively evaluate the performance of the probe, the present invention first conducted a detailed study on the absorption spectra of the GGT-probe probe before and after adding GGT. As Figure 4As shown, without the addition of GGT, the absorption spectrum of the probe shows an obvious absorption peak in the 550 - 600 nm region, which is consistent with the charge transfer characteristics in its closed state. After adding GGT, the absorption peak in the range of 550 - 600 nm was observed to be significantly weakened, while the absorption peak intensity at nearly 700 nm increased significantly. This change indicates that the removal of the protective peptide allows the charge transfer to be restored, thereby causing a significant change in the absorption characteristics, demonstrating that the probe has good responsiveness to GGT.

[0120] Fluorescence and photoacoustic spectroscopy analysis: Further, the present invention further confirmed the effect of the addition of GGT on the GGT - probe through fluorescence and photoacoustic spectroscopy analysis. As Figure 5 shown, in the experiment, fluorescence spectroscopy analysis showed that after adding GGT, the fluorescence intensity of the probe was significantly enhanced at a specific wavelength (such as nearly 700 nm), which corresponded to the change in the absorption spectrum. Similarly, photoacoustic spectroscopy analysis also showed a similar trend, that is, the addition of GGT significantly enhanced the photoacoustic signal of the probe at a specific wavelength, as Figure 6 shown, further verifying the high recognition ability and excellent responsiveness of the probe to GGT.

[0121] To further verify the selectivity of the GGT - probe of the present invention for GGT, a series of in vitro experiments were designed in the present invention to evaluate the influence of potential interfering factors on the experimental results. These potential interfering factors include various biological enzymes commonly found in the physiological environment.

[0122] In the specific experimental design, different potential interferents, such as biological enzymes, were added to each group to observe the results. In this experiment, different control groups were prepared, which were respectively added with alkaline phosphatase, aminopeptidase, carboxylesterase, matrix metalloproteinase and nitroreductase, and these control groups were compared with the experimental group containing GGT. The experimental results are as Figure 7 shown.

[0123] Figure 7 The experimental results of show that the GGT - probe has extremely high specificity in its response to GGT. In the experimental group added with GGT, a significant enhancement of the photoacoustic signal was observed, while in the experimental groups added with other potential interferents, there was no obvious change in the photoacoustic signal of the probe. This result indicates that despite the complex physiological environment with various potentially interfering factors, the GGT - probe can specifically recognize and respond to the activity of GGT, thus providing a highly selective tool in biomedical imaging and disease diagnosis.

[0124] In addition, the molecular design of the GGT-probe includes two hydrophilic sulfonic acid groups, which significantly enhances the biocompatibility of the probe. The hydrophilic characteristics of the sulfonic acid groups not only contribute to the dispersion of the probe in aqueous solutions but also effectively reduce the interaction between the probe and non-target substances in the body, minimizing possible toxic reactions. Therefore, the stability and biocompatibility of GGT-probe in vivo are greatly enhanced, making it more reliable and safe in complex physiological environments.

[0125] The above experiments not only verified the high selectivity of the GGT-probe but also demonstrated its application in complex biological samples.

[0126] The GGT-probe provided by the present invention detects the activity of GGT in an atherosclerotic mouse model through photoacoustic imaging.

[0127] The present invention combines photoacoustic imaging technology and GGT-probe to study the dynamic changes in GGT activity in an atherosclerosis (AS) model. As an enzyme that plays an important role in various physiological and pathological processes, GGT is widely present in various tissues, especially highly expressed in the liver and kidneys. Studies have shown that GGT is closely related to the development of atherosclerosis, lipid metabolism disorders, and various cardiovascular diseases. Therefore, monitoring the dynamic changes in GGT activity not only helps to understand the pathogenesis of these diseases but also contributes to early diagnosis and evaluation of treatment effects.

[0128] In the present invention, the GGT-probe adopted has a special structural design, which includes two hydrophilic sulfonic acid groups. The introduction of these sulfonic acid groups significantly improves the biocompatibility of the probe, reduces the mutual repulsion with cells and tissues in the body, and thus improves the targeting and detection performance of the probe. This design ensures that the probe can react more stably with GGT enzyme in vivo, increases the distribution efficiency of the probe in tissues, and enhances its detection ability in complex biological environments. Compared with traditional probes, the GGT-probe of the present invention has stronger hydrophilicity and lower cytotoxicity, greatly enhancing its application potential in the body.

[0129] In the present invention, two groups of experimental mouse models were used, representing different physiological and pathological states: a healthy control group (C57BL / 6 mice, conventionally raised for 16 weeks) and an atherosclerotic group (AS group, ApoE- / - mice, fed a high-fat diet for 16 weeks). After establishing these models, we systematically monitored the changes in GGT activity in mice after the use of the GGT-probe probe in combination with photoacoustic imaging. To ensure the accuracy and reproducibility of the experimental results, the present invention accurately calculated the injection dose of the GGT-probe probe based on the body weight of the mice and quantitatively analyzed the fluorescence intensity in the liver region, so as to more accurately quantify the changes in GGT activity in different groups.

[0130] During the experiment, photoacoustic (PA) images of each group of mice were obtained 10 minutes after the injection of the GGT-probe probe, as Figure 8 shown. The data showed that the PA signal of the healthy control group was low, while in the atherosclerotic group, the PA signal was significantly enhanced, reflecting the trend of GGT activity, indicating that the GGT activity was higher in the atherosclerotic mouse group, demonstrating the high level of GGT activity in the AS group of mice.

[0131] The GGT-probe probe detects the activity of GGT in an obese mouse model through photoacoustic imaging. In the present invention, for the study of the obese model, we particularly focused on the changes in GGT activity under the obese state and its impact on the development of the disease. By using C57BL / 6 mice and establishing an obese model by continuous high-fat diet for 16 weeks, the physiological conditions of human obesity can be simulated.

[0132] Experimental materials and animal models:

[0133] Experimental procedures:

[0134] In the experiment of the obese model, after C57BL / 6 mice were fed a high-fat diet, photoacoustic imaging was performed by intravenous injection of the GGT-probe probe to monitor the dynamic changes in GGT activity in the liver. The injection dose was accurately calculated according to the body weight of each mouse to ensure that each experimental individual received the same dose of the probe for subsequent comparison and analysis of the data.

[0135] During the experiment, photoacoustic images were collected 90 minutes after the injection of the GGT-probe probe. By quantitatively analyzing the fluorescence intensity in the liver region of obese mice, a significant signal enhancement could be observed, indicating an increase in GGT activity under the obese state, as Figure 9 shown.

[0136] Result analysis: Through these detailed analyses, it is clearly seen the increase in GGT activity and its dynamic changes under the obese state. This persistent high activity is directly associated with the risks of various obesity-related diseases, such as the development of lipid metabolism disorders and cardiovascular diseases. These findings not only help us understand how obesity promotes disease states by affecting the activity of key enzymes, but also provide possible biomarkers and therapeutic targets for early diagnosis and treatment.

[0137] Through this method, the present invention demonstrates how to quantitatively monitor GGT activity in an obesity model by using photoacoustic imaging technology in combination with the GGT-probe probe, providing a new tool and methodology for future obesity research and clinical applications.

[0138] The GGT-probe probe detects GGT activity in human serum samples through serum testing.

[0139] To further verify the potential of GGT-probe in clinical translational applications, the present invention uses GGT-probe to detect clinical samples.

[0140] In obese patients, there is often a higher risk of metabolic syndrome, such as diabetes, hypertension, cardiovascular diseases, etc. By differentiating between obese and non-obese patients, doctors can better assess the risk of complications and intervene early.

[0141] The GGT-probe of the present invention has two hydrophilic sulfonic acid groups, which significantly improves the biocompatibility and detection ability of the probe. Due to the presence of sulfonic acid groups, when the GGT-probe contacts biological fluids, it can effectively reduce non-specific binding, enhance the binding affinity with target molecules (such as GGT), ensuring high-sensitivity detection performance. In addition, the hydrophilic sulfonic acid groups help the probe to be evenly distributed in serum samples, enabling it to better bind to target molecules, thus greatly improving the detection accuracy.

[0142] Experimental steps 1. Sample preparation: Collect serum samples from people who meet the research criteria, and divide the patients into an obese group and a non-obese group according to the BMI value, as Figure 10 shown.

[0143] 2. Probe incubation: Incubate the GGT-probe probe with the serum samples of the two groups of people respectively.

[0144] 3. Fluorescence intensity measurement: Use a fluorescence spectrometer to measure the fluorescence intensity of the incubated samples, and compare the differences between the obese group and the non-obese group.

[0145] In this embodiment, according to the body mass index (BMI) value, the population was divided into a non-obese group and an obese group, and the serum samples of the two groups were incubated with GGT-probe. Among them, the BMI of the obese group patients was 29, and the BMI of the non-obese group was 21. We found that after incubation with the serum of the non-obese group, the fluorescence intensity of GGT-probe was weak, while after incubation with the serum of the obese group, the fluorescence intensity was significantly stronger, indicating that GGT-probe is expected to be used for detecting GGT in the serum of obese patients, such as Figure 11 shown.

[0146] Data analysis

[0147] By comparing the fluorescence intensities of the obese group and the non-obese group, the sensitivity and specificity of GGT-probe under different body weight states can be evaluated. The increase in GGT activity in the serum of obese group patients is reflected in the significant enhancement of fluorescence intensity, which is consistent with the clinical observation of the increased risk of metabolic syndrome in the obese state. This result further verifies the excellent performance of GGT-probe in detecting GGT in the serum of obese patients. In particular, the enhanced biocompatibility and detection ability provide scientific basis and experimental data support for the development of diagnostic methods based on this probe.

[0148] In summary, the successful development of the GGT-probe provided by the present invention successfully solves the problem of insufficient sensitivity and specificity of traditional GGT detection methods, and provides an efficient and real-time detection means. This probe contains two hydrophilic sulfonic acid groups, which significantly improve its biocompatibility in vivo, reduce the immune response to the biological system, and thus enhance its stability and long-term detection ability in vivo. In addition, the addition of hydrophilic sulfonic acid groups also improves the binding ability of the probe to target molecules, further enhancing the detection sensitivity. This will greatly promote the early diagnosis and treatment of related diseases and improve the prognosis of patients.

[0149] In addition, the probe technology also provides the possibility for the future development of detection methods for other biomarkers, with broad market application prospects and commercial potential. Through these detailed invention contents, the present invention not only demonstrates the development of a new type of probe, but also elaborates its important value in biomedical research and potential clinical applications.

[0150] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the drawings are only examples and do not limit the present invention. The object of the present invention has been fully and effectively achieved. The function and structural principle of the present invention have been shown and explained in the embodiments. Without departing from the said principle, the embodiments of the present invention can have any deformation or modification.

Claims

1. A method for preparing a probe for monitoring glutamyl transpeptidase activity, characterized in that: The following steps are involved: (S10) Preparing a hemicyanine photoacoustic group containing two hydrophilic sulfonic acid groups and a hemicyanine molecular solution, comprising the following steps: (S101) dissolving 2,2'-(2-methyl-2,3-dihydro-1H-benz[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) in acetonitrile to prepare compound 2; (S102) reacting compound 2 with an equivalent amount of N-(4-formyl-2,3-dihydro-1H-anthracen-6-yl)acetamide to prepare compound 4, wherein the prepared compound 4 contains two hydrophilic sulfonic acid groups; (S103) deacetylation of compound 4 to obtain the hemicyanine photoacoustic group; (S104) dissolving the hemicyanine photoacoustic group in dichloromethane to obtain a hemicyanine molecular solution, wherein the structures of compound 2 and compound 4 are as follows: (S20) dissolving N-Boc-Glu-OtBu and HATU in an organic solvent, adding DMAP, activating at 0-4°C, and adding the hemicyanine molecule solution and continuing to stir the reaction; (S30) removing unreacted raw materials and by-products through water washing and drying operations; (S40) obtaining the target product GGT-probe probe by acidic deprotection step and silica gel column chromatography, wherein the structure of the GGT-probe probe is Where n=2.

2. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 1, wherein in step (S101), 2,2'-(2-methyl-2,3-dihydro-1H-benzo[e]indole-1,1-diyl)bis(ethane-1-sulfonic acid) can also be dissolved in anhydrous dichloromethane or anhydrous tetrahydrofuran to prepare compound 2.

3. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 2, wherein in the step (S103), compound 4 is dissolved in an ethanol solution of sodium hydroxide, a methanol solution of sodium hydroxide, or an acetonitrile solution of sodium hydroxide, the reaction system is refluxed and stirred at 60°C, and the reaction is carried out at a temperature in the range of 50°C to 70°C for 3 to 5 hours. After the reaction is completed, the excess alkaline substances in the reaction mixture are neutralized with dilute hydrochloric acid, the reaction solution is separated by extraction or filtration, the organic solvent phase is collected, and the organic phase is washed multiple times with saturated brine to remove unreacted reagents and impurities, and then the collected organic phase is dried and deacetylated to obtain the hemicyanine photoacoustic phase.

4. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to any one of claims 1 to 3, wherein in step (S20), the organic solvent used is dichloromethane, carbon tetrachloride or dimethylformamide; and the stirring reaction conditions are 20-30°C and the reaction time is 10-15 hours.

5. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 4, wherein in step (S20), N-(tert-butyloxycarbonyl)-L-glutamate-1-tert-butyl ester and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in dichloromethane, and 4-dimethylaminopyridine is added as a catalyst, and the mixture is stirred and reacted at 0-4°C for 0.5-1 hour, and then the hemicyanine molecule solution in step (S10) is added to the solution prepared in step (S20), and the mixture is stirred and reacted at 25°C for 12 hours.

6. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 5, wherein in step (S30), first, the reaction solution is diluted with dichloromethane, and then washed twice with deionized water, 5 mL each time, to remove polar impurities in the solvent; then, the organic layer is dried over anhydrous sodium sulfate to remove moisture.

7. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 6, wherein the step (S40) comprises the following steps: (S401) deprotection reaction; and (S402) purified by silica gel column chromatography.

8. The method for preparing a probe for monitoring glutamyl transpeptidase activity according to claim 6, wherein the step (S40) comprises the following steps: (S401) The residue obtained in the step (S30) was dissolved in 10 mL of dichloromethane, and 5 mL of trifluoroacetic acid was added to perform an acidic deprotection reaction to remove the N-Boc protecting group. The mixture was stirred at room temperature for 0.5 hour to complete the deprotection reaction. (S402) The solvent was evaporated and the residue was purified by silica gel column chromatography using dichloromethane / methanol in a volume ratio of 95:5 as eluent to obtain the target product GGT-probe probe.

9. A probe for monitoring glutamyl transpeptidase activity prepared according to any one of claims 1 to 8, characterized in that: The probe is a GGT-probe probe, and its structure is: Wherein n=2, the GGT-probe probe comprises a photoacoustic group, which is a hemicyanine molecule with two hydrophilic sulfonic acid groups and is obtained by condensing a peptide chain containing a GGT recognition site and an amino group.

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