A gamma-glutamyl transferase and mitochondrial viscosity dual-responsive fluorescent probe, and a preparation method and application thereof

Abstract

The application discloses a gamma-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe and a preparation method and application thereof. The gamma-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe structure is shown as formula I. The fluorescent probe provided by the application takes coumarin and cyanine dye as two different emitting fluorophore parents, constructs a gamma-glutamyltransferase and viscosity dual-target recognition site through two strategies of hydroxyl protection-deprotection and introduction of a molecular rotor, and introduces a lipophilic cationic molecule to realize mitochondrial targeting by using the negative electricity of a mitochondrial inner membrane, has the advantages of anti-spectral crosstalk, mitochondrial targeting and simultaneous imaging of two analytes, and can effectively monitor the proliferation and invasion of liver cancer cells in the process that a liver precancerous lesion deteriorates into early hepatocellular carcinoma. The fluorescent probe can be applied to the medical technical field of diagnosis of liver precancerous lesion patients and the like, and creates conditions for the gamma-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe in diagnosis of liver precancerous lesions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical analysis technology, specifically relating to a dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity, its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the fastest-growing and most aggressive malignant tumors. Most HCC patients are diagnosed at an advanced stage, missing the opportunity for treatment at this precancerous stage. Since precancerous liver lesions are manageable and reversible, effective detection and treatment before progression to early-stage HCC can significantly reduce the incidence and mortality rates. Therefore, there is an urgent need to develop a non-invasive and reliable method for detecting precancerous liver lesions to prevent the formation of early-stage HCC.

[0003] Currently, histopathological examination is the gold standard for diagnosing precancerous liver lesions. However, this method is invasive, has a high false-negative rate, and carries the risk of cancer metastasis. Although various diagnostic techniques are available, such as blood biochemistry tests, ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT), they still lack sufficient sensitivity and specificity for diagnosing precancerous liver lesions. In recent years, fluorescent probes have played an important role in the diagnosis of liver-related diseases due to their high sensitivity, high spatiotemporal resolution, and real-time imaging capabilities. Fluorescent probes that rely on biomarkers are more likely to diagnose precancerous liver lesions. The process of precancerous liver lesions developing into early-stage hepatocellular carcinoma (HCC) is complex. Although numerous biomarkers have been reported, accurate diagnosis using a single biomarker is still not possible. Therefore, developing novel fluorescent probes that respond to multiple biomarkers simultaneously is of great significance for the diagnosis and regulation of precancerous liver lesions.

[0004] In the precancerous stage of liver disease, chronic liver injury triggers the activation of hepatic stellate cells, disrupting cellular redox balance and thus promoting the malignant transformation of precancerous liver disease into early-stage hepatocellular carcinoma (HCC). During this stage, activated hepatic stellate cells (aHSCs) promote angiogenesis, epithelial-mesenchymal transition (EMT), and immune escape by secreting cytokines and extracellular matrix components. This induces the proliferation and invasion of hepatocellular carcinoma cells within the precancerous tissue. In this process, hepatocellular carcinoma cells produce large amounts of reactive oxygen species (ROS), and mitochondrial viscosity increases with excessive ROS production. Simultaneously, hepatocellular carcinoma cells upregulate GGT, decompose extracellular glutathione (GSH), and the provided cysteine ​​(Cys) (the rate-limiting substrate for intracellular GSH biosynthesis) is transported into the cell to exert an antioxidant effect, maintaining the redox balance within the hepatocellular carcinoma cells and inhibiting their proliferation and invasion. When ROS levels exceed the cell's antioxidant capacity, the balance between oxidation and antioxidation is disrupted, leading to oxidative stress. This provides more favorable conditions for the activation of hepatic stellate cells and the proliferation and invasion of liver cancer cells, accelerating the malignant transformation of precancerous liver lesions into early-stage HCC. The applicant discovered that this process is accompanied by upregulation of GGT and an increase in mitochondrial viscosity. Real-time monitoring of the increase in GGT and mitochondrial viscosity would facilitate real-time tracking and effective regulation of the development of precancerous liver lesions into early-stage HCC, thereby preventing the development of early-stage HCC and inhibiting its occurrence at its source. Given that dual-target detection single-molecule fluorescent probes can avoid a series of problems caused by the combination of multiple fluorescent probes, such as signal crosstalk, localization, and metabolic differences, developing fluorescent probes that can simultaneously monitor GGT and mitochondrial viscosity to achieve the diagnosis of precancerous liver lesions is a pressing technical challenge that needs to be addressed at this stage. Summary of the Invention

[0005] The present invention aims to provide a dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity, its preparation method, and its application. This dual-response fluorescent probe can simultaneously perform fluorescence imaging of γ-glutamyl transferase and mitochondrial viscosity, enabling effective monitoring of precancerous liver lesions and thus facilitating the diagnosis and management of patients with precancerous liver lesions. The dual-response fluorescent probe provided by this invention has two emission wavelengths and displays blue (λ). em =450nm)-Red(λ) em With dual-channel emission at 720nm, the two signals do not interfere with each other. By monitoring the dynamic changes of γ-glutamyl transferase and mitochondrial viscosity in cells and living organisms in real time, it is possible to diagnose precancerous lesions of the liver.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a fluorescent probe that is dual-responsive to γ-glutamyl transferase and mitochondrial viscosity, having a structural formula as shown in Formula I:

[0008]

[0009] Where n is an integer from 1 to 20;

[0010] R1, R2 and R3 are each independently selected from any one of hydrogen, alkyl groups having 1 to 8 carbons, halogens, methoxy groups, trifluoromethyl groups, ester groups or amide groups;

[0011] R4 and R5 are substituents at indeterminate positions on the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyl, alkyl sulfonic acid group having 1-18 carbons, ester group having 1-18 carbons, amide group having 1-18 carbons, N,N-dimethylamino, N,N-diethylamino, trifluoromethyl, sulfonic acid group or sulfonate.

[0012] R6 is selected from any one of L3, L4, L5, L6, L7, L8 or L9;

[0013]

[0014] Y is selected from inorganic negative ions or organic negative ions.

[0015] A second aspect of the present invention provides a method for preparing a dual-responsive fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity, comprising the following steps:

[0016] 1) Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol and carbodiimide hydrochloride were dissolved in organic solvent A and reacted under ice bath conditions to obtain compound Z1;

[0017]

[0018] 2) Compound Z1 was dissolved in organic solvent A under ice bath conditions, and phosphorus tribromide was slowly added dropwise. The reaction was carried out under ice bath conditions to obtain compound Z2.

[0019]

[0020] 3) Compounds Z3 and Z4 were dissolved in dry organic solvent B and refluxed under nitrogen protection to obtain compound Z5;

[0021]

[0022] 4) Dissolve compounds Z5 and Z6 in organic solvent B, add piperidine dropwise, and reflux to obtain compound Z7;

[0023]

[0024] 5) Dissolve Z8, Michaelis acid and potassium carbonate in solvent C and react at room temperature to obtain compound Z9;

[0025]

[0026] 6) Compound Z9, N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were dissolved in dry organic solvent D, and then 1-Boc-piperazine was added. The reaction was carried out at room temperature to obtain compound Z10.

[0027]

[0028] 7) Compound Z10 was dissolved in dry organic solvent A, and trifluoroacetic acid was added under ice bath conditions. The reaction was carried out under ice bath conditions to obtain compound Z11.

[0029]

[0030] 8) Compound Z7, N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) were dissolved in dry organic solvent D, and then compound Z11 was added. The reaction was carried out at room temperature under nitrogen protection to obtain compound Z12.

[0031]

[0032] 9) Compound Z2, compound Z12 and potassium carbonate were dissolved in dry organic solvent B and reacted at room temperature under nitrogen protection to obtain compound Z13;

[0033]

[0034] 10) Compound Z13 was dissolved in dry organic solvent A, and trifluoroacetic acid was added under ice bath conditions. The reaction was carried out under ice bath conditions to obtain the compound shown in Formula I.

[0035]

[0036] The organic solvent A is at least one of dichloromethane, tetrahydrofuran, and diethyl ether; the organic solvent B is at least one of acetonitrile, methanol, and ethanol; the organic solvent C is at least one of water, methanol, and ethanol; and the organic solvent D is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and acetonitrile.

[0037] Furthermore, in step 1), the molar ratio of Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol, and carbodiimide hydrochloride (EDCI) is 1:(1-3):(1-5), preferably 1:(1-2):(2-3);

[0038] In step 2), the molar ratio of compound Z1 to phosphorus tribromide is 1:(1-10), preferably 1:(4-8);

[0039] In step 3), the molar ratio of compound Z3 to compound Z4 is (1-3):1, preferably (1-2):1;

[0040] In step 4), the molar ratio of compound Z5 to compound Z6 is 1:(0.5-2), preferably 1:(0.7-1.2);

[0041] In step 5), the molar ratio of Z8, Michaelis acid, and potassium carbonate is 1:(1-3), preferably 1:(1-2);

[0042] In step 6), the molar ratio of compound Z9, N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), and 1-Boc-piperazine is 1:(1-5):(1-5):(1-5), preferably 1:(1-2):(1-2):(1-2).

[0043] In step 7), the molar ratio of compound Z10 to trifluoroacetic acid is 1:(1-20), preferably 1:(1-10);

[0044] In step 8), the molar ratio of compound Z7, N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), and compound Z11 is 1:(1-5):(1-5):(1-5), preferably 1:(1-2):(1-2):(1-2);

[0045] In step 9), the molar ratio of compound Z2, compound Z12, and potassium carbonate is 1:(1-3):(1-3), preferably 1:(1-2):(1-2);

[0046] In step 10), the molar ratio of compound Z13 to trifluoroacetic acid is 1:(1-20), preferably 1:(1-10).

[0047] A third aspect of the present invention provides the use of a dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity, as shown in Formula I, in the preparation of a detection reagent and a fluorescent imaging agent for detecting γ-glutamyl transferase activity and mitochondrial viscosity.

[0048] The dual-response fluorescent probe for GGT and mitochondrial viscosity of the present invention exhibits high selectivity and high sensitivity for γ-glutamyl transferase and mitochondrial viscosity. It emits fluorescence of varying intensities depending on the γ-glutamyl transferase activity and viscosity of the detection system. A positive correlation is found between the fluorescence intensity emitted at 450 nm and the GGT activity, and a positive correlation is found between the fluorescence intensity emitted at 720 nm and the viscosity. Preferably, the GGT activity of the dual-response fluorescent probe for GGT in the detection system is 0-70 U / L, more preferably 5-50 U / L, and the viscosity in the detection system is preferably 0.5-1099 cP, more preferably 0.8-945 cP.

[0049] The detection system can be an in vitro system or an in vivo system. Preferably, the in vitro system can be an aqueous environment, isolated living cells or isolated biological tissues, etc., and the in vivo system can be in vivo living cells or in vivo tissues, etc.

[0050] In a fourth aspect, the invention provides the application of the aforementioned GGT and mitochondrial viscosity dual-response fluorescent probe in the preparation of a detection reagent for real-time visualization monitoring of early hepatocellular carcinoma cell proliferation and invasion.

[0051] In the above technical solution, the application method of the detection reagent includes the following steps:

[0052] (1) Given that GGT activity, viscosity standard samples, and GGT and mitochondrial viscosity dual-response fluorescent probes are dissolved in buffer solutions, the fluorescence intensity of the system is measured, and standard curves between GGT activity and system fluorescence intensity and viscosity and system fluorescence intensity are established respectively.

[0053] (2) The cells to be analyzed were mixed and cultured to induce the proliferation and invasion of liver cancer cells. The cells were incubated with GGT and mitochondrial viscosity dual-response fluorescent probe solution and imaged. The proliferation and invasion of liver cancer cells were monitored in real time by observing the dynamic fluctuations of GGT activity and mitochondrial viscosity. During the process of increasing GGT activity and mitochondrial viscosity, regulation was given and the regulation effect was monitored.

[0054] (3) The cells to be analyzed were mixed and cultured, and a cell suspension was prepared and injected into the subcutaneous tissue of mice to induce the proliferation and invasion of liver cancer cells. A dual-response fluorescent probe of GGT and mitochondrial viscosity was injected and imaged. The proliferation and invasion of liver cancer cells in vivo were monitored in real time by observing the dynamic fluctuations of GGT activity and mitochondrial viscosity. During the process of increasing GGT activity and mitochondrial viscosity, regulation was given and the regulatory effect was monitored.

[0055] (4) Imaging of GGT and mitochondrial viscosity in liver cancer and adjacent tissues in clinical samples enables rapid and accurate differentiation between liver cancer tissue and adjacent tissues, thereby achieving accurate diagnosis of precancerous lesions in adjacent tissues.

[0056] In the above technical solution, in step (1), the buffer solution is preferably an aqueous buffer solution. The standard curve can be established by conventional methods, such as preparing multiple standard samples with known GGT activity and viscosity at different concentrations and dissolving them with fluorescent probes of specified concentrations in appropriate buffer solutions, measuring the fluorescence intensity of each system, and thus establishing the standard curve.

[0057] In the above technical solution, the concentration of the GGT and mitochondrial viscosity dual-response fluorescent probe in the fluorescence emission system is 1.0-100.0 μM, preferably 10.0-50.0 μM.

[0058] The advantages and beneficial technical effects of this invention are:

[0059] 1. This invention provides a novel dual-response fluorescent probe for γ-glutamyl transferase (GGT) and mitochondrial viscosity, using coumarin and cyanine dyes as two different fluorophores. This fluorescent probe exhibits dual emission wavelengths without spectral crosstalk, demonstrating high selectivity and sensitivity for GGT activity and mitochondrial viscosity at emission wavelengths of 450 nm and 720 nm, respectively. The 270 nm difference in emission wavelength between the two fluorophores suppresses fluorescence energy resonance transfer (FRET), achieving complete independence of the two signals. Through a combination of hydroxyl protection-deprotection and the introduction of a "molecular rotor," dual target recognition sites for GGT and viscosity are constructed. Utilizing the negative charge of the mitochondrial inner membrane, a lipophilic cationic molecule is introduced to achieve mitochondrial targeting, and the localization ability of the carbon chain is enhanced by regulating its length, specifically responding to mitochondrial viscosity. This fluorescent probe possesses advantages such as resistance to spectral crosstalk, mitochondrial targeting, and simultaneous imaging of two analytes, creating conditions for the diagnosis of precancerous lesions of the liver using dual-response fluorescent probes for GGT and mitochondrial viscosity.

[0060] 2. The γ-glutamyl transferase and mitochondrial viscosity dual-responsive fluorescent probe provided by this invention utilizes the negative charge of the inner mitochondrial membrane to introduce lipophilic cationic molecules to achieve mitochondrial targeting, and enhances its localization ability in the inner mitochondrial membrane by regulating the length of the carbon chain. The colocalization coefficients of the above-mentioned GGT and mitochondrial viscosity dual-responsive fluorescent probe with commercial mitochondrial and lysosomal fluorescent probes in liver cancer cells are 0.92 and 0.68, respectively, indicating that the above-mentioned GGT and mitochondrial viscosity dual-responsive fluorescent probe has excellent targeting performance for mitochondria.

[0061] 3. The present invention also provides an application for rapid real-time visualization monitoring of precancerous liver lesions using the dual-response probe. This application uses the GGT and mitochondrial viscosity dual-response fluorescent probe of the present invention to perform real-time visualization monitoring of GGT and mitochondrial viscosity of liver cancer cells during the progression of precancerous liver lesions to early HCC. By monitoring the dynamic changes of GGT and mitochondrial viscosity in mouse subcutaneous tumors, rapid real-time visualization monitoring is achieved, and appropriate regulation is given in a timely manner to realize real-time monitoring of the diagnosis and regulation effects of precancerous liver lesions.

[0062] 4. The present invention also provides an application for rapid and accurate visualization of precancerous lesions in clinical samples using the dual-response probe. This application uses the GGT and mitochondrial viscosity dual-response fluorescent probe of the present invention to qualitatively and quantitatively analyze GGT and mitochondrial viscosity in liver cancer and adjacent tissues in clinical samples. By imaging GGT and mitochondrial viscosity in pathological sections of clinical samples, rapid and accurate visualization and differentiation of liver cancer tissue and adjacent tissues can be achieved, thereby realizing the accurate diagnosis of precancerous lesions in liver. Attached Figure Description

[0063] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0064] Figure 1 The fluorescence spectrum of the fluorescent probe I1 prepared in Example 1 of this invention reacts with GTT.

[0065] Figure 2 The fluorescence spectra of fluorescent probe I1 prepared in Example 1 of this invention after reacting with GTT of different activities are shown.

[0066] Figure 3 The graph shows the linear relationship between the fluorescent probes I1-I6 prepared in Example 1 of this invention and the GGT activity.

[0067] Figure 4 The fluorescence change histogram (emission wavelength 450 nm) of the fluorescent probe I1 prepared in Example 1 of this invention with the addition of different interfering analytes is shown.

[0068] Figure 5 The fluorescence spectra of the fluorescent probe I1 prepared in Example 1 of this invention respond to different viscosities.

[0069] Figure 6 The graph shows the linear relationship between the fluorescent probes I1 to I6 prepared in Example 1 of this invention and different viscosities.

[0070] Figure 7 The fluorescence change histogram (emission wavelength 720 nm) of the fluorescent probe I1 prepared in Example 1 of this invention with the addition of different interfering analytes is shown.

[0071] Figure 8 The image shows a bar chart of fluorescence changes of the fluorescent probe I1 prepared in Example 1 of this invention in different polar solvents (emission wavelength is 720 nm).

[0072] Figure 9 Line graphs showing the responses of fluorescent probes I1 to I6 prepared in Example 1 of this invention to different pH values.

[0073] Figure 10 The results of cytotoxicity assays for fluorescent probes I1-I6 prepared in Example 1 of this invention are shown.

[0074] Figure 11 The results of the colocalization experiment of the fluorescent probe I1 prepared in Example 1 of this invention are verified.

[0075] Figure 12 The fluorescent probe I1 prepared in Example 1 of this invention was used to test the results of endogenous GGT and mitochondrial adhesion in cells.

[0076] Figure 13 The fluorescent probe I1 prepared in Example 1 of this invention was used to image L02 cells, LX-2 cells and HepG2 cells.

[0077] Figure 14 The fluorescent probe I1 prepared in Example 1 of this invention is used for real-time imaging results of mixed cultured cells.

[0078] Figure 15 The fluorescent probe I1 prepared in Example 1 of this invention is used to image the real-time imaging results of the continuous proliferation and invasion process of liver cancer cells.

[0079] Figure 16 The fluorescent probe I1 prepared in Example 1 of this invention is used to image the real-time imaging results of inhibiting the proliferation and invasion of liver cancer cells.

[0080] Figure 17 The fluorescent probe I1 prepared in Example 1 of this invention was used to image the fluorescence intensity of mouse subcutaneous tumors over time.

[0081] Figure 18 The fluorescent probe I1 prepared in Example 1 of this invention is used to image the real-time detection results of the proliferation and invasion process of subcutaneous tumors in mice.

[0082] Figure 19 The fluorescent probe I1 prepared in Example 1 of this invention is used to image the real-time detection results of the inhibition of subcutaneous tumor proliferation and invasion in mice.

[0083] Figure 20 The fluorescent probe I1 prepared in Example 1 of this invention was used to image the test results of cancerous tissue and adjacent tissue in clinical samples. Detailed Implementation

[0084] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0085] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0086] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0087] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0088] The following examples provide a method for preparing a dual-response fluorescent probe I1 for GGT and mitochondrial viscosity, having the structure shown in Formula I. The preparation method is as follows:

[0089] 1) Synthesis of compound Z1: Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol, and carbodiimide hydrochloride (EDCI) were dissolved in dry dichloromethane and reacted in an ice bath for 12 h. After the solvent was evaporated, pure compound Z1 was obtained by column chromatography. The molar ratio of Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol, and carbodiimide hydrochloride (EDCI) was 1:(1-3):(1-3), preferably 1:(1-2):(1-2). The eluent for column chromatography was petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (1-3):1.

[0090] 2) Synthesis of compound Z2: Compound Z1 was dissolved in dry tetrahydrofuran, and phosphorus tribromide (PBr3) was added dropwise under ice bath conditions. The reaction was carried out for 2 hours under ice bath conditions. The mixture was extracted three times with dichloromethane and water. The organic layer was dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. After the solvent was evaporated, pure compound Z2 was obtained by column chromatography. The molar ratio of compound Z1 to phosphorus tribromide (PBr3) was 1:(1-3), preferably 1:(1-2). The eluent for column chromatography was petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (1-3):1.

[0091] 3) Synthesis of compound Z5: Compounds Z3 and Z4 were dissolved in dry acetonitrile and refluxed under nitrogen protection for 12 hours. After cooling to room temperature, the reaction solution was poured into diethyl ether, and gray crystals precipitated. The crude product was obtained by filtration. The crude product was washed repeatedly with petroleum ether in small amounts until the solid was grayish-white, thus obtaining pure compound Z5. The molar ratio of compounds Z3 and Z4 was (1-2):1-2, preferably (1-1.5):1.

[0092] 4) Synthesis of compound Z7: Compounds Z5 and Z6 were dissolved in anhydrous ethanol, and piperidine was added dropwise. The reaction was carried out under nitrogen protection and refluxed for 14 h. After the solvent was evaporated, pure compound Z7 was obtained by column chromatography. The molar ratio of compounds Z5 and Z6 was 1:(1-1.5), preferably 1:(1-1.2), and the eluent for column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (35-60):1.

[0093] 5) Synthesis of compound Z9: Compound Z8, Michaelis acid, and potassium carbonate were dissolved in deionized water and reacted at room temperature for 18 hours. The crude product was obtained by filtration. The crude product was washed repeatedly with small amounts of ice-cold ethanol until the solid was off-white, thus obtaining pure compound Z9. The molar ratio of compound Z8, Michaelis acid, and potassium carbonate was 1:(1-3):(1-3), preferably 1:(1-1.5):(1-2).

[0094] 6) Synthesis of compound Z10: Compound Z9, N,N'-dicyclohexylcarbodiimide (DCC), and 1-hydroxybenzotriazole (HOBt) were dissolved in dry N,N-dimethylformamide and reacted at room temperature for 0.5 h. Then, 1-Boc-piperazine was added. The reaction was carried out at room temperature for 24 h under nitrogen protection. After the solvent was evaporated, the product was dissolved in acetonitrile, and the solid was filtered off. After the filtrate was evaporated, pure compound Z10-1 was obtained by column chromatography. The molar ratio of compound Z9, N,N'-dicyclohexylcarbodiimide (DCC), and 1-hydroxybenzotriazole (HOBt) was 1:(1-3):(1-3), preferably 1:(1-2):(1-2). The eluent for column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (5-10):1.

[0095] 7) Synthesis of compound Z11: Compound Z10 was dissolved in dry dichloromethane, and trifluoroacetic acid was added under ice bath conditions. The reaction was carried out at room temperature for 4 hours under nitrogen protection. After evaporating the filtrate, pure compound Z11 was obtained by column chromatography. The molar ratio of compound Z10 to trifluoroacetic acid was 1:(1-20), preferably 1:(1-10). The eluent for column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (4-8):1.

[0096] 8) Synthesis of compound Z12: Compound Z7, N,N'-dicyclohexylcarbodiimide (DCC), and 1-hydroxybenzotriazole (HOBt) were dissolved in dry N,N-dimethylformamide and reacted at room temperature for 0.5 h. Then, compound Z11 was added. The reaction was carried out at room temperature for 24 h under nitrogen protection. After the solvent was evaporated, the product was dissolved in acetonitrile, and the solid was filtered off. After the filtrate was evaporated, pure compound Z12 was obtained by column chromatography. The molar ratio of compound Z7, N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), and compound Z11 was 1:(1-3):(1-3):(1-3), preferably 1:(1-2):(1-2):(1-2). The eluent for column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (15-30):1.

[0097] 9) Synthesis of compound Z13: Compounds Z2 and Z12, and potassium carbonate were dissolved in dry acetonitrile and reacted at room temperature for 48 h under nitrogen protection. The mixture was extracted three times with ethyl acetate and water. The organic layer was dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. After the solvent was evaporated, pure compound Z13 was obtained by column chromatography. The molar ratio of compound Z2, compound Z12, and potassium carbonate was 1:(1-3):(1-3), preferably 1:(1-2):(1-2). The eluent for column chromatography was dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (15-30):1.

[0098] 10) Synthesis of Compound I: Compound Z13 was dissolved in dry dichloromethane, and trifluoroacetic acid was added under ice bath conditions. The reaction was carried out under nitrogen protection in an ice bath for 4 hours. Saturated sodium bicarbonate solution and dichloromethane were added to the reaction solution, and the precipitated solid was collected to obtain pure Compound I. The molar ratio of compound Z13 to trifluoroacetic acid was 1:(1-20), preferably 1:(1-10).

[0099] It should be noted that, in some of the embodiments described below, compounds I1 (n=4), I2 (n=10), and I3 (n=16) refer to compounds whose structural formulas (compounds I) shown in the above synthetic route have n=4, n=10, and n=16, respectively. Similarly, other compounds described in some of the embodiments below can be deduced by analogy.

[0100] Example 1

[0101] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe, fluorescent probe I1 (n=4):

[0102] 1) Synthesis of compound Z1-1

[0103] Boc-L-glutamic acid-1-tert-butyl ester (1.0 g, 3.30 mmol), 4-aminobenzyl alcohol (398 mg, 3.23 mmol), and carbodiimide hydrochloride (EDCI) (632 mg, 3.30 mmol) were dissolved in dry dichloromethane (45 mL) and reacted in an ice bath for 12 h. The solvent was removed under reduced pressure, and the mixture was extracted three times with dichloromethane and water. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography using ethyl acetate / petroleum ether (2:1 v / v) as the eluent to obtain compound Z1-1. 1 H NMR (400MHz, Methanol-d4): 7.52(2H), 7.29(2H), 4.55(2H), 4.01(1H), 2.47(2H), 2.15(1H), 1.93(1H), 1.47(9H), 1.43(9H). HRMS(ESI)m / z calcd for C 21 H 33 N2O6 + (M+H) + 409.2333, found 409.2330.

[0104] 2) Synthesis of compound Z2-1

[0105] Compound Z1-1 (1.12 g, 2.74 mmol) was dissolved in dry tetrahydrofuran (50 mL), and phosphorus tribromide (PBr3) (4.06 g, 15 mmol) was added dropwise under ice bath conditions. The reaction was carried out for 2 h under ice bath conditions. The mixture was extracted three times with dichloromethane and water, and the organic layer was dried with anhydrous sodium sulfate. The crude product was obtained by rotary evaporation after filtration. Compound Z2-1 was purified by silica gel column chromatography using ethyl acetate / petroleum ether (5:1 v / v) as the eluent. 1H NMR (400MHz, Methanol-d4): 7.55(2H), 7.27(2H), 4.40(2H), 4.03(1H), 2.48(2H), 2.17(1H), 1.93(1H), 1.46(9H), 1.42(9H). HRMS(ESI)m / z calcd for C 21 H 32 BrN2O5(M+H) + 471.1489, found 471.1485.

[0106] 3) Synthesis of compound Z5-1

[0107] 6-Bromhexanoic acid Z3-1 (28.1 g, 144.10 mmol) and 2,3,3-trimethyl-4,5-benzo-3H-indole Z4-1 (20.0 g, 95.60 mmol) were dissolved in dry acetonitrile (100 mL) and refluxed under nitrogen protection for 12 h. After cooling to room temperature, the reaction solution was poured into diethyl ether (300 mL), and gray crystals precipitated. The crude product was obtained by filtration. The crude product was washed repeatedly with small amounts of petroleum ether until the solid was grayish-white, thus obtaining pure compound Z5-1. 1 HNMR(400MHz,DMSO-d6):12.03(1H),8.41(1H),8.34(1H),8.24(2H),7.82(1H ),7.75(1H),4.65(2H),3.02(3H),2.20(2H),1.92(2H),1.81(6H),1.48(4H). HRMS(ESI)m / z calcd for C 21 H 26 NO2 + (M) + 324.1958, found 324.1959.

[0108] 4) Synthesis of compound Z7-1 (n=4)

[0109] Compound Z5-1 (3.23 g, 10 mmol) and 4-dimethylaminocinnamaldehyde Z6-1 (1.75 g, 10 mmol) were dissolved in anhydrous ethanol (20 mL), and piperidine (100 μL) was added dropwise. The reaction was refluxed under nitrogen protection for 14 h. After the solvent was evaporated, the compound was purified by silica gel column chromatography with dichloromethane / methanol (50:1 v / v) as the eluent to obtain compound Z7-1. 1HNMR(400MHz,DMSO-d6):12.03(1H),8.46-8.36(2H),8.23(1H),8.18(1H),8.02(1H),7.81-7.73(2H),7.67(1H),7.61 (2H),7.35(1H),7.08(1H),6.85(2H),4.50(2H),3.10(6H),2.23(2H),1.97(6H),1.85(2H),1.62-1.54(2H),1.48(2H). HRMS(ESI)m / z calcd for C 32 H 37 N2O2 + (M) + 481.2842, found 481.2855.

[0110] 5) Synthesis of compound Z9-1

[0111] 2,4-Dihydroxybenzaldehyde Z8-1 (1.38 g, 10 mmol), Michaelis acid (1.44 g, 10 mmol), and potassium carbonate (0.14 g, 1.0 mmol) were dissolved in deionized water (50 mL) and reacted at room temperature for 18 h. The crude product was obtained by filtration. The crude product was washed repeatedly with small amounts of ice-cold ethanol until the solid was off-white, thus obtaining the pure product of compound Z9-1. 1 H NMR (400MHz, DMSO-d6): 8.38(1H), 7.58(1H), 6.70(1H), 6.53(1H). HRMS(ESI)m / z calcd for C 10 H7O5 + (M+H) + 207.0288, found 207.0282.

[0112] 6) Synthesis of compound Z10-1

[0113] Compound Z9-1 (0.44 g, 2 mmol), N,N'-dicyclohexylcarbodiimide (DCC) (0.82 g, 4 mmol), and 1-hydroxybenzotriazole (HOBt) (0.54 g, 4 mmol) were dissolved in dry N,N-dimethylformamide (15 mL) and reacted at room temperature for 0.5 h. Then, 1-Boc-piperazine (0.37 g, 2 mmol) was added. The reaction was carried out under nitrogen protection at room temperature for 24 h. After the solvent was evaporated, the solution was dissolved in acetonitrile (20 mL), and the solid was filtered off. After the filtrate was evaporated, the solution was purified by silica gel column chromatography using dichloromethane / methanol (10:1 v / v) as the eluent to obtain compound Z10-1. 1H NMR (400MHz, Methanol-d4): 7.97(1H), 7.45(1H), 6.74(1H), 6.62(1H), 3.69(2H), 3.47(6H), 1.46(9H).

[0114] 7) Synthesis of compound Z11-1

[0115] Compound Z10-1 (0.30 g, 0.80 mmol) was dissolved in dry dichloromethane (3 mL), and trifluoroacetic acid (3 mL) was added under ice bath conditions. The reaction was carried out at room temperature for 4 h under nitrogen protection. After evaporating the filtrate, compound Z11-1 was purified by silica gel column chromatography using dichloromethane / methanol (4:1 v / v) as the eluent. 1 H NMR (400MHz, DMSO-d6): 10.89(1H), 8.85(1H), 8.14(1H), 7.62(1H), 6.86(1H), 6.77(1H), 3.78(2H), 3.58(2H), 3.17(2H), 3.10(2H). HRMS(ESI)m / z calcd for C 14 H 15 N2O4 + (M+H) + 275.1025, found 275.1028.

[0116] 8) Synthesis of compound Z12-1 (n=4)

[0117] Compound Z7-1 (n=4) (1.12 g, 2 mmol), N,N'-dicyclohexylcarbodiimide (DCC) (0.82 g, 4 mmol), and 1-hydroxybenzotriazole (HOBt) (0.82 g, 4 mmol) were dissolved in dry N,N-dimethylformamide and reacted at room temperature for 0.5 h. Then, compound Z11-1 (0.55 g, 2 mmol) was added. The reaction was carried out under nitrogen protection at room temperature for 24 h. After the solvent was evaporated, the solution was dissolved in acetonitrile, and the solid was filtered off. After the filtrate was evaporated, the solution was purified by silica gel column chromatography using dichloromethane / methanol (20:1 v / v) as the eluent to obtain compound Z12-1. 1H NMR(400MHz, DMSO-d6):10.80(1H),8.49-8.35(2H),8.22(1H),8.16(1H),8.07(1H),8.02(1H),7.77(2H),7.66(1H),7.61(3H),7.42-7.31( 1H),7.09(1H),6.89-6.80(3H),6.77(1H),4.52(2H),3.45(6H),3.28 (2H),3.09(6H),2.34(2H),1.97(6H),1.86(2H),1.58(2H),1.47(2H). HRMS(ESI)m / z calcd for C 46 H 49 N4O5 + (M) + 737.3703, found 737.3700.

[0118] 9) Synthesis of compound Z13-1 (n=4)

[0119] Compound Z2-1 (0.94 g, 2 mmol), compound Z12-1 (1.63 g, 2 mmol), and potassium carbonate (0.33 g, 2.4 mmol) were dissolved in dry acetonitrile (10 mL) and reacted at room temperature for 48 h under nitrogen protection. The mixture was extracted three times with ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the crude product. After the solvent was evaporated, the crude product was purified by silica gel column chromatography using dichloromethane / methanol (15:1 v / v) as the eluent to obtain compound Z13-1. 1 H NMR(400MHz,DMSO-d6):9.95(1H),8.42(2H),8.22(1H),8.17(1H),8.11(1 H),8.01(1H),7.76(2H),7.68(2H),7.61(4H),7.41(2H),7.33(1H),7.21-7 .02(4H),6.83(2H),5.18(2H),4.51(2H),3.84(1H),3.53(9H),3.09(6H),2 .41(2H),2.34(2H),1.97(7H),1.87(2H),1.58(2H),1.50(2H),1.39(18H). HRMS(ESI)m / z calcd for C 67 H 79 N6O 10 + (M) + 1127.5858, found 1127.5846.

[0120] 10) Synthesis of compound I1 (n=4)

[0121] Compound Z13-1 (1.21 g, 1 mmol) was dissolved in dry dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added under ice bath conditions. The reaction was carried out under nitrogen protection in an ice bath for 4 h. Saturated sodium bicarbonate solution and dichloromethane were added to the reaction solution, and the precipitated solid was collected to obtain pure compound I1. 1 H NMR(400MHz,DMSO-d6):10.21(1H),8.41(3H),8.23(1H),8.16(1H),8.11(1H ),8.02(1H),7.80-7.74(2H),7.69(2H),7.62(4H),7.41(2H),7.34(1H),7.1 5-7.02(4H),6.83(2H),5.18(2H),4.52(2H),3.82(1H),3.53(8H),3.08(6H) ,2.57(2H),2.39-2.29(2H),2.16-2.01(4H),1.97(6H),1.87(2H),1.58(2H). HRMS(ESI)m / zcalcd for C 58 H 63 N6O8 + (M) + 971.4707, found 971.4693.

[0122]

[0123] Example 2

[0124] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe I2 (n=10):

[0125] The fluorescent probe I2 was prepared using a similar procedure to that in Example 1. 1 HNMR(400MHz,DMSO-d6):10.19(1H),8.42-8.19(6H),7.91(1H),7.82-7.71(4H),7.64(4H),7.44-7.40(2H),7.35(1H),7.20-7.07(4H),6.79( 2H),5.08(2H),4.21(2H),3.78(1H),3.48(8H),3.06(6H),2.36(2H),2 .18-2.27(4H),1.82(2H),1.70(6H),1.44-1.62(6H),1.42-1.33(10H). HRMS(ESI)m / zcalcd for C 64 H75 N6O8 + (M) + 1055.5641, found 1055.5643.

[0126] The difference from Example 1 is that in step 3), 6-bromohexanoic acid (Z3-1) was replaced with 12-bromohexanoic acid (Z3-2) to obtain compound Z5-2 (n=10). HRMS(ESI) m / z calcd for C 27 H 38 NO2 + (M) + 408.2897, found 408.2896; In step 4), compound Z5-1 (n=4) is replaced with compound Z5-2 (n=10) to obtain compound Z7-2 (n=10), HRMS(ESI) m / z calcd for C 38 H 49 N2O2 + (M) + 565.3788, found 565.3789; In step 8), compound Z7-1 (n=4) is replaced with compound Z7-2 (n=10) to obtain compound Z12-2, HRMS(ESI) m / z calcd for C 52 H 61 N4O5 + (M) + 821.4636, found 821.4630; In step 9), compound Z12-1 (n=4) is replaced with compound Z12-2 (n=10) to obtain compound Z13-2 (n=10), HRMS(ESI) m / z calcd for C 73 H 91 N6O 10 + (M) + 1211.6791,found1211.6796; In step 10), compound Z13-1 (n=4) is replaced with compound Z13-2 (n=10).

[0127]

[0128] Example 3

[0129] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe I3 (n=16):

[0130] The fluorescent probe I3 was prepared using a similar procedure to that in Example 1. 1HNMR(400MHz,DMSO-d6):10.13(1H),8.38-8.21(4H),8.12-8.04(2H),7.96(1H),7.87-7.73(4H),7.64-7.42(4H),7.35-7.19(5H),7.02( 2H),6.68(2H),5.19(2H),4.18(2H),3.52(8H),3.42(1H),33.33(6H),3.31(2H),2.14-2.11(4H),1.68(6H),1.64(2H),1.42-1.29(28H). HRMS(ESI)m / z calcd forC 70 H 87 N6O8 + (M) + 1139.6580, found 1139.6577.

[0131] The difference from Example 1 is that in step 3), 12-bromohexanoic acid (Z3-1) is replaced with 18-bromohexanoic acid (Z3-3) to obtain compound Z5-3. HRMS(ESI) m / z calcd for C 33 H 50 NO2 + (M) + 492.3836, found 492.3830; In step 4), compound Z5-1 is replaced with compound Z5-3 to obtain compound Z7-3, HRMS(ESI) m / z calcd for C 44 H 61 N2O2 + (M) + 649.4728, found 649.4722; In step 8), compound Z7-1 is replaced with compound Z7-3 to obtain compound Z12-3, HRMS(ESI) m / z calcd for C 58 H 73 N4O5 + (M) + 905.5575, found 905.5579; In step 9), compound Z12-1 is replaced with compound Z12-3 to obtain compound Z13-3, HRMS(ESI) m / z calcd for C 79 H 103 N6O 10 + (M) + 1295.7730, found 1295.7734; in step 10), compound Z13-1 is replaced with compound Z13-3.

[0132]

[0133] Example 4

[0134] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe I4:

[0135] The fluorescent probe I4 was prepared using a similar procedure to that in Example 1. 1 HNMR(400MHz, DMSO-d6):10.31(1H),8.62(2H),8.36(1H),7.76-7.62(9H),7.39-7.34(3H),7.14-7.12(2H),6.79-6.74(5H),5.36(1H),5. 12(2H),4.11(2H),3.52-3.48(5H),3.41(4H),3.12(6H),2.43(3H),2 .29(2H),2.20(2H),2.12(2H),1.64(6H),1.49(2H),1.38-1.34(4H). HRMS(ESI)m / z calcd forC 59 H 65 N6O8 + (M) + 985.4858, found 985.4856.

[0136] The difference from Example 1 is that in step 5), 2,4-dihydroxybenzaldehyde (Z8-1) is replaced with 2,4-dihydroxy-6-methylbenzaldehyde (Z8-4) to obtain compound Z9-4. HRMS(ESI) m / z calcd for C 11 H9O5 + (M+H) + 221.0444, found 221.0443; In step 6), compound Z9-1 (n=4) is replaced with compound Z9-4 to obtain compound Z10-4, HRMS(ESI) m / z calcd for C 20 H 25 N2O6 + (M+H) + 389.1707, found 389.1701; In step 7), compound Z10-1 is replaced with compound Z10-4 to obtain compound Z11-4, HRMS(ESI) m / z calcd for C 15 H 17 N2O4 + (M+H) +289.1183, found 289.1182; In step 8), compound Z11-1 is replaced with compound Z11-4 to obtain compound Z12-4, HRMS(ESI) m / z calcd for C 47 H 51 N4O5 + (M) + 751.3854, found 751.3858; In step 9), compound Z12-1 is replaced with compound Z12-4 to obtain compound Z13-4, HRMS(ESI) m / z calcd for C 68 H 81 N6O 10 + (M) + 1141.6009, found 1141.6008; in step 10), compound Z13-1 is replaced with compound Z13-4.

[0137]

[0138] Example 5

[0139] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe I5:

[0140] The fluorescent probe I5 was prepared using a procedure similar to that in Example 1. 1 HNMR(400MHz,DMSO-d6):10.01(1H),8.80(2H),8.38-8.31(3H),7.92-7.88(2H),7.70-7.63(6H),7.12-7.06(4H),7.11(1H),6.76-6.72(4H) ,5.34(1H),5.16(2H),4.12(2H),3.49(8H),3.32(1H),3.12(6H),2.35 (2H),2.22(2H),2.17(2H),1.69(2H),1.64(6H),1.38(2H),1.34(2H). HRMS(ESI)m / z calcdfor C 58 H 62 BrN6O8 + (M) + 1049.3807, found 1049.3802.

[0141] The difference from Example 1 is that in step 3), 2,3,3-trimethyl-4,5-benzo-3H-indole (Z1-5) was replaced with 7-bromo-1,1,2-trimethyl-1H-benzo[e]indole (Z4-5) to obtain compound Z5-5. HRMS(ESI) m / z calcd for C 21 H 25 BrNO2 + (M) + 402.1063, found 402.1069; In step 4), compound Z5-1 is replaced with compound Z5-5 to obtain compound Z7-5, HRMS(ESI) m / z calcd for C 32 H 36 BrN2O2 + (M) + 559.1955, found 559.1955; In step 8), compound Z7-1 is replaced with compound Z7-5 to obtain compound Z12-5, HRMS(ESI) m / zcalcd for C 46 H 48 BrN4O5 + (M) + 815.2803, found 815.2801; In step 9), compound Z12-1 is replaced with compound Z12-5 to obtain compound Z13-5, HRMS(ESI) m / z calcd for C 67 H 78 BrN6O 10 + (M) + 1205.4957, found 1205.4955; in step 10), replace compound Z13-1 with compound Z13-5.

[0142]

[0143] Example 6

[0144] Preparation of GGT and mitochondrial viscosity dual-responsive fluorescent probe I6:

[0145] The fluorescent probe I6 was prepared using a procedure similar to that in Example 1. 1HNMR(400MHz,DMSO-d6):10.17(1H),8.88(2H),8.43-8.41(2H),8.34-8.32(2H),8.08-8.01(5H),7.63-7.51(12H),7.35-7.32(2H),6.8 9(1H),6.81-6.78(2H),5.43(1H),5.34(2H),4.24(2H),3.53(8H),3.42(1H),2.32-2.26(6H),1.63(6H),1.56(2H),1.51(2H),1.48(2H). HRMS(ESI)m / z calcd forC 64 H 62 N5O8 + (M) + 1028.4593, found 1028.4591.

[0146] The difference from Example 1 is that in step 4), 4-dimethylaminocinnamaldehyde (Z6-1) is replaced with 3-(anthracite-9-yl)propenal (Z6-6) to obtain compound Z7-6. HRMS(ESI) m / z calcd for C 38 H 36 NO2 + (M) + 538.2741, found 538.2744; In step 8), compound Z7-1 is replaced with compound Z7-6 to obtain compound Z12-6, HRMS(ESI) m / zcalcd for C 52 H 48 N3O5 + (M) + 794.3588, found 794.3582; In step 9), compound Z12-1 is replaced with compound Z12-6 to obtain compound Z13-6, HRMS(ESI) m / z calcd for C 73 H 78 N5O 10 + (M) + 1184.5743,found1184.5741; In step 10), compound Z13-1 is replaced with compound Z13-6.

[0147]

[0148] Example 7

[0149] The response time of the fluorescent probe I1 prepared in Example 1 to GGT activity was determined.

[0150] The fluorescence emission spectra of fluorescent probe I1 in response to GGT were measured using a fluorescence spectrometer. The test results are shown in [Figure number missing]. Figure 1 A fluorescence spectrophotometer was used, with the excitation wavelength set to 405 nm and the scanning wavelength range to 425 nm–600 nm. The buffer solution used was PBS (20 mM, pH 7.2). The probe concentration was 10 μM. GGT with an activity of 50 U / L was added to the detection system, and its effect on the fluorescence intensity of the fluorescent probe I1 at different time points was observed. The results are as follows: Figure 1 As shown. (Through) Figure 1 It can be observed that as the time increases sequentially from 0 min to 60 min, the emission intensity at 450 nm gradually increases with time, and the fluorescence emission intensity reaches its maximum at 60 min.

[0151] Example 8

[0152] The responses of fluorescent probes I1-I6 prepared in Examples 1-6 to GGT assays with different activities were studied, and a linear relationship between GGT activity and fluorescence intensity was established.

[0153] The fluorescence emission spectra of fluorescent probe I1 in response to GGT were measured using a fluorescence spectrometer. The test results are shown in [Figure number missing]. Figure 2 A fluorescence spectrophotometer was used, with the excitation wavelength set to 405 nm and the scanning wavelength range to 425 nm–600 nm. The buffer solution used was PBS (20 mM, pH 7.2). The probe concentration was 10 μM. Different activities of GGT were added to the detection system, and their effects on the fluorescence intensity of fluorescent probes I1–I6 were observed. The results are as follows: Figure 3 As shown. (Through) Figure 2 It can be observed that when the GGT activity increases sequentially from 0, 5, 10, 15, 20, 25, 30, 40, 50, 70, to 90 U / L, the emission intensity at 450 nm gradually increases with the increase of GGT activity. The emission intensity and GGT activity show a good linear relationship between 0 and 50 U / L. Figure 3 As shown, the linear equations are: I1: y = 6353x + 70684, with a linear correlation coefficient of 0.9958; I2: y = 7224x + 34428, with a linear correlation coefficient of 0.9903; I3: y = 7498x + 44797, with a linear correlation coefficient of 0.9850; I4: y = 5763x + 23212, with a linear correlation coefficient of 0.9931; I5: y = 6145x + 61079, with a linear correlation coefficient of 0.9952; and I6: y = 6394x + 58562, with a linear correlation coefficient of 0.9959.

[0154] Example 9

[0155] Selective detection of GGT activity by fluorescent probe I1 prepared in Example 1:

[0156] The fluorescent probe I1 and the interfering analyte were added to PBS buffer (20 mM, pH 7.2) to prepare the test solution. The concentration of fluorescent probe I1 in the test solution was 10 μM, and the concentration of the interfering analyte was 100 μM. The interfering analyte was Na... + K + Ca 2+ Mg 2+ Fe 2+ Fe 3+ Cu 2+ Cu + Al 3+ Zn 2+ Cl - SO4 2- HSO3 - Each test solution contains the following fluorescent probes: glucose, alanine (Ala), threonine (Thr), lysine (Lys), cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), gycerol, and GGT.

[0157] The fluorescence intensity of each test solution was detected using a fluorescence spectrophotometer. The excitation wavelength was set to 405 nm, and the fluorescence intensity at the emission wavelength of 450 nm was collected. The detection results of fluorescent probe I1 are as follows: Figure 4 As shown. According to Figure 4 The results showed that fluorescence enhancement only occurred in the presence of GGT, while the fluorescence intensity of other interfering analytes did not change significantly. This demonstrates that fluorescent probe I1 exhibits good selectivity for GGT at an excitation wavelength of 405 nm and an emission wavelength of 450 nm.

[0158] Example 10

[0159] Examples 1-6 show the responses of fluorescent probes I1-I6 to different viscosities, establishing a linear relationship between viscosity and fluorescence intensity:

[0160] The fluorescence emission spectra of fluorescent probe I1 at different viscosities were measured using a fluorescence spectrometer. The test results are shown in [Figure 1]. Figure 5Using a fluorescence spectrophotometer, with the excitation wavelength set to 600 nm and the scanning wavelength range of 620 nm–900 nm, solutions of different volume ratios were prepared using glycerol and PBS (20 mM, pH 7.2) to obtain test solutions of different viscosities. These test solutions were then used to further dilute the fluorescent probe I1 to a final concentration of 10 μM. The effect of different viscosities of the test solutions on the fluorescence intensity of the fluorescent probe I1 was observed. The results are as follows: Figure 5 As shown. (Through) Figure 5 It can be observed that as the viscosity values ​​increase sequentially from 0.893, 1.221, 1.754, 2.505, 4.165, 6.963, 12.36, 27.73, 64.2, 241.2, to 945 cP, the emission intensity at 720 nm gradually increases with the increase of viscosity value. The logarithm of the emission intensity has a good linear relationship with the logarithm of the viscosity value between 0.893 and 945 cP. Figure 6 As shown, the linear equations are: I1: y = 0.85x + 2.85, with a linear correlation coefficient of 0.9962; I2: y = 0.90x + 2.41, with a linear correlation coefficient of 0.9816; I3: y = 0.88x + 2.57, with a linear correlation coefficient of 0.9967; I4: y = 0.87x + 2.67, with a linear correlation coefficient of 0.9861; I5: y = 0.66x + 1.92, with a linear correlation coefficient of 0.9907; and I6: y = 0.61x + 1.36, with a linear correlation coefficient of 0.9796.

[0161] Example 11

[0162] Selective detection of viscosity using fluorescent probe I1 prepared in Example 1:

[0163] The fluorescent probe I1 and the interfering analyte were added to PBS buffer (20 mM, pH 7.2) to prepare the test solution. The concentration of fluorescent probe I1 in the test solution was 10 μM, and the concentration of the interfering analyte was 100 μM. The interfering analyte was Na... + K + Ca 2+ Mg 2+ Fe 2+ Fe 3+ Cu 2+ Cu + Al 3+ Zn 2+ Cl - SO4 2- HSO3 -Each test solution contains the following fluorescent probe I1 and an interfering analyte: Glucosamine, Alanine (Ala), Threonine (Thr), Lysine (Lys), Cysteine ​​(Cys), Homocysteine ​​(Hcy), Glutathione (GSH), GGT, and Glycerol.

[0164] The fluorescence intensity of each test solution was detected using a fluorescence spectrophotometer. The excitation wavelength was set to 600 nm, and the fluorescence intensity at the emission wavelength of 720 nm was collected. The detection results of fluorescent probe I1 are as follows: Figure 7 As shown. According to Figure 7 The results showed that fluorescence enhancement only occurred in the presence of glycerol, while the fluorescence intensity of other interfering analytes did not change significantly. This demonstrates that fluorescent probe I1 exhibits good selectivity for viscosity at an excitation wavelength of 600 nm and an emission wavelength of 720 nm.

[0165] Example 12

[0166] Detection of the response of fluorescent probe I1 prepared in Example 1 to solvent polarity:

[0167] Fluorescent probe I1 was prepared into test solutions with a final concentration of 10 μM by adding it to solvents of different polarities (toluene, chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, water, PBS, and glycerol). The effect of test solutions of different polarities on the fluorescence intensity of fluorescent probe I1 was observed, and the results are as follows: Figure 8 As shown. According to Figure 8 The results showed that fluorescence enhancement only occurred in the presence of glycerol; the fluorescence intensity of other test solutions did not change significantly. This demonstrates that fluorescent probe I1, with an excitation wavelength of 600 nm and an emission wavelength of 720 nm, only responds to viscosity and not to solution polarity.

[0168] Example 13

[0169] Detection of the response of fluorescent probes I1-I6 prepared in Examples 1-6 to solution pH value:

[0170] Test solutions with different pH values ​​(pH = 4.0-10.0) were prepared for fluorescent probe I1, with a probe concentration of 10 μM. GGT with an activity of 50 U / L was added to the detection system, and the effect of different pH values ​​on the fluorescence intensity of fluorescent probes I1–I6 was observed. The results are as follows: Figure 9 As shown. (Through) Figure 9It was observed that the emission intensity at 450 nm was significantly enhanced when the pH was greater than 6, reaching its maximum at pH 7.4, after which the fluorescence intensity gradually decreased. In particular, the pH in vivo is around 7.4; therefore, pH has no effect on the application of the aforementioned fluorescent probes I1–I6.

[0171] Example 14

[0172] Cytotoxicity studies of fluorescent probes I1-I6 prepared in Examples 1-6 against different cell lines (L02, LX-2, HepG2, L929, Hela):

[0173] The biosafety of three fluorescent probes was tested using the MTT assay. Cells in logarithmic growth cycles (5 × 10⁻⁶ cells) were used. 5 Cells / well were seeded in 96-well plates and cultured at 37°C and 5% CO2. L02, LX-2, HepG2, and HeLa cells were cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h, while L929 cells were cultured in MEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. After removing the medium, fresh medium solution (serum-free) containing different concentrations (0, 2.5, 5, 10, 20, 40 μM) of fluorescent probes was added to each well. After co-incubation for 24 h, 10 μL of LTT reagent (5 mg / mL) was added to each well, and incubation continued for 4 h. After removing the medium, 100 μL of dimethyl sulfoxide was added to each well, and the cells were shaken. The OD value was measured at 490 nm using a microplate reader, and the cell viability (%) was calculated. The results are as follows: Figure 10 As shown, the cell survival rate was above 80%, proving that the six fluorescent probes I1 to I6 have low toxicity in the range of 0-40 μM and their effect on cell (survival rate) is negligible in the experiment.

[0174] The formula for calculating cell viability is:

[0175]

[0176] Example 15

[0177] The co-localization experiment of the fluorescent probe I1 prepared in Example 1 and the commercial mitochondrial probe MTG confirmed that the fluorescent probe I1 can be localized to mitochondria.

[0178] Cells in the logarithmic growth cycle (1×10⁻⁶) 6HepG2 cells / well were seeded in confocal microscopy dishes and cultured for 24 h in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) at 37 °C and 5% CO2. The culture medium was aspirated from the confocal microscopy dish, and 1 mL of fresh medium containing fluorescent probe I1 (10 μM) and commercial probe MTG (5 μM) was added, followed by incubation for 30 min. The co-stained HepG2 cells were then imaged using confocal microscopy. The excitation wavelength for the red channel of fluorescent probe I1 was 640 nm, and the fluorescence collection wavelength was 650-750 nm. The excitation wavelength for MTG was 488 nm, and the fluorescence collection wavelength was 498-550 nm. The excitation wavelength for LTG was 488 nm, and the fluorescence collection wavelength was 498-550 nm. The test results are shown below. Figure 11 As shown. From Figure 11 As can be seen, the co-localization coefficient of fluorescent probe I1 with the commercial mitochondrial localization reagent is 0.92, while the co-localization coefficient with the commercial lysosomal localization reagent is only 0.64, indicating that fluorescent probe I1 is localized in the mitochondria of the cell.

[0179] Example 16

[0180] Imaging of the fluorescent probe I1 prepared in Example 1 in live cells:

[0181] The activity of endogenous GGT and mitochondrial viscosity were monitored in L02 cells (normal human hepatocytes) using fluorescent probe I1. The specific method was as follows:

[0182] Cells in the logarithmic growth cycle (1×10⁻⁶) 6HepG2 cells / well were seeded in confocal dishes and cultured at 37°C and 5% CO2 in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. The medium was then removed, and fresh medium was added for incubation for 48 h as the control group. L02 cells were incubated for 48 h in the same amount of medium containing 1 mM sodium butyrate (NaBu) (GGT inducer) as the experimental group. L02 cells were incubated for 48 h in the same amount of medium containing 1 mM sodium butyrate (NaBu), and then the medium was aspirated, and L02 cells were incubated for 2 h in medium containing 1 mM acivicin (GGT inhibitor) as the experimental group. L02 cells were incubated for 2 h in the same amount of medium containing 10 μM acivicin (NaBu). L02 cells were incubated with nystatin (Nys) (a mitochondrial viscosity inducer) culture medium solution for 2 hours as the experimental group. The same amount of culture medium solution containing 1 mM sodium butyrate (NaBu) was added and incubated with L02 cells for 48 hours. Then, the culture medium was removed, and the same amount of culture medium solution containing 10 μM nystatin (Nys) was added and incubated with L02 cells for 2 hours as the experimental group. All five groups of cells were washed 2-3 times with PBS buffer, then incubated with 10 μM fluorescent probe I1 culture medium solution for 60 minutes, washed 2-3 times with PBS buffer, and the fluorescence in the cells was observed using a fluorescence microscope. The excitation wavelength for the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm. The excitation wavelength for the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0183] The results are as follows Figure 12 As shown, Figure 12 The fluorescence intensity of L02 cells in the GGT and viscosity channels is shown. It is evident that the addition of sodium butyrate (NaBu) significantly increased the fluorescence intensity of the blue channel, indicating that the fluorescent probe I1 reacted with NaBu-induced GGT, leading to the enhanced fluorescence intensity of the blue channel. However, when both NaBu and acivicin were added simultaneously, the fluorescence intensity of the blue channel did not change significantly, indicating that the fluorescent probe I1 did not react with GGT. These results demonstrate that the fluorescent probe I1 can detect endogenous GGT in live cells. The addition of nystatin (Nys) significantly increased the fluorescence intensity of the red channel, indicating that nystatin treatment increased mitochondrial viscosity in L02 cells, leading to a significant enhancement of the fluorescence intensity of the red channel of the fluorescent probe I1. Furthermore, when both NaBu and nystatin (Nys) were added simultaneously, the fluorescence intensities of both the blue and red channels significantly increased. These results demonstrate that the fluorescent probe I1 can detect mitochondrial viscosity in live cells.

[0184] Example 17

[0185] The fluorescent probe I1 prepared in Example 1 distinguishes between L02 cells (normal human hepatocytes), LX-2 cells (human hepatic stellate cells), and HepG2 cells:

[0186] L02 cells, LX-2 cells, and HepG2 cells (1×10⁻⁶) in logarithmic growth cycle were used to measure the growth cycle of L02 cells, LX-2 cells, and HepG2 cells. 6 Cells / wells were mixed using the direct contact method (①: L02 cells and LX-2 cells; ②: L02 cells and HepG2 cells; ③: HepG2 cells and LX-2 cells; ④: L02 cells, LX-2 cells and HepG2 cells), and seeded separately in confocal dishes. They were cultured for 24 h at 37℃ and 5% CO2 in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin). After removing the medium, the five groups of cells were washed 2-3 times with PBS buffer, and then incubated with 10 μM fluorescent probe I1 medium solution for 60 min. The cells were washed 2-3 times with PBS buffer. Fluorescence in the cells was observed using a fluorescence microscope. The excitation wavelength of the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm; the excitation wavelength of the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0187] The results are as follows Figure 13 As shown, the fluorescence intensity of the blue channel in L02 cells was significantly lower than that in LX-2 cells and HepG2 cells, and the fluorescence intensity of the red channel in LX-2 cells was significantly lower than that in LX-2 cells and HepG2 cells. The fluorescence intensity of the blue channel and the fluorescence intensity of the red channel in HepG2 cells were significantly higher than those in L02 cells and LX-2 cells. These results indicate that the fluorescent probe I1 can distinguish the three cell types by imaging the GGT activity and mitochondrial viscosity of the three cells.

[0188] Example 18

[0189] Imaging of fluorescent probe I1 prepared in Example 1 during mixed cell culture:

[0190] Real-time monitoring of endogenous GGT activity and mitochondrial viscosity during mixed cell culture was performed using fluorescent probe I1. The specific method was as follows:

[0191] Cells were co-cultured using the direct contact method. Cells in logarithmic growth cycles (1×10⁻⁶) were cultured together. 6The cells / well mixture was divided into four groups: ① L02 cells and LX-2 cells; ② L02 cells and HepG2 cells; ③ HepG2 cells and LX-2 cells; ④ L02 cells, LX-2 cells and HepG2 cells. The cells were seeded in confocal dishes and cultured at 37°C and 5% CO2 in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. The medium was removed at 0, 12, 24, 36, 48, 60 and 72 h, and a medium solution containing 20 μM Erastin (ferroptosis inducer) was added. Before laser confocal imaging, the mixed cells from four groups treated for different times were washed 2-3 times with PBS buffer, then incubated with 10 μM fluorescent probe I1 culture medium solution for 60 min, washed 2-3 times with PBS buffer, and the fluorescence in the cells was observed using a fluorescence microscope. The excitation wavelength of the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm. The excitation wavelength of the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0192] The results are as follows Figure 14 As shown, Figure 14 The figures show the fluorescence intensity of L02 and LX-2 cells, L02 and HepG2 cells, HepG2 and LX-2 cells, and L02, LX-2 and HepG2 cells, respectively. It can be seen that when L02 and LX-2 cells are co-cultured, the fluorescence intensity of the blue and red channels reaches its peak at 24 hours and then gradually decreases. When L02, LX-2 and HepG2 cells are co-cultured, the fluorescence intensity of the blue and red channels reaches its peak at 36 hours and then gradually decreases. In the other two groups of mixed cells, the fluorescence intensity of the blue and red channels shows no significant change within 72 hours. These results demonstrate that GGT and mitochondrial viscosity only increase significantly when L02 and LX-2 cells are co-cultured or when L02, LX-2 and HepG2 cells are co-cultured.

[0193] Example 19

[0194] The fluorescent probe I1 prepared in Example 1 was used for imaging during the early HCC proliferation and invasion monitoring in a cell model:

[0195] The fluorescent probe I1 was used to monitor the endogenous GGT activity and mitochondrial viscosity of HepG2 cells during continuous proliferation and invasion. The specific method was as follows:

[0196] Cells were co-cultured using the direct contact method. L02 cells, LX-2 cells, and HepG2 cells at logarithmic growth cycles (1×10⁶) were cultured together. 6Cells / well mixtures were seeded in confocal dishes and cultured at 37°C and 5% CO2 in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. The medium was removed, and cells were incubated with a medium solution containing 20 μM Erastin (ferroptosis inducer) for a specified time. The medium was removed again at 60 and 108 h, and the cells were incubated again with a medium solution containing 20 μM Erastin. Before laser confocal imaging, the mixed cells from the four treatment times were washed 2-3 times with PBS buffer, then incubated with 10 μM fluorescent probe I1 medium solution for 60 min, washed 2-3 times with PBS buffer, and the fluorescence in the cells was observed using a fluorescence microscope. The excitation wavelength of the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm; the excitation wavelength of the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0197] The results are as follows Figure 15 As shown, Figure 15 The fluorescence intensity of L02, LX-2, and HepG2 cells in a co-culture was analyzed. The fluorescence intensity of both the blue and red channels gradually increased, reaching its peak at 36 hours, and then gradually decreased. After a second induction with Erastin at 60 hours, the fluorescence intensity of both channels gradually increased, reaching its peak at 96 hours, and then gradually decreased. After a third induction with Erastin at 120 hours, the fluorescence intensity of both channels continued to increase, reaching its maximum at 156 hours and then stabilizing. These results indicate that... It can be seen that repeated stimulation of the mixed culture system when L02 cells, LX-2 cells and HepG2 cells are cultured together eventually increases the GGT and mitochondrial viscosity of HepG2 cells. This indicates that the death of L02 cells activates LX-2 cells. The activated LX-2 cells can induce the proliferation and invasion of HepG2 cells. In this process, the redox balance in the cells is out of control, reaching a high level of redox microenvironment. The significant increase in GGT and mitochondrial viscosity in HepG2 cells promotes their proliferation and invasion.

[0198] Example 20

[0199] The fluorescent probe I1 prepared in Example 1 was used for imaging during the monitoring of early HCC proliferation and invasion in a cell model:

[0200] The activity of endogenous GGT and mitochondrial viscosity in HepG2 cells were monitored in real time using fluorescent probe I1 during the inhibition of cell proliferation and invasion. The specific method was as follows:

[0201] Cells were co-cultured using the direct contact method. L02 cells, LX-2 cells, and HepG2 cells at logarithmic growth cycles (1×10⁶) were cultured together. 6 The cells / well mixture was seeded in a confocal dish and cultured at 37°C and 5% CO2 in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. The medium was removed, and a medium solution containing 20 μM Erastin (ferroptosis inducer) was added and incubated for a certain period of time. After 48 h, the medium was removed, and a medium solution containing 20 μM Erastin (ferroptosis inducer) and 1 mM glutathione (free radical scavenger) was added again for incubation. Before laser confocal imaging, the mixed cells from four groups treated for different times were washed 2-3 times with PBS buffer, then incubated with 10 μM fluorescent probe I1 culture medium solution for 60 min, washed 2-3 times with PBS buffer, and the fluorescence in the cells was observed using a fluorescence microscope. The excitation wavelength of the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm. The excitation wavelength of the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0202] The results are as follows Figure 16 As shown, Figure 16 The fluorescence intensity of L02, LX-2, and HepG2 cells in a co-culture was shown. The fluorescence intensity of both the blue and red channels gradually increased, reaching its peak at 36 hours, and then gradually decreased. After a second induction with Erastin at 48 hours, followed by glutathione treatment, the fluorescence intensity of both channels no longer increased. These results indicate that timely treatment before HepG2 cells proliferate and invade can maintain normal redox balance in the cells, thereby inhibiting their proliferation and invasion. This demonstrates that fluorescent probe I1 can monitor the early stages of HCC proliferation and invasion and its therapeutic effect.

[0203] Example 21

[0204] The fluorescent probe I1 prepared in Example 1 was used to image GGT activity and mitochondrial viscosity in a mouse model:

[0205] In tumor-bearing mice, endogenous GGT activity and mitochondrial viscosity were monitored in real time using fluorescent probe I1. The specific method was as follows:

[0206] Female BALB / c nude mice were selected as the in vivo imaging model, strictly adhering to relevant Chinese regulations on laboratory animal welfare and ethics, and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: Logarithmically growing L02 cells, LX-2 cells, and HepG2 cells were mixed to form a cell line suspension, and then... 6 Cells were injected into the right axilla of 5-week-old female BALB / c nude mice before tumor growth reached 30 mm. 3 When determining the volume, relevant experiments were conducted. The mice were fasted the day before the experiment, and 50 μM of fluorescent probe I1 was injected intratumorally. Subsequently, fluorescence in the subcutaneous tumors of mice was observed using a small animal in vivo imaging system (IVIS) at 0, 0.5, 1.5, 2, 4, 6, and 12 hours. After the experiment, the mice were euthanized. The excitation wavelength filter for the blue channel of fluorescent probe I1 was 420 nm, and the fluorescence collection wavelength filter was 520 nm. The excitation wavelength filter for the red channel was 640 nm, and the fluorescence collection wavelength filter was 720 nm.

[0207] The results are as follows Figure 17 As shown, the fluorescence intensity of the blue channel increases with time, reaching its strongest at 1.5h, then decreases from 4h to its lowest at 12h. Similarly, the fluorescence intensity of the red channel increases with time, reaching its strongest at 1.5h, then decreases from 4h to its lowest at 12h. This indicates that fluorescent probe I1 can image GGT activity and mitochondrial viscosity in vivo.

[0208] Example 22

[0209] The fluorescent probe I1 prepared in Example 1 was used to image the early HCC proliferation and invasion monitoring process in a mouse model:

[0210] The activity of endogenous GGT and mitochondrial viscosity of HepG2 cells during proliferation and invasion were monitored in real time using fluorescent probe I1 in tumor-bearing mice. The specific method was as follows:

[0211] Female BALB / c nude mice were selected as the in vivo imaging model, strictly adhering to relevant Chinese regulations on laboratory animal welfare and ethics, and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: Logarithmically growing L02 cells, LX-2 cells, and HepG2 cells were mixed to form a cell line suspension, and then... 6 Cells were injected into the right axilla of 5-week-old female BALB / c nude mice before tumor growth reached 30 mm. 3When determining the volume, relevant experiments were conducted. The mice were fasted the day before the experiment. 20 μM Erastin was injected intratumorally at specific times, and again at 60 and 108 hours. Before each in vivo imaging, 50 μM of fluorescent probe I1 was injected intratumorally. 1.5 hours later, fluorescence in the subcutaneous tumors of mice was observed using a small animal in vivo imaging system (IVIS). The excitation wavelength for the blue channel of fluorescent probe I1 was 420 nm, and the fluorescence collection wavelength was 520 nm; the excitation wavelength for the red channel was 640 nm, and the fluorescence collection wavelength was 720 nm.

[0212] The results are as follows Figure 18 As shown, the fluorescence intensity of the GGT channel and viscosity channel fluctuated with repeated Erastin induction, eventually ceasing to decrease, exhibiting the same trend as in cell experiments. This indicates that fluorescent probe I1 can perform real-time imaging of GGT and mitochondrial viscosity during the proliferation and invasion of HepG2 cells in vivo.

[0213] Example 23

[0214] The fluorescent probe I1 prepared in Example 1 was used for imaging during the monitoring of early HCC proliferation and invasion in a mouse model:

[0215] In tumor-bearing mice, the activity of endogenous GGT and mitochondrial viscosity during the inhibition of HepG2 cell proliferation and invasion were monitored in real time using fluorescent probe I1. The specific method was as follows:

[0216] Female BALB / c nude mice were selected as the in vivo imaging model, strictly adhering to relevant Chinese regulations on laboratory animal welfare and ethics, and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: Logarithmically growing L02 cells, LX-2 cells, and HepG2 cells were mixed to form a cell line suspension, and then... 6 Cells were injected into the right axilla of 5-week-old female BALB / c nude mice before tumor growth reached 30 mm. 3 When determining the volume, relevant experiments were conducted. The mice were fasted the day before the experiment. 20 μM Erastin was injected intratumorally at specific times. At 60 and 108 hours, 20 μM Erastin and 1 mM glutathione were injected intratumorally again. Before each in vivo imaging, 50 μM of fluorescent probe I1 was injected intratumorally. 1.5 hours later, fluorescence in the subcutaneous tumors of mice was observed using a small animal in vivo imaging system (IVIS). The excitation wavelength for the blue channel of fluorescent probe I1 was 420 nm, and the fluorescence collection wavelength was 520 nm; the excitation wavelength for the red channel was 640 nm, and the fluorescence collection wavelength was 720 nm.

[0217] The results are as follows Figure 19As shown, the fluorescence intensity of the blue and red channels gradually increased, reaching its peak at 36 hours, and then gradually decreased. After a second induction with Erastin at 48 hours, followed by glutathione treatment, the fluorescence intensity of the blue and red channels no longer increased. These results indicate that timely treatment before HepG2 cells proliferate and invade can maintain normal redox balance in vivo, thereby inhibiting their proliferation and invasion. This demonstrates that fluorescent probe I1 can monitor the early stages of HCC proliferation and invasion in vivo and its therapeutic effect.

[0218] Example 24

[0219] The fluorescent probe I1 prepared in Example 1 was used to distinguish between liver cancer and adjacent non-cancerous tissue in clinical samples:

[0220] The fluorescent probe I1 was used to image GGT and mitochondrial viscosity in clinical samples, specifically in the following manner:

[0221] Clinical liver tissue samples were collected from the First Affiliated Hospital of Gannan Medical University. All experiments were conducted in accordance with the regulations of the Medical Ethics Committee of the First Affiliated Hospital of Gannan Medical University, and informed written consent was obtained from all participants. Clinical tissue samples were embedded in OCT gel for fixation, prepared into frozen sections using a cryostat, and stained with fluorescent probe I1 (20 μM) for fluorescent staining, immunofluorescence staining, and H&E staining. Fluorescence in cells was observed using a fluorescence microscope. The excitation wavelength of the blue channel of fluorescent probe I1 was 405 nm, and the fluorescence collection wavelength was 420-500 nm; the excitation wavelength of the red channel was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0222] The results are as follows Figure 20 As shown, the fluorescence of cancerous tissue in both the blue and red channels is higher than that of adjacent normal tissue, indicating that the GGT and mitochondrial viscosity of cancerous tissue are higher than those of adjacent normal tissue. Furthermore, the immunofluorescence results are consistent with those obtained by fluorescent probe I1 and are completely consistent with the H&E analysis results. In summary, these results demonstrate the potential of fluorescent probe I1 to distinguish between cancerous and adjacent normal tissue.

[0223] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A γ-glutamyltransferase and mitochondria viscosity dual-responsive fluorescent probe, characterized in that, It has a structural formula as shown in formula I: Wherein, n is an integer of 4-16; R1 is selected from hydrogen or methyl, R2 and R3 are both hydrogen; R4 is halogen or hydrogen, R5 is hydrogen; R6 is selected from L3 or L8; Y is selected from halogen ion.

2. The γ-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe according to claim 1, characterized in that, The dual-response fluorescent probe is the following compound:

3. The method for preparing a γ-glutamyltransferase and mitochondria viscosity dual-responsive fluorescent probe according to any one of claims 1-2, characterized in that, The preparation method comprises the following steps: 1) Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol and carbodiimide hydrochloride are dissolved in organic solvent A, and the reaction is carried out under ice bath condition to obtain compound Z1; 2) Compound Z1 is dissolved in organic solvent A under ice bath condition, and phosphorus tribromide is slowly added dropwise, and the reaction is carried out under ice bath condition to obtain compound Z2; 3) Compound Z3 and compound Z4 are dissolved in dry organic solvent B, and the reflux reaction is carried out under nitrogen protection to obtain compound Z5; 4) Compound Z5 and Z6 are dissolved in organic solvent B, and piperidine is added dropwise, and the reflux reaction is carried out to obtain compound Z7; 5) Compound Z8, Mischler acid and potassium carbonate are dissolved in organic solvent C, and the reaction is carried out at room temperature to obtain compound Z9; 6) Compound Z9, N,N'-dicyclohexyl carbodiimide and 1-hydroxybenzotriazole are dissolved in dry organic solvent D, and then 1-Boc-piperazine is added, and the reaction is carried out at room temperature to obtain compound Z10; 7) Compound Z10 is dissolved in dry organic solvent A, and trifluoroacetic acid is added under ice bath condition, and the reaction is carried out under ice bath condition to obtain compound Z11; 8) Compound Z7, N,N'-dicyclohexyl carbodiimide and 1-hydroxybenzotriazole are dissolved in dry organic solvent D, and then compound Z11 is added, and the reaction is carried out at room temperature under nitrogen protection to obtain compound Z12; 9) Compound Z2, compound Z12 and potassium carbonate are dissolved in dry organic solvent B, and the reaction is carried out at room temperature under nitrogen protection to obtain compound Z13; 10) Compound Z13 is dissolved in dry organic solvent A, and trifluoroacetic acid is added under ice bath condition, and the reaction is carried out under ice bath condition to obtain the compound shown in formula I; The organic solvent A is at least one of dichloromethane, tetrahydrofuran and diethyl ether, the organic solvent B is at least one of acetonitrile, methanol and ethanol, the organic solvent C is at least one of water, methanol and ethanol, and the organic solvent D is at least one of N,N-dimethylformamide, dimethyl sulfoxide and acetonitrile.

4. The production method according to claim 3, characterized by, In step 1), the molar ratio of Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol and carbodiimide hydrochloride is 1:1-3:1-5; In step 2), the molar ratio of the compound Z1 and phosphorus tribromide is 1:1-10; In step 3), the molar ratio of the compound Z3 and compound Z4 is 1-3:1; In step 4), the molar ratio of the compound Z5 and compound Z6 is 1:0.5-2; In step 5), the molar ratio of Z8, Mischler acid and potassium carbonate is 1:1-3; In step 6), the molar ratio of the compound Z9, N,N'-dicyclohexyl carbodiimide, 1-hydroxybenzotriazole and 1-Boc-piperazine is 1:1-5:1-5:1-5; In step 7), the molar ratio of the compound Z10 and trifluoroacetic acid is 1:1-20; In step 8), the molar ratio of the compound Z7, N,N'-dicyclohexyl carbodiimide, 1-hydroxybenzotriazole and the compound Z11 is 1:1-5:1-5:1-5; In step 9), the molar ratio of the compound Z2, the compound Z12 and potassium carbonate is 1:1-3:1-3; In step 10), the molar ratio of the compound Z13 and trifluoroacetic acid is 1:1-20.

5. The use of the γ-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe according to any one of claims 1-2 in the preparation of a detection reagent for detecting the activity of γ-glutamyltransferase and mitochondrial viscosity.

6. Use according to claim 5, characterized in that, The detection range of the activity of γ-glutamyltransferase in the detection system is 0-70 U / L, and the detection range of mitochondrial viscosity is 0.5-1099 cP.

7. The use of the γ-glutamyltransferase and mitochondrial viscosity dual-responsive fluorescent probe according to any one of claims 1-2 in the preparation of a detection reagent for real-time visual monitoring of early liver cancer cell proliferation and invasion.

8. Use according to claim 7, characterized in that, The dual-responsive fluorescent probe in the detection reagent takes coumarin and cyanine dye as the parent fluorescent group, and the emission wavelength of the fluorescent probe is 450 nm and 720 nm.

Images