Gamma-glutamyltransferase and mitochondrial viscosity dual-response fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a gamma-glutamyltransferase and mitochondrial viscosity dual-response fluorescent probe as well as a preparation method and application thereof. The structure of the gamma-glutamyltransferase and mitochondrial viscosity dual-response fluorescent probe is as shown in a formula I in the specification. According to the fluorescent probe provided by the invention, coumarin and cyanine dyes are taken as two fluorophore parents with different emission, gamma-glutamyltransferase and viscosity double-target recognition sites are constructed through two strategies of hydroxyl protection-deprotection and molecular rotor introduction, and the gamma-glutamyltransferase and viscosity double-target recognition sites are identified by utilizing electronegativity of mitochondrial inner membranes. By introducing lipophilic cationic molecules to realize mitochondrial targeting and specific response to mitochondrial viscosity, the probe has the advantages of spectrum crosstalk resistance, mitochondrial targeting and simultaneous imaging of two analytes, and can effectively monitor proliferation and invasion of liver cancer cells in the process that precancerous lesions of the liver deteriorate into early hepatocellular carcinoma. The fluorescent probe disclosed by the invention can be applied to the technical field of medicines such as diagnosis of patients with precancerous lesions of the liver, and creates conditions for the gamma-glutamyltransferase and mitochondrial viscosity dual-response fluorescent probe to diagnose precancerous lesions of the liver. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical analysis, and specifically relates to a gamma-glutamyl transferase and mitochondrial viscosity dual-response fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the fastest growing and most invasive malignant tumors. Most HCC patients are diagnosed only in the middle and late stages and lose the opportunity for treatment at the stage of liver precancerous lesions. Since liver precancerous lesions are controllable and reversible, if they can be detected and effectively treated before they progress to early HCC, the morbidity and mortality of early HCC patients can be significantly reduced. Therefore, there is an urgent need to develop a non-invasive and reliable method for detecting liver precancerous lesions to prevent the formation of early HCC.

[0003] At present, the gold standard for diagnosing liver precancerous lesions is histopathological examination, but this method is invasive, has a high false negative rate, and has the risk of cancer metastasis. Although there are currently a variety of diagnostic techniques, such as blood biochemical index detection, ultrasound, magnetic resonance imaging (MRI) and computed tomography (CT), the diagnosis of liver precancerous lesions still lacks sufficient sensitivity and specificity. In recent years, fluorescent probes have played an important role in the diagnosis of liver-related diseases due to their many excellent characteristics such as high sensitivity, high spatiotemporal resolution and real-time imaging capabilities. Fluorescent probes that rely on biomarkers are more likely to achieve the diagnosis of liver precancerous lesions. The process of liver precancerous lesions developing into early HCC is relatively complex. Although many related markers have been reported, it is still impossible to achieve accurate diagnosis of this development process through a single marker. Therefore, the development of new fluorescent probes that respond to multiple markers simultaneously is of great significance for the diagnosis and regulation of liver precancerous lesions.

[0004] In the precancerous stage of liver, chronic liver damage triggers the activation of hepatic stellate cells, destroys the cellular redox balance, and thus promotes the malignant transformation of precancerous liver lesions into early HCC. At this stage, activated hepatic stellate cells (aHSC) promote angiogenesis, epithelial-mesenchymal transition (EMT) and immune escape by secreting cytokines and extracellular matrix components, inducing the proliferation and invasion of liver cancer cells in precancerous liver lesions. In this process, liver cancer cells produce a large amount of reactive oxygen species (ROS), and the viscosity of mitochondria increases with the excessive production of ROS. At the same time, liver cancer cells upregulate GGT to decompose extracellular glutathione (GSH), and the provided cysteine ​​(Cys) (the rate-limiting substrate for intracellular GSH biosynthesis) is transported into the cell to play an antioxidant role, maintaining the redox balance in liver cancer cells and inhibiting the proliferation and invasion of liver cancer cells. When the ROS level exceeds the cellular antioxidant capacity, the balance between oxidation and antioxidant is broken, and oxidative stress occurs, which provides more favorable conditions for the activation of hepatic stellate cells and the proliferation and invasion of liver cancer cells, and accelerates the speed of malignant transformation of liver precancerous lesions into early HCC. The applicant found that the above process is accompanied by the upregulation of GGT and the increase of mitochondrial viscosity. If the increase of GGT and mitochondrial viscosity can be monitored in real time, it will help to track the process of liver precancerous lesions developing into early HCC in real time and effectively regulate it, so as to prevent the development of early HCC in time and inhibit the occurrence of early HCC from the source. In view of the fact that the dual-target detection of single-molecule fluorescent probes can avoid a series of problems such as signal crosstalk, positioning and metabolic differences caused by the combination of multiple fluorescent probes, the development of fluorescent probes that can simultaneously monitor GGT and mitochondrial viscosity to achieve the diagnosis of liver precancerous lesions is a technical problem that needs to be solved at this stage. Summary of the invention

[0005] The purpose of the present invention is to provide a dual-response fluorescent probe of γ-glutamyl transferase and mitochondrial viscosity and its preparation method and application, wherein the dual-response fluorescent probe can simultaneously perform fluorescent imaging of γ-glutamyl transferase and mitochondrial viscosity, and is used to effectively monitor the stage of liver precancerous lesions, thereby being used for the diagnosis and regulation of patients with liver precancerous lesions. The dual-response fluorescent probe provided by the present invention has two emission wavelengths and displays blue (λ em =450nm)-red(λ em =720nm) dual-channel emission, the two signals do not interfere with each other, and the diagnosis of liver precancerous lesions can be achieved by real-time monitoring of the dynamic changes of γ-glutamyl transferase and mitochondrial viscosity in cells and living bodies.

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

[0007] In a first aspect of the present invention, a dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity is provided, having a structural formula as shown in Formula I:

[0008]

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

[0010] R1, R2 and R3 are each independently selected from any one of hydrogen, an alkyl group having 1 to 8 carbon atoms, a halogen, a methoxy group, a trifluoromethyl group, an ester group or an amide group;

[0011] R4 and R5 are substituents at undetermined positions of the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyl, alkylsulfonic acid group having 1-18 carbon atoms, ester group having 1-18 carbon atoms, amide group having 1-18 carbon atoms, 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 anions or organic anions.

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

[0016] 1) dissolving Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol and carbodiimide hydrochloride in an organic solvent A, and reacting in an ice bath to obtain compound Z1;

[0017]

[0018] 2) Compound Z1 is dissolved in organic solvent A under ice bath conditions, phosphorus tribromide is slowly added dropwise, and the mixture is reacted under ice bath conditions to obtain compound Z2;

[0019]

[0020] 3) dissolving compound Z3 and compound Z4 in a dry organic solvent B, and performing a reflux reaction under nitrogen protection to obtain compound Z5;

[0021]

[0022] 4) Compounds Z5 and Z6 are dissolved in an organic solvent B, piperidine is added dropwise, and the mixture is refluxed to obtain compound Z7;

[0023]

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

[0025]

[0026] 6) Compound Z9, N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) are dissolved in a dry organic solvent D, 1-Boc-piperazine is added, and the mixture is reacted at room temperature to obtain compound Z10;

[0027]

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

[0029]

[0030] 8) Compound Z7, N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) are dissolved in a dry organic solvent D, and compound Z11 is added, and the reaction is carried out at room temperature under nitrogen protection to obtain compound Z12;

[0031]

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

[0033]

[0034] 10) Compound Z13 is dissolved in dry organic solvent A, trifluoroacetic acid is added under ice bath conditions, and the mixture is reacted under ice bath conditions to obtain a compound of 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] Further, 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 the 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 the 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 the compound Z13 to trifluoroacetic acid is 1:(1-20), preferably 1:(1-10).

[0047] The 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 has high selectivity and high sensitivity to γ-glutamyl transferase and mitochondrial viscosity, and emits fluorescence of different intensities according to the γ-glutamyl transferase activity and viscosity of the detection system. There is a positive correlation between the fluorescence intensity emitted at 450 nm and the activity of GGT, and there is a positive correlation between the fluorescence intensity emitted at 720 nm and the viscosity. The dual-response fluorescent probe for GGT and mitochondrial viscosity of the present invention preferably has an activity of 0-70 U / L for GGT in the detection system, more preferably 5-50 U / L, and preferably has a viscosity of 0.5-1099 cP in the detection system, 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 system, 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] The fourth aspect of the present invention provides the use of the GGT and mitochondrial viscosity dual-response fluorescent probe in the preparation of a detection reagent for real-time visual monitoring of early liver cancer cell proliferation and invasion.

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

[0052] (1) The known GGT activity, viscosity standard sample, GGT and mitochondrial viscosity dual-response fluorescent probe are dissolved in a buffer solution, the fluorescence intensity of the system is measured, and a standard curve between GGT activity and system fluorescence intensity and a standard curve between viscosity and system fluorescence intensity are established respectively;

[0053] (2) The cells to be analyzed are mixed and cultured to induce liver cancer cell proliferation and invasion, and a dual-response fluorescent probe solution of GGT and mitochondrial viscosity is added for incubation and imaging. The proliferation and invasion of liver cancer cells are monitored in real time by observing the dynamic fluctuations of GGT activity and mitochondrial viscosity. During the increase of GGT activity and mitochondrial viscosity, regulation is given to monitor the regulatory effect;

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

[0055] (4) Imaging of GGT and mitochondrial viscosity in liver cancer and adjacent tissues in clinical samples can achieve rapid and accurate differentiation of liver cancer tissues from adjacent tissues, thereby enabling accurate diagnosis of liver precancerous lesions in adjacent tissues.

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

[0057] In the above technical solution, the concentration of 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 the present invention are:

[0059] 1. A new γ-glutamyl transferase and mitochondrial viscosity dual-response fluorescent probe provided by the present invention uses coumarin and cyanine dyes as two different emission fluorophore matrices, and the fluorescent probe has dual emission wavelengths, and no spectral crosstalk, and at emission wavelengths of 450nm and 720nm, it has high selectivity and high sensitivity to GGT activity and mitochondrial viscosity, respectively. The emission wavelengths of the two fluorophores differ by 270nm, thereby inhibiting fluorescence resonance energy transfer (FRET), achieving complete independence of the two signals, and constructing GGT and viscosity dual-target recognition sites through the protection-deprotection of hydroxyl groups and the introduction of "molecular rotors". The negative charge of the mitochondrial inner membrane is utilized to introduce lipophilic cationic molecules to achieve mitochondrial targeting, and the positioning ability of the mitochondrial inner membrane is enhanced by regulating the length of the carbon chain, and the specific response to mitochondrial viscosity. The fluorescent probe of the present invention has the advantages of anti-spectral crosstalk, mitochondrial targeting and simultaneous imaging of two analytes, creating conditions for the diagnosis of GGT and mitochondrial viscosity dual-response fluorescent probes in liver precancerous lesions.

[0060] 2. The γ-glutamyl transferase and mitochondrial viscosity dual-response fluorescent probe provided by the present invention utilizes the negative charge of the mitochondrial inner membrane to introduce lipophilic cationic molecules to achieve mitochondrial targeting, and enhances its localization ability in the mitochondrial inner membrane by regulating the length of the carbon chain. The co-localization coefficients of the above-mentioned GGT and mitochondrial viscosity dual-response 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-response fluorescent probe has excellent targeting performance for mitochondria.

[0061] 3. The present invention also provides an application of rapid real-time visual monitoring of liver precancerous lesions using the dual-response probe. The application uses the GGT and mitochondrial viscosity dual-response fluorescent probe of the present invention to perform real-time visual monitoring of GGT and mitochondrial viscosity of liver cancer cells during the deterioration of liver precancerous lesions into early HCC. By monitoring the dynamic changes of GGT and mitochondrial viscosity in subcutaneous tumors of mice, rapid real-time visual monitoring is achieved, and appropriate regulation is given in time to achieve real-time monitoring of the diagnosis and regulation effects of liver precancerous lesions.

[0062] 4. The present invention also provides an application of using the dual-response probe to quickly and accurately visualize precancerous lesion tissue in clinical samples. The application uses the GGT and mitochondrial viscosity dual-response fluorescent probe of the present invention to qualitatively and quantitatively analyze the GGT and mitochondrial viscosity in liver cancer and adjacent tissues in clinical samples. By imaging the 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 achieving accurate diagnosis of precancerous lesion tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0064] Figure 1 This is a fluorescence spectrum diagram of the reaction time of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention and GTT.

[0065] Figure 2 This is a fluorescence spectrum diagram of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention after reacting with GTTs of different activities.

[0066] Figure 3 This is a linear relationship diagram between the fluorescent probes Ⅰ1~I6 prepared in Example 1 of the present invention and the activity of GGT.

[0067] Figure 4 The fluorescence change bar graph of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention when different interfering analytes are added (the emission wavelength is 450nm).

[0068] Figure 5 This is a fluorescence spectrum diagram of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention in response to different viscosities.

[0069] Figure 6 This is a linear relationship diagram of the responses of fluorescent probes I1-I6 prepared in Example 1 of the present invention to different viscosities.

[0070] Figure 7 The fluorescence change bar graph of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention when different interfering analytes are added (emission wavelength is 720 nm).

[0071] Figure 8 This is a bar graph of the fluorescence changes of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention in solvents of different polarities (emission wavelength is 720 nm).

[0072] Fig. 9 This is a line graph showing the responses of the fluorescent probes I1 to I6 prepared in Example 1 of the present invention to different pH values.

[0073] Fig.10 These are the cytotoxicity test results of the fluorescent probes I1 to I6 prepared in Example 1 of the present invention.

[0074] Fig.11 This is the co-localization experimental verification result of the fluorescent probe Ⅰ1 prepared in Example 1 of the present invention.

[0075] Fig.12 The fluorescent probe I1 prepared in Example 1 of the present invention is used to image the test results of endogenous GGT and mitochondrial adhesion in cells.

[0076] Fig.13 The fluorescent probe I1 prepared in Example 1 of the present invention is used to image L02 cells, LX-2 cells and HepG2 cells.

[0077] Fig.14 This is the real-time imaging result of using the fluorescent probe I1 prepared in Example 1 of the present invention for mixed culture cells.

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

[0079] Fig.16 The real-time imaging result of the fluorescent probe I1 prepared in Example 1 of the present invention being used to image and inhibit the proliferation and invasion of liver cancer cells.

[0080] Fig.17 The fluorescent probe I1 prepared in Example 1 of the present invention is used to image the test results of the change of fluorescence intensity of mouse subcutaneous tumor over time.

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

[0082] Fig.19 The fluorescent probe I1 prepared in Example 1 of the present invention is used to image the real-time detection results of the process of inhibiting the proliferation and invasion of subcutaneous tumors in mice.

[0083] Fig. 20 The fluorescent probe I1 prepared in Example 1 of the present invention is used to image the test results of cancer tissue and adjacent tissue in clinical samples. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

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

[0086] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

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

[0088] The following examples provide a method for preparing a dual-responsive fluorescent probe I1 for GGT and mitochondrial viscosity having a structural formula as 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 for 12 hours under ice bath conditions. After the solvent was spin-dried, the pure compound Z1 was separated 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), and the eluent of the column chromatography was petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate was (1-3):1.

[0090] 2) Synthesis of compound Z2: Dissolve compound Z1 in dry tetrahydrofuran, drop phosphorus tribromide (PBr3) in an ice bath, and react for 2 hours in an ice bath to complete the reaction. Extract with dichloromethane and water three times, add anhydrous sodium sulfate to the organic layer and dry it, filter and evaporate to obtain a crude product. After the solvent is dried, column chromatography is performed to obtain a pure compound Z2. The molar ratio of compound Z1 to phosphorus tribromide (PBr3) is 1:(1-3), preferably 1:(1-2), and the eluent for column chromatography is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is (1-3):1.

[0091] 3) Synthesis of compound Z5: Compound Z3 and compound Z4 were dissolved in dry acetonitrile and refluxed for 12 hours under nitrogen protection. After cooling to room temperature, the reaction solution was poured into ether to precipitate gray crystals, which were filtered to obtain a crude product. The crude product was washed with petroleum ether in small amounts for multiple times until the solid was off-white, thereby obtaining a pure compound Z5. The molar ratio of the compound Z3 to Z4 is (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 mixture was refluxed under nitrogen protection for 14 hours. After the solvent was dried, the pure compound Z7 was separated by column chromatography. The molar ratio of the compounds Z5 and Z6 was 1:(1-1.5), preferably 1:(1-1.2), and the eluent for column chromatography was dichloromethane and methanol, and the volume ratio of dichloromethane and methanol was (35-60):1.

[0093] 5) Synthesis of compound Z9: Compound Z8, Michaelis acid and potassium carbonate were dissolved in deionized water, reacted at room temperature for 18 hours, and filtered to obtain a crude product. The crude product was washed with icy ethanol in small amounts for multiple times until the solid was off-white, thereby obtaining a 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, reacted at room temperature for 0.5h, and then 1-Boc-piperazine was added. The reaction was carried out at room temperature for 24h under nitrogen protection. After the solvent was dried, it was dissolved in acetonitrile and the solid was filtered out. After the filtrate was dried, the pure compound Z10-1 was obtained by column chromatography. The molar ratio of the compound Z9, N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) is 1:(1-3):(1-3), preferably 1:(1-2):(1-2). The eluent for column chromatography is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (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 the filtrate was dried, the pure compound Z11 was separated 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, and the volume ratio of dichloromethane to methanol was (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, reacted at room temperature for 0.5h, and then compound Z11 was added. The reaction was carried out at room temperature for 24h under nitrogen protection. After the solvent was dried, it was dissolved in acetonitrile and the solid was filtered out. After the filtrate was dried, the pure compound Z12 was separated by column chromatography. The molar ratio of compound Z7, N,N'-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt) and compound Z11 is 1:(1-3):(1-3):(1-3), preferably 1:(1-2):(1-2):(1-2). The eluent for column chromatography is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is (15-30):1.

[0097] 9) Synthesis of compound Z13: Compound Z2, compound Z12 and potassium carbonate were dissolved in dry acetonitrile and reacted at room temperature for 48 hours under nitrogen protection. The mixture was extracted with ethyl acetate and water three times, anhydrous sodium sulfate was added to the organic layer for drying, and the crude product was obtained by filtration and rotary evaporation. After the solvent was dried, the 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, and the volume ratio of dichloromethane and methanol was (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 in an ice bath under nitrogen protection for 4 hours. Saturated sodium bicarbonate solution and dichloromethane were added to the reaction solution, and the precipitated solid was collected to obtain a pure compound I. The molar ratio of the compound Z13 to trifluoroacetic acid is 1:(1-20), preferably 1:(1-10).

[0099] It should be noted that compounds Ⅰ1 (n=4), Ⅰ2 (n=10), and Ⅰ3 (n=16) described in some of the following examples refer to compounds (compound Ⅰ) shown in formula Ⅰ in the above synthesis route, wherein n in the structural formula is n=4; n=10 and n=16. Similarly, other similar expressions such as compounds described in some of the following examples can be used by analogy for other compounds.

[0100] Example 1

[0101] Preparation of GGT and mitochondrial viscosity dual-response 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 for 12 h in an ice bath. The solvent was removed under reduced pressure, extracted three times with dichloromethane and water, and the organic layer was dried over anhydrous sodium sulfate. It was purified by silica gel column chromatography using ethyl acetate / petroleum ether (volume ratio of 2:1) 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, and the reaction was completed under ice bath for 2 h. The mixture was extracted with dichloromethane and water three times, anhydrous sodium sulfate was added to the organic layer for drying, and the crude product was obtained by filtration and rotary evaporation. Compound Z2-1 was obtained after purification by silica gel column chromatography using ethyl acetate / petroleum ether (volume ratio of 5:1) 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] Dissolve 6-bromohexanoic 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) in dry acetonitrile (100 mL) and reflux for 12 h under nitrogen protection. Cool to room temperature, pour the reaction solution into ether (300 mL), precipitate gray crystals, and filter to obtain a crude product. Wash the crude product with petroleum ether in small amounts several times until the solid is off-white, and obtain the 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 mixture was refluxed for 14 h under nitrogen protection. After the solvent was dried, it was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio of 50:1) 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] Dissolve 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) in deionized water (50 mL), react at room temperature for 18 h, and filter to obtain a crude product. Wash the crude product with ice ethanol in small amounts several times until the solid is off-white, and the pure compound Z9-1 is obtained. 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), reacted at room temperature for 0.5 h, and then 1-Boc-piperazine (0.37 g, 2 mmol) was added. The reaction was carried out at room temperature for 24 h under nitrogen protection. After the solvent was dried, it was dissolved in acetonitrile (20 mL) and the solid was filtered out. After the filtrate was dried, it was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio of 10:1) as 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 the filtrate was dried, it was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio of 4:1) as the eluent to obtain compound Z11-1. 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) (1.12g, 2mmol), N, N'-dicyclohexylcarbodiimide (DCC) (0.82g, 4mmol) and 1-hydroxybenzotriazole (HOBt) (0.82g, 4mmol) were dissolved in dry N, N-dimethylformamide, reacted at room temperature for 0.5h, and then compound Z11-1 (0.55g, 2mmol) was added. The reaction was carried out at room temperature for 24h under nitrogen protection. After the solvent was dried, it was dissolved in acetonitrile and the solid was filtered out. After the filtrate was dried, it was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio of 20:1) as 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, anhydrous sodium sulfate was added to the organic layer and dried, and the crude product was filtered and evaporated. After the solvent was dried, it was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio of 15:1) 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] Dissolve compound Z13-1 (1.21 g, 1 mmol) in dry dichloromethane (2 mL), add trifluoroacetic acid (2 mL) under ice bath conditions, react in an ice bath under nitrogen protection for 4 h. Add saturated sodium bicarbonate solution and dichloromethane to the reaction solution, collect the precipitated solid, and 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-response fluorescent probe Ⅰ2 (n=10):

[0125] The pure fluorescent probe I2 was prepared by similar steps as 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) is replaced by 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) was 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) was replaced with compound Z7-2 (n=10) to obtain compound Z12-2, HRMS (ESI) m / zcalcd for C 52 H 61 N4O5 + (M) + 821.4636, found 821.4630; In step 9), compound Z12-1 (n = 4) was 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, found 1211.6796; In step 10), compound Z13-1 (n=4) was replaced with compound Z13-2 (n=10).

[0127]

[0128] Example 3

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

[0130] The fluorescent probe I3 pure product was prepared by the similar steps as 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 by 18-bromohexanoic acid (Z3-3) to obtain compound Z5-3. HRMS (ESI) m / z calculated for C 33 H 50 NO2 + (M) + 492.3836, found 492.3830; In step 4), compound Z5-1 was 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 was 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 was 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 was replaced with compound Z13-3.

[0132]

[0133] Example 4

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

[0135] The pure fluorescent probe I4 was prepared by the steps similar to those 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 by 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) was 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 was replaced with compound Z10-4 to obtain compound Z11-4, HRMS (ESI) m / z calculated for C 15 H 17 N2O4 + (M+H) +289.1183, found 289.1182; in step 8), compound Z11-1 was 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 was replaced with compound Z12-4 to obtain compound Z13-4, HRMS (ESI) m / z calculated for C 68 H 81 N6O 10 + (M) + 1141.6009, found 1141.6008; In step 10), compound Z13-1 was replaced with compound Z13-4.

[0137]

[0138] Example 5

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

[0140] The pure fluorescent probe I5 was prepared by similar steps as 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) is replaced by 7-bromo-1,1,2-trimethyl-1H-benzo[e]indole (Z4-5) to obtain compound Z5-5. HRMS (ESI) m / z calculated for C 21 H 25 BrNO2 + (M) + 402.1063, found 402.1069; in step 4), compound Z5-1 was replaced with compound Z5-5 to obtain compound Z7-5, HRMS (ESI) m / z calcd for C 32 H 36 BrN2O2 + (M) + 559.1955, found559.1955; in step 8), compound Z7-1 was 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 was 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), compound Z13-1 was replaced with compound Z13-5.

[0142]

[0143] Example 6

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

[0145] The pure fluorescent probe I6 was prepared by similar steps as 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 by 3-(anthracen-9-yl)acrolein (Z6-6) to obtain compound Z7-6. HRMS (ESI) m / z calculated for C 38 H 36 NO2 + (M) + 538.2741, found 538.2744; in step 8), compound Z7-1 was 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 was 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, found 1184.5741; In step 10), compound Z13-1 was replaced with compound Z13-6.

[0147]

[0148] Example 7

[0149] Determination of the response time of the fluorescent probe Ⅰ1 prepared in Example 1 to GGT activity:

[0150] The fluorescence emission spectrum of the fluorescent probe Ⅰ1 after reacting with GGT was tested by fluorescence spectrometer. The test results are shown in Figure 1 . Using a fluorescence spectrophotometer, set the excitation wavelength to 405nm, the scanning wavelength range to 425nm-600nm, and the buffer solution used was PBS (20mM, pH=7.2) buffer. The probe concentration was 10μM, and GGT with an activity of 50U / L was added to the detection system to observe its effect on the fluorescence intensity of the fluorescent probe Ⅰ1 at different times. The results are as follows Figure 1 As shown. Figure 1 It can be observed that when the time increases from 0 min to 60 min, the emission intensity at 450 nm gradually increases with time, and the fluorescence emission intensity reaches the maximum at 60 min.

[0151] Example 8

[0152] The responses of the fluorescent probes I1 to I6 prepared in Examples 1-6 to the determination of different GGT activities were used to establish a linear relationship between GGT activity and fluorescence intensity:

[0153] The fluorescence emission spectrum of the fluorescent probe Ⅰ1 after reacting with GGT was tested by fluorescence spectrometer. The test results are shown in Figure 2 Using a fluorescence spectrophotometer, the excitation wavelength was set to 405nm, the scanning wavelength range was 425nm-600nm, and the buffer solution used was PBS (20mM, pH=7.2) buffer. The probe concentration was 10μM, and GGT with different activities was added to the detection system to observe its effect on the fluorescence intensity of fluorescent probes Ⅰ1~I6. The results are as follows Figure 3 As shown. Figure 2 It can be observed that when the activity of GGT increases successively from 0, 5, 10, 15, 20, 25, 30, 40, 50, 70, and 90 U / L, the emission intensity at 450 nm gradually increases with the increase of GGT activity, and the emission intensity has a good linear relationship with GGT activity between 0 and 50 U / L, such as Figure 3 As shown, the linear equation of I1 is: y=6353x+70684, and the linear correlation coefficient is 0.9958; the linear equation of I2 is: y=7224x+34428, and the linear correlation coefficient is 0.9903; the linear equation of I3 is: y=7498x+44797, and the linear correlation coefficient is 0.9850; the linear equation of I4 is: y=5763x+23212, and the linear correlation coefficient is 0.9931; the linear equation of I5 is: y=6145x+61079, and the linear correlation coefficient is 0.9952; the linear equation of I6 is: y=6394x+58562, and the linear correlation coefficient is 0.9959.

[0154] Example 9

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

[0156] Fluorescent probe Ⅰ1 and interfering analyte were added to PBS buffer solution (20mM, pH=7.2) to prepare test solution. The concentration of fluorescent probe Ⅰ1 in the test solution was 10μM, and the concentration of interfering analyte was 100μM. The interfering analytes were Na + , K + , Ca 2+ Mg 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Cu + 、Al 3+ 、Zn 2+ , Cl - 、SO4 2- 、HSO3 - , Glucose, alanine (Ala), threonine (Thr), lysine (Lys), cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), Glycerol, GGT, that is, each test solution includes the above-mentioned fluorescent probe Ⅰ1 and an interfering analyte.

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

[0158] Example 10

[0159] The responses of the fluorescent probes I1 to I6 prepared in Examples 1-6 to different viscosities were used to establish a linear relationship between viscosity and fluorescence intensity:

[0160] The fluorescence emission spectra of the fluorescent probe Ⅰ1 at different viscosities were tested by fluorescence spectrometer. The test results are shown in Figure 5Using a fluorescence spectrophotometer, the excitation wavelength was set to 600nm, the scanning wavelength range was 620nm-900nm, and solutions with different volume ratios were prepared with glycerol (Glycerol) and PBS (20mM, pH=7.2) to obtain test solutions with different viscosity values. The test solution was used to further dilute the fluorescent probe Ⅰ1 to a final concentration of 10μM. The effect of the test solutions with different viscosity values ​​on the fluorescence intensity of the fluorescent probe Ⅰ1 was observed. The results are shown in Figure 2. Figure 5 As shown. Figure 5 It can be observed that when the viscosity values ​​increase successively to 0.893, 1.221, 1.754, 2.505, 4.165, 6.963, 12.36, 27.73, 64.2, 241.2, and 945 cP, the emission intensity at 720 nm gradually increases with the increase of the viscosity value, and the logarithm of the emission intensity and the logarithm of the viscosity value have a good linear relationship between 0.893 and 945 cP, as shown in Fig. Figure 6 As shown, the linear equation of I1 is: y=0.85x+2.85, and the linear correlation coefficient is 0.9962; the linear equation of I2 is: y=0.90x+2.41, and the linear correlation coefficient is 0.9816; the linear equation of I3 is: y=0.88x+2.57, and the linear correlation coefficient is 0.9967; the linear equation of I4 is: y=0.87x+2.67, and the linear correlation coefficient is 0.9861; the linear equation of I5 is: y=0.66x+1.92, and the linear correlation coefficient is 0.9907; the linear equation of I6 is: y=0.61x+1.36, and the linear correlation coefficient is 0.9796.

[0161] Embodiment 11

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

[0163] Fluorescent probe Ⅰ1 and interfering analyte were added to PBS buffer solution (20mM, pH=7.2) to prepare test solution. The concentration of fluorescent probe Ⅰ1 in the test solution was 10μM, and the concentration of interfering analyte was 100μM. The interfering analytes were Na + , K + , Ca 2+ Mg 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Cu + 、Al 3+ 、Zn 2+ , Cl - 、SO4 2- 、HSO3 -, Glucose, alanine (Ala), threonine (Thr), lysine (Lys), cysteine ​​(Cys), homocysteine ​​(Hcy), glutathione (GSH), GGT, glycerol (Glycerol), that is, each test solution includes the above-mentioned fluorescent probe Ⅰ1 and an interfering analyte.

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

[0165] Example 12

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

[0167] Fluorescent probe Ⅰ1 was added into solvents of different polarities (toluene, chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, water, PBS and glycerol) to prepare a test solution of fluorescent probe Ⅰ1 with a final concentration of 10 μM. The effects of test solutions of different polarities on the fluorescence intensity of fluorescent probe Ⅰ1 were observed. The results are shown in FIG. Figure 8 As shown. Figure 8 The results showed that fluorescence enhancement occurred only in the presence of glycerol, and the fluorescence intensity of other test solutions did not change significantly. This proved that the fluorescent probe Ⅰ1 only responded to viscosity when the excitation wavelength was 600nm and the emission wavelength was 720nm, but not to the polarity of the solution.

[0168] Embodiment 13

[0169] Response detection of fluorescent probes I1-I6 prepared in Examples 1-6 to solution pH value:

[0170] The fluorescent probe Ⅰ1 was prepared into test solutions with different pH values ​​(pH = 4.0-10.0) and the probe concentration was 10 μM. GGT with an activity of 50 U / L was added to the detection system to observe its effect on the fluorescence intensity of fluorescent probes Ⅰ1~I6 at different pH values. The results are as follows Fig. 9 As shown. Fig. 9It can be observed that when the pH is greater than 6, the emission intensity at 450nm is significantly enhanced, and the fluorescence emission intensity reaches a maximum when the pH is equal to 7.4, and then the fluorescence intensity gradually decreases. In particular, the pH in the body is around 7.4, so pH has no effect on the application of the above fluorescent probes Ⅰ1~I6.

[0171] Embodiment 14

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

[0173] The biosafety of the three fluorescent probes was tested using the MTT method. 5 cells / well) were inoculated in a 96-well plate. 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, and L929 was cultured in MEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) for 24 h. The medium was removed, and fresh medium solutions (without serum) containing different concentrations (0, 2.5, 5, 10, 20, and 40 μM) of fluorescent probes were added, respectively. After incubation with the cells for 24 h, 10 μL MTT reagent (5 mg / mL) was added to each well, and the cells were incubated in the incubator for another 4 h. The medium was removed, and 100 μL dimethyl sulfoxide was added to each well. After shaking, the OD value was measured at 490 nm by an enzyme reader, and the cell survival rate (%) was calculated. The results are shown in Fig.10 As shown, the cell survival rates were all above 80%, proving that the six fluorescent probes I1 to I6 had low toxicity within the range of 0-40 μM and had negligible effects on cells (survival rate) in the experiment.

[0174] The cell viability was calculated as follows:

[0175]

[0176] Embodiment 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 in mitochondria:

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

[0179] Example 16

[0180] The fluorescent probe Ⅰ1 prepared in Example 1 was imaged in living cells:

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

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

[0183] The results are as follows Fig.12 As shown, Fig.12 The fluorescence of L02 cells in the GGT channel and viscosity channel is shown in Figure 2. After adding sodium butyrate (NaBu), the fluorescence intensity of the blue channel increased significantly, indicating that the fluorescence probe Ⅰ1 reacted with GGT induced by sodium butyrate (NaBu), causing the fluorescence intensity of the blue channel to increase. At the same time, when sodium butyrate (NaBu) and acivicin (Acivicin) were added at the same time, the fluorescence intensity of the blue channel did not change significantly, indicating that the fluorescence probe Ⅰ1 did not react with GGT. The above results show that the fluorescence probe Ⅰ1 can detect endogenous GGT in living cells. After adding nystatin (Nys), the fluorescence intensity of the red channel increased significantly, indicating that the viscosity of mitochondria in L02 cells treated with nystatin (Nys) increased, causing the fluorescence intensity of the red channel of the fluorescent probe Ⅰ1 to increase significantly. At the same time, when sodium butyrate (NaBu) and nystatin (Nys) were added at the same time, the fluorescence intensities of the blue and red channels were significantly increased. The above results show that the fluorescence probe Ⅰ1 can detect the viscosity of mitochondria in living cells.

[0184] Embodiment 17

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

[0186] L02 cells, LX-2 cells and HepG2 cells (1×10 6 cells / well) were mixed by 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), respectively inoculated in confocal dishes, cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, 100 U / mL streptomycin) at 37°C and 5% CO2 for 24 h, the medium was removed, the five groups of cells were washed 2-3 times with PBS buffer, and 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 with a fluorescence microscope, wherein the blue channel excitation wavelength of fluorescent probe Ⅰ1 is 405 nm, the fluorescence collection wavelength is 420-500 nm, the red channel excitation wavelength is 640 nm, and the fluorescence collection wavelength is 650-750 nm.

[0187] The results are as follows Fig.13 As shown, it can be seen that the blue channel fluorescence intensity of L02 cells is significantly lower than that of LX-2 cells and HepG2 cells, the red channel fluorescence intensity of LX-2 cells is significantly lower than that of LX-2 cells and HepG2 cells, and the blue channel fluorescence intensity and red channel fluorescence intensity of HepG2 cells are significantly higher than those of L02 cells and LX-2 cells. The above results show that fluorescent probe Ⅰ1 can distinguish the three cells by imaging the GGT activity and mitochondrial viscosity of the three cells.

[0188] Embodiment 18

[0189] The fluorescent probe I1 prepared in Example 1 was imaged 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 is as follows:

[0191] Direct contact method was used to mix cells. Cells in logarithmic growth cycle (1×10 6cells / well) were mixed and divided into the following 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 were seeded in a confocal dish and cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, 100 U / mL streptomycin) at 37°C and 5% CO2 for 24 h. The medium was removed at 0, 12, 24, 36, 48, 60 and 72 h, and a culture medium solution containing 20 μM Erastin (ferroptosis inducer) was added. Before laser confocal imaging, the four groups of mixed cells with different treatment times were washed 2-3 times with PBS buffer, then incubated with a 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 blue channel excitation wavelength of the fluorescent probe Ⅰ1 was 405 nm, the fluorescence collection wavelength was 420-500 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0192] The results are as follows Fig.14 As shown, Fig.14 In the figure, they are ①: L02 cells and LX-2 cells; ②: L02 cells and HepG2 cells; ③: HepG2 cells and LX-2 cells; ④: fluorescence intensity presentation of L02 cells, LX-2 cells and HepG2 cells. It can be seen that when L02 cells and LX-2 cells were mixed cultured, the fluorescence intensity of the blue channel and the red channel reached the highest at 24h and then gradually decreased. When L02 cells, LX-2 cells and HepG2 cells were mixed cultured, the fluorescence intensity of the blue channel and the red channel reached the highest at 36h and then gradually decreased. In the other two groups of mixed cells, the fluorescence intensity of the blue channel and the red channel did not change significantly within 72h. The above results prove that GGT and mitochondrial viscosity will only increase significantly when L02 cells and LX-2 cells are mixed cultured or L02 cells, LX-2 cells and HepG2 cells are mixed cultured.

[0193] Embodiment 19

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

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

[0196] The cells were mixed by direct contact method. L02 cells, LX-2 cells and HepG2 cells (1×10 6cells / well) were mixed and inoculated in a confocal dish. They were cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) at 37°C and 5% CO2 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. The medium was removed at 60 and 108 h, and a medium solution containing 20 μM Erastin (ferroptosis inducer) was added again for incubation. Before laser confocal imaging, the mixed cells of the four groups with different treatment times were washed 2-3 times with PBS buffer, and then incubated with a medium solution of fluorescent probe Ⅰ1 with a concentration of 10 μM for 60 min, washed 2-3 times with PBS buffer, and the fluorescence in the cells was observed with a fluorescence microscope. The blue channel excitation wavelength of fluorescent probe Ⅰ1 was 405 nm, the fluorescence collection wavelength was 420-500 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0197] The results are as follows Fig.15 As shown, Fig.15 The fluorescence intensity of the mixed culture of L02 cells, LX-2 cells and HepG2 cells is presented. It can be seen that the fluorescence intensity of the blue channel and the red channel gradually increased, reached the highest at 36h, and then gradually decreased. After the second induction with Erastin at 60h, the fluorescence intensity of the blue channel and the red channel gradually increased, reached the highest at 96h, and then gradually decreased. After the third induction with Erastin at 120h, the fluorescence intensity of the blue channel and the red channel continued to increase, reached the maximum at 156h and tended to stabilize. The above results can be It can be seen that when L02 cells, LX-2 cells and HepG2 cells are co-cultured, repeated stimulation of the co-culture system will eventually increase the GGT and mitochondrial viscosity of HepG2 cells, indicating that the death of L02 cells activates LX-2 cells, and 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 and reaches a higher level of redox microenvironment. The significant increase of GGT and mitochondrial viscosity in HepG2 cells promotes their proliferation and invasion.

[0198] Embodiment 20

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

[0200] Real-time monitoring of endogenous GGT activity and mitochondrial viscosity during the inhibition of HepG2 cell proliferation and invasion was performed using fluorescent probe Ⅰ1. The specific method is as follows:

[0201] The cells were mixed by direct contact method. L02 cells, LX-2 cells and HepG2 cells (1×10 6 cells / well) were mixed and inoculated in a confocal dish. They were cultured in DMEM complete medium (containing 10% fetal bovine serum, 100 mg / L penicillin, and 100 U / mL streptomycin) at 37°C and 5% CO2 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. The medium was removed after 48 h and a medium solution containing 20 μM Erastin (ferroptosis inducer) and 1 mM glutathione (free radical scavenger) was added and incubated again. Before laser confocal imaging, the four groups of mixed cells with different treatment times were washed 2-3 times with PBS buffer, then incubated with a 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 blue channel excitation wavelength of the fluorescent probe Ⅰ1 was 405 nm, the fluorescence collection wavelength was 420-500 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0202] The results are as follows Fig.16 As shown, Fig.16 The fluorescence intensity of L02 cells, LX-2 cells and HepG2 cells mixed culture is presented. It can be seen that the fluorescence intensity of the blue channel and the red channel gradually increased, reaching the highest at 36h, and then gradually decreased. After the second induction with Erastin at 48h and treatment with glutathione, the fluorescence intensity of the blue channel and the red channel no longer increased. The above results show that if timely treatment is given before the proliferation and invasion of HepG2 cells, the normal redox balance in the cells can be maintained, thereby inhibiting their proliferation and invasion. This shows that fluorescent probe Ⅰ1 can monitor the early stage of HCC proliferation and invasion and its treatment effect.

[0203] Embodiment 21

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

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

[0206] Female BALB / c nude mice were selected as the in vivo imaging model, and the relevant regulations on experimental animal welfare and experimental animal ethics in my country were strictly followed and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: L02 cells, LX-2 cells, and HepG2 cells in the logarithmic growth phase were mixed into a cell line suspension and cultured at 5×10 6 The cells were injected into the right axilla of 5-week-old female BALB / c nude mice. 3 The mice were fasted one day before the experiment, and 50 μM fluorescent probe Ⅰ1 was injected intratumorally. Then, the fluorescence in the subcutaneous tumor of the mice was observed with a small animal in vivo imaging system (IVIS) at 0, 0.5, 1.5, 2, 4, 6, and 12 h. After the experiment, the mice were euthanized. The blue channel excitation wavelength filter of the fluorescent probe Ⅰ1 was 420 nm, the fluorescence collection wavelength filter was 520 nm, the red channel excitation wavelength filter was 640 nm, and the fluorescence collection wavelength filter was 720 nm.

[0207] The results are as follows Fig.17 As shown, it can be seen that the fluorescence intensity of the blue channel increases with time, reaches the highest at 1.5 hours, begins to decrease from 4 hours, and reaches the lowest at 12 hours. The fluorescence intensity of the red channel also increases with time, reaches the highest at 1.5 hours, begins to decrease from 4 hours, and reaches the lowest at 12 hours, indicating that fluorescent probe Ⅰ1 can image GGT activity and mitochondrial viscosity in vivo.

[0208] Embodiment 22

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

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

[0211] Female BALB / c nude mice were selected as the in vivo imaging model, and the relevant regulations on experimental animal welfare and experimental animal ethics in my country were strictly followed and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: L02 cells, LX-2 cells, and HepG2 cells in the logarithmic growth phase were mixed into a cell line suspension and cultured at 5×10 6 The cells were injected into the right axilla of 5-week-old female BALB / c nude mice. 3The mice were fasted the day before the experiment, and 20 μM Erastin was injected intratumorally at a certain time, and 20 μM Erastin was injected intratumorally again at 60 and 108 h. Before each in vivo imaging, 50 μM fluorescent probe Ⅰ1 was injected intratumorally, and the fluorescence in the subcutaneous tumor of the mice was observed 1.5 h later using a small animal in vivo imaging system (IVIS), where the blue channel excitation wavelength of the fluorescent probe Ⅰ1 was 420 nm, the fluorescence collection wavelength was 520 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 720 nm.

[0212] The results are as follows Fig.18 As shown, it can be seen that the fluorescence intensity of the GGT channel and the viscosity channel fluctuates with the repeated induction of Erastin, and finally stops decreasing, which has the same trend as the cell experiment, indicating that the fluorescent probe Ⅰ1 can perform real-time imaging of GGT and mitochondrial viscosity during the proliferation and invasion of HepG2 cells in vivo.

[0213] Embodiment 23

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

[0215] The fluorescent probe Ⅰ1 was used to monitor the endogenous GGT activity and mitochondrial viscosity in real time during the inhibition of HepG2 cell proliferation and invasion in tumor-bearing mice. The specific method is as follows:

[0216] Female BALB / c nude mice were selected as the in vivo imaging model, and the relevant regulations on experimental animal welfare and experimental animal ethics in my country were strictly followed and approved by the Animal Ethics Review Committee of Gannan Medical University. Subcutaneous tumor preparation: L02 cells, LX-2 cells, and HepG2 cells in the logarithmic growth phase were mixed into a cell line suspension and cultured at 5×10 6 The cells were injected into the right axilla of 5-week-old female BALB / c nude mice. 3 The mice were fasted the day before the experiment, and 20 μM Erastin was injected intratumorally at a certain time. 20 μM Erastin and 1 mM glutathione were injected intratumorally again at 60 and 108 h. Before each in vivo imaging, 50 μM fluorescent probe Ⅰ1 was injected intratumorally. After 1.5 h, the fluorescence in the subcutaneous tumor of the mice was observed with a small animal in vivo imaging system (IVIS). The blue channel excitation wavelength of the fluorescent probe Ⅰ1 was 420 nm, the fluorescence collection wavelength was 520 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 720 nm.

[0217] The results are as follows Fig.19As shown, the fluorescence intensity of the blue channel and the red channel gradually increased, reaching the highest at 36h, and then gradually decreased. After the second induction with Erastin at 48h and treatment with glutathione, the fluorescence intensity of the blue channel and the red channel no longer increased. The above results show that if timely treatment is given before the proliferation and invasion of HepG2 cells, the normal redox balance in the cells can be maintained in vivo, thereby inhibiting their proliferation and invasion. This shows that fluorescent probe Ⅰ1 can monitor the early stage of HCC proliferation and invasion and its treatment effect in vivo.

[0218] Embodiment 24

[0219] The fluorescent probe I1 prepared in Example 1 distinguishes liver cancer and adjacent tissues in clinical samples:

[0220] Using fluorescent probe Ⅰ1, GGT and mitochondrial viscosity of cancer tissue and adjacent tissue were imaged in clinical samples. The specific method is as follows:

[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 subjects. Clinical tissue samples were embedded in OCT gel and fixed, and frozen sections were prepared using a freezing microtome. The tissue sections were stained with fluorescent probe Ⅰ1 (20 μM) for fluorescence, immunofluorescence, and H&E staining. Fluorescence in cells was observed using a fluorescence microscope, where the blue channel excitation wavelength of fluorescent probe Ⅰ1 was 405 nm, the fluorescence collection wavelength was 420-500 nm, the red channel excitation wavelength was 640 nm, and the fluorescence collection wavelength was 650-750 nm.

[0222] The results are as follows Fig. 20 As shown in the figure, the fluorescence of cancer tissue in the blue channel and the red channel are higher than those of adjacent tissue, indicating that the GGT and mitochondrial viscosity of cancer tissue are higher than those of adjacent tissue. At the same time, the results obtained by immunofluorescence are consistent with those of fluorescent probe Ⅰ1, which is completely consistent with the results of H&E analysis. In general, these results show that fluorescent probe Ⅰ1 has the potential to distinguish cancer tissue from adjacent tissue.

[0223] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity, characterized in that: It has the structural formula shown in Formula I: Wherein, n is an integer from 1 to 20; R1, R2 and R3 are each independently selected from any one of hydrogen, an alkyl group having 1 to 8 carbon atoms, a halogen, a methoxy group, a trifluoromethyl group, an ester group or an amide group; R4 and R5 are substituents at undetermined positions of the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyl, alkylsulfonic acid group having 1-18 carbon atoms, ester group having 1-18 carbon atoms, amide group having 1-18 carbon atoms, N,N-dimethylamino, N,N-diethylamino, trifluoromethyl, sulfonic acid group or sulfonate; R6 is selected from any one of L3, L4, L5, L6, L7, L8 or L9; Y is selected from inorganic anions or organic anions.

2. The γ-glutamyl transferase and mitochondrial viscosity dual-response fluorescent probe according to claim 1, characterized in that: n is an integer from 2 to 18; R1, R2 and R3 are each independently selected from any one of hydrogen, methyl, halogen, methoxy, trifluoromethyl, ester or amide; R4 and R5 are substituents at undetermined positions of the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyl, N,N-diethylamino, trifluoromethyl or sulfonic acid; R6 is selected from any one of L3, L4, L5, L6 or L8; Y is selected from halogen ions, ClO 4- , BF 4- 、CH3COO - CF3COO - or Ots - Any one of .

3. The γ-glutamyl transferase and mitochondrial viscosity dual-response fluorescent probe according to claim 2, characterized in that: n is an integer from 4 to 16; R1, R2 and R3 are each independently selected from any one of hydrogen, methyl, halogen and methoxy; R4 and R5 are substituents at undetermined positions of the benzene ring, each independently selected from any one of hydrogen, halogen, and methoxy; R6 is selected from any one of L3, L5 and L8; Y is selected from halogen ions, ClO 4- and CH3COO - Any one of .

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

5. The method for preparing the dual-response fluorescent probe of γ-glutamyl transferase and mitochondrial viscosity according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: 1) dissolving Boc-L-glutamic acid-1-tert-butyl ester, 4-aminobenzyl alcohol and carbodiimide hydrochloride in an organic solvent A, and reacting in an ice bath to obtain compound Z1; 2) Compound Z1 is dissolved in organic solvent A under ice bath conditions, phosphorus tribromide is slowly added dropwise, and the mixture is reacted under ice bath conditions to obtain compound Z2; 3) dissolving compound Z3 and compound Z4 in a dry organic solvent B, and performing a reflux reaction under nitrogen protection to obtain compound Z5; 4) Compounds Z5 and Z6 are dissolved in an organic solvent B, piperidine is added dropwise, and the mixture is refluxed to obtain compound Z7; 5) dissolving compound Z8, Michaelis' acid and potassium carbonate in organic solvent C and reacting at room temperature to obtain compound Z9; 6) Compound Z9, N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole are dissolved in a dry organic solvent D, and 1-Boc-piperazine is added to react at room temperature to obtain compound Z10; 7) Compound Z10 is dissolved in dry organic solvent A, trifluoroacetic acid is added under ice bath conditions, and the reaction is carried out under ice bath conditions to obtain compound Z11; 8) Compound Z7, N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole are dissolved in a dry organic solvent D, and 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 a dry organic solvent B, and reacted at room temperature under nitrogen protection to obtain compound Z13; 10) Compound Z13 is dissolved in dry organic solvent A, trifluoroacetic acid is added under ice bath conditions, and the reaction is carried out under ice bath conditions to obtain a compound represented by 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.

6. The preparation method according to claim 5, characterized in that: 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 to phosphorus tribromide is 1:1-10; In step 3), the molar ratio of compound Z3 to compound Z4 is 1-3:1; In step 4), the molar ratio of compound Z5 to compound Z6 is 1:0.5-2; In step 5), the molar ratio of Z8, Michaelis acid and potassium carbonate is 1:1-3; In step 6), the molar ratio of the compound Z9, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, and 1-Boc-piperazine is 1:1-5:1-5:1-5; In step 7), the molar ratio of the compound Z10 to trifluoroacetic acid is 1:1-20; In step 8), the molar ratio of the compound Z7, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, and the compound Z11 is 1:1-5:1-5:1-5; In step 9), the molar ratio of compound Z2, compound Z12 and potassium carbonate is 1:1-3:1-3; In step 10), the molar ratio of the compound Z13 to trifluoroacetic acid is 1:1-20.

7. Use of the dual-response fluorescent probe for γ-glutamyl transferase and mitochondrial viscosity according to any one of claims 1 to 4 in the preparation of a detection reagent for detecting γ-glutamyl transferase activity and mitochondrial viscosity.

8. The use according to claim 7, characterized in that: In the detection system, the detection range of γ-glutamyl transferase activity is 0-70 U / L, and the detection range of mitochondrial viscosity is 0.5-1099 cP.

9. Use of the dual-response fluorescent probe of γ-glutamyl transferase and mitochondrial viscosity according to any one of claims 1 to 4 in the preparation of a detection reagent for real-time visual monitoring of early liver cancer cell proliferation and invasion.

10. The use according to claim 9, characterized in that: The dual-response fluorescent probe in the detection reagent uses coumarin and cyanine dye as fluorophore matrices, and the emission wavelengths of the fluorescent probe are 450nm and 720nm.

Images