Pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, preparation method and application thereof

Abstract

The present invention discloses a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase and its preparation method and application. The fluorescent probe (Py-GGT-1) uses (E)-4-styryl pyridine fragment as a fluorescent signal reporter group, uses aniline to modify the fluorescent group, introduces tert-butyloxycarbonyl-L-glutamate 1-tert-butyl fat (Boc-Glu-OtBu) by the condensation reaction of amino and carboxyl groups, and uses trifluoroacetic acid to remove the Boc protecting group as the enzyme cleavage site of γ-glutamyl transpeptidase. The fluorescent probe Py-GGT-1 with stronger fluorescence produces a weakly fluorescent metabolite Py-1-NH2 under the catalytic enzymolysis of γ-glutamyl transpeptidase. The present invention can be used for qualitative and quantitative detection of γ-glutamyl transpeptidase in serum, and it has the advantages of good selectivity, high detection sensitivity, and fast detection speed.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a pyridine fluorescent probe for rapid in vitro detection of gamma-glutamyl transpeptidase, and a preparation method and application thereof. Background Art

[0002] Cancer, a major public health issue, poses a serious threat to the health of the global population. To detect and treat cancer promptly, we must develop early diagnostic methods. Tumor markers are characteristic substances produced directly by tumor cells or induced by non-tumor cells through tumor cells, and they are of practical significance for early cancer screening.

[0003] γ-Glutathione (GST), a tumor marker, has been shown to be closely associated with numerous diseases, including malignancies, diabetes, and drug resistance. It is a cell surface enzyme that regulates the homeostasis of γ-glutamyl (γ-Glu), glutathione (GSH), and cysteine (Cys) within cells. γ-Glutathione (GSH) is overexpressed on the cell surface of several malignant tumors, including liver, neck, and ovarian tumors. Furthermore, studies have shown that γ-Glutathione (GST) is upregulated in response to harmful stimuli from the internal and external environments through cellular oxidative stress.

[0004] To date, the detection of γ-glutamyl transpeptidase has primarily relied on methods such as chromatography, electrochemiluminescence, surface-enhanced resonance Raman scattering, and colorimetry. However, these methods require complex instrumentation and are cumbersome to operate. Fluorescent probes, due to their high resolution and good cell compatibility, have become an effective alternative for tumor marker detection. Among them, organic fluorescent probes have attracted considerable attention in tumor marker detection (Li, X.; Yin, C.; Zhang, L.; Huo, F., Dyes and Pigments. 2023, 219, 111-631). In recent years, the detection of γ-glutamyltransferase has mainly focused on cyanines (Gui, L.; Li, W.; Pan, Y.; Zhao, J.; Kong, X.; Liu, J.; Zuo, K.; Yan, J.; Ling, Y.; Ling, C.; Li, R.; Yuan, Z., Sens. Actuators B. Chem., 2024, 398, 134718.), BODIPYs (Wang, F.; Zhu, Y.; Zhou, L.; Pan, L.; Cui, Z.; Fei, Q. ; Luo, S.; Pan, D.; Huang, Q.; Wang, RJAC, Angew. Chem. Int. Ed., 2015, 127, 7457-7461.), benzobithiadiazoles (Kim, YJ; Park, SJ; Lim, CS; Lee, DJ; Noh, C.-K.; Lee, K.; Shin, SJ; Kim, HM, Anal. Chem., 2019, 91, 9246-9250.) and other fluorescent dyes are used to design and synthesize organic fluorescent probes for detecting γ-glutamyl transpeptidase. In addition to these traditional fluorescent dyes, we also noticed that the development of new fluorescent dyes based on pyridine skeletons is attracting attention (Batalin, S., Dyes and Pigments, 2022, 208, 110850). Pyridine, as an important nitrogen-containing heterocyclic compound, has a wide range of applications in medicine and pesticides. Because of its strong electron-withdrawing effect, the charge transfer within the probe molecule is enhanced, which significantly changes the probe intensity and emission wavelength. Therefore, the development of a high-performance fluorescent probe based on the pyridine skeleton for the detection of γ-glutamyl transpeptidase in serum samples has practical significance and may also provide the possibility for early diagnosis of late-stage cancer. Summary of the Invention

[0005] The purpose of the present invention is to provide a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0006] The object of the present invention is achieved through the following technical solution: a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, including a fluorescent probe Py-GGT-1, the structural formula of which is:

[0007]

[0008] The invention discloses a method for preparing a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, comprising: using a (E)-4-phenylvinylpyridine fragment as a fluorescent signal reporter group in a fluorescent probe (Py-GGT-1), modifying the fluorescent group with aniline, introducing tert-butyloxycarbonyl-L-glutamic acid 1-tert-butyl ester through a condensation reaction between an amino group and a carboxyl group, and removing the Boc protecting group with trifluoroacetic acid to serve as an enzyme cleavage site for γ-glutamyl transpeptidase.

[0009] The preparation method of a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase specifically comprises the following steps:

[0010] In the first step, 1,4-dioxane and water are used as the reaction medium, 2-bromo-4-chloropyridine and (4-((tert-butyloxycarbonyl)amino)phenyl)boric acid are used as reactants in an argon atmosphere, tetrakis(triphenylphosphine palladium) is used as a catalyst, and sodium carbonate is used as a reaction base. The reaction is refluxed at 85°C for 12 hours. After completion of the reaction, the mixture is extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as the eluent to obtain tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate as a white solid;

[0011] In the second step, dichloromethane is used as a reaction medium, and the tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate obtained in step (1) is dissolved in dichloromethane, trifluoroacetic acid is slowly added dropwise to the dichloromethane solution, and the mixture is stirred at room temperature until the reaction is completed. The solvent and most of the trifluoroacetic acid are then distilled off under reduced pressure, and a saturated aqueous sodium bicarbonate solution is added to adjust the alkaline environment. After water extraction, the mixture is separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as eluent to obtain 4-(4-chloropyridin-2-yl)aniline as a white solid;

[0012] In the third step, tert-butyloxycarbonyl-L-glutamate 1-tert-butyl ester (Boc-Glu-OtBu), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) were dissolved in DMF and stirred at room temperature for 15 minutes. A DMF solution of 4-(4-chloropyridin-2-yl)aniline was added and the mixture was reacted at room temperature for 24 hours. After completion of the reaction, the mixture was extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as eluent to obtain tert-butyl N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate as a white solid.

[0013] The fourth step is to use 1,4-dioxane and water as the reaction medium, N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate tert-butyl ester and (E)-styrene boronic acid as reactants in an argon atmosphere, tetrakis(triphenylphosphine)palladium as a catalyst, and sodium carbonate as a reaction base, and reflux at 105°C for 12 hours. After completion of the reaction, the mixture is extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as an eluent to obtain (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-styrylpyridin-2-yl)phenyl)glutamate tert-butyl ester as a white solid;

[0014] In the fifth step, using dichloromethane as the reaction medium, (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamate tert-butyl ester was dissolved in dichloromethane, and trifluoroacetic acid diluted with dichloromethane was slowly added dropwise. The reaction was carried out at room temperature for 6 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the mixture was separated by a C18 column using methanol / water as the eluent to obtain a pyridine target fluorescent probe.

[0015] Furthermore, the molar ratio of the 2-bromo-4-chloropyridine to 4-((tert-butyloxycarbonyl)amino)phenyl)boric acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1:(1.0-2.0):(0.04-0.06):(2.0-3.0);

[0016] The molar ratio of the tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate to trifluoroacetic acid is 1:(13-14).

[0017] Furthermore, the molar ratio of the 4-(4-chloropyridin-2-yl)aniline to Boc-Glu-OtBu, HATU, and DIPEA is 1:(1.0-2.0):(1.0-2.0):(1.0-2.0);

[0018] The molar ratio of the N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate to (E)-styreneboric acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1:(1.0-2.0):(0.04-0.06):(2.0-3.0);

[0019] The molar ratio of the (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylvinylpyridin-2-yl)phenyl)glutamate to trifluoroacetic acid is 1:(20.0-30.0).

[0020] A fluorescent probe or a prepared fluorescent probe is used to selectively identify gamma-glutamyl transpeptidase in PBS. The synthesized pyridine skeleton fluorescent probe Py-GGT-1 can be used to detect gamma-glutamyl transpeptidase in the serum of liver cancer patients.

[0021] The invention discloses an application of a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase. The fluorescent probe Py-GGT-1 generates a weakly fluorescent metabolite Py-1-NH2 under the catalytic enzymatic hydrolysis of γ-glutamyl transpeptidase. The γ-glutamyl transpeptidase is overexpressed in the bodies of liver cancer patients. The synthesized pyridine skeleton fluorescent probe can be used to detect γ-glutamyl transpeptidase in the serum of liver cancer patients.

[0022] Furthermore, in the γ-glutamyl transpeptidase detection, the γ-glutamyl transpeptidase and the fluorescent probe were reacted by incubating them together in PBS with a pH of 7.4 at 37° C. for 30 min.

[0023] Furthermore, the reaction concentration of the fluorescent probe was 10 μmol·L-1.

[0024] The reaction equation for the synthesis of the fluorescent probe is as follows:

[0025]

[0026] For the preparation of fluorescent probe reaction, various raw material ratios were obtained through creative experiments, among which (E)-N 2 -(tert-Butyloxycarbonyl)-N 5 The ratio of tert-butyl-(4-(4-phenylvinylpyridin-2-yl)phenyl)glutamate to trifluoroacetic acid is particularly important because trifluoroacetic acid affects the removal of the protecting group, which is related to whether the reaction can proceed smoothly.

[0027] In addition, characterization by hydrogen nuclear magnetic resonance spectroscopy, mass spectrometry and other methods respectively indicated that the fluorescent probe had been successfully synthesized.

[0028] The optimal reaction concentration of the synthetic fluorescent probe was 10 μmol·L -1During the detection process, if the concentration of the fluorescent probe used is lower than 10 μmol·L -1 , the probe’s own luminescence and its response signal are weak, which may weaken the detection sensitivity to a certain extent.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention uses a synthetic fluorescent probe for the identification and detection of gamma-glutamyl transpeptidase. Because its structure contains the recognition site glutamine bond of gamma-glutamyl transpeptidase, (E)-4-phenylvinylpyridine is used as a fluorescent signal reporter group, and the fluorescent group is modified with aniline. Boc-Glu-OtBu is introduced through a condensation reaction of amino and carboxyl groups. After the Boc protecting group is removed with trifluoroacetic acid, it serves as an enzyme cleavage site for gamma-glutamyl transpeptidase. The probe Py-GGT-1 has strong fluorescence and catalyzes the hydrolysis of the glutamine bond under the action of gamma-glutamyl transpeptidase to form Py-1-NH2 with weak fluorescence. The present invention can be used to detect the content of gamma-glutamyl transpeptidase in vitro, and can also be quantitatively detected by fluorescence method, with advantages such as good selectivity, high detection sensitivity, and fast detection speed.

[0031] The pyridine fluorescent probe disclosed in the present invention contains a recognition group for γ-glutamyl transpeptidase, which has strong fluorescence. After adding γ-glutamyl transpeptidase, the probe catalyzes the hydrolysis of glutamine bonds under the action of transglutaminase, forming a compound with weak fluorescence. Through this process of fluorescence attenuation, the purpose of detecting γ-glutamyl transpeptidase is achieved. In addition, the method and strategy for detecting γ-glutamyl transpeptidase disclosed in the present invention has great market application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Attachment Figure 2 1H NMR and 13CNMR spectra of the fluorescent probe Py-GGT-1 of the present invention;

[0033] Figure 3 Attachment Figure 4 1H NMR and 13CNMR spectra of the fluorescent probe intermediate Py-1-NH2 of the present invention;

[0034] Figure 5 The accompanying drawings show the UV absorption (a) and fluorescence emission (b) spectra of the fluorescent probe of the present invention before and after the reaction with γ-glutamyl transpeptidase;

[0035] Figure 6 The accompanying figure shows the selectivity of the fluorescent probe of the present invention for γ-glutamyl transpeptidase among different interferents;

[0036] Figure 7 The accompanying figure shows the detection of endogenous γ-glutamyl transpeptidase in liver cancer cells (HepG2) by the fluorescent probe of the present invention;

[0037] Figure 8 The accompanying drawing is a curve showing the fluorescence intensity at 445 nm and the activity of γ-glutamyl transpeptidase after the fluorescent probe of the present invention was incubated with different concentrations of γ-glutamyl transpeptidase in normal human serum diluted ten times with PBS. DETAILED DESCRIPTION

[0038] 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, rather than all the embodiments. 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.

[0039] The present invention discloses a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, the structural formula of the fluorescent probe is:

[0040]

[0041] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0042] Specific implementation 1: Preparation of fluorescent probe Py-GGT-1:

[0043] The specific steps include:

[0044] In the first step, under an argon atmosphere, 2-bromo-4-chloropyridine (0.58 g, 3 mmol), 4-((tert-butyloxycarbonyl)amino)phenyl)boric acid (0.85 g, 3.6 mmol), tetrakis(triphenylphosphine)palladium (0.17 g, 0.15 mmol), and sodium carbonate (0.79 g, 7.5 mmol) were added to a 100 mL round-bottom flask, followed by 18 mL of 1,4-dioxane and 9 mL of water, and the mixture was refluxed at 85 ° C for 24 h. After the reaction, 30 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water three times (3×30 mL) and with salt once (30 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was then separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain white solid tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate Py-1-1 with a yield of 80%. The reaction process is shown in Formula (1).

[0045]

[0046] In the second step, the (4-(4-chloropyridin-2-yl)phenyl)carbamic acid tert-butyl ester Py-1-1 (0.61 g, 2 mmol) obtained in step (1) was dissolved in 6 mL of dichloromethane, and trifluoroacetic acid (2 mL) was slowly added dropwise. After the reaction was completed, the solvent was distilled off under reduced pressure, saturated sodium bicarbonate solution was added to adjust the solution to be weakly alkaline, and an appropriate amount of ethyl acetate was added to the solution for extraction. The solution was washed with salt once (30 mL), dried over anhydrous Na2SO4, and the solvent was distilled off under reduced pressure. After separation by 200-300 mesh silica gel column chromatography, petroleum ether / ethyl acetate was used as eluent to obtain white solid 4-(4-chloropyridin-2-yl)aniline Py-1-2 with a yield of 95%. The reaction process is shown in formula (2);

[0047]

[0048] In the third step, Boc-Glu-OtBu (0.45 g, 1.5 mmol), HATU (0.57 g, 1.5 mmol), and DIPEA (0.28 mL) were dissolved in 10 mL of dry DMF and stirred at room temperature for 15 min. Subsequently, a solution of 4-(4-chloropyridin-2-yl)aniline Py-1-2 (0.24 g, 1 mmol) in DMF (3 mL) was added and the mixture was reacted at room temperature for 24 h. 30 mL of ethyl acetate was added, and the mixture was washed with water three times (3×30 mL) and with salt once (30 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was then separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain white solid N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamic acid tert-butyl ester Py-1-3 in a yield of 40%. The reaction process is shown in Formula (3).

[0049]

[0050] In the fourth step, in an argon atmosphere, N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate tert-butyl ester Py-1-3 (0.24 g, 0.5 mmol), (E)-phenyleneboronic acid (0.089 g, 0.6 mmol), tetrakis(triphenylphosphine)palladium (0.029 g, 0.025 mmol), and sodium carbonate (0.13 g, 1.25 mmol) were added to a 50 mL round-bottom flask in sequence, and then 6 mL of 1,4-dioxane and 3 mL of water were added, and the mixture was refluxed at 105 ° C for 12 h. After the reaction, 20 mL of ethyl acetate was added to the reaction solution, and the mixture was washed with water three times (3×30 mL) and with salt once (30 mL). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was then separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain white solid (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamic acid tert-butyl ester Py-1-4 with a yield of 70%. The reaction process is shown in Formula (4);

[0051]

[0052] In the fifth step, (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamate tert-butyl ester Py-1-4 (0.11 g, 0.1 mmol) was dissolved in 2 mL of dichloromethane, and trifluoroacetic acid (0.2 mL) diluted with 0.5 mL of dichloromethane was slowly added dropwise. The reaction was carried out at room temperature for 6 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the product was separated by a C18 column using methanol / water as the eluent to obtain Py-GGT-1 as a white solid with a yield of 62%. The reaction process is shown in Formula (5);

[0053]

[0054] Specific Example 2: Preparation of fluorescent probe Py-GGT-1:

[0055] The specific steps include:

[0056] In the first step, under an argon atmosphere, 2-bromo-4-chloropyridine (0.96 g, 5 mmol), (4-((tert-butoxycarbonyl)amino)phenyl)boric acid (1.42 g, 6 mmol), tetrakis(triphenylphosphine)palladium (0.29 g, 0.25 mmol), and sodium carbonate (1.32 g, 12.5 mmol) were added to a 100 mL round-bottom flask, followed by 30 mL of 1,4-dioxane and 15 mL of water, and the mixture was refluxed at 85 ° C for 24 h. After the reaction, 50 mL of ethyl acetate was added to the reaction solution, and the mixture was washed three times with deionized water (3×20 mL) and once with a saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain white solid tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate Py-1-1 with a yield of 77%. The reaction process is shown in Formula (1).

[0057]

[0058] In the second step, the (4-(4-chloropyridin-2-yl)phenyl)carbamic acid tert-butyl ester Py-1-1 (0.49 g, 1 mmol) obtained in step (1) was dissolved in 4 mL of dichloromethane, and 2 mL of trifluoroacetic acid was slowly added dropwise. After the reaction was completed, the solvent was distilled off under reduced pressure, a saturated sodium bicarbonate solution was added to adjust the solution to be weakly alkaline, and an appropriate amount of ethyl acetate was added to the solution for extraction. The organic phase was washed once with a saturated NaCl solution and dried over anhydrous Na2SO4. The solvent was distilled off under reduced pressure and then separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain a white solid 4-(4-chloropyridin-2-yl)aniline Py-1-2 with a yield of 93%. The reaction process is shown in Formula (2).

[0059]

[0060] In the third step, Boc-Glu-OtBu (1.50 g, 4.95 mmol), HATU (1.88 g, 4.95 mmol), and DIPEA (0.6 mL) were dissolved in 20 mL of dry DMF and stirred at room temperature for 15 min. Subsequently, a solution of 4-(4-chloropyridin-2-yl)aniline Py-1-2 (0.67 g, 3.3 mmol) in DMF (10 mL) was added and the mixture was reacted at room temperature for 24 h. 30 mL of ethyl acetate was added, and the mixture was washed three times with deionized water (3×30 mL), and once with a saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain white solid N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamic acid tert-butyl ester Py-1-3 in a yield of 38%. The reaction process is shown in Formula (3);

[0061]

[0062] In the fourth step, in an argon atmosphere, N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate tert-butyl ester Py-1-3 (0.49 g, 1 mmol), (E)-phenyleneboronic acid (0.18 g, 1.2 mmol), tetrakis(triphenylphosphine)palladium (0.058 g, 0.05 mmol), and sodium carbonate (0.26 g, 2.5 mmol) were added to a 100 mL round-bottom flask in sequence, and then 20 mL of 1,4-dioxane and 10 mL of water were added, and the mixture was refluxed at 105 ° C for 12 h. After the reaction, 40 mL of ethyl acetate was added to the reaction solution, and the mixture was washed three times with deionized water (3×30 mL) and once with a saturated NaCl solution. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by distillation under reduced pressure. The mixture was then separated by 200-300 mesh silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain white solid (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamic acid tert-butyl ester Py-1-4 in a yield of 68%. The reaction process is shown in Formula (4).

[0063]

[0064] In the fifth step, (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamate tert-butyl ester Py-1-4 (0.11 g, 0.2 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) diluted with dichloromethane (0.5 mL) was slowly added dropwise. The mixture was reacted at room temperature for 6 h. After completion of the reaction, the solvent was removed by distillation under reduced pressure, and the mixture was separated by a C18 column using methanol / water as the eluent to obtain Py-GGT-1 as a white solid with a yield of 60%. The reaction process is shown in Formula (5);

[0065]

[0066] The following will further verify the superior effect of the technical solution of the present invention compared with the existing technology, the specific content is as follows:

[0067] Figure 1 , Figure 2 This is the NMR spectrum of the fluorescent probe Py-GGT-1. The specific spectral peaks are: 1H NMR (400MHz, DMSO-d6) δ10.30 (s, 1H), 8.60 (d, J = 5.1Hz, 1H), 8.21-8.00 (m, 4H), 7.85-7.57 (m, 6H), 7.50-7.27 (m, 6H), 3.85 (d, J = 6.8Hz, 1H), 2.70-2.54 (m, 2H), 2.17-2.03 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 171.4, 170.7, 156.6, 150.3, 145.8, 140.5, 136.7, 133.8, 133.5, 129.4, 129.1, 127.5, 126.7, 119.7, 119.5, 117.1, 52.5, 32.3, 26.4. This corresponds to the probe group, proving that the probe was successfully synthesized. The mass spectrometry data obtained was HRMS (ESI) calcd for C24H24N3O3+: 402.1818 (M+H+), found: 402.1825. This further verified the successful synthesis of the probe. In summary, the NMR and HRMS results confirmed the chemical structure of Py-GGT-1.

[0068] Figure 3 , Figure 4To explore the NMR spectrum of the intermediate Py-1-NH2, the specific spectral peaks are: 1H NMR (400MHz, CDCl3) δ8.58 (dd, J = 5.2, 0.8Hz, 1H), 7.90-7.84 (m, 2H), 7.68 (dd, J = 1.6, 0.8Hz, 1H), 7.58-7.52 (m, 2H), 7.43-7.30 (m, 4H), 7.23 (dd, J = 5.2, 1.7Hz, 1H), 7.07 (d, J = 16.3Hz, 1H), 6.80-6.75 (m, 2H), 3.84 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 158.0, 149.8, 147.5, 145.2, 136.4, 132.7, 129.8, 128.9, 128.6, 128.1, 127.0, 126.6, 118.1, 117.0, 115.1. The mass spectrum data obtained was HRMS (ESI) calcd for C19H17N2+: 273.1392 (M+H+), found: 273.1405.

[0069] The ability of the fluorescent probe Py-GGT-1 to react with γ-glutamyl transpeptidase in buffer solution was tested: A 10.0 mmol / L solution of the synthesized fluorescent probe Py-GGT-1 was prepared in DMSO. 2 μL of the solution was added to a centrifuge tube containing 1 mL of PBS (10 mmol / L, pH 7.4) for a fluorescent probe concentration of 20 μmol / L. 100 μL of the probe was added to a centrifuge tube containing 96 μL of PBS. 4 μL of γ-glutamyl transpeptidase (10 U / mL) was added to a final concentration of 0.2 U / mL. After 30 minutes, changes in the UV absorption and fluorescence emission spectra were measured. Fluorescence measurement conditions were λex = 310 nm (EX Slit: 10 nm, EM Slit: 10 nm). Figure 5 The UV absorption (a) and fluorescence emission (b) spectra of the fluorescent probe Py-GGT-1 before and after the reaction with γ-glutamyl transpeptidase. A represents the fluorescent probe (10 μmol·L-1), and B represents the mixture of the fluorescent probe (10 μmol·L-1) and γ-glutamyl transpeptidase (0.2 U·mL-1). Figure 5 (a) It can be seen that the absorption peak of the solution is enhanced after the reaction. Figure 5 (b) It was found that the fluorescence intensity of the reaction solution decreased due to the addition of γ-glutamyl transpeptidase. This indicates that γ-glutamyl transpeptidase can react with the fluorescent probe. The fluorescence test conditions were λex = 310 nm (EX Slit: 10 nm, EM Slit: 10 nm).

[0070] Selectivity of fluorescent probe Py-GGT-1 for γ-glutamyl transpeptidase in PBS buffer: In order to demonstrate the specific recognition of γ-glutamyl transpeptidase by fluorescent probe Py-GGT-1, some amino acids, inorganic salts [Thr, Glu, Ile, Cys, Met, Pro, Asp, Ser, Val, Phe, Ala, Arg, Leu, Lys, Gln, NaCl, KI, FeSO4, FeCl3, CaCl2, Al(NO3)3, ZnI2] and enzymes contained in organisms (AchE, PPO, β-gal, ALP, Esterase) and human serum albumin (HSA) were selected, 200 μL PBS buffer solution was selected as the test system, and probe Py-GGT-1 and different analytes were added to the system. Figure 6 As shown, 1 is the blank group with only the probe added, 2 is the control group with GGT added, Figure 6 (a) 3-30 is a component to which only interfering substances are added. Figure 6 (b) 3-30 is the addition of interfering components and γ-glutamyl transpeptidase. The prepared solution was incubated at 37 ° C for 30 minutes, and each experimental group was measured three times. The measurement results showed that after adding other interfering components, the intensity of the fluorescent probe was basically consistent with the blank sample, and after adding γ-glutamyl transpeptidase, the fluorescence intensity of the fluorescent probe was weakened and basically consistent with the control group, indicating that the probe has strong anti-interference ability and can be used for biological sample detection. Fluorescence test conditions λex = 310nm (EX Slit: 10nm, EMSlit: 10nm)

[0071] Fluorescent probe Py-GGT-1 was used to detect endogenous γ-glutamyl transpeptidase in HepG 2 cells: HepG 2 cells were cultured in DMEM medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2 concentration. In order to obtain fluorescence imaging of endogenous γ-glutamyl transpeptidase, HepG 2 cells were incubated with 10 μM Py-GGT-1 for different times (15, 30, 50, 80, 100, 120 min), then treated with 4% paraformaldehyde (20 min), washed 3 times with PBS, and imaged using a Leica STELLARIS 5 laser confocal microscope. Figure 7 As shown, with the increase of incubation time, the fluorescence signal gradually weakened, indicating that the probe can be used to detect endogenous γ-glutamyl transpeptidase.

[0072] Determination of the minimum detection limit of the fluorescent probe Py-GGT-1 for γ-glutamyl transpeptidase in normal human serum: At 37°C, in normal human serum diluted ten times with PBS, the fluorescence emission spectrum and the titration experiment of the fluorescent probe on γ-glutamyl transpeptidase showed that the synthesized fluorescent probe has high sensitivity for detecting γ-glutamyl transpeptidase in human serum, indicating that the probe has potential application value in the efficient detection of γ-glutamyl transpeptidase in the serum of liver cancer patients in vitro. And through Figure 8 The results showed that the fluorescence intensity of the fluorescent probe at 420 nm was linearly correlated with the concentration of GGT in the range of 0-60 mU·mL-1 (linear correlation coefficient R2=0.981). The fluorescence test conditions were λex=310 nm (EX Slit: 10 nm, EM Slit: 10 nm).

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0074] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase, characterized by: It includes a fluorescent probe Py-GGT-1, the structural formula of which is:

2. The method for preparing a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 1, wherein: The invention comprises a fluorescent probe (Py-GGT-1) using an (E)-4-phenylvinylpyridine fragment as a fluorescent signal reporter group, modifying the fluorescent group with aniline, introducing tert-butyloxycarbonyl-L-glutamate 1-tert-butyl ester through a condensation reaction between the amino group and the carboxyl group, and removing the Boc protecting group with trifluoroacetic acid to serve as an enzyme cleavage site for γ-glutamyl transpeptidase.

3. The method for preparing a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 2, wherein: The specific steps include: In the first step, 1,4-dioxane and water are used as the reaction medium, 2-bromo-4-chloropyridine and (4-((tert-butyloxycarbonyl)amino)phenyl)boric acid are used as reactants in an argon atmosphere, tetrakis(triphenylphosphine palladium) is used as a catalyst, and sodium carbonate is used as a reaction base. The reaction is refluxed at 85°C for 12 hours. After completion of the reaction, the mixture is extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as the eluent to obtain tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate as a white solid. In the second step, dichloromethane is used as a reaction medium, and the tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate obtained in step (1) is dissolved in dichloromethane, trifluoroacetic acid is slowly added dropwise to the dichloromethane solution, and the mixture is stirred at room temperature until the reaction is completed. The solvent and most of the trifluoroacetic acid are then distilled off under reduced pressure, and a saturated aqueous sodium bicarbonate solution is added to adjust the alkaline environment. After water extraction, the mixture is separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as eluent to obtain 4-(4-chloropyridin-2-yl)aniline as a white solid; In the third step, tert-butyloxycarbonyl-L-glutamate (Boc-Glu-OtBu), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) were dissolved in DMF and stirred at room temperature for 15 minutes. A DMF solution of 4-(4-chloropyridin-2-yl)aniline was added and the mixture was reacted at room temperature for 24 hours. After the reaction was completed, the mixture was extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as eluent to obtain tert-butyl N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate as a white solid. The fourth step is to use 1,4-dioxane and water as the reaction medium, N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate tert-butyl ester and (E)-styrene boronic acid as reactants in an argon atmosphere, tetrakis(triphenylphosphine)palladium as a catalyst, and sodium carbonate as a reaction base, and reflux at 105°C for 12 hours. After completion of the reaction, the mixture is extracted with water and separated by 200-300 mesh silica gel column chromatography using ethyl acetate / petroleum ether as an eluent to obtain (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-styrylpyridin-2-yl)phenyl)glutamate tert-butyl ester as a white solid; In the fifth step, using dichloromethane as the reaction medium, (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylpyridin-2-yl)phenyl)glutamate tert-butyl ester was dissolved in dichloromethane, and trifluoroacetic acid diluted with dichloromethane was slowly added dropwise. The reaction was carried out at room temperature for 6 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and the mixture was separated by a C18 column using methanol / water as the eluent to obtain a pyridine target fluorescent probe.

4. The method for preparing a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 3, wherein: The molar ratio of the 2-bromo-4-chloropyridine to 4-((tert-butyloxycarbonyl)amino)phenyl)boric acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1:(1.0-2.0):(0.04-0.06):(2.0-3.0); The molar ratio of the tert-butyl (4-(4-chloropyridin-2-yl)phenyl)carbamate to trifluoroacetic acid is 1:(13-14).

5. The method for preparing a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 3, characterized in that: The molar ratio of the 4-(4-chloropyridin-2-yl)aniline to Boc-Glu-OtBu, HATU, and DIPEA is 1:(1.0-2.0):(1.0-2.0):(1.0-2.0); The molar ratio of the N2-(tert-butyloxycarbonyl)-N5-(4-(4-chloropyridin-2-yl)phenyl)glutamate to (E)-styreneboric acid, tetrakis(triphenylphosphine)palladium, and sodium carbonate is 1:(1.0-2.0):(0.04-0.06):(2.0-3.0); The molar ratio of the (E)-N2-(tert-butyloxycarbonyl)-N5-(4-(4-phenylvinylpyridin-2-yl)phenyl)glutamate to trifluoroacetic acid is 1:(20.0-30.0).

6. Use of the fluorescent probe according to claim 1 for selectively identifying γ-glutamyl transpeptidase in PBS for non-diagnostic purposes, characterized in that: The synthesized pyridine skeleton fluorescent probe Py-GGT-1 can be used to detect γ-glutamyl transpeptidase in the serum of liver cancer patients.

7. Use of the pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 1, characterized in that: The fluorescent probe Py-GGT-1 γ Under the catalytic enzymatic hydrolysis of γ-glutamyl transpeptidase, a weakly fluorescent metabolite Py-1-NH2 is produced. The γ-glutamyl transpeptidase is over-expressed in the body of liver cancer patients. The synthesized pyridine skeleton fluorescent probe can be used for non-diagnostic detection of γ-glutamyl transpeptidase in the serum of liver cancer patients.

8. The use of a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 7, characterized in that: In the γ-glutamyl transpeptidase assay, the γ-glutamyl transpeptidase and the fluorescent probe were incubated at 37°C in PBS with a pH of 7.4 for 30 min.

9. The use of a pyridine fluorescent probe for rapid in vitro detection of γ-glutamyl transpeptidase according to claim 8, characterized in that: The reaction concentration of the fluorescent probe is 10 μmol·L-1.

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