A glutaryltranspeptidase fluorescent probe, and a preparation method and application thereof

Abstract

The application belongs to the field of fluorescent probes, and particularly relates to a glutamyl transpeptidase fluorescent probe and a preparation method and application thereof. In order to provide a fluorescent probe for accurately determining GGT activity and visualizing the position thereof, the application develops a glutamyl transpeptidase (GGT) fluorescent probe Cy5-GGT by using the characteristics that the conversion of the center position "ester group -> carboxyl group" of pentamethine cyanine dye can cause a significant fluorescence off-on response of the dye. When excited by 633 nm light, the probe itself has negligible fluorescence; after acting with GGT, the glutamyl bond in the probe is first cut by GGT, then 1,6-elimination reaction occurs, and finally the strong fluorescent center position carboxyl-substituted pentamethine cyanine dye Cy5-COO is released. The probe can utilize the characteristics that cancer cells have excessive expression of GGT on the surface, and can distinguish cancer cells / tissues and normal cells / tissues with high contrast.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, specifically relating to a glutamyl transpeptidase fluorescent probe, its preparation method, and its application. Background Technology

[0002] Gamma-glutamyl transferase (GGT, EC 2.3.2.2) is a cell surface enzyme that catalyzes the degradation of glutathione and its conjugates in vivo, producing cysteylglycine and gamma-glutamyl residues. It is a key enzyme in the gamma-glutamyl cycle and can also selectively catalyze the cleavage of other gamma-glutamyl compounds. GGT is widely distributed in human tissues, mainly found in the kidneys, liver, and pancreas. Serum GGT primarily originates from the hepatobiliary system, with a normal concentration of 3-50 μ / L. GGT participates in various important physiological processes, and its abnormal activity is closely related to many diseases. Diseases such as acute hepatitis, chronic active hepatitis, alcoholic liver disease, fatty liver, cirrhosis, and biliary obstruction can cause elevated GGT expression levels. Therefore, detecting serum GGT activity is an effective means of clinical diagnosis of various hepatobiliary diseases. Furthermore, studies have found that GGT is overexpressed in various cancer cells; primary or metastatic cancers can induce hepatocytes to produce large amounts of GGT, with concentrations sometimes exceeding 10 times the normal value. Therefore, accurate determination of GGT activity and visualization of its location are of great significance for diagnosing hepatobiliary diseases or guiding cancer treatment. Summary of the Invention

[0003] This invention utilizes the characteristic that the "ester group → carboxyl group" transition at the central position of pentamethine cyanide dye can induce a significant fluorescence on / off response in the dye, and develops a fluorescent probe for gamma-glutamyl transferase (GGT), Cy5-GGT. When excited with 633 nm light, the probe itself exhibits negligible fluorescence; upon interaction with GGT, the glutamyl bond in the probe is first cleaved by GGT, followed by a 1,6-elimination reaction, ultimately releasing the strongly fluorescent centrally carboxyl-substituted pentamethine cyanide dye Cy5-COO. Considering the overexpression of GGT in various cancer cells, this probe can be further used for high-contrast differentiation between cancer cells / tissues and normal cells / tissues, demonstrating great application potential in fluorescence-guided tumor surgery.

[0004] To achieve the above objectives, the present invention employs the following technical solution:

[0005] A fluorescent probe for gamma-glutamyl transferase has the following structural formula:

[0006]

[0007] A method for preparing a fluorescent probe for gamma-glutamyl transferase includes the following steps:

[0008]

[0009] Step 1: Under N2 environment, DMF is dissolved in anhydrous dichloromethane, and then oxalyl chloride is gradually added. The reaction is stirred at room temperature. After the reaction is completed, the solvent is completely evaporated under reduced pressure to obtain compound 1, which is a white solid. It is used directly in the next reaction without purification.

[0010] Step 2: Compound 1 and monomethyl malonate were dissolved in anhydrous dichloromethane and refluxed. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate were added to the mixture in sequence and stirred. The mixture was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain compound Cy5-COOM, which was a dark blue solid.

[0011] Step 3: Dissolve compound Cy5-COOM in a mixed solution of MeOH and NaOH, stir and react, cool, dilute with water and extract with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, then purify by vacuum distillation and column chromatography to obtain compound Cy5-COO, which is a dark blue solid.

[0012] Step 4: Cy5-COO, compound 3, and potassium carbonate were dissolved in anhydrous N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was redissolved in a mixed solution of trifluoroacetic acid and dichloromethane. After further stirring, the mixture was purified by vacuum distillation and column chromatography to obtain compound Cy5-GGT, which is a dark blue solid.

[0013] Furthermore, in step 1, the molar ratio of DMF to oxaloyl chloride is 30:35, and the stirring reaction time is 2 hours.

[0014] Furthermore, in step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40°C, and the reaction time is overnight; the molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate is 1:2:3; the stirring reaction temperature is 90°C, and the reaction time is 4 hours; the developing solvent for column chromatography separation and purification is dichloromethane / methanol = 10 / 1 (v / v).

[0015] Furthermore, in step 3, the stirring reaction temperature is 43°C, the time is 3 hours, and the developing solvent for column chromatography separation and purification is 10-50% methanol / dichloromethane (v / v).

[0016] Furthermore, in step 4, the molar ratio of Cy5-COO, compound 3, and potassium carbonate is 1:3:2, the stirring temperature is 48°C, the time is 3 hours, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1, the further stirring temperature is room temperature, the time is 20 minutes, and the developing solvent for column chromatography separation and purification is CH2Cl2 / MeOH = 8:1 (v / v).

[0017] An application of a fluorescent probe for gamma-glutamyl transferase in distinguishing between normal cells / tissues and cancer cells / tissues.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] Currently, various methods for detecting GGT activity have been reported, including high-performance liquid chromatography (HPLC), electrochemical methods, and colorimetric methods. These methods typically suffer from poor sensitivity, are time-consuming, and cannot provide real-time imaging of live cells. In contrast, fluorescence imaging methods offer significant advantages such as high sensitivity, ease of operation, and non-destructive sample handling. They can detect GGT activity in vitro and visualize the location and activity of GGT in live samples. The GGT fluorescent probe developed in this invention utilizes the characteristic that the conversion of the central substituent of pentamethine cyanine dye from an ester group to a carboxyl group causes a large on / off change in dye fluorescence. This probe can distinguish normal cells / tissues from cancer cells / tissues with high contrast, thus possessing potential application value in surgical navigation. Attached Figure Description

[0020] Figure 1 The NMR and HRMS spectra of compound Cy5-COO are shown.

[0021] Figure 2 The NMR and HRMS spectra of compound Cy5-COOM are shown.

[0022] Figure 3 The NMR and HRMS spectra of compound Cy5-GGT are shown.

[0023] Figure 4 In the figure, (A) shows the UV-Vis absorption spectra of Cy5-GGT (5μM) and GGT (100U / L) before and after the reaction at 37℃ and under PBS (10mM, pH=7.4); (B) shows the fluorescence spectrum changes and the fluorescence intensity at 662nm over time after the reaction of Cy5-GGT (5μM) and GGT (100U / L) at 37℃ and under PBS (10mM, pH=7.4).

[0024] Figure 5The HPLC and HRMS chromatograms before and after the reaction of Cy5-GGT with GGT are shown. HPLC conditions: C18 column (2.1×100 mm); mobile phase: MeCN / H2O (3:7 to 9:1, v / v, containing 0.1% formic acid); flow rate: 0.2 mL / min.

[0025] Figure 6 In the figure, (A) shows the fluorescence spectra of Cy5-GGT (5 μM) before and after reaction with an increased concentration of GGT (0-10 U / L) under the conditions of 37℃, PBS (10 mM, pH=7.4); (B) shows the linear correlation between fluorescence intensity at 662 nm and GGT concentration.

[0026] Figure 7 To determine the reaction conditions at 37°C, PBS (10 mM, pH 7.4), Cy5-GGT (5 μM) in DMEM, or with (B) NADPH (500 μM), (C) GSH (1 mM), (D) Cys (200 μM), (E) H2O2 (100 μM), and (F) O2, respectively. ·- UV-Vis absorption spectrum changes after reaction with (100 μM), (G)APN (50 ng / mL), and (H)NTR (2.0 μg / mL, containing 0.5 mM NADPH) (detected for 30 minutes respectively);

[0027] Figure 8 Cell viability of A549 cells after treatment with different concentrations (0 μM, 2.0 μM, 4.0 μM, 6.0 μM, 8.0 μM and 10.0 μM) of Cy5-GGT for 24 hours;

[0028] Figure 9 Cell images of cancer cells (HepG2 and A549 cells) and normal cells (BEAS-2B and LO2 cells) loaded with Cy5-GGT (2.0 μM), respectively; for cancer cells, cells were incubated with the probe for 60 minutes, or pre-incubated with the inhibitor (GGsTop, 100 μM, 60 minutes) before incubation with the probe for 60 minutes; for normal cells, cells were incubated with the probe only for 60 minutes; images were collected at wavelengths of 640-750 nm (λ). ex =633nm), scale bar: 20μm.

[0029] Figure 10 In the image, (A) shows confocal images of HepG2 tumor tissue and leg muscle tissue loaded with Cy5-GGT (2.0 μM), respectively. For the former, the tissue was incubated with the probe for 60 minutes, or pre-incubated with the inhibitor (GGsTop, 100 μM, 60 minutes) before incubating the probe for 60 minutes. For the latter, the tissue was incubated with the probe only for 60 minutes. The collection wavelength was 640–750 nm (λ).ex =633nm), scale bar: 20μm; (B) is the fluorescence quantitative chromatogram of (A);

[0030] Figure 11 In vivo imaging of HepG2 tumor-bearing mice after intratumoral injection of Cy5-GGT (10 μM, 100 μL); using a 610 nm excitation filter and a 700 nm emission filter. Detailed Implementation

[0031] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0032] Example 1

[0033] A fluorescent probe for gamma-glutamyl transferase has the following structural formula:

[0034]

[0035] A method for preparing a fluorescent probe for gamma-glutamyl transferase includes the following steps:

[0036] Step 1: Under N2 environment, DMF (2.3 mL, 30 mmol) and anhydrous dichloromethane (15 mL) were added to a dry flask, followed by the gradual addition of oxaloyl chloride (3.0 mL, 35 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was evaporated completely under reduced pressure to obtain compound 1 (3.6 g, 94.7%), a white solid, which was used directly in the next reaction without purification.

[0037] Step 2: Compound 1 (1.2 g, 10 mmol) and monomethyl malonate (0.59 g, 5 mmol) were dissolved in anhydrous dichloromethane (20 mL) and refluxed overnight at 40 °C. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride (10 mL), 1,2,3,3-tetramethyl-3H-indole iodide (3.01 g, 10 mmol) and anhydrous sodium acetate (1.23 g, 15 mmol) were added sequentially to the solution. The mixture was stirred at 90 °C for 4 hours. The solution was then diluted with water and extracted with dichloromethane. The organic layers were combined and purified by drying with anhydrous sodium sulfate, vacuum distillation, and column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound Cy5-COOM (1.1 g, 38.7%), which was a dark blue solid. 1 H NMR(600Hz,CD3Cl)δ8.52(d,J=14.4Hz,2H),7.45(t,J=7.2Hz,4H),7.33(t,J =8.4Hz,4H),7.03(d,J=15.0Hz,4H),3.99(s,6H),3.93(s,3H),1.79(s,12H);13 C NMR (150MHz, CD3Cl) δ177.7,167.1,142.5,141.2,128.9,126.4,122.3,111.9,102.1,52.0,50.2,34.3,28.2; ESI-MS[M] + :calcd for441.2537,Found 441.2526.

[0038] Step 3: The compound Cy5-COOM (852 mg, 1.5 mmol) was dissolved in a mixed solution of MeOH (40 mL) and NaOH (2 mM, 60 mL), and the mixture was stirred at 43 °C for 3 hours. After cooling, the mixture was diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography (10-50% methanol / dichloromethane, v / v) to obtain the compound Cy5-COO (318 mg, 38.3%), which was a dark blue solid. 1 H NMR (600Hz, CD3OD) δ8.29(s,2H),7.45(m,8H),6.90(s,2H),3.72(s,6H),1.76(s,12H); 13 C NMR (150MHz, CD3OD) δ142.7,141.4,128.4,125.5,121.9,111.0,101.7,60.1,49.4,30.6,26.7,19.5,13.1; ESI-MS[M] + :calcdfor427.2380,Found 427.2389.

[0039] Step 4: Cy5-COO (554 mg, 1.0 mmol), compound 3 (1410 mg, 3.0 mmol), and potassium carbonate (276 mg, 2.0 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The mixture was stirred at 48 °C for 3 hours. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was redissolved in a mixed solution of trifluoroacetic acid (10 mL) and dichloromethane (10 mL). The mixture was stirred at room temperature for 20 minutes. After purification by distillation under reduced pressure and column chromatography (dichloromethane / methanol = 8 / 1, v / v), compound Cy5-GGT was obtained as a dark blue solid (329 mg, yield 42.3%). 1H NMR (600Hz, CD3OD) δ8.41(s,2H),7.64(d,J=7.2Hz,2H),7.57(d,J=5.4Hz,2H),7.53(d,J=7.2Hz,2H),7.48(d,J=6.6Hz,2H),7.43(d,J=6 .0Hz,2H),7.37(t,J=7.8Hz,2H),7.02(d,J=14.4Hz,2H),5.34(s,2H),4.05(m,1H),3.66(s,6H),2.76(m,2H),2.29(m,2H),1.74(s,12H); 13 C NMR (150MHz, CD3OD) δ177.5,171.2,166.3,142.4,141.5,138.7,131.9,129.3,128.5,126 .0,121.9,120.1,111.4,101.6,66.1,53.4,49.7,31.8,30.7,29.3,26.6,25.6; ESI-MS[M] + :calcd for 661.3384,Found 661.3373.

[0040] Example 2

[0041] 1. Study of photophysical properties

[0042] First, we investigated the absorption and emission spectra of the probe Cy5-GGT and GGT before and after reaction in PBS (10 mM, pH = 7.4) at 37 °C. The results are as follows: Figure 4 As shown. Figure 4 As shown in (A), the maximum absorption peak of the probe in PBS is 610 nm. After interaction with GGT, the absorption peak of the probe at 610 nm red-shifts to 635 nm. Figure 4 As shown in (B), when excited by 633nm light, the probe itself has a weak emission peak. After interacting with GGT, the emission peak at 662nm gradually rises and reaches its maximum value at 20 minutes, with fluorescence enhancement of 10.5 times.

[0043] Figure 5 The images show HPLC and HRMS chromatograms of Cy5-GGT before and after the reaction with GGT; HPLC conditions: C18 column (2.1 × 100 mm); mobile phase: MeCN / H2O (3:7 to 9:1, v / v, containing 0.1% formic acid); flow rate: 0.2 mL / min. HPLC-MS analysis showed that Cy5-GGT reacted with GGT to generate a strongly fluorescent Cy5-COO dye.

[0044] Figure 6 (A) shows the fluorescence spectra of Cy5-GGT (5 μM) before and after reaction with an increased concentration of GGT (0-10 U / L) under PBS (10 mM, pH = 7.4) conditions at 37℃; (B) shows the linear correlation between fluorescence intensity at 662 nm and GGT concentration. The fluorescence titration experiment showed that the fluorescence intensity of the probe Cy5-GGT at 662 nm increased with increasing GGT concentration. The calculated detection limit for GGT was 0.019 U / L.

[0045] To further verify the high selectivity of Cy5-GGT for GGT, we tested the stability of the probe Cy5-APN in DMEM medium (a commonly used cell culture medium containing various biologically relevant substances such as cations, anions, various amino acids, glucose, and pyruvate) or in PBS containing NADPH, GSH, Cys, H2O2, O2·-, APN, and NTR, respectively. Figure 7 As shown, the probe is stable in all of the above substances. These results demonstrate that the probe Cy5-GGT is a highly selective and sensitive fluorescence-enhanced GGT fluorescent probe.

[0046] 2. Cell imaging research

[0047] Next, we will verify the ability of the probe Cy5-GGT to perform GGT imaging in biological systems.

[0048] First, the cytotoxicity of the probe was tested using a CCK8 cell proliferation assay, and the results were as follows: Figure 8 As shown, the cell survival rate of the probe was greater than 90% in the concentration range of 0-10 μM, which proves the biosafety of the probe.

[0049] Because GGT is overexpressed in various cancer cells, we selected two cancer cell lines (HepG2 cells and A549 cells) and two normal cell lines (LO2 cells and BEAS-2B cells) to verify the ability of the probe Cy5-GGT to distinguish between cancer cells and normal cells. Figure 9 As shown, cancer cells loaded with Cy5-GGT exhibited a significant red fluorescent signal, while the fluorescent signal in normal cells loaded with the probe was negligible. Furthermore, no fluorescent signal was observed in cancer cells pre-loaded with a GGT inhibitor, indicating that the red fluorescent signal in cancer cells is indeed caused by GGT. These results demonstrate that Cy5-GGT can utilize the overexpression of GGT on the surface of cancer cells to distinguish cancer cells from normal cells with high contrast.

[0050] Finally, we evaluated the ability of the probe Cy5-GGT to distinguish between cancerous and normal tissues at the in vivo level. Figure 10(A) shows confocal imaging images of HepG2 tumor tissue and leg muscle tissue loaded with Cy5-GGT (2.0 μM), respectively. For the former, the tissue was incubated with the probe for 60 minutes, or pre-incubated with the inhibitor (GGsTop, 100 μM, 60 minutes) before incubating with the probe for 60 minutes. For the latter, the tissue was incubated with the probe only for 60 minutes. The collection wavelength was 640-750 nm (λex = 633 nm), and the scale bar was 20 μm. (B) is the quantitative fluorescence image of (A). Figure 10 As shown in (A), HepG2 tumor tissue sections loaded with Cy5-GGT exhibit a bright fluorescence signal in the red channel, which can be suppressed by the GGT inhibitor GGsTop; normal muscle tissue sections loaded with Cy5-GGT show a negligible fluorescence signal in the red channel. Figure 10 As shown in (B), the average fluorescence intensity of cancerous tissue is 9.0 times that of normal tissue, which is far greater than the clinically acceptable threshold of 2.

[0051] Furthermore, we investigated the ability of Cy5-GGT to perform in situ real-time imaging of HepG2 tumor-bearing mice, and in vivo imaging such as... Figure 11 As shown. By Figure 11 The results showed that when the probe was injected in situ into the tumor and the leg, respectively, the fluorescence intensity in the tumor area gradually increased and reached its maximum at 150 minutes, while the fluorescence in the normal tissue area was negligible throughout the detection time, with a maximum signal-to-noise ratio (T / N) of 10. These results indicate that Cy5-GGT can distinguish between cancerous and normal tissues with high contrast, thus possessing potential application value.

[0052] In summary, this invention constructs a highly selective and sensitive GGT fluorescent probe based on the "ester group → carboxyl group" conversion strategy of pentamethine cyanine dye. The detection limit of this probe for GGT is 0.019 U / L. Utilizing the characteristic of cancer cells overexpressing GGT, the probe can distinguish cancer cells / tissues from normal cells / tissues with high contrast, thus showing great promise for applications in surgical navigation.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fluorescent probe for gamma-glutamyl transferase, characterized in that, Its structural formula is: 。 2. A method for preparing the fluorescent probe for gamma-glutamyl transferase as described in claim 1, characterized in that, Includes the following steps: Step 1: Under N2 environment, DMF is dissolved in anhydrous dichloromethane, and then oxalyl chloride is gradually added. The reaction is stirred at room temperature. After the reaction is completed, the solvent is completely evaporated under reduced pressure to obtain compound 1, which is a white solid. It is used directly in the next reaction without purification. Step 2: Compound 1 and monomethyl malonate were dissolved in anhydrous dichloromethane and refluxed. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate were added to the mixture in sequence and stirred. The mixture was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain compound Cy5-COOM, which was a dark blue solid. Compound 1 is: Compound 2 is: The compound Cy5-COOM is: ; Step 3: Dissolve compound Cy5-COOM in a mixed solution of MeOH and NaOH, stir and react, cool, dilute with water and extract with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, then purify by vacuum distillation and column chromatography to obtain compound Cy5-COO, which is a dark blue solid. The compound Cy5-COO is: ; Step 4: Cy5-COO, compound 3, and potassium carbonate were dissolved in anhydrous N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was redissolved in a mixed solution of trifluoroacetic acid and dichloromethane. After further stirring, the mixture was purified by vacuum distillation and column chromatography to obtain compound Cy5-GGT, which is a dark blue solid. The compound 3 is: .

3. The method for preparing a fluorescent probe for gamma-glutamyl transferase according to claim 2, characterized in that, In step 1, the molar ratio of DMF to oxaloyl chloride is 30:35, and the stirring reaction time is 2 hours.

4. The method for preparing a fluorescent probe for gamma-glutamyl transferase according to claim 2, characterized in that, In step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40°C, and the reaction time is overnight. The molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate is 1:2:

3. The stirring reaction temperature is 90°C, and the reaction time is 4 hours. The developing solvent for column chromatography separation and purification is dichloromethane / methanol = 10 / 1, v / v.

5. The method for preparing a fluorescent probe for gamma-glutamyl transferase according to claim 2, characterized in that, In step 3, the stirring reaction temperature is 43℃ ​​and the time is 3h. The developing solvent for column chromatography separation and purification is 10-50% methanol / dichloromethane, v / v.

6. The method for preparing a fluorescent probe for gamma-glutamyl transferase according to claim 2, characterized in that, In step 4, the molar ratio of Cy5-COO, compound 3, and potassium carbonate is 1:3:

2. The stirring temperature is 48°C, the reaction time is 3 h, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1, the further stirring temperature is room temperature, the reaction time is 20 minutes, and the developing solvent for column chromatography separation and purification is CH2Cl2 / MeOH = 8:1, v / v.

7. The application of the fluorescent probe for gamma-glutamyl transferase according to claim 1, characterized in that, Used to prepare reagents that can distinguish between normal cells / tissues and cancer cells / tissues.

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