Fluorescent probe and application thereof in detection of glioma idh mutation

Abstract

The application discloses a fluorescent probe and application thereof in glioma IDH mutation detection, and the fluorescent probe BOD-G constructed in the application can sensitively, specifically and rapidly detect H2O2, and the fluorescence response of BOD-G to H2O2 gradually increases with the increase of pH. Since the ROS level and pH of IDH mutant glioma are higher than those of IDH wild-type glioma, the fluorescent probe can rapidly distinguish IDH mutant glioma from wild-type glioma, and greatly shortens the detection time.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a fluorescent probe and its application in the detection of IDH mutations in gliomas. Background Technology

[0002] High-frequency mutations in isocitrate dehydrogenase (IDH) primarily occur in gliomas. IDH mutations (mutIDH) play a crucial role in the development and progression of gliomas. In 2016, the World Health Organization (WHO) included IDH mutations as an important molecular marker for glioma classification. IDH mutations have the potential for targeted therapy of gliomas and participate in guiding radiotherapy or alkylating agents. Patients with IDH-mutant gliomas generally have a good prognosis, with a median survival of up to 7 years, compared to only 1.7 years for wild-type gliomas. Therefore, IDH mutations are an important biomarker for gliomas.

[0003] In gliomas, IDH mutations primarily occur when arginine at position 132 of the IDH1 catalytic site is replaced by histidine (R132H), accounting for approximately 90% of all IDH mutations. Other types include IDH1 (R132C, R132L, R132S, R132G) and IDH2 (R172K). IDH has three isoenzymes; mutations in IDH1 or IDH2 reduce their ability to oxidize isocitrate to α-ketoglutarate (α-KG), accompanied by a decrease in NADPH production. Simultaneously, the mutated IDH acquires a new enzymatic function, reducing α-KG to 2-hydroxyglutarate (2-HG), a process that also consumes NADPH. Studies have found that IDH mutations alter the tumor microenvironment of gliomas. The large accumulation of 2-HG can enhance oxidative stress in the brain, increasing reactive oxygen species (ROS) levels, and can also degrade intracellular hypoxia-inducible factor 1 (HIF1), leading to reduced lactate accumulation and an increased pH in glioma tissue. IDH mutations lead to NADPH depletion, which hinders the synthesis of intracellular glutathione (GSH) and the clearance of ROS, thus increasing intracellular ROS levels. Therefore, IDH-mutant gliomas exhibit higher levels of ROS and pH.

[0004] Given the significant impact of IDH mutations on gliomas, detecting these mutations is crucial for diagnosis, personalized treatment, and clinical prognosis. Currently, researchers have developed various methods for detecting IDH mutations in gliomas. Immunohistochemistry (IHC) and Sanger sequencing are standard clinical methods. IHC utilizes IDH1 R132H-specific antibodies to detect IDH1 R132H gliomas, providing accurate mutation information, but typically takes 3 days. However, due to limitations in antibody types, IHC can only detect IDH1 R132H, and the antibodies are expensive, resulting in high testing costs. Sanger sequencing analyzes gene sequencing results to determine the mutation type, theoretically capable of detecting all types of IDH mutations. However, most hospitals lack sequencing platforms, and sequencing is usually sent to companies, taking 5-7 days, which is time-consuming and costly. Nuclear magnetic resonance spectroscopy (MRS) indirectly reflects IDH mutations in gliomas by detecting the IDH mutation metabolite 2-HG. This method has low sensitivity, typically requiring a 2-HG concentration greater than 1 mM for detection, and is easily interfered with by other brain metabolites. Positron emission tomography (PET) imaging, which uses radioactive tracers specific to the IDH1 mutant enzyme for non-invasive detection of gliomas, is radioactive and suffers from poor metabolic stability of the tracers themselves, as well as insufficient affinity and selectivity for the IDH1 mutant enzyme. Therefore, developing an efficient method for detecting IDH mutations in gliomas is of great significance.

[0005] Fluorescent probes possess advantages such as high sensitivity, good selectivity, rapid response, simple operation, no radiation, and in-situ real-time detection, making them powerful tools for the detection of important biomolecules in living systems. In recent years, based on the tumor-specific microenvironment, such as decreased extracellular pH and increased intracellular GSH and ROS levels, various organic molecular fluorescent probes have been applied to tumor imaging. However, fluorescent probes for the detection of IDH-mutant gliomas have not yet been reported. Therefore, this application aims to design fluorescent probes based on the changes in the glioma microenvironment caused by IDH mutations and propose a simple and rapid new method for the detection of IDH-mutant gliomas. Summary of the Invention

[0006] The technical problem to be solved by this invention is: how to provide a new fluorescent probe BOD-G and use it to achieve efficient and rapid differentiation between IDH mutant and IDH wild-type gliomas.

[0007] The technical solution of the present invention is: the compound shown in formula (Ⅰ), wherein the compound BOD-G has BODIPY fluorescent parent as the fluorescent group, benzoyl as the H2O2 recognition group, and oligoethylene glycol structure as the water-soluble group.

[0008]

[0009] A kit containing the compounds described above.

[0010] The use of the above-described compounds in the preparation of reagents for detecting IDH mutations in gliomas.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The fluorescent probe BOD-G constructed in this invention can sensitively, specifically, and rapidly detect H2O2. The fluorescence response of BOD-G to H2O2 gradually increases with increasing pH. Since the ROS level and pH of IDH mutant gliomas are higher than those of IDH wild-type gliomas, this fluorescent probe can quickly distinguish between IDH mutant and wild-type glioma tissues, greatly shortening the detection time. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the synthetic route for BOD-G;

[0014] Figure 2 This is a schematic diagram of the response mechanism of BOD-G to H2O2.

[0015] Figure 3 Spectral performance analysis of BOD-G response to H2O2;

[0016] Figure 4 Selectivity analysis of BOD-G for H2O2;

[0017] Figure 5 The effect of pH on the reaction of BOD-G with H2O2;

[0018] Figure 6 Cytotoxicity analysis for BOD-G;

[0019] Figure 7 Fluorescence imaging detection of H2O2 in U87MG-mut cells using BOD-G;

[0020] Figure 8 Comparison of fluorescence imaging of BOD-G on mutIDH1 and wtIDH glioma cells;

[0021] Figure 9 Comparison of fluorescence imaging of BOD-G on mutIDH1 and wtIDH glioma tissues;

[0022] Figure 10 The proton NMR spectrum confirming the BOD-G structure ( 1 H NMR);

[0023] Figure 11 The carbon NMR spectrum confirming the BOD-G structure ( 13 C NMR);

[0024] Figure 12 High-resolution mass spectra (HRMS) confirming the structure of BOD-G. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0026] Example 1: Synthesis of BOD-G

[0027] The fluorescent probe BOD-G is synthesized via a three-step organic reaction, as shown in the following synthetic route: Figure 1 As shown:

[0028] (1) Synthesis of compound 3

[0029] Compound 1 (0.20 g, 0.75 mmol), K₂CO₃ (0.52 g, 3.77 mmol), and KI (0.02 g, 0.08 mmol) were dissolved in 6 mL of acetone. Compound 2 (0.51 g, 1.89 mmol) was slowly added dropwise to the mixture, and the mixture was heated and stirred at 60 °C for 12 h. After the reaction was complete, the mixture was extracted three times with CH₂Cl₂ / H₂O. The organic phase was dried over anhydrous MgSO₄, filtered, and the solvent was removed by vacuum distillation. Finally, the product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:1, v / v) to give a colorless liquid product 3 (0.29 g, 85%).

[0030] (2) Synthesis of compound 5

[0031] Compound 3 (0.13 g, 0.29 mmol) and compound 4 (0.10 g, 0.29 mmol) were dissolved in 9 mL of ethyl acetate and 12 mL of triethylamine. PdCl2(PPh3)2 (0.04 g, 0.05 mmol) and CuI (0.02 g, 0.09 mmol) were added under continuous N2 purging, and the mixture was heated and stirred at 70 °C for 3.5 h. After the reaction was complete, the mixture was extracted three times with CH2Cl2 / H2O. The organic phase was dried over anhydrous MgSO4, filtered, and the solvent was removed by vacuum distillation. Finally, the mixture was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1, v / v) to give red solid product 5 (0.13 g, 66%).

[0032] (3) Synthesis of compound BOD-G

[0033] Compound 5 (0.05 g, 0.07 mmol) was dissolved in 2 mL DMSO, and PdCl2 (0.02 g, 0.11 mmol) was added. The mixture was heated and stirred at 65 °C for 3.5 h. After the reaction was complete, the mixture was extracted three times with CH2Cl2 / H2O. The organic phase was dried over anhydrous MgSO4, filtered, and the solvent was removed by vacuum distillation. Finally, the mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1, v / v) to give the red solid product BOD-G (0.03 g, 62%). The structure of BOD-G was determined by... 1 H NMR, 13 Fully characterized by C NMR and high-resolution mass spectrometry (HRMS). 1 H NMR (400MHz, CDCl3): δ (ppm) 8.48 (d, J = 2.2Hz, 1H), 8.23 ​​(dd, J = 8.9, 2.2Hz, 1H) ,8.13(d,J=8.2Hz,2H),7.50(d,J=8.2Hz,2H),7.28(d,J=9.0Hz,1H),5.99(s,2 H),4.42–4.35(t,J=4.6Hz,2H),3.97–3.91(t,J=4.6Hz,2H),3.76–3.71(m,2H) ,3.67–3.60(m,8H),3.55–3.51(m,2H),3.36(s,3H),2.54(s,6H),1.37(s,6H). 13 C NMR (101MHz, CDCl3): δ (ppm) 191.96, 190.05, 157.20, 156.52, 142.95, 142.36, 140.03, 139.45, 135.71, 133.07, 130.96, 130.84, 129.38, 128.03,125.45,121.87,115.17,72.07,71.31,70.78,70.74,70.71,70.65,70.33,69.18,59.22,14.93.HRMS(ESI):m / z,730.2701[M+Na] + ,calcd forC 36 H 40 BF2N3O9Na, 730.2717.

[0034] Example 2: Response mechanism of BOD-G to H2O2

[0035] The response mechanism of BOD-G to H2O2 is as follows: Figure 2As shown. Due to the strong electron-withdrawing ability of nitrobenzene, the charge within the BOD-G molecule transfers from the BODIPY fluorophore to the nitrobenzene site, resulting in photoinduced electron transfer (PET), which leads to very weak fluorescence in BOD-G itself. When H2O2 selectively oxidizes the benzoylayl structure of BOD-G, the PET effect is blocked, generating a new product BODIPY1 with strong fluorescence properties.

[0036] Example 3: Spectral performance analysis of BOD-G's response to H2O2

[0037] BOD-G and H2O2 were tested in a PBS (0.1M, pH=7.4) buffer solution containing 5% DMSO. Figure 3 ).

[0038] Figure 3 a represents the UV-Vis absorption spectra of BOD-G and H2O2 before and after 2 hours of reaction. The absorption spectrum of BOD-G (5 μM) is relatively broad, with a maximum absorption wavelength of 517 nm. When BOD-G (5 μM) and H2O2 (2 mM) react fully at 37 °C for 2 hours, the absorption spectrum narrows and becomes higher, with the maximum absorption wavelength blue-shifted to 498 nm.

[0039] Figure 3 b shows the fluorescence intensity changes of BOD-G reacting with different concentrations of H2O2. Due to the effect of PET, the fluorescence of the BOD-G solution is very weak. When BOD-G (5 μM) reacts with H2O2 (100 μM-1 mM) at 37 °C for 1 h, the probe solution emits obvious green fluorescence with a maximum fluorescence wavelength of 508 nm, and the fluorescence intensity increases continuously with the increase of H2O2 concentration.

[0040] Figure 3 c represents the kinetic study of the reaction between BOD-G and H2O2 over time. The fluorescence changes of BOD-G (0.5 μM) and H2O2 (2 mM) were monitored in real time at 37 °C for 1 h. The fluorescence intensity of BOD-G at 508 nm increased continuously with increasing reaction time, reaching maximum equilibrium at 30 min, while BOD-G itself did not show significant changes in fluorescence intensity.

[0041] Figure 3 d represents the linear correlation analysis of the fluorescence intensity of BOD-G at 508 nm with the concentration of H2O2. BOD-G (5 μM) and H2O2 showed a linear relationship in the concentration range of 2.5-17.5 μM, with a detection limit of 1.07 μM (3σ / s, n=11).

[0042] Therefore, the fluorescent probe BOD-G constructed in this invention can detect H2O2 sensitively and rapidly, and has good stability.

[0043] Example 4: Selectivity analysis of BOD-G for H2O2

[0044] The selectivity of BOD-G for H2O2 was analyzed by comparing its response performance to various reactive oxygen species. BOD-G (5 μM) was compared with 500 μM H2O2 and O2. ·- , 1 O2, ·OH, ·NO, ONOO - 、·O t Bu and TBHP were reacted at 37℃ for 1 h. The reaction of BOD-G with H2O2 showed a significant enhancement of fluorescence signal, while the reaction with other reactive oxygen species only showed a slight change in fluorescence. Figure 4 Therefore, the fluorescent probe BOD-G constructed in this invention has good selectivity for H2O2.

[0045] Example 5: Effect of pH on the reaction of BOD-G with H2O2

[0046] BOD-G (5 μM) and H2O2 (500 μM) were reacted in buffer solutions with different pH values ​​(5.0, 6.0, 6.6, 7.0, 7.4, 8.0) at 37 °C for 1 h, and the fluorescence signal at 508 nm was collected. Figure 5 As shown, BOD-G reacts slowly with H2O2 in acidic solution. When the pH is 6, BOD-G and H2O2 begin to react weakly. As the pH increases, the fluorescence intensity gradually increases, indicating that the reaction between BOD-G and H2O2 is faster. This may be due to the stronger nucleophilicity of H2O2 under alkaline conditions.

[0047] Example 6: Cytotoxicity of BOD-G

[0048] The toxic effects of BOD-G on wild-type glioma cells (U87MG) and IDH1R132H mutant glioma cells (U87MG-mut) were analyzed using the CCK-8 toxicity assay. Within the concentration range of 0-30 μM, the survival rate of U87MG cells after 12 h of BOD-G incubation was higher than 90%, and the survival rate of U87MG-mut cells after 12 h of BOD-G incubation was higher than 85%. Figure 6 The fluorescent probe BOD-G constructed in this invention has minimal toxicity to both types of cells and can be used for studies at the live cell level.

[0049] Example 7: Fluorescence Imaging Detection of Intracellular H2O2 in U87MG-mut Cells by BOD-G

[0050] U87MG-mut with 5.0×10 4Cells were seeded at a concentration of 100 μM and cultured in 35 mm laser confocal microscopy dishes for 24 h. The culture medium was removed, and the cells were washed three times with PBS buffer, then incubated with BOD-G and imaged under a laser confocal microscope. U87MG-mut cells themselves did not exhibit fluorescence. After incubation with BOD-G (1 μM) for 5 min, significant green fluorescence appeared within the cells. To verify that the fluorescence enhancement was caused by H2O2, U87MG-mut cells were pretreated with the H2O2 scavenger NAC (2 mM) for 1 h, followed by incubation with BOD-G (1 μM) for 5 min. The fluorescence intensity within the cells was found to be significantly reduced. After NAC pretreatment, exogenous H2O2 (100 μM) was added, followed by incubation with BOD-G (1 μM) for 5 min. The fluorescence signal within the cells was found to be significantly enhanced again. Figure 7 The fluorescent probe BOD-G constructed in this invention can be used for fluorescence imaging detection of H2O2 in IDH mutant glioma cells.

[0051] Example 8: Fluorescence Imaging Analysis of BOD-G on mutIDH1 and wtIDH Glioblastoma Cells

[0052] Incubation of U87MG cells with BOD-G (1 μM) for 5 min revealed very weak intracellular fluorescence signals. Comparison of fluorescence signals between U87MG-mut and U87MG cells showed that the fluorescence intensity of U87MG-mut cells was 10.8 times that of U87MG cells, indicating that the H2O2 level in U87MG-mut cells was significantly higher than that in U87MG cells. Figure 8 The fluorescent probe BOD-G constructed in this invention can be used to rapidly distinguish between mutIDH1 glioma cells and wtIDH glioma cells.

[0053] Example 9: Fluorescence Imaging Analysis of BOD-G on mutIDH1 and wtIDH Glioma Tissues

[0054] Finally, BOD-G was applied to the detection of clinical tissue samples of IDH-mutant gliomas. Fresh tissue samples from patients diagnosed with mutIDH1 glioma and wtIDH glioma by immunohistochemistry and gene sequencing were selected. The tissues were cut into 100 μM thick sections using a vibratory microtome, and then incubated with BOD-G (5 μM) at room temperature for 15 min before imaging under a laser confocal microscope. BOD-G-treated mutIDH1 glioma tissue showed a very strong fluorescence signal, while BOD-G-treated wtIDH glioma tissue showed almost no fluorescence signal. Figure 9The significant difference in fluorescence signals between the two is mainly attributed to the higher levels of H2O2 and pH in mutIDH1 gliomas compared to wtIDH gliomas. The fluorescent probe BOD-G constructed in this invention can rapidly distinguish between mutIDH1 gliomas and wtIDH gliomas at the tissue level.

Claims

1. The use of the compound represented by formula (Ⅰ) in the preparation of reagents for detecting glioma IDH mutations:

Images