Molecular fluorescent probe for detecting peroxynitrite in brain of mouse and preparation method of molecular fluorescent probe
By designing a near-infrared small molecule fluorescent probe P3CF3, which contains specific probes and targeting groups, the problem of the existing technology being difficult to penetrate the blood-brain barrier to detect the ONOO-level of mice brains is solved, and a high sensitivity and specific detection effect is achieved.
Patent Information
- Application Number
- CN202510323073.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-06-20
AI Technical Summary
Existing fluorescent probes are difficult to penetrate the blood-brain barrier and cannot detect the peroxygen nitrite (ONOO-) levels in mice with high sensitivity and specific sensitivity.
A near-infrared small molecule fluorescent probe P3CF3 is designed, which contains 2-fluoro-5-nitrobenzoic acid as the probe site, which has specific recognition ability for ONOO-, and trifluoromethyl is a targeting group, which can penetrate the blood-brain barrier.
A sensitive and specific detection of ONOO-level in the mouse brain is achieved, with good biocompatibility and cell permeability, and imaging detection at ONOO-expression level can be achieved in the brain tissue of PD mice.
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Figure CN120172879A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of organic molecular fluorescence probes, and specifically discloses a molecular fluorescence probe capable of sensitively and specifically detecting peroxynitrite in the brain of mice, a preparation method thereof, and an application thereof. Background Art
[0002] Parkinson's disease (PD) is the most common neurodegenerative disease. The pathological features of PD include the progressive loss of dopaminergic neurons, leading to motor dysfunction, including involuntary tremors and muscle stiffness. Studies have shown that in the brains of PD patients and PD model mice in vivo, the level of peroxynitrite (ONOO － ) is abnormally expressed. ONOO － is a typical reactive nitrogen species, which is formed by the diffusion-limited reaction of nitric oxide and superoxide in a biological environment. ONOO － is considered an important neurotoxic factor and plays an important role in the pathogenesis of PD and other neurodegenerative diseases, and can be used as a potential biomarker for early prediction of PD. However, the potential biological role of ONOO － in the mechanism of the occurrence and development of PD has not been fully clarified. Therefore, accurately monitoring the level of ONOO － in the brain is of great research value for studying the pathogenesis of PD.
[0003] Fluorescence imaging technology, especially the use of small molecule fluorescence probes to detect and label biomarkers in a physiological environment, has the characteristics of sensitivity, specificity, non-invasiveness, and in-situ real-time detection. Currently, there have been reports of fluorescence probes for detecting ONOO － . However, the blood-brain barrier (BBB), which is composed of brain capillary endothelial cells, basement membrane, and astrocyte processes, hinders the entry of about 99% of small molecules. Therefore, few fluorescence probes can have the advantages of high sensitivity, high selectivity, good biocompatibility, and penetrating the BBB to achieve non-destructive detection of ONOO － in the brain. Summary of the Invention
[0004] In order to solve the above problems, the present invention provides a molecular fluorescence probe capable of penetrating the blood-brain barrier (BBB) and a preparation method thereof. The probe has good biocompatibility and a large Stokes shift, and can be used for sensitively and specifically detecting peroxynitrite (ONOO － ) in the brain of mice.
[0005] To achieve the above object, the present invention discloses the following technical solutions:
[0006] In the first aspect, a near-infrared small molecule fluorescent probe capable of specifically detecting ONOO － is provided, denoted as P3CF3, and its molecular structural formula is shown as follows: Among them, the 2-fluoro-5-nitrobenzoic acid part is the probe part of the P3CF3 fluorescent probe, which has good specific recognition for ONOO － ; the trifluoromethyl group is a targeting group for penetrating the BBB, which can enable the probe molecule to penetrate the BBB and accumulate in the mouse brain.
[0007] In the second aspect, the preparation method of the molecular fluorescent probe P3CF3 includes the following steps:
[0008] The first step: Add malononitrile, isophorone, piperidine, and acetic acid to the ethanol solution in sequence, protect with nitrogen, reflux and stir at 80 °C for 8 - 12 hours. After cooling to room temperature, rotate and evaporate to remove the solvent, and obtain a white solid CF3 through silica gel column chromatography purification;
[0009] The second step: Add CF3, 4-hydroxy-3-trifluoromethylbenzaldehyde, and piperidine to the ethanol solution in sequence, protect with nitrogen, reflux and stir at 80 °C for 8 - 12 hours, rotate and evaporate to remove the solvent, and obtain an orange-red solid F3CF3 through silica gel column chromatography purification;
[0010] The third step: Add the fluorophore F3CF3, 2-fluoro-5-nitrobenzoic acid, 4-dimethylaminopyridine, and dicyclohexylcarbodiimide to the dichloromethane solution in sequence, protect with nitrogen, stir at 0 °C for 0.5 hour, then raise the temperature to room temperature and stir for 6 - 8 hours. After rotating and evaporating to remove the organic reagent, obtain the final yellow-green solid product P3CF3 through silica gel column chromatography purification.
[0011] In the third aspect, a use of the P3CF3 fluorescent probe is provided.
[0012] Specifically, the P3CF3 fluorescent probe has a low detection limit, good selectivity, and the potential to penetrate the BBB.
[0013] Specifically, the P3CF3 fluorescent probe can be used for the detection of ONOO － in the PD Caenorhabditis elegans model.
[0014] Specifically, the P3CF3 fluorescent probe can be used for the detection of ONOO － in the brains of PD mice.
[0015] The beneficial effects of the present invention are as follows:
[0016] The design and synthesis method of the present invention is simple, and it has good specific recognition for ONOO －It has selective response ability, high biosafety of the probe, good cell permeability, and can detect ONOO in cells. － Level changes, and can achieve imaging detection of the expression level of ONOO in the brain tissue of PD mice. － This work provides a new detection method for clarifying the role of oxidative stress-regulated ONOO － in the PD pathology.
[0017] The advantages include the following points:
[0018] 1. The present invention aims at the preparation and synthesis method of the ONOO － fluorescent probe. The synthesis route is simple, the cost is low, the raw material utilization rate is high, and it is suitable for industrial production;
[0019] 2. Low detection limit: In in vitro tests, the fluorescent probe has good response ability to low concentrations of ONOO － . Through theoretical calculation, it is judged that the probe has good sensitivity and low detection limit. The detection limit reaches 9.27 nM, the sensitivity is high, and the linear range of the fluorescence intensity and the ONOO - concentration is in the range of 1 - 5 μM.
[0020] 3. Good selectivity: In a complex physiological environment, the probe needs to have good selectivity. By selecting interferents related to ONOO - to simulate the complexity of the biological environment, the reaction selectivity of the probe molecule to the target substrate is verified. Through testing, it is also proved that the probe has excellent selectivity in the presence of 29 common interferents.
[0021] 4. High biosafety: We carried out cytotoxicity experiments on the fluorescent probe molecule, and the results showed that the fluorescent probe molecule is non-toxic to cells and organisms and has good biocompatibility.
[0022] 5. Brain imaging of the PD in vivo model: We detected the PD nematodes and mouse models, and the probe can well perform fluorescence imaging on the overexpression of ONOO － in the PD model. In particular, the entry of the trifluoromethyl group in the probe makes it have high lipophilicity, and it can efficiently cross the BBB of the mouse brain to achieve highly sensitive detection of ONOO in the brain － , with significant brain-targeting characteristics, which will provide a simple and effective method for studying the mechanism of action of ONOO － in the course of PD patients. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to make the above and / or other purposes, features, and advantages of the present invention more obvious and understandable, the description of the accompanying drawings is as follows:
[0024] Figure 1 Synthetic route of the molecular fluorescence probe P3CF3;
[0025] Figure 2 1H NMR spectrum (i) and 13C NMR spectrum (ii) of the molecular fluorescence probe P3CF3, respectively;
[0026] Figure 3 UV-visible absorption spectra of probe P3CF3 before and after reacting with peroxynitrite (ONOO - );
[0027] Figure 4 Time kinetic performance test of probe P3CF3 for in vitro detection of peroxynitrite (ONOO - );
[0028] Figure 5 Concentration gradient test of probe P3CF3 for in vitro detection of peroxynitrite (ONOO - );
[0029] Figure 6 Response test of probe P3CF3 for in vitro detection of peroxynitrite (ONOO - ) and 29 common interfering substances;
[0030] Figure 7 Calculation curve of the detection limit (LOD) of probe P3CF3 for peroxynitrite (ONOO - );
[0031] Figure 8 Lipophilicity test of probe P3CF3;
[0032] Figure 9 Cytotoxicity experiment of probe P3CF3 at different concentrations;
[0033] Figure 10 Imaging of the expression level of peroxynitrite (ONOO - ) in cells by probe P3CF3 and its corresponding fluorescence intensity: (a) normal cell control group (b) group treated with exogenous ONOO － generating reagent SIN-1; (c) group treated with lipopolysaccharide and inflammatory factor IFN-γ to stimulate endogenous ONOO － generation; (d) group treated with uric acid, an ONOO － scavenger, in inflammatory cells; (e) group treated with TEMPO, a superoxide anion scavenger, the precursor of ONOO － in inflammatory cells; (f) group treated with L-NAME, a nitric oxide synthase inhibitor, in inflammatory cells;
[0034] Figure 11Fluorescence imaging of peroxynitrite (ONOO - ) by probe P3CF3 in normal nematodes and a PD Caenorhabditis elegans model;
[0035] Figure 12 Fluorescence imaging of peroxynitrite (ONOO - ) in the brain of normal mice and a PD mouse disease model by probe P3CF3 (a) and photos of its fluorescence distribution in different organs (b). Detailed implementation mode
[0036] The present invention is described in detail below. Specifically, in combination with the accompanying drawings of the specification of the present invention, the embodiments of the present invention are described more clearly and completely, without limiting its content. The embodiments described in the present invention are only a part, rather than all the embodiments, and all of them are within the protection scope of the present invention.
[0037] Example 1 Preparation of probe P3CF3. The synthetic route is as Figure 1 shown, specifically as follows:
[0038] First step: Malononitrile (1.68 g, 6.00 eq), isophorone (2.87 g, 5.00 eq), piperidine (0.50 mL, 0.10 eq), and acetic acid (0.50 mL, 0.10 eq) were successively added to 40 mL of an ethanol solution. Under nitrogen protection, the mixture was refluxed and stirred at 80 °C for 8 - 12 hours. After cooling to room temperature, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography to obtain a white solid CF3 (2.91 g, yield 75.39%).
[0039] Second step: CF3 (0.19 g, 1.00 eq), 4-hydroxy-3-trifluoromethylbenzaldehyde (0.20 g, 1.00 eq), and piperidine (0.25 mL, 0.10 eq) were successively added to 20 mL of an ethanol solution. Under nitrogen protection, the mixture was refluxed and stirred at 80 °C for 8 - 12 hours. The solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography to obtain a red solid F3CF3 (0.27 g, yield 71.81%).
[0040] Third step: The fluorophore F3CF3 (0.38 g, 1.00 eq), 2-fluoro-5-nitrobenzoic acid (0.38 g, 2.00 eq), 4-dimethylaminopyridine (0.01 g, 0.10 eq), and dicyclohexylcarbodiimide (0.02 g, 0.10 eq) were successively added to 10 mL of a dichloromethane solution. Under nitrogen protection, the mixture was stirred at 0 °C for 0.5 hour, and then stirred at room temperature for 6 - 8 hours. Water was added to terminate the reaction and extraction was carried out. After the organic phase was evaporated under reduced pressure to remove the organic reagent, the final yellow-green solid probe P3CF3 (0.14 g, yield 25.45%) was obtained by silica gel column chromatography.
[0041] Structural Characterization of Probe P3CF3 in Example 2
[0042] The structure of the probe P3CF3 prepared in the present invention was confirmed by characterization such as Figure 2 1H NMR spectrum, 13C NMR spectrum and mass spectrum shown below. 1 H NMR (400 MHz, Chloroform-d) δ 9.01 (s, 1H), 8.53 (d, J = 8.8 Hz, 1H), 7.84 (s, 1H), 7.78 (d, J = 10.2 Hz, 1H), 7.49~7.38 (m, 2H), 7.04 (s, 2H), 6.91 (s, 1H), 5.29 (s, 1H), 2.62 (s, 2H), 2.47 (s, 2H), 1.09 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 169.03, 152.59, 134.68, 133.78, 131.48, 130.96, 130.85, 128.77, 126.29, 126.24, 125.01, 124.98, 43.03, 39.27, 32.16, 30.21, 29.79, 28.11, 14.22. HRMS (m / z): calculated － for C 27 H 19 F4N3O4: 524.4596; found, 524.1231 (attached Figure 2 ).
[0043] In Vitro Spectroscopic Performance Test of Probe P3CF3 in Example 3
[0044] The reaction system for in vitro test was as follows: A 384-well microplate was used, and the total volume of the test liquid in each well was 90 μL, among which the content of PBS buffer (10 mM, pH = 7.4) was 99.8%, the content of DMSO was 0.2%, the final concentration of the probe in the test solution was 20 μM, and the test concentration of ONOO － was 20 μM. The above reaction system was incubated at 37 °C on an incubator shaker with a rotation speed of 300 rpm for 40 min in the dark. The specific experimental results are as follows:
[0045] (1) As Figure 3 shown, through the UV-visible absorption test of the microplate reader, under the condition that the concentration of ONOO － was 20 μM, the absorption peak of the probe P3CF3 at 400 nm decreased, and at the same time, an absorption peak at 485 nm began to appear, indicating that under the action of ONOO － , the probe molecule reacted with ONOO －A reaction occurred and fluorophores were released. The data indicated that the probe P3CF3 had the ability to recognize ONOO － .
[0046] (2) As Figure 4 shown, the kinetic response of the probe P3CF3 to ONOO － was tested by a microplate reader. The probe solution was prepared according to the above conditions, and 20 μM of ONOO － solution was immediately added for time kinetic testing. The test was carried out under the excitation of an excitation light with a wavelength of 485 nm, and the fluorescence intensity at the emission wavelength of 650 nm was recorded. The test results are shown in the appendix Figure 4 . After the addition of the ONOO － solution, the fluorescence intensity of the probe P3CF3 immediately increased and reached the maximum value after 40 min, indicating a short response time.
[0047] (3) As Figure 5 shown, it was a concentration gradient response test of the probe to ONOO － . Specifically, in a PBS buffer solution with pH = 7.4 (0.2% DMSO), the concentration of the probe P3CF3 was 20 μM, and different concentrations of peroxynitrite (0 - 20 μM) were added. Then, the reaction was carried out at 37 °C for 40 min to obtain the response results of the final concentration gradient (the excitation wavelength was 485 nm). The experimental results showed that the fluorescence intensity of the probe P3CF3 increased with the increase in the concentration of ONOO － .
[0048] (4) As Figure 6 shown, the anti-interference performance of the probe P3CF3 against 29 common potential interferents in the physiological system was tested. Specifically, in a PBS buffer solution with pH = 7.4 (0.2% DMSO), the concentration of the probe P3CF3 was 20 μM, the concentration of ONOO － was 25 μM, and the other interferents were 1 - 28: Na + , Mg 2+ , Ca 2+ , K + , Fe 2+ , Cu 2+ , Zn 2+ , Al 3+ , Fe 3+ , SO3 2- , SO4 2- , CO3 2- , SH - , S 2- , NO2 2- , NO3 2- , NO, ·OH, HSO 3- , 1O 2,ROO·,H2O2,ClO － ,Hcy,GSH,Cys,Vitamin C,Vitamin E, and the corresponding concentrations are 50 μM respectively. Experiments have shown that the probe P3CF3 has a very excellent specific response to ONOO － and can adapt to the complex environment in vivo.
[0049] (5) As Figure 7 shown, the detection limit (LOD) of the probe P3CF3 for ONOO － was studied and calculated through a concentration gradient. Using the formula LOD = 3δ / k, where δ is the systematic error obtained from 21 blank experiments, and k is the slope obtained from the linear relationship between the concentration of ONOO － and the change in the fluorescence intensity of P3CF3, the detection limit of the probe P3CF3 was calculated to be 9.27 nM.
[0050] (6) As Figure 8 shown, by preparing octanol solutions with different concentrations of the probe P3CF3 (0, 50, 100, 150, 200, 250, 300, 400 μM), a standard concentration curve was plotted under the test of an ultraviolet-visible spectrophotometer. Using the system of octanol:water = 9:1 to dissolve a fixed mass of the probe P3CF3, the lipophilicity of the probe was determined to be 9.67×10 -4 , and the data indicate that the probe has significant lipophilicity and can penetrate the blood-brain barrier (BBB) to achieve brain imaging.
[0051] Example 4 Real-time testing of ONOO by the probe P3CF3 in cells －
[0052] (1) As Figure 9 shown, through two types of cells, PC12 and SH-SY5Y, the toxicity of the probe P3CF3 at different concentrations (0, 1, 2, 5, 10, 20, 30, 40, 50 μM, 0.5% DMSO) to the cells was investigated. After adding the probe P3CF3 at the above concentrations and incubating in a cell culture incubator (37 °C, 5% CO2 + 95% air) for 24 h, a CCK-8 kit was used for detection. It was found that at concentrations of 0 - 40 μM, the survival rates of both types of cells were higher than 90%, indicating that this type of probe has good biosafety in the cell environment.
[0053] (2) We selected human neuroblastoma cells (SH-SY5Y) as the cell model to simulate the oxidative stress state under inflammatory conditions. Attached Figure 10 Figure a is the normal cell control group; to increase the exogenous ONOO － content in the cells, we added exogenous ONOO －Incubate with the reagent SIN-1 (10 μM) for 30 min (Appendix Figure 10 b); To increase the endogenous ONOO － content in cells, we added lipopolysaccharide (LPS, 20 μg / mL) and inflammatory factor IFN-γ (0.3 μg / mL) to the cells and continued to stimulate the cells for 12 h to make the cells in an inflammatory state and release endogenous ONOO － (Appendix Figure 10 c); Then add the probe P3CF3 and incubate for 40 min, followed by confocal imaging. Collect fluorescence images at 600 - 670 nm under 488 nm laser excitation. For the inhibitor groups, they are: As shown in Appendix Figure 10 d, add the ONOO － scavenger uric acid (600 μM) to the above-mentioned inflammatory cells and incubate for 30 min; As shown in Appendix Figure 10 e, add the ONOO － precursor superoxide anion scavenger TEMPO (1080 μM) to the inflammatory cells and incubate for 12 h; As shown in Appendix Figure 10 f, add the nitric oxide synthase inhibitor L-NAME (3 mM) to the inflammatory cells and incubate for 12 h. The imaging results show that the fluorescence of the probe P3CF3 in inflammatory cells is significantly enhanced, indicating that the probe has a good response to endogenous ONOO － in inflammatory cells. After inhibiting ONOO － in inflammatory cells, the fluorescence intensity of all inhibitor groups decreased. All of the above indicate that the probe P3CF3 has a specific response to ONOO － in cells.
[0054] Example 5 Fluorescence Imaging of P3CF3 in the Parkinson's (PD) Caenorhabditis elegans Model
[0055] Use wild-type nematode N2 as the experimental control group (Normal), and the Parkin-null nematode VC1024 of the PD model as the experimental group (PD model). The Parkin-null nematode VC1024 of the PD model was pretreated with uric acid (3 mM) for 3 minutes as the inhibitor group (Inhibitor). The above three types of nematodes were incubated in the probe P3CF3 solution (20 μM) for 50 minutes respectively, followed by confocal imaging. Collect fluorescence images at 600 - 670 nm under 488 nm laser excitation. As shown in Appendix Figure 11 , the results show that the probe P3CF3 shows a very obvious fluorescence intensity in the PD nematode model, while the fluorescence is significantly reduced in the normal group and the inhibitor treatment group. Therefore, this probe P3CF3 can preliminarily judge PD nematodes by fluorescence imaging of the ONOO － level.
[0056] Example 6 Fluorescent Imaging of the Brain in a Living Mouse Model of Parkinson's Disease (PD) Using Probe P3CF3
[0057] During the development of PD, it has been found that ONOO is overexpressed in the brains of patients — , and the probe P3CF3 of the present invention creatively introduces a trifluoromethyl group as a brain-targeting group, which enables the probe to penetrate the BBB for brain imaging. To study whether the probe can be used to image the changes in the endogenous ONOO － level in the brains of PD patients, a C57BL / 6 mouse (6 weeks old, male) was modeled using an MPTP modeling agent. By continuously injecting MPTP (25 mg / kg) for 7 days, subsequent open field tests, standing tests, grip ability tests, and pole climbing tests were performed to test the behavior of the mice and determine whether the PD mouse model was established. This is also a widely used method for modeling PD mouse models. Subsequently, after intraperitoneal injection of the probe P3CF3 saline solution (200 μM, containing 1% Tween 80 + 2% DMSO), the in vivo imaging of the mice at different times was recorded, and imaging pictures were collected under 488 nm laser excitation. As shown in Figure 12 Figure a, we can see that the fluorescent signal in the brains of PD mice is significantly higher than that in the control group (the control group are all healthy mice only injected with the probe), and reaches the maximum value after 1.5 hours. We took out the brains and other organs of the mice for imaging observation and found that the fluorescent signal in their brains was also significantly higher than that in the normal group ( Figure 12 Figure b). Therefore, this probe has good ability to penetrate the BBB and can monitor the overexpression level of ONOO in the brains of PD mice in real time, showing an application prospect for the auxiliary diagnosis of PD. －
[0058] The materials, methods, and examples described herein are merely exemplary and not restrictive. Those skilled in the art can draw on the content of this article and appropriately replace and / or modify process parameters. However, it should be particularly noted that all such similar replacements and / or modifications are obvious to those skilled in the art and will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims. Therefore, they are all considered to be included in the present invention.
Claims
1. A method for detecting peroxynitrite (ONOO) in the brain of Parkinson's mice － ) molecular fluorescent probe, characterized in that The 2-fluoro-5-nitrobenzoic acid part is the recognition site of the fluorescent probe, and the trifluoromethyl group is the targeting group that penetrates the blood-brain barrier. Its structural formula is as follows:
2. The method for preparing a molecular fluorescent probe for detecting ONOO according to claim 1, characterized in that: The preparation scheme is as follows: Step 1: Add malononitrile, isophorone, piperidine and acetic acid to an ethanol solution in sequence, under nitrogen protection, reflux and stir at 80°C for 8 to 12 hours, cool to room temperature, remove the solvent by rotary evaporation, and purify by silica gel column chromatography to obtain a white solid CF3; Step 2: CF3, 4-hydroxy-3-trifluoromethylbenzaldehyde and piperidine are added to the ethanol solution in sequence, and the mixture is refluxed and stirred at 80°C for 8 to 12 hours under nitrogen protection, and the solvent is evaporated under reduced pressure to obtain orange-red solid F3CF3 after purification by silica gel column chromatography; Step 3: Add the fluorophore F3CF3, 2-fluoro-5-nitrobenzoic acid, 4-dimethylaminopyridine and dicyclohexylcarbodiimide to the dichloromethane solution in sequence, protect with nitrogen, stir at 0°C for 0.5 hours, then heat to room temperature and stir for 6 to 8 hours. After removing the organic reagent by evaporation under reduced pressure, purify by silica gel column chromatography to obtain the final yellow-green solid product P3CF3.
3. The method of claim 1 having ONOO － The use of a molecular fluorescent probe with recognition function is characterized by: The molecular fluorescent probe can be used to prepare a qualitative or quantitative detection of ONOO － of detection reagents.
4. The use of the molecular fluorescent probe according to claim 1, characterized in that: ONOO in the Parkinson's disease (PD) model of Caenorhabditis elegans can be monitored by fluorescence intensity changes － Expression level monitoring.
5. The use of the molecular fluorescent probe according to claim 1, characterized in that: Molecular probes can efficiently cross the blood-brain barrier in Parkinson's (PD) mice, enabling highly sensitive detection of ONOO in the brain － , with significant brain-targeting features, enabling detection of ONOO in the brain of PD mice － application.