Small-molecule fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probe preparation, in particular to a small-molecule fluorescent probe and a preparation method thereof. The structural formula of the micromolecular fluorescent probe is shown in the specification. The micromolecular fluorescent probe provided by the invention can be used for detecting hydrogen peroxide, can respond to hypoxia cells and tissues, realizes in-situ fluorescence imaging, and can effectively penetrate through a blood brain barrier.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe preparation technology, and in particular to a small molecule fluorescent probe, its preparation method, and its application. Background Technology

[0002] Small molecule fluorescent probes are a class of small organic molecules that can detect specific targets by generating fluorescent signals. They typically consist of a fluorophore that generates a fluorescent signal and a recognition group that targets a specific target. They can be used to detect specific biomolecules, cells, or tissue structures in organisms.

[0003] Reactive oxygen species (ROS) are products of oxygen metabolism and are crucial for maintaining the balance of oxidative stress in organisms. However, under various pathological conditions, the level of ROS in the body can become abnormal, playing a double-edged sword effect. They may exacerbate or eliminate necrotic tissue cells, such as in cancer, cardiovascular disease, hypoxic-ischemic encephalopathy, and central nervous system damage like Alzheimer's disease. The main forms of ROS present in humans include singlet oxygen, hydroxyl radicals, hydrogen peroxide (H₂O₂), and superoxide anions.

[0004] However, there are currently few small molecule fluorescent probes for monitoring H2O2 that can be applied to both neonatal disease models and adult or even elderly disease models.

[0005] Therefore, the existing technology still needs further improvement and enhancement. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new small molecule fluorescent probe that can respond to hypoxic cells and tissues, achieve in situ fluorescence imaging, and effectively cross the blood-brain barrier.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, a small molecule fluorescent probe, wherein the structural formula of the small molecule fluorescent probe is as follows: .

[0008] Secondly, a method for preparing the small molecule fluorescent probe described in the first aspect, comprising: Compound I and Compound II were dissolved in an organic solvent, and piperidine was added to obtain a first mixed solution; The first mixed solution was heated in an inert atmosphere to obtain the small molecule fluorescent probe; The structural formula of compound I is: The structural formula of compound II is: .

[0009] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. The purpose and beneficial effects of the present invention can be better achieved and realized through the following preferred technical solutions.

[0010] As a preferred technical solution, the method for preparing the small molecule fluorescent probe involves heating the first mixed solution at a temperature of 90℃-110℃ for 3-5 hours.

[0011] As a preferred technical solution, the method for preparing the small molecule fluorescent probe, wherein the preparation method of compound II includes: 4-Formylbenzoic acid and 4-(hydroxymethyl)phenylboronic acid pinacol ester were dissolved in 4-dioxane, and TEA and diphenyl azidophosphate were added to obtain a second mixed solution; The second mixed solution was heated in an inert atmosphere to obtain compound II.

[0012] As a preferred technical solution, the method for preparing the small molecule fluorescent probe involves heating the first mixed solution at a temperature of 90℃-110℃ for 2-3 hours.

[0013] As a preferred technical solution, in the method for preparing the small molecule fluorescent probe, the molar ratio of compound I to compound II is 1:1 to 1:1.2.

[0014] As a preferred technical solution, in the method for preparing the small molecule fluorescent probe, the mass ratio of 4-formylbenzoic acid to 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:1.5-2.

[0015] As a preferred technical solution, in the method for preparing the small molecule fluorescent probe, the inert gas is nitrogen or argon.

[0016] As a preferred technical solution, in the method for preparing the small molecule fluorescent probe, the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol.

[0017] Thirdly, the application of the aforementioned small molecule fluorescent probe in the detection of hydrogen peroxide.

[0018] Beneficial effects: Compared with the prior art, the small molecule fluorescent probe provided by the present invention can be used to detect hydrogen peroxide. The small molecule fluorescent probe can respond to hypoxic cells and tissues to achieve in situ fluorescence imaging and can effectively cross the blood-brain barrier. Attached Figure Description

[0019] Figure 1 is a confocal microscope image of BV2 cells after OGD treatment and incubation with hydrogen peroxide probe; Figure 2 shows the TTC staining results of mouse brain tissue 3 days after HIE modeling. Figure 3 These are confocal microscopy images of brain tissue sections from mice injected intraperitoneally with hydrogen peroxide probe 4 h, 6 h, and 12 h 3 days after HIE modeling. Figure 4 shows confocal microscopy images of brain tissue sections after intraperitoneal injection of hydrogen peroxide probe in a perinatal infected animal model at 4 h, 6 h, and 12 h. Figure 5 It involves genotyping mice after they are born. Figure 6 These are confocal microscopy images of brain tissue sections from 10-month-old AD mice after intraperitoneal injection of hydrogen peroxide probe for 6 h and 12 h. Detailed Implementation

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form as well. Furthermore, it should be understood that when the terms "comprising" and / or "package" are used in this specification… When "includes", it indicates the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. Techniques, methods, and apparatus known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and apparatus should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0023] This invention provides a small molecule fluorescent probe, the structural formula of which is as follows: .

[0024] In this embodiment, one of the key characteristics of hypoxic cells / tissues is the imbalance of reactive oxygen species (ROS) metabolism. Hydrogen peroxide (H2O2), as an important biomarker of hypoxia-related pathological processes, shows a significant increase in its content during hypoxia. The small molecule fluorescent probe (MH-1) contains a pinacol ester phenylboronic acid group, which is a specific recognition site for H2O2. In the hypoxic inflammatory microenvironment, the concentration of H2O2 in brain tissue increases, which can react with the pinacol ester phenylboronic acid, causing the group to break down. This triggers a structural change in the probe molecule, releasing the fluorophore M.

[0025] MH-1's molecular structure has a moderate molecular weight and a certain degree of lipophilicity, which facilitates its crossing of the blood-brain barrier via passive diffusion. The benzene ring and borate ester groups in its structure enhance its lipophilicity and promote transmembrane transport.

[0026] Based on the same inventive concept, the present invention also provides the following preparation route for the above-mentioned small molecule fluorescent probe:

[0027] Specifically, the synthesis of compounds 3-3, 3-4 and 3-5 can be found in the master's thesis of Fang Jieqin, a 2019 graduate of Yunnan University, entitled "Design, Synthesis and Basic Properties Study of Fluorescent Probes for Detecting Small Molecular Substances in Biological Organisms".

[0028] 4-Formylbenzoic acid (1 g, 6.8 mmol) and 4-(Hydroxymethyl)benzeneboronic acid pinacol ester (1.7 g, 7.3 mmol) were dissolved in 13.4 mL of 1,4-dioxane. TEA (1 mL, 7.2 mmol) and diphenylphosphorylazide (DPPA, 1.6 mL, 7.3 mmol) were added. The reaction was carried out at 100 °C for 2.5 h under nitrogen protection. After the reaction, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (PE / EtOAc, V / V = 2 / 3) as the eluent. This yielded a white solid compound 3-6 (2.1 g, yield 81.1%).

[0029] Compounds 3-5 (208 mg, 1 mmol) and 3-6 (381 mg, 1 mmol) were dissolved in 10 mL of acetonitrile (ACN), and piperidine (PIP, 300 μL, 3 mmol) was added. The reaction solution was reacted at 100 °C for 4 h under nitrogen protection. After the reaction was completed, an orange precipitate was formed. The orange precipitate was filtered and washed three times with distilled water to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (DCM / MeOH, V / V = 250 / 1) as the eluent. The orange solid obtained was probe MH-1 (400 mg, yield 70.1%). The following specific experimental examples will further explain the small molecule fluorescent probes provided by this invention.

[0030] Experimental Example 1 Constructing a cellular oxygen and glucose deprivation (OGD) model: BV2 cell lines were constructed at a rate of 1×10⁻⁶. 6 Cells were seeded in a T25 cell culture flask and cultured in DMEM high-glucose medium. The night before the glucose-oxygen deprivation experiment, the cell line was digested with 0.25% trypsin and cultured at a concentration of 1×10⁻⁶ cells / flask. 5 Cells were seeded at the specified density in 12-well plates (DMEM high glucose medium). After 12 h, the DMEM high glucose medium was replaced with serum-free medium, and the 12-well plates were placed in an anoxic chamber and cultured under anoxic conditions of 37°C, 5% CO2 + 95% N2 for 4 h. This group was designated as the OGD group. A normal control group was also set up. The cells in the control group were cultured in normal DMEM high glucose medium under normal oxygen conditions in a cell culture incubator without any special treatment.

[0031] Fluorescent probe incubation: Immunofluorescence staining was performed 0 h after the completion of the BV2 cell OGD experiment. The specific steps were as follows: aspirate the culture medium → wash with PBS for 5 min (3 washes) → fix with 4% paraformaldehyde for 30 min → wash with PBS for 5 min (3 washes) → block with 5% donkey serum at room temperature for 50 min → add the corresponding primary antibody and incubate overnight at 4℃ (IBa-1) → wash with PBS for 5 min (3 washes) → add the complex of the fluorescently tagged secondary antibody and hydrogen peroxide probe and incubate at room temperature for 2 h → wash with PBS for 5 min (3 washes) → mount with DAPI fluorescent anti-quencher, store at 4℃, and observe using a Zeiss confocal fluorescence microscope imaging system. Subsequent image data analysis and processing were performed using its built-in analysis software. Results are as follows: Figure 1As shown, IBa-1 refers to a marker for microglia, H2O2 refers to a hydrogen peroxide probe, DAPI refers to a nuclear marker, and Merge refers to a merging channel. Compared to the Control group, BV2 cells were round after OGD, and the red fluorescence signal of the H2O2 channel was significantly enhanced, indicating that the novel hydrogen peroxide probe successfully responded in the in vitro hypoxic cell model.

[0032] Experiment Example 2 Establishment of a HIE animal model: C57BL / 6 mice 3 days after HIE modeling were selected. HIE model establishment: Wild-type mice 9-11 days old were anesthetized with an appropriate amount of isoflurane (30 μl / mouse) and quickly immobilized. A left-sided incision was made in the middle of the neck skin. After incision, the left anterior cervical muscles were meticulously dissected using ophthalmic straight and curved forceps to fully expose the carotid sheath. The left common carotid artery was separated from the vagus nerve and ligated with absorbable surgical needle-inlaid sutures. The wound was sutured after no blood flow was observed. 1.5 h after surgery, the newborn mice were placed in a modified isothermal hypoxia model experimental device. N2 was injected into the device at a uniform rate, and the mice were kept in a hypoxic chamber filled with a mixture of 8% O2 and 92% N2 at an ambient temperature of 34℃ for 40 min. A sham-operated group (Sham) was established among littermates. After thorough anesthesia, only the left common carotid artery was dissected without ligation, and no subsequent hypoxia treatment was performed. Three days after model establishment, the scalp of mice in the model group was cut open in vivo to observe the damage to the left brain tissue. Mice in the HIE group with white liquefied infarct foci in the damaged side of the brain tissue were assigned to the HIS group; mice in the model group with no obvious characteristics of the damaged side of the brain tissue were assigned to the HIL group.

[0033] TTC staining to verify lesions: Dissolve TTC powder in physiological saline to prepare a 1% TTC solution, using freshly prepared solution. Add 3 ml of the mixed 1% TTC solution to each well of a six-well plate, and preheat the plate in a water bath for half an hour beforehand. Three days after modeling, mice from the HIE group and age-matched Sham group were anesthetized with an intraperitoneal injection of 10% chloral hydrate solution (3 ml / kg). The brain tissue was completely removed and placed directly on a glass culture dish with a ruler. The brain tissue was frozen at -20℃ for 13-15 min. After removal, the culture dish containing the brain tissue was placed on crushed ice. The forebrain was evenly sliced ​​into 1 mm thick slices along the coronal plane. The brain slices were added to the six-well plate and incubated at 37℃ for 30 min in the dark. After staining, the TTC solution was aspirated, and the tissue was fixed overnight with 4% paraformaldehyde solution. Images were taken and analyzed the next day. The TTC staining results are as follows: Figure 2As shown, white represents the infarct area. Sham: sham-operated group; HIL: mild injury group; HIS: severe injury group. The results indicate that compared with the Sham group, the HIS group mice showed a large area of ​​white infarct lesions on the left side of the brain tissue, indicating that the HIE model was successfully established. The results are as follows... Figure 2 As shown.

[0034] Prepare the working solution for the fluorescent probe MH-1: Select the MH-1 probe and prepare a 20 μmol / L working solution using DMSO.

[0035] Preparation of brain tissue sections: For mice of different groups (Sham, HIL, HIS) with established models, MH-1 probe (2 mg / kg) was injected intraperitoneally. The mouse brains were dissected at 4 h, 6 h and 12 h respectively and sectioned directly in a cryostat. After short fixation with anhydrous ethanol (10 s), the sections were mounted directly.

[0036] Imaging and Analysis: The PWM region and cortical region of the slides were imaged and observed using a confocal microscope, and fluorescence signals were recorded. Immunofluorescence results are as follows: Figure 3 As shown in Figure 3, brain tissue sections from mice injected intraperitoneally with the MH-1 probe 3 days after HIE modeling were captured by confocal microscopy at time points of 4 h, 6 h, and 12 h (scale bar: 20 μm). Observations at different time points after intraperitoneal injection of the MH-1 probe revealed that, compared to the Sham group, the HIS group showed significant fluorescence signals in the periventricular white matter (PWM) and cortical injury areas, while the HIL group showed only weak fluorescence signals in the PWM and cortex. These results indicate that the probe can cross the blood-brain barrier, respond to hydrogen peroxide in ischemic and hypoxic brain tissue, and release fluorescent groups, thereby achieving fluorescence tracing of the targeted area.

[0037] Experimental Example 3 Establishment of a perinatal infection animal model: C57BL / 6 mice were selected for the perinatal infection model. The mice were randomly divided into a lipopolysaccharide (LPS) group and a saline control group. Mice in the LPS group received intraperitoneal injections of 1.2 mg / kg LPS once daily for seven consecutive days (P 3 d, P 9 d) to establish the perinatal infection model. Mice in the NaCl group received an equal volume of saline intraperitoneal injections once daily for seven consecutive days (P 3 d, P 9 d).

[0038] Prepare the working solution for the fluorescent probe MH-1: Select the MH-1 probe and prepare a 20 μmol / L working solution using DMSO.

[0039] Preparation of brain tissue sections: In mice of different groups with established models (Sham, HIL, HIS), MH-1 (2 mg / kg) was injected intraperitoneally. The brains of the mice were dissected at 4 h, 6 h, and 12 h, respectively, and sections were directly prepared in a cryostat. After brief fixation with anhydrous ethanol (10 s), the sections were mounted directly. Imaging and Analysis: The PWM region and cortical region of the slides were imaged and observed using a confocal microscope, and fluorescence signals were recorded. Results are as follows: Figure 4 As shown in Figure 4, brain tissue sections were captured by confocal microscopy at 4 h, 6 h, and 12 h after intraperitoneal injection of a hydrogen peroxide probe in a perinatal infection animal model. The scale bar is 20 μm. Observations at different time points after intraperitoneal injection revealed significant fluorescence signals in the periventricular white matter (PWM) and cortical regions of the LPS group mice compared to the Control group. These results indicate that the probe can cross the blood-brain barrier in a perinatal infection model, respond to hydrogen peroxide in the diseased brain tissue, and release fluorescent groups, thus achieving fluorescent tracing of infection-related pathological areas.

[0040] Experiment Example 4 Experimental animals: 10-month-old APP-PS1-TAU transgenic AD mice were selected.

[0041] Genotyping: A 1 mm mouse toe was placed in a centrifuge tube, and 150 μl of 50 mM NaOH solution was added to each tube. The tube was boiled in a metal bath at 99°C for 30 min. After cooling to room temperature, 150 μl of 1 M Tris-HCl (pH: 7.4) solution was added to each tube. The mixture was vortexed and centrifuged at 12000 rpm for 10 min at room temperature. The supernatant was used for subsequent PCR amplification experiments. A 1% agarose gel was prepared using 1×TAE. After amplification, agarose gel electrophoresis was performed, and the results were detected using a BIO-RAD Chemical XRS+ imaging system. Results are as follows: Figure 5 As shown, the mice included in the project were AD model mice that met the requirements.

[0042] The PCR primers are as follows:

[0043] The PCR system is as follows:

[0044] The PCR amplification procedure is as follows:

[0045] Preparation of brain tissue sections: For mice of different groups (AD mice and wild-type mice) with established models, MH-1 (2 mg / kg) was injected intraperitoneally. The mouse brains were dissected at 6 h and 12 h respectively and sectioned directly in a cryostat. After short fixation with anhydrous ethanol (10 s), the sections were mounted directly.

[0046] Imaging and Analysis: Confocal microscopy was used to image and observe the HIP region and cortical region of the sections, and fluorescence signals were recorded. Results are as follows: Figure 6 As shown, this probe can not only cross the blood-brain barrier of newborn mice, but also the blood-brain barrier of adult mice. It can also respond to hydrogen peroxide in the brain tissue of AD model and release fluorescent groups. At the same time, it can effectively distinguish the pathological differences between the AD group and the WT group, providing a visualized molecular detection tool for related research on Alzheimer's disease.

[0047] In summary, the present invention provides a solution.

[0048] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A small molecule fluorescent probe, characterized in that, The structural formula of the small molecule fluorescent probe is as follows: 。 2. A method for preparing the small molecule fluorescent probe according to claim 1, characterized in that, include: Compound I and Compound II were dissolved in an organic solvent, and piperidine was added to obtain a first mixed solution; The first mixed solution was heated in an inert atmosphere to obtain the small molecule fluorescent probe; The structural formula of compound I is: The structural formula of compound II is: .

3. The method for preparing the small molecule fluorescent probe according to claim 2, characterized in that, The first mixed solution is heated at a temperature of 90℃-110℃ for 3-5 hours.

4. The method for preparing the small molecule fluorescent probe according to claim 2, characterized in that, The preparation method of compound II includes: 4-Formylbenzoic acid and 4-(hydroxymethyl)phenylboronic acid pinacol ester were dissolved in 4-dioxane, and TEA and diphenyl azidophosphate were added to obtain a second mixed solution; The second mixed solution was heated in an inert atmosphere to obtain compound II.

5. The method for preparing the small molecule fluorescent probe according to claim 4, characterized in that, The first mixed solution is heated at a temperature of 90℃-110℃ for 2-3 hours.

6. The method for preparing the small molecule fluorescent probe according to claim 2, characterized in that, The molar ratio of compound I to compound II is 1:1 to 1:1.

2.

7. The method for preparing the small molecule fluorescent probe according to claim 4, characterized in that, The mass ratio of 4-formylbenzoic acid to 4-(hydroxymethyl)phenylboronic acid pinacol ester is 1:1.5-2.

8. The method for preparing the small molecule fluorescent probe according to claim 2, characterized in that, The inert gas is nitrogen or argon.

9. The method for preparing the small molecule fluorescent probe according to claim 2, characterized in that, The organic solvent is selected from one or more of acetonitrile, methanol, and ethanol.

10. The application of the small molecule fluorescent probe according to claim 1 in the detection of hydrogen peroxide.

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