Fluorescent probe with bis (cyanoisocyanide) ketone boric acid alkyl structure as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe with a bis (cyanoisocyanide) ketone boric acid alkyl structure. The structure of the probe is shown in the specification. The probe provided by the invention can fix a fluorescence signal through covalent cross-linked protein. The probe prepared by the invention can be used for effectively detecting the change of endogenous and exogenous H2O2 in cells. The probe is used for constructing a high-throughput screening platform, and the antioxidant capable of effectively adjusting the ROS level and the 5-HT balance can be rapidly screened out. The fluorescent probe provided by the invention provides a new strategy and tool for researching ROS signal transduction and abnormal modification of 5-HT related protein in nervous system diseases such as epilepsy, and indicates that the effect of 5-hydroxytryptamine in the nervous system diseases such as epilepsy is researched in a new way by combining a dual chemical sensing and marking method.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry technology and relates to a fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure, a preparation method and an application thereof, and in particular to a H2O2-activated serotonin precursor probe that reveals the interaction between 5-HT and adjacent proteins under oxidative stress, a preparation method and an application thereof. Background Art

[0002] Neurotransmitters are important signaling molecules that mediate neuronal communication and regulate diverse brain functions, thereby maintaining neurophysiological homeostasis. Among them, 5-hydroxytryptamine (5-HT) is crucial for regulating various neuropsychological processes in the central nervous system, including mood regulation, aggression, and memory. Serotonin exerts its effects primarily by activating 5-HT receptors, which are widely distributed throughout the brain. 5-HT receptors are primarily synthesized by neurons in the cerebral cortex and synapses, extending to key regions such as the hippocampus. The diverse functions of 5-HT are mediated by 12 different subtypes of G protein-coupled receptors (GPCRs), making serotonin receptor agonists an important therapeutic option for treating neurodegenerative diseases such as depression, schizophrenia, epilepsy, and Alzheimer's disease (AD). For example, the crucial role of serotonergic neurotransmitters in the pathophysiology of epilepsy is increasingly recognized, as evidenced by clinical findings of altered serotonin levels in patients with epilepsy. Increasing 5-HT levels through pharmacological or neuromodulatory techniques can reduce seizure frequency and severity, highlighting the potential of targeting the serotonergic system as a new therapeutic approach for epilepsy. In addition to its receptor-mediated effects, it is noteworthy that emerging evidence suggests that 5-HT can also act beyond receptor activation to directly alter chromatin through post-translational modifications targeting histones. These studies highlight the critical role of the neurotransmitter 5-HT in maintaining physiological homeostasis in the brain, and that antagonizing these functions or disrupting this homeostasis can lead to deleterious effects.

[0003] Oxidative stress is a pathological hallmark of many neurological disorders, including epilepsy. It stems from an imbalance between reactive oxygen species (ROS) production and the brain's antioxidant defenses, leading to intracellular lipid peroxidation, DNA damage, and neuroinflammation. Serotonin exhibits antioxidant properties by inhibiting excessive ROS production through activation of the 5-HT1A receptor, which contributes to its neuroprotective effects. Furthermore, 5-HT plays a central role in regulating neuroimmune and neuroinflammatory responses, which are crucial in the pathogenesis of epilepsy. Notably, 5-HT has been shown to modulate the activity of microglia and astrocytes—two key cell types involved in neuroinflammation—and influences T cell activation, proliferation, and differentiation under inflammatory conditions. The complex nature of serotonin's role in regulating cytokine production varies across cell types, further highlighting the complex nature of serotonin's involvement in neuroinflammation. Drugs such as selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, exhibit anti-inflammatory effects, further linking serotonin signaling to the regulation of chronic neuroinflammation. Despite these insights, our understanding of how 5-HT homeostasis changes in response to oxidative stress in neuronal cells, particularly in the context of excessive ROS accumulation, remains limited. The complex interplay between ROS, 5-HT, and functional intracellular proteins in the pathological context of epilepsy remains poorly understood, necessitating further investigation to elucidate the underlying mechanisms. However, the lack of targeted research tools has significantly hampered progress in this area, and expanding research methods and tools is therefore crucial to advancing research in this area and opening up new therapeutic avenues. Summary of the Invention

[0004] Purpose of the Invention: To address the problems of the prior art, the present invention provides a fluorescent probe with a bis(cyanoisocyanate)ketone boronic acid alkyl structure, designated HOP. This probe, designed using a tandem sensing and labeling strategy, emits a fluorescent signal in neurons activated by oxidative stress, covalently crosslinks with nearby proteins, and simultaneously releases 5-HT in situ, effectively preventing diffusion of the fluorophore. Mass spectrometry analysis confirmed that the locally released 5-HT further interacts with nearby functional proteins, including 5-HT-mediated covalent modification of proteins under oxidative stress. In vitro and in vivo fluorescence spectroscopy and fluorescence imaging studies demonstrate that the probe can effectively detect endogenous and exogenous H2O2. Furthermore, the present invention utilizes HOP for high-throughput screening to identify hesperetin, an antioxidant that effectively regulates H2O2 levels and 5-HT balance. HOP imaging was used in vitro and in vivo to verify the distribution of H2O2 in a mouse model of epilepsy. This not only deepens our understanding of small molecule biomarkers of widespread brain oxidative stress in neurological disorders such as epilepsy, but also provides technical support for the innovation of diagnostic tools and the optimization of therapeutic strategies. The present invention effectively solves the problems of the existing fluorescent probe technology, such as short emission wavelength, easy diffusion of fluorescent signals and insufficient specificity.

[0005] Technical solution: In order to achieve the above purpose, the present invention provides a fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure, the structure of which is shown below:

[0006]

[0007] The method for preparing the fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure of the present invention comprises the following steps:

[0008] A mixture of 4-hydroxybenzaldehyde and 2-(3,5,5-trimethylcyclohex-2-enylidene)malononitrile was dissolved in an organic solvent, piperidine was added and refluxed, and after completion of the reaction, the solvent was removed under reduced pressure and purified to obtain compound 2;

[0009] Hexamethylenetetramine was added to the solution containing compound 2, and the mixture was refluxed. After the reaction, the mixture was cooled to room temperature, and the reaction mixture was poured into ice water. The precipitate was collected by filtration and purified to obtain compound 3;

[0010] Compound 3 was dissolved in an organic solvent, triethylamine was added under stirring in an ice bath, and then trifluoromethanesulfonic anhydride was added. The reaction mixture was allowed to warm to room temperature and stirred for further reaction. The reaction was quenched and then extracted. The combined organic layer was dried, filtered, concentrated under reduced pressure, and purified to obtain compound 4;

[0011] Compound 4 was dissolved in an organic solvent and cooled, and sodium borohydride was added under stirring. The reaction mixture was warmed to room temperature and stirred for reaction. After the reaction was quenched, extraction was performed, and the combined organic layer was dried, filtered, concentrated under reduced pressure, and purified to obtain compound 5;

[0012] Compound 5 was dissolved in an organic solvent and cooled, carbonyldiimidazole was added, and the reaction mixture was stirred for reaction. 3-(2-aminoethyl)-1H-indol-5-ol was added, and the reaction mixture was stirred for reaction at room temperature. After the reaction was completed, the organic layer of the reaction mixture was washed, dried, concentrated under reduced pressure, and purified to obtain compound 6;

[0013] Compound 6, potassium acetate, palladium dichloride and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine were dissolved in an organic solvent, and the reaction mixture was heated and stirred under an inert atmosphere. After the reaction was completed, it was cooled to room temperature, and the organic layer was washed, dried, filtered, and concentrated under reduced pressure to obtain the probe HOP;

[0014] The reaction formula is as follows:

[0015]

[0016] Preferably, the above (a) piperidine, ethanol, 0°C, 8 hours; (b) trifluoroacetic acid, 72.4°C, 2 hours; (c) dichloromethane, triethylamine, trifluoromethanesulfonic anhydride, room temperature, 30 minutes; (d) methanol, sodium borohydride, room temperature, 30 minutes; (e) tetrahydrofuran, N,N'-carbonyldiimidazole, 0°C, 1.5 hours; (f) acetic acid, 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine), 1,4-dioxane, 90°C, 5 hours.

[0017] The fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure of the present invention is used in the preparation of a reagent or tool for detecting changes in endogenous and exogenous H2O2 in cells.

[0018] Among them, the fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure is used in the preparation of reagents or tools for detecting changes in endogenous H2O2 in primary neuronal cells under the action of glutamate and kainic acid (KA, a known commonly used epilepsy inducer).

[0019] Among them, the fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure is used in the preparation of a reagent or tool for transcellular detection of changes in endogenous H2O2 in microglia-neuron co-culture.

[0020] The fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure of the present invention is used in the preparation of an in vivo imaging reagent or tool for detecting changes in H2O2 in the brain.

[0021] The fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure of the present invention is used in the preparation of reagents or tools that can effectively screen antioxidants that regulate H2O2 levels and 5-HT balance.

[0022] The invention discloses an application of a fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure in the preparation and screening of therapeutic drugs for regulating epileptic oxidative stress.

[0023] The fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure of the present invention is used in screening hesperetin as an antioxidant for regulating H2O2 levels and 5-HT balance.

[0024] The probe prepared by the invention is a hydrogen peroxide-responsive neurotransmitter precursor fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure, and is also a H2O2-activated serotonin precursor probe that reveals the interaction between 5-HT and adjacent proteins under oxidative stress.

[0025] The probe HOP prepared by the present invention can be used as a dual-effect probe for H2O2 sensing and protein labeling under oxidative stress. The precursor of the probe HOP releases 5-HT under oxidative stress and changes adjacent functional proteins. The HOP probe can be used for high-throughput screening of natural products.

[0026] Furthermore, the probe HOP was used to perform in vivo imaging on epileptic mice to detect changes in H2O2 in the brain.

[0027] The design principle of the fluorescent probe HOP of the present invention: The probe utilizes the excellent fluorescence and remarkable photophysical properties of alkyl bis(cyanoisocyanate)ketone boronic acid derivatives. Using this structure, they discovered that under conditions of oxidative stress in neural cells, abnormal increases in ROS (particularly H2O2) trigger activation of HOP in a localized microenvironment within the cell where stress is concentrated. Because the small molecule probe freely distributes and diffuses within the cell, this activation not only reveals the spatiotemporal distribution of intracellular oxidative stress through the release of a fluorescent response signal, but also converts the boronic acid group into the corresponding phenolic derivative, generating highly reactive electrophilic intermediates. These intermediates covalently bind to nucleophilic amino acid residues on nearby proteins. This binding anchors the generated fluorophore to the protein macromolecule, thereby limiting its diffusion and improving localization accuracy. Simultaneously, the probe releases the active neurotransmitter 5-HT into the local microenvironment. This dual function of HOP is particularly important because it enables precise imaging of changes in H2O2 balance within the local microenvironment. Furthermore, the simultaneous release of 5-HT within this local microenvironment provides valuable information on whether 5-HT interacts specifically with nearby proteins under conditions of oxidative stress. These interactions may be the key to regulating the physiological response of oxidative stress, thereby providing a new method for understanding the dynamics of neurotransmitters and neurochemical-related pathophysiology under oxidative stress. Therefore, the probe prepared by the present invention will become a powerful tool for studying the dynamics of neurotransmitters and neurochemical-related pathophysiology under oxidative stress. The present invention has developed a new fluorescent probe that can effectively trace endogenous H2O2 signals. The probe prepared by the present invention can not only detect H2O2, but also fix the signal of the fluorophore through covalent modification with the protein, which is not easy to diffuse in the cell. This provides a good idea for the development of detection tools for small molecules in the oxidative stress process of most diseases. At the same time, the probe can be used to screen out natural products that can relieve and treat epilepsy, which is an effective detection of H2O2 produced in epilepsy based on the probe.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] The present invention constructs a H2O2-activated serotonin precursor probe that can reveal the interaction between 5-HT and neighboring proteins under oxidative stress. The dual function of the probe, namely the local interaction between 5-HT released after H2O2 activation and nearby proteins, not only limits the diffusion of the fluorophore itself and improves the accuracy of intracellular H2O2 mapping, but also facilitates the in situ study of the interaction between 5-HT and neighboring proteins under such stress conditions, revealing new insights into 5-HT-mediated protein interactions under oxidative stress.

[0030] The present invention uses the natural antioxidant hesperidin screened by the probe HOP to regulate H2O2 levels and 5-HT homeostasis in epilepsy models, providing a new platform for in situ monitoring of H2O2 signaling in living primary neuronal cells and epilepsy mouse models. It also provides a new strategy for exploring abnormal protein modifications related to 5-HT under oxidative stress, while controlling the release of 5-HT, and is expected to provide a powerful chemical tool for advancing imaging research in other neurodegenerative disease models such as AD.

[0031] The preparation and synthesis method of the fluorescent probe for HOP of the present invention has a novel synthesis route, is simple and easy to implement, has low cost, has high raw material utilization rate and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A is the ultraviolet absorption spectrum of the HOP listed in the present invention before and after the reaction with H2O2; B is the fluorescence spectrum of the HOP listed in the present invention before and after the reaction with H2O2; C and D are the fluorescence spectra of the HOP listed in the present invention after reacting with H2O2 at different times; E and F are the fluorescence spectra of the HOP listed in the present invention after reacting with H2O2 at different concentrations; G is a fluorescence intensity comparison diagram of the HOP listed in the present invention with different active oxygen species, metal cations, anions, amino acids and H2O2; F is the fluorescence intensity of the HOP listed in the present invention after reacting with H2O2 under different pH conditions.

[0033] Figure 2 Figure A shows the HOPs listed in the present invention incubated with BSA in the presence of 1 mM H2O2 for 20 minutes, separated by SDS-PAGE, and stained with In-gel fluorescence and Coomassie Brilliant Blue; Figure B shows the peptide segments modified by HOPs in the BSA protein identified by mass spectrometry analysis; and Figure C shows the computational simulation of the modified binding sites on BSA.

[0034] Figure 3 This is the MTT diagram of the cytotoxicity of HOP listed in the present invention;

[0035] Figure 4 Flow cytometric analysis of live cell labeling and quantitative analysis of relative fluorescence intensity of GL261 cells using the HOP listed in the present invention;

[0036] Figure 5 Confocal imaging analysis of GL261 cells labeled with HOP listed in the present invention after different treatments such as LPS, PMA and NAC, and quantitative analysis of the relative fluorescence intensity of the cells;

[0037] Figure 6 The HOP listed in the present invention is used to perform confocal imaging and quantitative analysis of relative fluorescence intensity on primary neural cells that have been treated with PMA, DPI and ML171 in succession;

[0038] Figure 7 Confocal imaging analysis of live cell labeling and relative quantification of cell fluorescence intensity of primary neurons, microglia and astrocytes treated with KA, Glu and ML171 by HOP listed in the present invention;

[0039] Figure 8 Confocal imaging analysis of live cell labeling and quantification of cell fluorescence intensity of primary neurons treated with KA, Glu and ML171 by the HOP listed in the present invention;

[0040] Figure 9 The HOP listed in the present invention is used to perform flow cytometric analysis of live cell labeling and quantification of relative fluorescence intensity of cells on primary neural cells treated with Glu and resveratrol;

[0041] Figure 10 The confocal imaging of the HOP listed in the present invention on the co-culture system of primary neurons and primary microglia treated with Glu and resveratrol;

[0042] Figure 11 Western immunoblotting analysis and relative gray value analysis of the HOP listed in the present invention on SIRT1 expression in HT22 cells treated with Glu and resveratrol;

[0043] Figure 12 The HOP listed in the present invention is used to perform high-throughput natural product screening imaging analysis and cell relative fluorescence intensity quantification on KA-induced HT22 mouse neuronal cells;

[0044] Figure 13 The relative fluorescence intensity analysis of the 12 natural products initially screened by high-throughput screening was performed by HOP listed in the present invention after flow cytometry analysis;

[0045] Figure 14 The HOP listed in the present invention was incubated with MPO and SIRT1 for 20 minutes and then with BSA for 20 minutes in the presence of 1 mM H2O2, and then separated by SDS-PAGE and stained with In-gel fluorescence and Coomassie brilliant blue;

[0046] Figure 15 To identify the peptides modified by HOP and their binding sites in MPO protein by mass spectrometry;

[0047] Figure 16 To identify the peptides modified by HOP and their binding sites in SIRT1 protein by mass spectrometry;

[0048] Figure 17 To analyze the three different binding sites of MPO and HOP through computer simulation;

[0049] Figure 18 To analyze the three different binding sites of SIRT1 and HOP through computer simulation;

[0050] Figure 19 The immunofluorescence co-localization of HOP, MPO and SIRT1 antibodies listed in the present invention is shown.

[0051] Figure 20 HPLC quantitative analysis of 5-HT levels in primary neuronal cells treated with KA;

[0052] Figure 21 The in vivo fluorescence imaging diagram of the HOP listed in the present invention on the brain of KA-induced epileptic mice and the fluorescence intensity change over time in the mouse brain;

[0053] Figure 22 The in vitro fluorescence imaging of the brain of KA-induced epileptic mice by HOP listed in the present invention and the relative fluorescence signal analysis compared with the control group;

[0054] Figure 23 In vivo fluorescence imaging of the brain of epileptic mice induced by Ptz and the change of fluorescence intensity in the mouse brain over time

[0055] Figure 24 The in vitro fluorescence imaging of the brain of epileptic mice induced by Ptz and the relative fluorescence signal analysis compared with the control group are shown in the HOP of the present invention;

[0056] Figure 25 The in vivo fluorescence images and the quantitative analysis of the relative ratio of fluorescence intensities of the HOP listed in the present invention collected at different times after intravenous injection into mice;

[0057] Figure 26 The in vitro fluorescence imaging images of the brains of mice with epilepsy induced by Ptz and mice treated with hesperidin and the relative fluorescence signal analysis compared with the control group are shown in the following figures:

[0058] Figure 27 To perform immunofluorescence staining of MPO and SIRT1 proteins in frozen brain sections of normal mice, Ptz-induced epileptic mice, and hesperidin-treated epileptic mice, and to quantitatively analyze the fluorescence signals;

[0059] Figure 28 Western Blot was used to detect the expression levels of MPO and SIRT1 in the cortex and hippocampus tissues of the treated and control group mice and the relative gray value analysis was performed.

[0060] Figure 29 is the HOP nuclear magnetic resonance hydrogen spectrum;

[0061] Figure 30 is the HOP carbon NMR spectrum;

[0062] Figure 31 This is the HOP high-resolution mass spectrum. DETAILED DESCRIPTION

[0063] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0064] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods without specific conditions specified in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0065] Example 1

[0066] A method for preparing a probe HOP, the preparation process of which is as follows:

[0067]

[0068] A mixture of 4-hydroxybenzaldehyde (5 g, 41 mmol) and 2-(3,5,5-trimethylcyclohex-2-enylidene)malononitrile (7.6 g, 41 mmol) was dissolved in 400 mL of ethanol and heated under reflux in the presence of 4 mL of piperidine for 8 hours. After completion of the reaction as confirmed by thin-layer chromatography, the solvent was removed under reduced pressure. The crude product was purified by recrystallization from ethanol to obtain the desired compound 2.

[0069] Hexamethylenetetramine (3.48 g, 12.4 mmol) was added to a 30 mL trifluoroacetic acid solution containing compound 2 (3.6 g, 12.4 mmol). The reaction mixture was heated under reflux for 2 hours until the starting material was completely consumed. After cooling to room temperature, the reaction mixture was poured into ice water and the precipitate was collected by filtration. Column chromatography was performed using PE and EA in a 1:1 ratio to obtain compound 3 as a red solid.

[0070] Compound 3 (3 g, 9.4 mmol) was dissolved in an organic solvent dichloromethane solution (100 mL) and cooled to 0 ° C in an ice bath. Triethylamine (1.31 mL, 9.4 mmol) was added with stirring, followed by trifluoromethanesulfonic anhydride (1.57 mL, 9.4 mmol). The reaction mixture was allowed to warm to room temperature and stirred for another 30 minutes. Saturated NaHCO3 solution was added to quench the reaction. The aqueous layer was extracted with dichloromethane, and the combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography using a 1:1 mixture of PE and EA as eluent to give compound 4 as an orange solid;

[0071] Compound 4 (3.2 g, 7.1 mmol) was dissolved in an organic solvent methanol (50 mL) and cooled to 0 ° C. Sodium borohydride (NaBH4, 269.6 mg, 7.1 mmol) was added in small amounts while stirring. The reaction mixture was warmed to room temperature and stirred for 30 minutes. Water was added to quench the reaction, and the aqueous phase was extracted with an organic solvent dichloromethane. The combined organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. A 2:1 mixture of PE and EA was used as the eluent and purified by column chromatography to obtain a yellow solid compound 5;

[0072] Compound 5 (2 g, 4.42 mmol) was dissolved in an organic solvent tetrahydrofuran (35 mL) and cooled to 0 ° C. N, N'-carbonyldiimidazole (716 mg, 4.42 mmol) was added, and the reaction mixture was stirred at 0 ° C for 30 minutes. 3-(2-aminoethyl)-1H-indol-5-ol (778 mg, 4.42 mmol) was then added, and the reaction mixture was stirred at room temperature for another hour. After the reaction was completed, the reaction mixture was diluted with water and then extracted with ethyl acetate. The organic layer was washed with water and brine in sequence, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Using a 1:1 mixture of PE and EA as the eluent, it was purified by silica gel column chromatography to obtain an orange solid compound 6;

[0073] Compound 6 (1 g, 1.53 mmol), potassium acetate (450 mg, 4.59 mmol), palladium dichloride (100 mg, 0.153 mmol) and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (583 mg, 2.295 mmol) were dissolved in dioxane (20 mL) and degassed by alternating three vacuum and argon purges. The reaction mixture was stirred at 90 ° C for 5 hours under argon. After the reaction was completed, it was cooled to room temperature, diluted with water, and then extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The orange solid HOP was purified by silica gel column chromatography using a 1:1 mixture of PE and EA as the eluent.

[0074] 1H NMR (300MHz, DMSO-d6) δ10.46(s,1H),8.58(s,1H),7.72(d,J=7.4Hz,2H),7.67( s,1H),7.47(d,J=16.1Hz,1H),7.36-7.22(m,2H),7.12(d,J=8.6Hz,1H),7.04(s, 1H),6.92(s,1H),6.83(s,1H),6.59(d,J=6.7Hz,1H),5.23(s,2H),3.26(d,J=6.9 Hz,2H),2.84-2.68(m,2H),2.61(s,2H),2.54(s,2H),1.30(s,12H),1.01(s,6H). 13 C NMR(75MHz,DMSO-d6)δ170.70,156.52,155.79,150.63,143.87,138.81,137.19,136.42,131.26,128.33,123.89,123.46 ,114.19,113.37,112.09,111.73,111.08,102.63,84.16,77.48,65.47,41.55,32.11,27.88,25.06.HRMS(m / z)calcd.for C 37 H 41 BN4O5[M+H] + 632.3170, found 633.3234.

[0075] The HOP probe prepared in this example has a hydrogen nuclear magnetic resonance spectrum, a carbon nuclear magnetic resonance spectrum, and a high-resolution mass spectrum. Figures 29-31 shown.

[0076] Example 2

[0077] Synthesis and preliminary evaluation of the probe

[0078] The final structure of the probe HOP prepared in Example 1 is determined by 1 H and 13C NMR spectroscopy and mass spectrometry fully confirmed the results. Preliminary in vitro testing was then conducted in PBS buffer (pH 7.4, 1% DMSO). An appropriate amount of HOP solid powder was weighed into a 1.5 mL centrifuge tube and dissolved in DMSO to a 10 mM stock solution. The solution was then aliquoted and stored at -80°C until further use. The 10 mM probe stock solution was diluted to 1 mM with DMSO. 2 μL of the 1 mM probe solution was added to 200 μL of PBS (10 mM, pH 7.4) buffer to prepare a 10 μM pure probe test solution. In another 1.5 mL EP tube, 2 μL of a 10 mM H2O2 solution was added to the reaction test solution while the above preparation was being performed. Three replicates were prepared for each test system. After incubation at 37°C on a shaker for 30 minutes, the system was transferred to a quartz test dish for UV spectrophotometric analysis. The probe itself exhibited a distinct absorption peak at approximately 410 nm ( Figure 1 A). In the absence of H2O2, the emission peak of HOP is around 580 nm. However, after H2O2 treatment, a significant increase in fluorescence intensity was observed at 680 nm, accompanied by a 100 nm red shift ( Figure 1 B). The fluorescence intensity increased approximately 60-fold within 10 minutes and reached a plateau after 30 minutes ( Figure 1 C and 1D). Meanwhile, with the increase of H2O2 concentration, the fluorescence signal gradually increased in the range of 0-200μM ( Figure 1 E and 1F).

[0079] The specific selectivity of the probe HOP for H2O2 was further studied. The HOP concentrated stock (10mM) was diluted to 1mM with DMSO. 2μL of 1mM HOP solution was added to PBS buffer with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 to prepare control test solutions. In addition, a batch of test solutions identical to the above system was prepared, and 2μL of 10mM H2O2 solution was added to ep tubes to prepare reaction test solutions. After thorough vortexing, the solution was incubated in a shaker at 37°C for 20 minutes before fluorescence spectrum testing. Three groups of parallel samples were set up for both test systems. Metal ion compounds (Na + , K + Mg 2+ , Ca 2+ 、Al 3+ 、Sn 2+ 、Ba 2+ 、Ni 2+ 、Fe 3+ 、Fe 2+ Cr 3+ 、Mn 2+ 、Cd2+ 、Ag + , Pb 2+ 、Zn 2+ 、Cu 2+ 、Cu + ), ROS and RNS compounds ( 1 O2、ClO - 、ONOO - ), sulfur-containing substances (Cys, GSH, Hcy) and H2O2 analytes are used for selective spectroscopic testing of HOP. + , K + Mg 2+ , Ca 2+ The final reaction concentrations of 1 mM for GSH and 200 μM for Cys, and 100 μM for all other analytes. The metal ion compounds, ROS and RNS compounds, and sulfur-containing substances were mixed to prepare metal ion mixtures, ROS and RNS mixtures, and sulfur-containing mixtures. Control test solutions were prepared by adding the metal ion mixtures, ROS and RNS mixtures, and sulfur-containing mixtures to 2 μL of 1 mM H2O2 solution in PBS buffer. A separate set of control solutions with the same system was prepared, and 2 μL of 10 mM H2O2 solution was added to each to prepare interference test solutions. After the selectivity and interference test systems were configured, they were incubated on a shaker at 37°C for 20 minutes before fluorescence spectroscopy was performed. Test conditions: excitation and emission slits of 10 nm and 10 nm, respectively, with a voltage of 650 V. The excitation wavelength was set to 561 nm, and the emission wavelength range was 520-800 nm. The probe has higher selectivity for H2O2 than other reactive oxygen species, metal cations, anions and amino acids ( Figure 1 G). It exhibited a significant fluorescence response in the pH range of 6.0 to 11.0, indicating that HOP is very suitable for biological applications ( Figure 1 These results demonstrate that HOP is sensitive and selective under physiological conditions and has great potential for sensing and monitoring H2O2 levels in biological systems.

[0080] Example 3

[0081] HOP for sensing H2O2 and labeling proteins under oxidative stress

[0082] In order to determine whether HOP would be anchored on the protein surface by cross-linking with adjacent proteins after being activated in a microenvironment in the presence of H2O2, bovine serum albumin (BSA) was used as a model protein substrate to study the protein labeling behavior of HOP in the presence and absence of H2O2 in vitro. In a buffer containing 10mM sodium phosphate (pH7.4), H2O2 and bovine serum albumin (BSA) were added, and HOP (50μM) was incubated at 37°C for 30 minutes. The protein samples were loaded into 8% or 10% SDS-PAGE protein gels for conversion (80V, 30 minutes) and separation (120V, 1-2 hours). After electrophoresis, the protein bands were excised from the gel and cut into small pieces for protein gel imaging. The experiment found that the strong fluorescent band corresponding to BSA could only be observed in the presence of H2O2 ( Figure 2 A), which confirms that HOP specifically labels proteins in a H2O2-dependent manner.

[0083] In order to further elucidate the detailed mechanism of its protein labeling, the covalent modification sites were identified by enzymatically digesting the HOP-treated BSA sample (i.e., the protein band in the previous step) and analyzing it using mass spectrometry. The imaged gel was destained with 50% acetonitrile (ACN) / 50mM ammonium bicarbonate (NH4HCO3) to destain the gel fragments. The gel fragments were incubated with 55mM iodoacetamide in 25mM NH4HCO3 to alkylate the digested protein fragments. Subsequently, the gel fragments were digested with 200ng of trypsin (Promega Sequence Grade Modified) in 50mM NH4HCO3 for 4 hours. The resulting peptide recovery solution was desalted with a C18 pipette tip, and the treated peptide sample was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The experiment found that in the presence of H2O2, the fluorophore released by HOP was covalently cross-linked with the cysteine ​​residues on BSA, while the released 5-HT was cross-linked with the tyrosine residues ( Figure 2 B). These findings indicate that HOP is activated in the presence of H2O2, and in this oxidative stress microenvironment, the released fluorophore and 5-HT covalently cross-link with neighboring proteins. In addition, molecular docking simulations were performed to observe the spatial changes in the interaction between HOP and BSA. The docking results showed the covalent binding mode of the probe upon H2O2 activation ( Figure 2 C) This example verifies that the probe fluorescence signal is fixed by covalently modifying the protein, further demonstrating that the probe HOP of the present invention can react with and be activated by H2O2 after entering the cell, thus achieving excellent imaging effects.

[0084] Example 4

[0085] HOP detects changes in endogenous and exogenous H2O2 at the cellular level

[0086] Prior to the imaging studies, the cytotoxicity of HOP itself on living cells was investigated. The cytotoxicity of GL261 cells was assessed by MTT assay. GL261 cells were plated at 5 × 10 cells per well. 3 The cells were cultured at a density of 5000 cells / well in a 96-well plate and placed in a 37°C incubator (5% CO2) for 24 hours. After removing the culture medium from the 96-well plate, the cells were washed with PBS and then treated with different concentrations of HOP (0, 0.01, 0.1, 1, 10, and 100 μM) in the 96-well plate and cultured for 24 hours. Subsequently, the 96-well plate was removed from the incubator, and 10 μL of 5 mg / mL MTT was added to the 96-well plate and cultured in the incubator for another 4 hours. The supernatant was then discarded, 150 μL of DMSO was added, and the cells were incubated with The absorbance of samples in 96-well plates was read at 570 nm (reference wavelength 630 nm) using a M200 Pro multi-mode microplate reader (Tecan, Switzerland). GL261 cells were cultured with different concentrations of HOP (1-10 μM) and no obvious cytotoxic effect was observed ( Figure 3 ).

[0087] Next, we attempted to investigate the responsiveness of HOP to endogenous and exogenous H2O2 in living cells. GL261 cells were plated at 5 × 10 5 Cells were cultured at a density of 10 μg / mL in 4 wells of a 6-well plate and placed in a conditioned incubator at 37°C and 5% CO2 for 12 h. The four wells were treated as follows: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 50 μM H2O2 for 30 min, and then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 100 μM H2O2 for 30 min, and then incubated with 5 μM HOP for 20 min; (4) cells were pretreated with 200 μM H2O2 for 30 min, and then incubated with 5 μM HOP for 20 min. Endogenous H2O2 flow cytometry analysis: GL261 and SHSY5Y cells were cultured at a density of 10 μg / mL in each well. 5Cells were cultured at a density of 100 μg / mL in a 6-well plate and placed in a conditioned incubator at 37°C and 5% CO2 for 12 h. Each well of the 6-well plate was treated with the following different treatments: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 1 μg / mL LPS for 12 h, and then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 1 μg / mL LPS for 12 h, and then treated with 2 mM NAC, 5 μM Ebselen, and 5 μM DPI for 30 min, respectively, and then incubated with 5 μM HOP for 20 min; (4) Pretreat the cells with 1 μg / mL PMA for 1 h, then incubate the cells with 5 μM HOP for 20 min; (5) Pretreat the cells with 1 μg / mL PMA for 1 h, then treat the cells with 2 mM NAC, 5 μM Ebselen, and 5 μM DPI for 30 min, then incubate the cells with 5 μM HOP for 20 min; (6) Treat the cells with 2 mM NAC for 30 min, then incubate the cells with 5 μM HOP for 20 min.

[0088] After all probes have been incubated, rinse the cells twice with PBS. Add an appropriate amount of trypsin to digest the cells for 2 minutes, then add the corresponding amount of culture medium containing 2% serum to terminate the digestion. Collect the cells into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Remove the supernatant, resuspend the cell pellet with PBS, and centrifuge again, repeating twice. Finally, add 500 μL of PBS to resuspend the cells and place on ice for testing. Flow cytometer ( Figure 4 ) and confocal imaging ( Figure 5 ) analysis showed that PMA-stimulated cells showed a significant increase in fluorescence. Addition of N-acetylcysteine ​​(NAC, a H2O2 scavenger) inhibited this fluorescence enhancement. The application of HOP in primary neuronal cells under PMA-induced oxidative stress conditions was further evaluated. Imaging of neurons after 1 hour of PMA stimulation showed a corresponding increase in endogenous H2O2 and a significant increase in fluorescence compared to the control group. In contrast, treatment with DPI and ML171 significantly reduced baseline fluorescence and PMA-induced fluorescence enhancement ( Figure 6 In conclusion, all these results confirm that HOP, as a dual-activity-based probe, can effectively detect changes in endogenous and exogenous H2O2 using microscopy and flow cytometry under stimulated conditions in living cells.

[0089] Example 5

[0090] HOP is used to detect changes in endogenous H2O2 produced by glutamate and KA.

[0091] Since HOP can effectively monitor endogenous H2O2 produced in cells, its application has been extended to studying oxidative stress signaling in primary neurons. Oxidative stress, especially the accumulation of reactive oxygen species (ROS) (such as myeloperoxidase (MPO)-related H2O2), is believed to be closely related to neurological diseases such as epilepsy. Primary neurons, microglia, and astrocytes were cultured at 5×10 4 Cells were cultured at a density of 100 μg / mL in 5 wells of a 24-well plate and incubated in a conditioned incubator at 37°C and 5% CO2 for 7 days. Each well of the 24-well plate was treated with the following different treatments: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 500 μM KA for 1 h, then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 500 μM KA for 1 h, then treated with 10 μM ML171 for 30 min, then incubated with 5 μM HOP for 20 min; (4) cells were pretreated with 2 mM Glu for 1 h, then incubated with 5 μM HOP for 20 min; (5) cells were pretreated with 2 mM Glu for 1 h, then treated with 10 μM ML171 for 30 min, then incubated with 5 μM HOP for 20 min. After the probe incubation was completed, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, and then washed twice with PBS before preparation of slides. Fluorescence imaging was then performed under a Leica SP8 confocal microscope. ex =561nm,λ em =650-750 nm. Compared with the control group, cells treated with glutamate and KA showed significantly brighter fluorescence signals, indicating that HOP is highly sensitive to elevated oxidative stress.

[0092] Primary neuronal cells were cultured at a rate of 5 × 10 4Cells were cultured at a density of 100 μg / mL in 5 wells of a 24-well plate and incubated in a conditioned incubator at 37°C and 5% CO2 for 7 days. Each well of the 24-well plate was treated with the following different treatments: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 500 μM KA for 1 h, then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 500 μM KA for 1 h, then treated with 500 μM 4-ABAH for 30 min, then incubated with 5 μM HOP for 20 min; (4) cells were pretreated with 2 mM Glu for 1 h, then incubated with 5 μM HOP for 20 min; (5) cells were pretreated with 2 mM Glu for 1 h, then treated with 500 μM 4-ABAH for 30 min, then incubated with 5 μM HOP for 20 min. After the probe incubation, the cells were washed twice with PBS, fixed with 4% paraformaldehyde, and then washed twice with PBS before preparation of slides. Fluorescence imaging was then performed under a Leica SP8 confocal microscope. Figure 7 ) and the MPO inhibitor 4-ABAH ( Figure 8 ) can also prevent the fluorescence enhancement of HOP. These findings provide direct evidence that neurons and glial cells produce excessive endogenous H2O2 in response to stimulation by glutamate and its analog KA, and that the neurotransmitter precursor-based probe HOP can specifically recognize and detect endogenous H2O2 produced in primary neurons.

[0093] Example 6

[0094] HOP observes changes in H2O2 across cells in a microglia-neuron co-culture model

[0095] Deacetylase SIRT1 plays an important role in oxidative stress damage. Resveratrol (a known SIRT1 activator) can exert anti-inflammatory effects by reducing the excessive accumulation of ROS. Therefore, resveratrol is considered as a potential H2O2 regulator for chemical regulation or intervention in subsequent H2O2 transcellular signaling studies. First, cells stimulated with Glu and resveratrol were detected by flow cytometry. Primary neuronal cells were cultured at 8×10 cells per well. 5The cells were cultured at a density of 100 μg / mL in a 6-well plate coated with PDL and placed in a conditioned incubator at 37°C and 5% CO2 for 7 days. Each well of a 6-well plate was treated as follows: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 2 mM Glu for 1 h, and then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 1 μM resveratrol for 1 h, and then incubated with 5 μM HOP for 20 min; (4) cells were pretreated with 2 mM Glu for 1 h, 1 μM resveratrol was added for another 1 h, and then incubated with 5 μM HOP for 20 min; (5) cells were pretreated with 1 μM resveratrol for 1 h, 2 mM Glu was added for another 1 h, and then incubated with 5 μM HOP for 20 min; (6) cells were co-treated with 2 mM Glu and 1 μM resveratrol for 1 h, and then 5 μM HOP was added for another 20 min. After the probe incubation is completed, the cells are rinsed twice with PBS, 1 mL of PBS is added to the well plate, and the cells in the well plate are scraped with a 23 cm cell scraper. After blowing, the cells are collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant is removed, the cell pellet is resuspended with PBS and centrifuged again, and repeated twice. Finally, 500 μL of PBS is added to resuspend the cells and placed on ice for testing. The results showed that the cell fluorescence under Glu stimulation increased significantly, indicating that the intracellular H2O2 level increased, while resveratrol treatment alleviated this reaction ( Figure 9 These findings suggest that resveratrol can alleviate the abnormal accumulation of intracellular H2O2 by chemically regulating neuronal cells.

[0096] Next, primary neuronal cells were cultured at 5 × 10 4 Microglial cells were cultured at a density of 1×10 in 3 wells of a 24-well plate and placed in a conditioned incubator at 37°C and 5% CO2 for 7 days. 5 The cells were seeded onto neuronal cultures at a density of 100 cells / mL. Each well of a 24-well plate was treated with the following different treatments: (1) cells were incubated with 5 μM HOP for 20 min; (2) cells were pretreated with 2 mM Glu for 1 h, and then incubated with 5 μM HOP for 20 min; (3) cells were pretreated with 1 μM resveratrol for 1 h, and then incubated with 5 μM HOP for 20 min. After the probe incubation, the cells were rinsed twice with PBS, fixed with 4% paraformaldehyde, and then washed twice with PBS before preparation of slides. Fluorescence imaging was then performed under a Leica SP8 confocal microscope. HOP: λ ex =561nm,λ em =650-750nm. A significant decrease in the fluorescence signal ratio of neurons / microglia was observed in the co-culture stimulated by Glu ( Figure 10 ), which indicates that Glu-induced stress disrupts the redox balance of neuronal cells. The increase in microglial H2O2 levels observed in neuronal stress responses confirms that HOP can effectively capture and monitor intracellular H2O2 flux, thereby visualizing transcellular stress signals between neurons and microglia in complex co-culture models. In contrast, it was also observed that the fluorescence ratio of the resveratrol-treated experimental group tended to be stable, which further indicated that resveratrol eliminated excess H2O2. To further verify that resveratrol eliminates H2O2 by activating SIRT1, Western blot analysis was performed using a SIRT1-specific antibody, and the results showed that Glu stimulation significantly reduced the expression of SIRT1, while co-treatment with resveratrol restored its expression level ( Figure 11 These findings suggest a role for resveratrol in SIRT1-mediated antioxidant processes and demonstrate the utility of HOP as a versatile probe to track dynamic H2O2 fluxes in neuronal cells.

[0097] Example 7

[0098] High-throughput imaging

[0099] The present invention further developed a fluorescence-based high-throughput screening platform that integrates high-content screening (HCS) and high-content analysis (HCA). 4 Cells were seeded into PerkinElmer CellCarrier-96 plates (500 μM cells / well) and pretreated with KA (500 μM) for 1 h. Cells were then treated with various anti-inflammatory agents (20 μM) or different antioxidants (20 μM) (including cannabidiol, aminolipin, α-asarone, 7-hydroxycoumarin, naringenin, hesperidin, piperine, kaurenic acid, purpurogenol, caryophyllene, 3,4,5-trimethoxycinnamic acid, apigenin, quercetin, ibuprofen, melatonin, rutin, coumarin, podophyllotoxin, β-salicylic acid, artemisinin, and carbamazepine) for 1 h, and then incubated with HOP (5 μM) and the nuclear dye Hoechst (1 μg / mL) for 20 minutes. After treatment, cells were rinsed three times with PBS to remove excess reagents. Imaging was performed using a high-throughput fluorescence imaging system. The excitation filter wavelength used for imaging and quantitative analysis was 561 nm, and the emission collection range was 650-720 nm. The data were quantitatively analyzed using Image J software ( Figure 12 ). The results showed that most of the selected natural products induced a significant decrease in the HOP fluorescence signal, indicating that they downregulated the production of H2O2. In addition, flow cytometric analysis was performed to further confirm the reduction in H2O2 levels ( Figure 13Hesperidin, a classic flavonoid with diverse biological activities, effectively inhibited HOP fluorescence compared to both the untreated negative control and the KA pre-treated positive control, demonstrating its potent antioxidant activity in neurons. Together, these results highlight the utility of HOP as a high-throughput screening tool for identifying natural products that modulate oxidative stress in living neurons. Hesperidin's ability to reduce H2O2 levels offers new opportunities for developing therapeutic strategies against oxidative stress-related neuronal damage.

[0100] Example 8

[0101] HOP precursor releases 5-HT under oxidative stress and alters adjacent functional proteins

[0102] The above examples demonstrate that HOP can selectively detect elevated intracellular H2O2 levels under conditions of oxidative stress induced by various stimuli. High-throughput screening identified hesperidin, a natural product with potent antioxidant properties, as a potent inhibitor of elevated H2O2 levels in KA-stimulated neurons. Next, we sought to further investigate whether 5-HT under oxidative stress can label functional proteins within its microenvironment and whether the modulatory compound hesperidin can help restore 5-HT homeostasis. MPO and SIRT1 are considered key regulatory proteins influencing 5-HT receptor activity, particularly in neurodegenerative diseases. This study utilized an H2O2-dependent assay in which HOP was incubated with MPO or SIRT1 protein, followed by SDS-PAGE analysis of the resulting protein labeling. H2O2 and myeloperoxidase (MPO) or sirtuin 1 (SIRT1) were added to a buffer containing 10 mM sodium phosphate (pH 7.4) and incubated with HOP (50 μM) at 37°C for 30 minutes. Protein samples were loaded onto 8% or 10% SDS-PAGE protein gels for conversion (80V, 30 minutes) and separation (120V, 1-2 hours). After electrophoresis, the protein bands of interest were excised from the gel and cut into small pieces for protein gel imaging. In the presence of H2O2, fluorescent bands corresponding to MPO and SIRT1 were observed ( Figure 14 ), which indicates that the protein has been selectively modified. Mass spectrometry analysis further confirmed that the fluorophore part of HOP is similar to that of MPO ( Figure 15 ) and SIRT1( Figure 16 ) covalently binds to the cysteine ​​residues in the 5-HT moiety, while the 5-HT moiety forms covalent bonds with the tyrosine residues in these functional proteins. Similarly, detailed molecular docking studies structurally verified these covalent interactions, revealing that HOP, after being activated by H2O2, binds to MPO ( Figure 17 ) and SIRT1( Figure 18 )'s precise binding configuration.

[0103] In addition, HT22 cells were plated at 5 × 10 4 Cells were cultured at a density of 100 μg / mL in two wells of a 24-well plate and incubated in a conditioned incubator at 37°C and 5% CO2 for 24 hours. Cells were first incubated with 5 μM HOP for 20 minutes, then fixed with freshly prepared 4% paraformaldehyde in 1×PBS for 10 minutes. After fixation, they were rinsed three times with PBS for 5 minutes each. Next, the cells were incubated with freshly prepared 1×PBST solution for 5 minutes to permeabilize the cells. The cells were then rinsed three times with PBS for 5 minutes each. Next, the cells were covered with 3% BSA solution and incubated at 37°C for 30-60 minutes. The blocking solution was then aspirated, and 2 μL of MPO antibody and 2 μL of SIRT1 antibody were added to two ep tubes with 0.5 mL of 3% BSA solution, respectively. After mixing, the cells were added to the cell wells to cover the cells and incubated at 4°C overnight. After the primary antibody incubation was completed, the cells were rinsed three times with 1×PBST for 5 minutes each. Add 2 μL of Alexa Fluor 488 rabbit antibody to the ep tube with 1 mL of 3% BSA solution to prepare the immunofluorescence secondary antibody incubation solution, add it to the cell well plate to cover the cells, and incubate at 37°C for 1 hour. After completion, rinse the cells three times with 1×PBST for 5 minutes each time. Drop a drop of anti-fluorescence quenching blocking agent on the slide, carefully remove the cell slide, and cover the cell surface with the anti-fluorescence quenching agent. Microscopic imaging was performed using a Leica TCS SP8 MP confocal fluorescence microscope. The immunofluorescence channel was excited at a wavelength of 488 nm, and the emission spectrum was collected in the wavelength range of 500-550 nm. The HOP channel was excited at a wavelength of 561 nm, and the emission spectrum was collected in the wavelength range of 650-720 nm. Further immunofluorescence co-localization imaging of HOP with MPO and SIRT1 also showed that the localization of HOP and protein was relatively good ( Figure 19). These findings suggest that under oxidative stress, HOP is covalently fixed to protein macromolecules in the local microenvironment and promotes the release of the neurotransmitter 5-HT through interactions with neighboring proteins. This mechanism reflects the dynamic changes in redox balance observed in the stressed intracellular environment. In this oxidative stress microenvironment, abnormal interactions between 5-HT precursors and functional proteins may disrupt normal neurotransmitter function, thereby potentially contributing to the occurrence and development of neurodegenerative diseases. More importantly, this process inevitably leads to a large depletion of intracellular 5-HT, indicating that under oxidative conditions, the balance of 5-HT is severely disrupted. To further investigate the changes in 5-HT homeostasis during oxidative stress and the potential role of regulators in regulating its homeostasis, 5-HT levels were measured in KA-stimulated cells and compared with cells treated with hesperetin. Well-grown primary neuronal cells were divided into the following groups: (1) a control group without any treatment; (2) an experimental group in which cells were treated with 500 μM KA for 12 h; and (3) an experimental group in which cells were pretreated with 500 μM KA for 12 h and then incubated with 20 μM hesperidin for 12 h. After treatment, the three groups of cells were removed from the incubator, rinsed twice with PBS, added with 1 mL of purified water, and scraped from the well plate with a cell scraper. The collected cell suspension was centrifuged at 1,500 rpm for 8 min to collect the cell pellet. The supernatant was discarded and the cell pellet was resuspended in 200 μL of purified water. Cell lysis was performed by freeze-thaw method and the samples were stored at -80°C until further analysis. For 5-HT detection, 50 μL aliquots of cell homogenate were mixed with 20 μL of trifluoroacetic acid to precipitate proteins. The mixture was vortexed for 30 s and allowed to stand at room temperature for 5 min. After standing, the cells were centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was collected and centrifuged again for 10 min. Finally, the supernatant was carefully collected and analyzed by HPLC. 5-HT levels were significantly reduced in cells treated with KA, while hesperetin treatment partially restored 5-HT levels ( Figure 20 Taken together, these observations suggest that HOP has a dual role as both a sensor and a reactive neurotransmitter precursor, providing a foundation for the development of tools to study changes in neurotransmitters during oxidative stress and offering insights into the diagnosis and treatment of clinical diseases.

[0104] Example 9

[0105] In vivo imaging of H2O2 flux in the brain of epileptic mice

[0106] To evaluate the potential of HOP as an in vivo imaging tool for endogenous H2O2 flux in neurological disease models, 5-week-old BALB / c mice were first weighed to confirm their body weight, which was approximately 20 g per mouse. An acute epilepsy model was induced by ip injection of 120 μL of KA (1 mg / mL). 12 hours later, the mice were placed in an anesthesia box containing isoflurane for 5 minutes, and then injected with 60 μL of a 1 mg / mL HOP solution by iv injection. In vivo imaging showed that the fluorescence intensity of the epileptic mouse brain at different time points (0, 10, 20, 30, 45 and 60 minutes) was significantly increased compared with the PBS control group. The increase in fluorescence indicates that HOP effectively crossed the blood-brain barrier and tracked the upregulation of H2O2 in the brain ( Figure 21 Furthermore, in vitro fluorescence imaging of brain tissue from these mice validated these in vivo observations, confirming the accuracy of the HOP-based oxidative stress assay ( Figure 22 ).

[0107] To further verify the reliability of the HOP application, an acute epileptic mouse model induced by pentylenetetrazol (Ptz) was used, which is another classic epileptic seizure model. First, 5-week-old BALB / c mice were weighed to confirm their body weight, which was approximately 20g per mouse. An acute epileptic model was induced by ip injection of 120μL of 240μL Ptz (5mg / mL). After 12h, the mice were placed in an anesthesia box containing isoflurane for 5min, and then 60μL of a 1mg / mL HOP solution was injected through the tail vein. Like the KA model, HOP can also detect elevated H2O2 levels in the brains of epileptic mice under Ptz-induced oxidative stress conditions ( Figure 23 and Figure 24 Consistent with previous research, the fluorescence signal in the epilepsy model group was significantly enhanced, exceeding that in the PBS group. These in vivo and in vitro studies collectively demonstrate that HOP has excellent imaging performance and is a promising tool for real-time monitoring of dynamic changes in endogenous H2O2 within the brains of living epileptic mouse models.

[0108] In order to further explore whether hesperidin can play an antioxidant role in epileptic mouse models, HOP imaging technology was used to observe the effect of hesperidin in classic Ptz-induced epileptic mice. First, 5-week-old BALB / c mice were weighed to confirm their body weight, which was approximately 20g per mouse. An acute epilepsy model was induced by ip injection of 240μL of Ptz (5mg / mL). 12h later, 328μL of the antioxidant hesperidin (2mM) was injected iv to treat Ptz-induced acute epilepsy. After 12h, the mice were placed in an anesthesia box containing isoflurane for 5 minutes, and then 60μL of HOP solution with a concentration of 1mg / mL was injected through the tail vein. In vivo imaging showed that the brain fluorescence signal of epileptic mice treated with hesperidin was significantly weakened compared with mice treated with Ptz alone ( Figure 25 In vitro brain imaging further validated these findings ( Figure 26 ). These results indicate that hesperidin can effectively regulate the excessive production of H2O2 in the brains of epileptic mice. In addition, to elucidate the underlying molecular mechanisms, the expression of SIRT1 and MPO proteins, key markers of oxidative stress regulation, in the brains of Ptz-treated and hesperidin-treated mice was investigated. Immunofluorescence staining of brain sections revealed that Ptz-induced epilepsy was accompanied by increased MPO expression and decreased SIRT1 expression. In contrast, hesperidin treatment resulted in a significant upregulation of SIRT1, accompanied by a decrease in MPO levels ( Figure 27 ). These findings suggest that decreased SIRT1 activity is associated with increased MPO expression, which in turn exacerbates H2O2-mediated oxidative stress. Hesperidin appears to counteract this process by activating SIRT1, inhibiting MPO, and clearing excess H2O2. Furthermore, Western blotting of brain tissue from the cerebral cortex and hippocampus confirmed these results, showing that hesperidin treatment upregulated SIRT1 expression while downregulating MPO ( Figure 28 Together, these findings suggest that stress in the brains of epileptic mice leads to upregulation of MPO and decreased SIRT1 activity, and that hesperetin may be a potent SIRT1 activator that antagonizes this process. Our newly designed neurotransmitter precursor, HOP, allows for the observation of changes and regulation of H2O2 balance in the brain during this process, providing a powerful chemical tool for studying oxidative stress regulation in neurological disease models. This study highlights the utility of HOP for real-time monitoring of oxidative stress in vivo and underscores the potential of hesperetin as a therapeutic agent for modulating oxidative stress pathways in epilepsy.

Claims

1. A fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure, characterized in that: The structure of the probe is shown below:

2. A method for preparing a fluorescent probe having a bis(cyanoisocyanate)ketoneboronic acid alkyl structure according to claim 1, characterized in that: Preferably, the method comprises the following steps: A mixture of 4-hydroxybenzaldehyde and 2-(3,5,5-trimethylcyclohex-2-enylidene)malononitrile was dissolved in an organic solvent, piperidine was added and refluxed, and after completion of the reaction, the solvent was removed under reduced pressure and purified to obtain compound 2; Hexamethylenetetramine was added to the solution containing compound 2, and the mixture was refluxed. After the reaction, the mixture was cooled to room temperature, and the reaction mixture was poured into ice water. The precipitate was collected by filtration and purified to obtain compound 3; Compound 3 was dissolved in an organic solvent, triethylamine was added under stirring in an ice bath, and then trifluoromethanesulfonic anhydride was added. The reaction mixture was allowed to warm to room temperature and stirred for further reaction. The reaction was quenched and then extracted. The combined organic layer was dried, filtered, concentrated under reduced pressure, and purified to obtain compound 4; Compound 4 was dissolved in an organic solvent and cooled, and sodium borohydride was added under stirring. The reaction mixture was warmed to room temperature and stirred for reaction. After the reaction was quenched, extraction was performed, and the combined organic layer was dried, filtered, concentrated under reduced pressure, and purified to obtain compound 5; Compound 5 was dissolved in an organic solvent and cooled, carbonyldiimidazole was added, and the reaction mixture was stirred for reaction. 3-(2-aminoethyl)-1H-indol-5-ol was added, and the reaction mixture was stirred for reaction at room temperature. After the reaction was completed, the organic layer of the reaction mixture was washed, dried, concentrated under reduced pressure, and purified to obtain compound 6; Compound 6, potassium acetate, palladium dichloride and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine were dissolved in an organic solvent, and the reaction mixture was heated and stirred under an inert atmosphere. After the reaction was completed, it was cooled to room temperature, and the organic layer was washed, dried, filtered, and concentrated under reduced pressure to obtain the probe HOP; The reaction formula is as follows:

3. Use of the fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure according to claim 1 in the preparation of a reagent or tool for detecting changes in endogenous and exogenous H2O2 in cells.

4. The use according to claim 3, characterized in that The fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure is used in the preparation of a reagent or tool for detecting changes in endogenous H2O2 in primary neuronal cells under the action of glutamic acid and kainic acid.

5. The use according to claim 3, characterized in that The fluorescent probe having a bis(cyanoisocyanate)ketone boric acid alkyl structure is used in the preparation of a reagent or tool for transcellular detection of changes in endogenous H2O2 in microglia-neuron co-culture.

6. Use of the fluorescent probe having a bis(cyanoisocyanate)ketone boronate alkyl structure according to claim 1 in the preparation of an in vivo imaging reagent or tool for detecting changes in H2O2 in the brain.

7. Use of the fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure according to claim 1 in the preparation of a reagent or tool for effectively screening antioxidants that regulate H2O2 levels and 5-HT balance.

8. Use of the fluorescent probe having a bis(cyanoisocyanate)ketoneboronic acid alkyl structure according to claim 1 in the preparation and screening of therapeutic drugs for regulating oxidative stress in epilepsy.

9. Use of the fluorescent probe having a bis(cyanoisocyanate)ketone boronic acid alkyl structure according to claim 1 in screening hesperetin as an antioxidant for regulating H2O2 levels and 5-HT balance.

Images