A class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles, preparation method and application thereof

Abstract

The application belongs to the field of fluorescent probes, and discloses a kind of fluorescent probe for monitoring dynamic change of hydrogen peroxide in different subcellular organelles, preparation method and application thereof, the probe is with flavonol as parent nucleus, and hydrogen peroxide ratio type fluorescent probe PML is targeted to lysosome, and hydrogen peroxide ratio type fluorescent probe PZD is targeted to lipid droplet.The application also provides the preparation method and application of the probe.The probe provided by the application has novel structure, simple synthesis method, good recognition effect on hydrogen peroxide, especially hydrogen peroxide in subcellular organelles, fast response speed, high sensitivity, reliable inspection result, good biocompatibility, subcellular organelle positioning function, so that they can be used for monitoring hydrogen peroxide in intracellular microenvironment, and it has great significance to apply probe technology to practical related pathological model to develop the mechanism of hydrogen peroxide in its pathology, especially in the process of ferroptosis.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, and in particular to a class of fluorescent probes for monitoring dynamic changes of hydrogen peroxide in different subcellular organelles, their preparation methods, and their applications. Background Technology

[0002] Hydrogen peroxide (H2O2), an important reactive oxygen species (ROS) molecule in cells, plays a crucial role in physiological processes such as immune responses, signal transduction, and cell proliferation. However, excessive accumulation of H2O2 can trigger oxidative stress, leading to cell damage and being associated with various pathological processes, including cancer and neurodegenerative diseases. Lysosomes, as degradation centers and signaling hubs, lack catalase, making them prone to accumulating H2O2. In acidic environments, H2O2 reacts with stored iron ions (Fe2+) via the Fenton reaction. 2+ The generation of highly reactive hydroxyl radicals (·OH) by H2O2 disrupts lysosomal membrane stability, triggering ferroptosis. However, the molecular mechanism and concentration-dependent effect of H2O2 regulating lysosomal function remain unclear, necessitating the development of specific probes capable of real-time monitoring of H2O2 dynamics within lysosomes. On the other hand, lipid droplets, as core organelles of lipid metabolism, store polyunsaturated fatty acids that are prone to oxidation, closely related to lipid peroxidation associated with ferroptosis. However, existing fluorescent probes (such as BODIPY or Nile Red) suffer from small Stokes shifts, poor selectivity, and are prone to decreased sensitivity due to aggregation-induced quenching, making it difficult to accurately detect fluctuations in H2O2 within lipid droplets and limiting further research into the mechanisms of ferroptosis.

[0003] Current methods for H2O2 detection, such as electrochemical methods or mass spectrometry, cannot achieve in vivo dynamic imaging. Existing fluorescent probes often suffer from drawbacks such as poor water solubility, significant background interference, or inability to target specific subcellular organelles. For example, in the prior art, CN105001856 A discloses a type of fluorescent probe for monitoring lipid peroxidation processes in different subcellular organelles. This probe involves the reaction of a fluoroboron pyrrole derivative with cinnamaldehyde, using piperidine as a catalyst and a 4Å molecular sieve as a dehydrating agent. The resulting reactant is then quaternized to obtain the fluorescent probe molecule. However, this technique can only detect lipid peroxidation (LPO) and cannot directly detect hydrogen peroxide. Furthermore, this probe utilizes the oxidizing power of ROS to oxidize the probe's double bonds, resulting in a shorter fluorescence wavelength and a lack of specificity, requiring further improvement. CN 106928263 A discloses an open-type fluorescent probe for hydrogen peroxide and its preparation and application. The hydrogen peroxide fluorescent probe compound has the structure of Formula I. However, this technical solution can detect hydrogen peroxide, but it lacks subcellular organelle localization function and cannot distinguish the subcellular origin of hydrogen peroxide. At the same time, it relies on single-wavelength fluorescence enhancement (495 nm) and is easily affected by factors such as concentration and environment.

[0004] While some aryl borate-based probes have been used for H2O2 detection, they lack targeting groups on lysosomes or lipid droplets, making it difficult to accumulate in specific organelles. Furthermore, the acidic environment of lysosomes may cause premature hydrolysis of the borate groups, interfering with the accuracy of H2O2 detection. Traditional lipid droplet dyes (such as Nile Red) are prone to aggregation-induced quenching, while BODIPY-based probes may lack specificity due to polar sensitivity, further limiting their application in complex biological systems.

[0005] In summary, existing technologies have the following shortcomings: First, they cannot target specific cells: traditional probes (such as BODIPY and Nile Red) lack lysosomal / lipodroplet targeting groups, making it impossible to accumulate in target organelles; second, they lack sensitivity: due to the small Stokes shift, aggregation-induced quenching (ACQ), and the easy hydrolysis and failure of borate ester groups in acidic environments, the signal-to-noise ratio is low; third, dynamic monitoring is lacking: electrochemical / mass spectrometry methods require the destruction of cell structures, making it impossible to achieve real-time imaging of live cells. The root cause of these shortcomings is that existing technologies have not integrated subcellular organelle targeting groups and molecular designs that resist environmental interference (such as acid hydrolysis and ACQ), making it difficult to meet the need to reveal the changes in H2O2 during ferroptosis and provide support for the early diagnosis of related diseases. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing hydrogen peroxide fluorescent probes, this invention provides a class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles, their preparation method, and their applications. This invention develops a novel class of fluorescent probes that possess subcellular organelle targeting capabilities, high sensitivity, high specificity, and a high signal-to-noise ratio, thereby revealing the changes in H2O2 during ferroptosis and providing technical support for the early diagnosis of related diseases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles is characterized by having a flavonol as the parent nucleus, with pinacol phenylboronic acid linked at position 3 and a targeting group -OR linked at position 5. The probe has the following general structural formula:

[0009]

[0010] In the general formula, R= PML (Probe for H2O2 monitoring in lysosomal), a hydrogen peroxide ratiometric fluorescent probe targeting lysosomes.

[0011] R= PZD (Probe for H2O2 monitoring in lipid droplets), a hydrogen peroxide ratiometric fluorescent probe targeting lipid droplets.

[0012] It can also replace the flavonol core in the general formula with a coumarin or cyanobenzopyran-type ESIPT fluorophore;

[0013] Replace the enzyme targeting group in the general structural formula with morpholino, triphenylphosphine salt or galactose derivative;

[0014] Replace the lipid droplet targeting group in the general structural formula with a cholesterol group or a fatty acid chain;

[0015] Replace the borate pinacol ester in the general structural formula with arylboronic acid or tellurium / selenium compound.

[0016] The preparation method of the fluorescent probe PML is as follows:

[0017]

[0018] The preparation method of the fluorescent probe PZD is as follows:

[0019] .

[0020] One type of fluorescent probe has been applied to monitor the dynamic changes of hydrogen peroxide in different subcellular organelles. Among them, the fluorescent probe PML is used for the detection of hydrogen peroxide in lysosomal organelles. Its molecular structure contains a morpholine group, which is protonated in the acidic environment of lysosomes (pH 4.5–5.0). Through lysosomal-specific enrichment under the ion trapping effect, it can be targeted to lysosomes to monitor the dynamic changes of hydrogen peroxide in lysosomes during RSL3-induced ferroptosis. It can also be used for localized lysosomal fluorescence imaging.

[0021] Among them, the fluorescent probe PZD is used to detect hydrogen peroxide in lipid droplet organelles. By modifying long-chain hexyl groups, it can be embedded in the hydrophobic core of lipid droplets to target and locate lipid droplets, thereby monitoring the dynamic changes of hydrogen peroxide in cellular lipid droplets during RSL3-induced ferroptosis. It can also be applied to the localization of lipid droplet fluorescence imaging.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention provides a novel class of fluorescent probes, specifically the fluorescent probes PML and PZD, which target lysosomes / lipid droplets. These probes possess novel structures and exhibit sub-organelle targeting capabilities, high sensitivity, high specificity, and a high signal-to-noise ratio. They can reveal the changes in H2O2 during ferroptosis, providing technical support for the early diagnosis of related diseases. The fluorescent probes PML and PZD demonstrate good responsiveness to hydrogen peroxide in both solution and cells. Cytotoxicity tests show that the fluorescent probes PML / PZD have good cell compatibility. Fluorescence imaging experiments show that the fluorescent probes PML / PZD can be effectively localized to lysosomes / lipid droplets, making them suitable for lysosome / lipid droplet fluorescence imaging and in-situ detection of hydrogen peroxide. They can be used to detect in-situ changes in hydrogen peroxide within lysosomes and lipid droplets induced by lipopolysaccharide, and can also be used to monitor changes in cell viscosity during RSL3-induced ferroptosis.

[0024] 2. Based on the ESIPT principle, this invention provides methods for synthesizing highly specific and sensitive ratiometric fluorescent probes (PML / PZD) that can target multiple organelles. These methods are simple, use mild synthesis conditions, and exhibit good recognition of hydrogen peroxide, especially in subcellular organelles. They also demonstrate fast response, high sensitivity, and reliable detection results. This approach overcomes the drawbacks of existing probe synthesis methods, such as complexity and stringent synthesis conditions, while also possessing advantages like low toxicity and good biocompatibility. Furthermore, it provides subcellular organelle localization capabilities, enabling their application in monitoring hydrogen peroxide within the intracellular microenvironment.

[0025] 3. The PML and PZD prepared in this invention can achieve specific labeling of lysosomes and lipid droplets, respectively. Among them, the probe PML has the best effect in localizing lysosomes. Applying PML to lysosome-related physiological or pathological models can provide support for studying the role mechanism of hydrogen peroxide in the process of ferroptosis. PML / PZD also has great application potential in exploring the bioimaging of organelles affected by ferroptosis.

[0026] 4. The fluorescent probe PML / PZD provided by this invention can directly monitor hydrogen peroxide, a key upstream signaling molecule of LPO; and utilizes the hydrogen peroxide-specific recognition group to specifically recognize hydrogen peroxide; overcoming the shortcomings of a type of fluorescent probe disclosed in CN105001856 A for monitoring lipid peroxidation processes in different subcellular organelles, which only supports the detection of lipid peroxidation (LPO) and uses the oxidizing ability of ROS to oxidize the probe double bonds, thus shortening the fluorescence wavelength and lacking specificity.

[0027] 5. The fluorescent probe PML / PZD provided by this invention has made a breakthrough in subcellular organelle targeting capability: through the design of specific groups, it achieves precise targeting of subcellular organelles such as lysosomes and lipid droplets; it adopts ratiometric detection (ESIPT dual-channel ratio change) to reduce background interference and make quantitative detection more reliable; it overcomes the shortcomings of CN 106928263 A, which can detect hydrogen peroxide but lacks subcellular organelle localization function, cannot distinguish the subcellular source of hydrogen peroxide, and relies on single-wavelength fluorescence enhancement (495 nm), which is easily affected by concentration, environmental and other factors.

[0028] 6. This invention achieves high-precision dynamic monitoring of hydrogen peroxide (H2O2) in lysosomes and lipid droplets. In particular, it successfully revealed the real-time changes of H2O2 in subcellular organelles in a ferroptosis pathological model. The detection limits of the probes PML and PZD reached 85.6 nM and 65.2 nM, respectively, with excellent linear response (R²>0.98) and high selectivity for 14 interfering substances.

[0029] The above is an overview of the invention's technical solution. The invention will be further described below in conjunction with specific embodiments and accompanying drawings. Attached Figure Description

[0030] Figure 1 This is a synthetic route diagram of the fluorescent probe PML used in this invention.

[0031] Figure 2 This is a synthetic route diagram of the fluorescent probe PZD used in this invention.

[0032] Figure 3 The emission spectrum of the fluorescence intensity of the fluorescent probe PML as a function of hydrogen peroxide concentration and the linear relationship between the fluorescence intensity ratio (1535nm / 1440nm) and the hydrogen peroxide concentration are shown in the embodiments of the present invention.

[0033] Figure 4 The emission spectrum of the fluorescent probe PZD as a function of hydrogen peroxide concentration and the linear relationship between the fluorescence intensity ratio (1535nm / 1420nm) and the hydrogen peroxide concentration are shown in the embodiments of the present invention.

[0034] Figure 5 This is a selectivity diagram of the fluorescent probe PML for hydrogen peroxide in an embodiment of the present invention;

[0035] Figure 6 This is a selectivity diagram of the fluorescent probe PZD for hydrogen peroxide in an embodiment of the present invention;

[0036] Figure 7 This is a graph showing the cytotoxicity test results of the fluorescent probe PML in this invention.

[0037] Figure 8 This is a graph showing the cytotoxicity test results of the fluorescent probe PZD in this invention.

[0038] Figure 9 This is a diagram showing the co-localization effect of the fluorescent probe PML and the lysosome-targeting commercial dye Lyso-Tracker Red in the implementation of this invention.

[0039] Figure 10 This is a diagram showing the co-localization effect of the fluorescent probe PZD and the lipid droplet-targeting commercial dye Nile Red in the implementation of this invention;

[0040] Figure 11 This is a cellular imaging diagram of exogenous H2O2 produced by the fluorescent probe PML in an embodiment of the present invention.

[0041] Figure 12 This is a cellular imaging diagram of exogenous H2O2 produced by the fluorescent probe PZD in an embodiment of the present invention.

[0042] Figure 13 This is a cellular imaging image of endogenous H2O2 produced by the fluorescent probe PML in an embodiment of the present invention.

[0043] Figure 14 This is a cellular imaging image of endogenous H2O2 produced by the fluorescent probe PZD in an embodiment of the present invention.

[0044] Figure 15 Characterization diagram of the RSL3-induced ferroptosis model in HepG2 cells successfully established in this embodiment of the invention;

[0045] Figure 16 This invention provides fluorescence imaging of H2O2 during ferroptosis using the fluorescent probe PML.

[0046] Figure 17 This invention provides fluorescence imaging of H2O2 during ferroptosis using the fluorescent probe PZD.

[0047] Figure 18 This is a schematic diagram illustrating the detection principle of the probe PML in an embodiment of the present invention. Detailed Implementation

[0048] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. In the following embodiments, M, representing concentration, is an abbreviation for mol / L.

[0049] Basic Implementation

[0050] A class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles is characterized by having a flavonol as the parent nucleus, with pinacol phenylboronic acid linked at position 3 and a targeting group -OR linked at position 5. The probe has the following general structural formula:

[0051]

[0052] In the general formula, R= The target lysosome is a hydrogen peroxide ratiometric fluorescent probe PML (Probe for H2O2 monitoring in lysosomal).

[0053] R= The probe targeting lipid droplets is PZD (Probe for H2O2 monitoring in lipid droplet), a ratiometric fluorescent probe for hydrogen peroxide.

[0054] The preparation method of the fluorescent probe PML is as follows:

[0055] .

[0056] The preparation method of the fluorescent probe PZD is as follows:

[0057] .

[0058] One type of fluorescent probe has been applied to monitor the dynamic changes of hydrogen peroxide in different subcellular organelles. Among them, the fluorescent probe PML is used for the detection of hydrogen peroxide in lysosomal organelles. Its molecular structure contains a morpholine group, which is protonated in the acidic environment of lysosomes (pH 4.5–5.0). Through lysosomal-specific enrichment under the ion trapping effect, it can be targeted to lysosomes to monitor the dynamic changes of hydrogen peroxide in lysosomes during RSL3-induced ferroptosis. It can also be used for localized lysosomal fluorescence imaging.

[0059] Among them, the fluorescent probe PZD is used to detect hydrogen peroxide in lipid droplet organelles. By modifying long-chain hexyl groups, it can be embedded in the hydrophobic core of lipid droplets to target and locate lipid droplets, thereby monitoring the dynamic changes of hydrogen peroxide in cellular lipid droplets during RSL3-induced ferroptosis. It can also be applied to the localization of lipid droplet fluorescence imaging.

[0060] The lysosome-targeting probe PML provided in this embodiment contains a morpholine group in its molecular structure. This group is protonated in the acidic environment of the lysosome (pH 4.5–5.0) and achieves lysosome-specific enrichment through the ion trapping effect.

[0061] The lipid droplet targeting probe PZD provided in this embodiment is modified with long-chain hexyl groups and utilizes hydrophobicity to embed into the hydrophobic core of lipid droplets to achieve targeted positioning of lipid droplets.

[0062] The basic principle of the inspection in this invention is the ESIPT fluorescence response mechanism, which achieves subcellular organelle targeting and high-precision H2O2 detection through the synergistic action of the following components:

[0063] (1) Flavonol core: As a fluorescent signal source, its enol form (440 nm blue light) and keto form (535 nm green light) tautomerism form constitutes the basis for ratio detection;

[0064] (2) Targeting group:

[0065] Morpholine group (PML): After protonation, it becomes positively charged and specifically binds to the acidic membrane of lysosomes (pH≈4.5).

[0066] Long alkyl chain (PZD): Hydrophobic driving probe embedding into the neutral lipid core of lipid droplets;

[0067] (3) Boron ester pinacol ester: as an H2O2-responsive switch, it is specifically broken under the action of H2O2, which relieves the steric hindrance to ESIPT and triggers the change in fluorescence ratio;

[0068] (4) Anti-interference design: Pinarol ester protects borate ester bond to resist acidic hydrolysis of lysosome; large conjugated flavonol skeleton provides >100nm Stokes shift to avoid aggregation quenching.

[0069] See appendix Figure 18 The detection principle is as follows: the probe core uses a flavonol core and operates based on the excited-state intramolecular proton transfer (ESIPT) mechanism. When not responding to H₂O₂, benzyloxyphenylboronic acid pinacol ester hinders ESIPT, and the probe only emits enol fluorescence. After responding to H₂O₂: hydrogen peroxide hydrolyzes the borate ester group, restoring the ESIPT effect, and the probe switches to emitting keto fluorescence (535 nm yellow-green light). Ratio detection is achieved through the fluorescence intensity ratio.

[0070] The following is in conjunction with the appendix Figure 1-18 The following detailed description includes several specific embodiments. In the examples below, M, representing concentration, is an abbreviation for mol / L.

[0071] Example 1

[0072] Based on the basic embodiment, this embodiment provides a class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles. Specifically, it provides a fluorescent probe PML that specifically detects hydrogen peroxide targeting lysosomes, as well as a method for synthesizing PZD targeting lipid droplets and a probe thereof.

[0073] The synthetic route of the fluorescent probe PML is as follows: Figure 1 As shown, the synthesis steps are as follows:

[0074] (1) Weigh p-hydroxybenzaldehyde (concentration 1.0 mmol / L) and 1,6-dibromohexane (concentration 2.5 mmol / L) and place them in a 100 mL round-bottom flask. Add acetone as solvent, then add anhydrous potassium carbonate (molar ratio of 1:1, concentration 1 mmol / L). Place the reaction system in an oil bath at 60 °C and stir overnight. Monitor the reaction progress by TLC. After the reaction is complete, cool the reaction solution to room temperature, extract with ethyl acetate, wash with water and saturated brine, and then rotary evaporate to obtain a yellow liquid. Then, obtain a white solid compound 1 by silica gel column chromatography (PE:EA=50:1).

[0075] (2) Weigh out compound 1 and morpholine (molar ratio 1:1.5, compound 1 concentration 1 mmol / L) and add to 9 mL of acetonitrile to dissolve, then add anhydrous potassium carbonate. Place the reaction system in an oil bath at 70℃ and stir for 24 hours. Monitor the reaction progress by TLC. After the reaction is complete, cool the reaction solution to room temperature, extract with ethyl acetate, wash with saturated brine, and then rotary evaporate to obtain yellow liquid compound 2; the concentrations of anhydrous potassium carbonate and acetonitrile are 1 mmol / L and 1.3 mmol / L, respectively;

[0076] (3) Weigh 2-hydroxyacetophenone and compound 2 (molar ratio 1:1) and dissolve them in 10 mL of methanol. Then add NaOH and stir the reaction in an oil bath at 60℃ for 12 h. Then add 3 mL of 40% NaOH solution (dropping rate about 1.0 mL / min) and 6 mL of 6% H2O2 solution (dropping rate about 1.0 mL / min) and react overnight at 4℃±1℃. The reaction progress is monitored by TLC. After the reaction is completed, pour the reaction solution into ice water, add dilute hydrochloric acid solution until pH=7, extract with ethyl acetate, wash with saturated brine, remove the solvent with a rotary evaporator to obtain a yellow solid mixture, recrystallize with anhydrous ethanol to obtain yellow solid compound ML;

[0077] (4) Weigh compound ML and pinacol 4-bromomethylphenylboronic acid (molar ratio 3:4) into a 50 mL round-bottom flask. Add 14 mL of N,N-dimethylformamide (DMF) and sonicate to dissolve. Then add anhydrous sodium carbonate and place the reaction system in an oil bath at 70℃±3℃ and stir for 24 hours. Monitor the reaction progress by TLC. After the reaction is complete, cool the reaction solution to room temperature, extract with ethyl acetate, wash with saturated brine, remove the solvent with a rotary evaporator to obtain a light yellow liquid, and purify to obtain a light yellow solid compound PML (500 mg, 46%). 1H NMR (500 MHz, Chloroform-d) δ 8.27(dd, J = 8.0, 1.7 Hz, 1H), 8.03 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 8.0 Hz,2H), 7.68 – 7.64 (m, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.39 (dd, J = 18.6, 8.0Hz, 3H), 6.94 (d, J = 9.0 Hz, 2H), 5.16 (s, 2H), 4.03 (t, J = 6.5 Hz, 2H), 3.72 (t, J = 4.7 Hz, 4H), 2.45 (s, 4H), 2.37 – 2.33 (m, 2H), 1.86 – 1.80 (m,2H), 1.58 – 1.48 (m, 4H), 1.41 (d, J = 8.8 Hz, 2H), 1.34 (s, 12H). 13 C NMR(126 MHz, Chloroform-d) δ 174.99, 161.05, 156.38, 155.17, 139.89, 139.22,134.72, 133.25, 130.58, 127.86, 125.76, 124.61, For C 38 H 46 BNO 7, [M+H + ] 640.34, found 640.3385.

[0078] The synthetic route of the fluorescent probe PZD is as follows: Figure 2 As shown, the synthesis steps are as follows:

[0079] (1) After placing p-hydroxybenzaldehyde and 1-bromohexane (molar ratio 1:1) into a 50 mL round-bottom flask, 10 mL of N,N-dimethylformamide (DMF) was added as a solvent, followed by anhydrous potassium carbonate. The reaction system was placed in an oil bath at 70℃±3℃ and stirred overnight. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature, extracted with ethyl acetate, washed with water and brine, and then rotary evaporated to obtain a pale yellow liquid. Then, the solution was purified by silica gel column chromatography (PE:EA=50:1) to obtain a white solid compound 3.

[0080] (2) 2-hydroxyacetophenone and compound 3 (molar ratio 1.1:1) were added to a 50 mL round-bottom flask, dissolved in 7.5 mL of methanol, and NaOH was added. The mixture was stirred in an oil bath at 60 °C for 12 h. Then, 2 mL of 40% NaOH solution (dropping rate approximately 1.0 mL / min) and 5 mL of 6% H2O2 solution (dropping rate approximately 1.0 mL / min) were added dropwise, and the mixture was reacted overnight at 4 °C ± 1 °C. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into ice water, and dilute hydrochloric acid solution was added dropwise until the pH reached 7. The mixture was then extracted with ethyl acetate, washed with saturated brine, and the solvent was removed by rotary evaporation to obtain a yellow solid mixture. The mixture was recrystallized from methanol to obtain a yellow solid compound ZD.

[0081] (3) 339 mg of compound ZD and 300 mg of pinacol 4-bromomethylphenylboronic acid (molar ratio 1:1.1) were added to a 50 mL round-bottom flask, dissolved in 7 mL of DMF, and anhydrous sodium carbonate (2.8 mmol / L) was added. The reaction system was placed in an oil bath at 70℃±3℃ and stirred for 24 hours. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature, extracted with ethyl acetate, washed with saturated brine, and the solvent was removed by rotary evaporation to obtain a light yellow liquid. After purification, a light yellow solid compound PML (310 mg, 55.9%) was obtained. 1H NMR (500 MHz, Chloroform-d) δ 8.28 (dd,J = 8.0, 1.7 Hz, 1H), 8.03 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 8.1 Hz, 2H), 7.66 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.39 (dd, J =18.2, 8.0 Hz, 3H), 6.95 (d, J = 9.0 Hz, 2H), 5.16 (s, 2H), 4.03 (t, J = 6.6Hz, 2H), 1.87 – 1.79 (m, 2H), 1.53 – 1.45 (m, 2H), 1.39 – 1.35 (m, 4H), 1.34 (s, 12H), 0.96 – 0.90 (m, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 175.04,161.14, 156.52, 155.19, 139.86, 139.20, 134.71, 133.26, 130.58, 127.89,125.77, 124.62, 124.18, 122.99, 117.93, 114.28, 83.80, 68.20, 31.60, 29.15,25.72, 24.88, 22.63, 14.07. ESI-TOF MS (m / z) calcd. for C 34 H 39 BO6, [M+H + ]555.49found 555.2543.

[0082] Example 2

[0083] This embodiment is based on Example 1, but with adjustments to the proportions of some components to obtain a fluorescent probe with the same general formula structure and performance.

[0084] The preparation method of the fluorescent probe PML specifically includes the following steps:

[0085] (1) p-hydroxybenzaldehyde and 1,6-dibromohexane were dissolved in acetone, anhydrous potassium carbonate was added, and the mixture was stirred at 60°C for 12 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with pure water and saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was further purified by silica gel column chromatography to obtain a white solid compound 1. The molar ratio of p-hydroxybenzaldehyde to 1,6-dibromohexane was 1:3, the molar ratio of anhydrous potassium carbonate to acetone was 0.8:1, and the concentration of anhydrous potassium carbonate was 0.8 mmol / L.

[0086] (2) White solid compound 1, morpholine and anhydrous potassium carbonate were added to acetonitrile and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain yellow liquid compound 2. The molar ratio of white solid compound 1 to morpholine was 1:1.8, the molar ratio of anhydrous potassium carbonate to acetonitrile was 1:1.7, and the concentration of anhydrous potassium carbonate was 1 mmol / L.

[0087] (3) 2-hydroxyacetophenone, compound 2 and sodium hydroxide were added to methanol and stirred at 60°C for 12 hours under nitrogen protection. Then, 40% NaOH solution and 6% H2O2 solution were slowly added dropwise and the reaction was continued at 4°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to 7 with dilute hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain crude product. The crude product was recrystallized from anhydrous ethanol to obtain yellow solid compound ML. The molar ratio of 2-hydroxyacetophenone to compound 2 was 1.2:1, the molar ratio of sodium hydroxide to methanol was 1:2.5, and the concentration of sodium hydroxide was 1 mmol / L.

[0088] (4) Compound ML, 4-bromomethylphenylboronic acid pinacol ester and anhydrous sodium carbonate were added to DMF and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The product was purified by silica gel column chromatography to obtain a light yellow solid compound PML. The molar ratio of ML to 4-bromomethylphenylboronic acid pinacol ester was 0.6:1, the molar ratio of anhydrous potassium carbonate to DMF was 0.3:1, and the concentration of anhydrous potassium carbonate was 0.3 mmol / L.

[0089] The preparation method of the fluorescent probe PZD specifically includes the following steps:

[0090] (1) p-hydroxybenzaldehyde, 1-bromohexane and anhydrous potassium carbonate were added to DMF and stirred at 70°C for 12 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with pure water and saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a white solid compound 3. The molar ratio of p-hydroxybenzaldehyde to 1-bromohexane was 1:1, the molar ratio of anhydrous potassium carbonate to DMF was 0.5:1, and the concentration of anhydrous potassium carbonate was 0.5 mmol / L.

[0091] (2) 2-hydroxyacetophenone, compound 3 and sodium hydroxide were added to methanol and stirred at 60°C for 12 hours under nitrogen protection. Then, 40% NaOH solution and 6% H2O2 solution were slowly added dropwise and the reaction was continued at 4°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to 7 with dilute hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was recrystallized from methanol to obtain the yellow solid compound ZD. The molar ratio of 2-hydroxyacetophenone to compound 3 was 0.9:1 and the molar ratio of sodium hydroxide to methanol (mmol / L) was 0.4:1.

[0092] (3) Compound ZD, 4-bromomethylphenylboronic acid pinacol ester and anhydrous sodium carbonate were added to DMF and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a light yellow solid compound PZD. The molar ratio of compound ZD to 4-bromomethylphenylboronic acid pinacol ester was 1.0:1.2, and the molar ratio (mmol / L) of anhydrous sodium carbonate to DMF was 0.5:1.

[0093] Example 3

[0094] This embodiment is based on Embodiment 1 and Embodiment 2, with adjustments made to the proportions of some components to obtain a fluorescent probe with the same general formula structure and performance. The preparation method of the fluorescent probe PML specifically includes the following steps:

[0095] (1) p-hydroxybenzaldehyde and 1,6-dibromohexane were dissolved in acetone, anhydrous potassium carbonate was added, and the mixture was stirred at 60°C for 12 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with pure water and saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was further purified by silica gel column chromatography to obtain a white solid compound 1. The molar ratio of p-hydroxybenzaldehyde to 1,6-dibromohexane was 1.0:2.3, and the molar ratio (mmol / L) of anhydrous potassium carbonate to acetone was 1.2:1.

[0096] (2) White solid compound 1, morpholine and anhydrous potassium carbonate were added to acetonitrile and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain yellow liquid compound 2. The molar ratio of white solid compound 1 to morpholine was 1:1.3, and the molar ratio (mmol / L) of anhydrous potassium carbonate to acetonitrile was 1:1.3.

[0097] (3) 2-hydroxyacetophenone, compound 2 and sodium hydroxide were added to methanol and stirred at 60°C for 12 hours under nitrogen protection. Then, 40% NaOH solution and 6% H2O2 solution were slowly added dropwise and the reaction was continued at 4°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to 7 with dilute hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain crude product. The product was recrystallized from anhydrous ethanol to obtain yellow solid compound ML. The molar ratio of 2-hydroxyacetophenone to compound 2 was 0.8:1, and the molar ratio of sodium hydroxide to methanol (mmol / L) was 1:1.8.

[0098] (4) Compound ML, 4-bromomethylphenylboronic acid pinacol ester and anhydrous sodium carbonate were added to DMF and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The product was purified by silica gel column chromatography to obtain a light yellow solid compound PML. The molar ratio of ML to 4-bromomethylphenylboronic acid pinacol ester was 0.9:1, and the corresponding concentrations were 0.9 mmol / L and 1 mmol / L. The concentration ratio of anhydrous potassium carbonate to DMF was 0.8:1, and the corresponding concentrations were 0.8 mmol / L and 1 mmol / L.

[0099] The changes in the proportions of compounds used in Examples 1, 2, and 3 within the recorded range resulted in products with identical molecular structures and properties.

[0100] Example 4

[0101] This embodiment is based on Example 1, specifically verifying the relationship between the fluorescence intensity of fluorescent probes PML and PZD and the hydrogen peroxide concentration.

[0102] The fluorescent molecular probe PML prepared in Example 1 was prepared into a stock solution with a concentration of 1 mmol / L in acetonitrile solution. 20 μL of this stock solution was dissolved in 980 μL each of acetonitrile and PBS buffer solution (0.01 mol / L, pH = 7.4). Then, 0 µL, 15 µL, 30 µL, 45 µL, 60 µL, 75 µL, 90 µL, 105 µL, 120 µL, and 150 µL of hydrogen peroxide stock solution (1 mmol / L) were added to the test system, resulting in a probe concentration of 10 μM and hydrogen peroxide concentrations of 0 µM, 5 µM, 10 µM, 15 µM, 20 µM, 25 µM, 30 µM, 40 µM, and 50 µM, respectively. After incubation at room temperature for 20 min to allow sufficient response, the fluorescence spectra were analyzed using a fluorescence spectrophotometer. The obtained fluorescence spectra are shown below. Figure 3 (A). Through Figure 3 (A) It can be seen that as the concentration of hydrogen peroxide increases, the fluorescence intensity (I) at 535 nm decreases. 535 nm The fluorescence intensity gradually increases, reaching a peak at 440 nm (If). 440nm () Gradually weakens. From Figure 3 (C) shows that a good linear relationship is observed in the 0-30 μM range (R0). 2 =0.99415), and the calculated detection limit is 85.6 nM.

[0103] Similarly, such as Figure 4 As shown, with increasing hydrogen peroxide concentration added to the probe PZD, the fluorescence intensity of the probe gradually decreases at 420 nm, while the fluorescence intensity gradually increases at 535 nm. Furthermore, the ratio of its fluorescence intensity at 535 nm to that at 420 nm exhibits a good linear relationship within the hydrogen peroxide concentration range of 0 to 30 µM, with R0... 2 The value is 0.9817, and the calculated detection limit is 65.2 nM.

[0104] Taking the PML probe as an example, its detection principle utilizes the excited-state intramolecular proton transfer (ESIPT) mechanism. The pinacol ester of benzyloxyphenylboronic acid inhibits the ESIPT activity of the probe, causing it to emit only enol form emission. Upon the addition of hydrogen peroxide, the ESIPT activity is restored, leading to keto emission and ultimately achieving ratiometric detection of hydrogen peroxide. The detection mechanism is detailed in the attached figure. Figure 18 As shown.

[0105] Example 5

[0106] This embodiment is based on Example 1, specifically verifying the selectivity of fluorescent molecular probes PML and PZD in response to hydrogen peroxide.

[0107] The fluorescent molecular probe PML prepared in Example 1 was prepared into a stock solution with a concentration of 1 mmol / L in acetonitrile solution. 20 μL of this stock solution was dissolved in 980 μL each of acetonitrile and PBS buffer solution (0.01 M, pH = 7.4) in the test system. 20 μL of a 1 mmol / L hydrogen peroxide stock solution and 20 μL of a 1000 μM cation exchange solution (Na₂O₃) were also added. + K + Ca 2+ Mg 2+ Cu 2+ Fe 2+ Zn 2+ Cs 2+ Zr 2+ ), 20 μL of reactive oxygen species at a concentration of 1000 μM ( 1 O2, ONOO - ,ClO - NO 2- The mother liquor of Na+ and OH- ensures that the final detection system contains a probe concentration of 10 μM, a hydrogen peroxide concentration of 10 μM, and cations (Na+). + K + Ca 2+ Mg 2+ Cu 2+ Fe 2+ Zn 2+ Cs 2+ Zr 2+ ) and reactive oxygen species ( 1 O2, ONOO - ,ClO - NO2 - The concentrations of , and ·OH were 10 μM. After incubation at room temperature for 20 min to allow sufficient response, analysis was performed using a fluorescence spectrophotometer. The obtained fluorescence spectra are shown below. Figure 5 As shown. (Through) Figure 5 It can be seen that the probe PML only has a significant response to hydrogen peroxide, while other ions show no change.

[0108] Similarly, such as Figure 6 As shown, when 14 other interfering substances were added to the probe PZD solution, all of them exhibited blue fluorescence at 535 nm, while the addition of hydrogen peroxide changed the fluorescence from blue to yellow-green. This indicates that the probe PZD is effective against ClO₂. - There was no response from any of the 14 interfering substances, indicating that it has good specificity for hydrogen peroxide.

[0109] Example 6

[0110] This embodiment is based on Example 1, specifically verifying the cytotoxicity study of the fluorescent molecular probes PML and PZD.

[0111] After seeding healthy HepG2 cells at 5000 cells / well in 96-well plates and incubating overnight, 100 µL of culture medium containing probe concentrations of 5 µM, 10 µM, 15 µM, 20 µM, 25 µM, and 30 µM were added to each well, with 3-4 replicates per group (including a cell-free blank control). After incubation in a CO2 incubator for 24 hours, the probe-containing culture medium was aspirated, and the cells were washed three times with PBS buffer. 100 µL of 10% CCK8 dilution was added to each well, and the cells were incubated in a CO2 incubator in the dark for 2-3 hours. The absorbance of each well was then measured at 450 nm using a microplate reader, and the data were analyzed. Figure 7 As shown in the CCK8 experiment, when the concentration of PML was increased to 30 µM, the cell viability of HepG2 cells remained above 80%, indicating that PML has no significant cytotoxicity to HepG2 cells at concentrations below 30 µM and has good biocompatibility.

[0112] Similarly, such as Figure 8 As shown, when the concentration of PZD was increased to 200 µM, the cell viability of HepG2 cells remained above 80%, indicating that PML has no significant cytotoxicity to HepG2 cells at concentrations below 200 µM and has excellent biocompatibility.

[0113] Example 7

[0114] This embodiment is based on Embodiment 1, and performs cell localization tests on fluorescent probes PML and PZD that target lysosomes / lipodroplets.

[0115] The fluorescent probe PML prepared in Example 1 (10 μM, with 20 μL of hydrogen peroxide stock solution added simultaneously) was co-incubated with the commercial lysosomal localization reagent Lyso-Tracker Red for 1 hour, and real-time cell imaging was achieved using a smart cell imaging system. Figure 9 As shown, HepG2 cells exhibited bright green fluorescence in the green channel, and its green fluorescence showed good overlap with the red fluorescence of commercial lysosomal probes, with a Pearson correlation coefficient as high as 0.93, demonstrating that PML has excellent lysosomal targeting ability.

[0116] Similarly, we investigated the subcellular organelle localization ability of the probe PZD. We co-incubated PZD with Nile Red, a commercially available lipid droplet-targeting red dye, to explore the lipid droplet targeting effect of PZD. Figure 10 As shown, the red fluorescence in the lipid droplet red fluorescence detection kit has good overlap with the green fluorescence of the fluorescent group ZD, and the localization correlation coefficient between the red fluorescence and the green fluorescence is 0.91, indicating that the probe PZD can be used as a lipid droplet targeting probe.

[0117] Example 8

[0118] This embodiment is based on Example 1. Well-cultured HepG2 cells were further divided into four groups and seeded into 12-well plates at appropriate densities. After incubation for 24 hours, reactive oxygen species in the HepG2 cells were depleted. Then, the cells were incubated with 0, 10, 20, and 40 μM hydrogen peroxide / PBS solutions for 30 minutes each, followed by incubation with a 10 μM probe solution for 1 hour. After washing three times with PBS buffer, fluorescence signals were collected using a smart cell imaging system and analyzed using ImageJ software.

[0119] like Figure 11 As shown, when HepG2 cells were treated with only 10 µM probe PML, only fluorescence in the blue channel was observed. However, when HepG2 cells were first incubated with 10 µM, 20 µM, and 40 µM H2O2 for 30 minutes, followed by incubation with 10 µM probe PML, the fluorescence in the blue channel gradually weakened, while fluorescence emission was observed in the green channel, and the fluorescence intensity gradually increased. Moreover, when the H2O2 concentration reached 40 µM, no obvious blue fluorescence signal was detected in the blue channel, while the fluorescence intensity of the green channel was significantly enhanced compared to the 20 µM response. These results indicate that probe PML can be used for imaging changes in hydrogen peroxide concentration in cells.

[0120] Similarly, such as Figure 12 As shown, HepG2 cells incubated solely with probe PZD exhibited only blue fluorescence emission, while HepG2 cells pretreated with 10 µM, 20 µM, and 40 µM hydrogen peroxide showed green fluorescence upon incubation with probe PZD. Furthermore, the blue fluorescence gradually weakened while the green fluorescence gradually strengthened with increasing hydrogen peroxide concentration. When the hydrogen peroxide concentration reached 40 µM, only bright green fluorescence was observed. This indicates that PZD can visually detect the concentration of intracellular hydrogen peroxide.

[0121] Example 9

[0122] This embodiment, based on Embodiment 1, further investigates the ability of fluorescent probes PML and PZD targeting lysosomes / lipodroplets to detect hydrogen peroxide in live cells.

[0123] Healthy HepG2 cells were divided into three groups and seeded at appropriate densities into 12-well plates. After incubation for 24 hours, reactive oxygen species in the HepG2 cells were depleted. One group was incubated with 0 μg / mL lipopolysaccharide for 2 hours; another group was incubated with 10 μg / mL lipopolysaccharide for 2 hours; and the third group was incubated with 10 μg / mL lipopolysaccharide + 1 mmol / L NAC for 2 hours. After washing three times with PBS buffer, fluorescence signals were collected using a smart cell imaging system and analyzed using ImageJ software.

[0124] Lipopolysaccharide (LPS) can stimulate cells to produce a large number of inflammatory factors, inducing oxidative stress in cells and thus generating various reactive oxygen species (ROS), including hydrogen peroxide. Figure 13 As shown, when HepG2 cells were first incubated with 10 µg / mL LPS for 2 hours, and then incubated with the probe PML, the fluorescence in the blue channel was weakened compared to those incubated with PML alone, while a bright green fluorescence was observed in the green channel. Furthermore, when HepG2 cells were co-incubated with the reactive oxygen species scavenger N-acetyl-L-cysteine ​​(NAC, 1 mmol / L) and LPS for 2 hours, and then incubated with the probe PML, only blue fluorescence in the blue channel was observed. Therefore, these results indicate that the fluorescence signal of the probe PML is triggered by hydrogen peroxide produced by HepG2 cells inducing by lipopolysaccharide, and that the probe PML has the ability to detect endogenous hydrogen peroxide.

[0125] Similarly, such as Figure 14 As shown, in HepG2 cells incubated only with probe PZD, only blue fluorescence in the blue fluorescence channel was observed. However, when cells were first incubated with 10 µg / mL LPS and then with a certain concentration of PZD, the blue fluorescence in the blue channel was weakened, while bright green fluorescence was observed in the green channel. Furthermore, when cells were co-incubated with a certain concentration of NAC and LPS before being incubated with PZD, the green fluorescence in the green channel was significantly weakened, while the fluorescence in the blue channel remained unchanged. In conclusion, probe PZD can be used for imaging detection of endogenous hydrogen peroxide in cells.

[0126] Example 10

[0127] This embodiment, based on Example 1, investigates the ability of fluorescent probes PML and PZD, which target lysosomes / lipid droplets, to detect hydrogen peroxide in a ferroptosis cell model.

[0128] Before the experiment, HepG2 cells were induced with the ferroptosis inducer RSL3 at a concentration of 3 µM to establish a ferroptosis model. The expression of related proteins GPX4 and Nrf2 was detected by Western blotting (WB), and the levels of ferrous ions, MDA, and other related molecules were detected using an MDA assay kit and a ferrous colorimetric assay kit to determine whether the ferroptosis tumor cell model was successfully induced. Figure 13 As shown, we successfully established the RSL3-induced ferroptosis model.

[0129] Cells were then divided into three groups. The first group was incubated with 10 µM PML or PZD probe solution for 1 hour. The second group was incubated with 3 µM RSL3 solution for 3 hours, followed by incubation with 10 µM PML or PZD probe solution for 1 hour. The third group was treated with 3 µM RSL3 + 15 µM Fer-1 for 3 hours, followed by incubation with 10 µM PML or PZD probe solution for 1 hour. Cells were then washed three times with PBS buffer, and fluorescence signals were collected under a fluorescence microscope for analysis using ImageJ software.

[0130] like Figure 16 As shown, after incubating cells with 3 µM RSL3 and 15 µM Fer-1 for 4 hours, followed by incubation with the probe PML, almost no fluorescence was observed in the green channel, while fluorescence was observed in the blue channel. This indicates that RSL3-induced ferroptosis can lead to a large amount of endogenous hydrogen peroxide production in cells. In conclusion, the probe PML can be used to detect hydrogen peroxide during ferroptosis.

[0131] Similarly, such as Figure 17 As shown, when cells were treated with probe PZD alone, only blue fluorescence was observed. However, in the RSL3-induced ferroptosis model, after incubating cells with probe PZD, both blue and green fluorescence were observed. Furthermore, the green fluorescence intensity in the 6-hour treatment group with the ferroptosis agonist RSL3 was significantly stronger than that in the 3-hour group. Subsequently, when cells were co-incubated with the ferroptosis inhibitor Fer-1 and RSL3, followed by incubation with probe PZD, blue fluorescence was observed, and no green fluorescence was detected, which was consistent with our expectations.

[0132] The above results indicate that probes PML and PZD can be used to detect hydrogen peroxide during ferroptosis and are expected to become effective tools for assessing the ferroptosis process.

[0133] The probes provided in the above embodiments of the present invention have novel structures, simple synthesis methods, and good recognition effects on hydrogen peroxide, especially hydrogen peroxide in subcellular organelles. They have fast response speed, high sensitivity, reliable test results, and good biocompatibility. They also have the function of subcellular organelle localization, enabling them to be used for monitoring hydrogen peroxide in the intracellular microenvironment. PML and PZD can achieve specific labeling of lysosomes and lipid droplets, respectively. Among them, the probe molecule PML has the best localization effect on lysosomes. This is of great significance for applying PML to actual lysosome-related pathological models to study the role mechanism of hydrogen peroxide in its pathology, especially in the process of ferroptosis.

[0134] In other embodiments, the following alternative solutions can also be used, and the probes obtained all have the technical effects described in this invention and are all within the protection scope of this invention:

[0135] Fluorescent core adjustment: The flavonol core is replaced with a coumarin or cyanobenzopyran-based ESIPT fluorophore to adjust the emission wavelength;

[0136] Targeting group replacement: Replace the lysosomal targeting group from morpholino with triphenylphosphine salt (targeting mitochondria) or galactose derivative (targeting Golgi apparatus).

[0137] Replace the lipid droplet targeting group with a cholesterol group or a fatty acid chain to enhance hydrophobicity.

[0138] Response unit modification: Replacing borate pinacol ester with arylboronic acid (direct response) or tellurium / selenium compound to improve response speed, but increasing toxicity risk.

[0139] It should be noted that, within the range of material ratios and process parameters described in this invention, specific values ​​can be selected independently, and the resulting probes will all achieve the technical effects described in this invention. Therefore, this invention will not list them one by one.

[0140] While specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of the invention. Various modifications or variations that can be made by those skilled in the art based on the disclosed content of the invention without creative effort are still within the protection scope of the present invention.

Claims

1. A class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles, characterized in that, Specifically, it is a hydrogen peroxide ratiometric fluorescent probe PML, which has the following general structural formula: 。 2. A method for preparing a fluorescent probe according to claim 1 for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles, characterized in that, Its preparation method is as follows: ； Specifically, the steps include the following: (1) Dissolve p-hydroxybenzaldehyde and 1,6-dibromohexane in acetone, add anhydrous potassium carbonate, and stir the reaction at 60°C for 12 hours under nitrogen protection. After the reaction is completed, cool to room temperature, extract with ethyl acetate, combine the organic phases, wash with pure water and saturated brine, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation to obtain crude product. The crude product is further purified by silica gel column chromatography to obtain white solid compound 1. (2) Add white solid compound 1, morpholine and anhydrous potassium carbonate to acetonitrile, and stir at 70°C for 24 hours under nitrogen protection. After the reaction is completed, cool to room temperature, extract with ethyl acetate, combine the organic phases and wash with saturated brine, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation to obtain crude product. Purify the crude product by silica gel column chromatography to obtain yellow liquid compound 2. (3) 2-hydroxyacetophenone, compound 2 and sodium hydroxide were added to methanol and stirred at 60°C for 12 hours under nitrogen protection. Then, 40% NaOH solution and 6% H2O2 solution were slowly added dropwise and the reaction was continued at 4°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to 7 with dilute hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was recrystallized from anhydrous ethanol to obtain the yellow solid compound ML. (4) Compound ML, pinacol 4-bromomethylphenylboronic acid and anhydrous potassium carbonate were added to DMF and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the light yellow solid compound PML.

3. The method for preparing a fluorescent probe for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles according to claim 2, characterized in that: In step (1), the molar ratio of p-hydroxybenzaldehyde to 1,6-dibromohexane is 1.0:(2.3~3.0), and the molar ratio of anhydrous potassium carbonate to acetone is (0.8~1.2):1; In step (2), the molar ratio of white solid compound 1 to morpholine is 1:(1.3~1.8), and the molar ratio of anhydrous potassium carbonate to acetonitrile is 1:(1.3~1.7). In step (3), the molar ratio of 2-hydroxyacetophenone to compound 2 is (0.8~1.2):1, and the molar ratio of sodium hydroxide to methanol is 1:(1.8~2.5). In step (4), the molar ratio of ML to pinacol 4-bromomethylbenzylboronic acid is (0.6~0.9):1, and the molar ratio of anhydrous potassium carbonate to DMF is (0.3~0.8):

1.

4. A class of fluorescent probes for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles, characterized in that, Specifically, it is the hydrogen peroxide ratiometric fluorescent probe PZD, which has the following general structural formula: 。 5. The method for preparing a fluorescent probe for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles according to claim 4, characterized in that, Its preparation method is as follows: ； Specifically, the steps include the following: (1) p-hydroxybenzaldehyde, 1-bromohexane and anhydrous potassium carbonate were added to DMF and stirred at 70°C for 12 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with pure water and saturated brine. The mixture was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a white solid compound 3. (2) 2-hydroxyacetophenone, compound 3 and sodium hydroxide were added to methanol and stirred at 60°C for 12 hours under nitrogen protection. Then, 40% NaOH solution and 6% H2O2 solution were slowly added dropwise and the reaction was continued at 4°C for 12 hours. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to 7 with dilute hydrochloric acid, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was recrystallized from methanol to obtain the yellow solid compound ZD. (3) Compound ZD, 4-bromomethylphenylboronic acid pinacol ester and anhydrous potassium carbonate were added to DMF and stirred at 70°C for 24 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined and washed with saturated brine. The mixture was dried with anhydrous sodium sulfate and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the light yellow solid compound PZD.

6. The method for preparing a fluorescent probe for monitoring the dynamic changes of hydrogen peroxide in different subcellular organelles according to claim 5, characterized in that, In step (1), the molar ratio of p-hydroxybenzaldehyde to 1-bromohexane is (1.0~1.2):1, and the molar ratio of anhydrous potassium carbonate to DMF is (0.4~0.6):

1. In step (2), the molar ratio of 2-hydroxyacetophenone to compound 3 is (0.9~1.2):1, and the molar ratio of sodium hydroxide to methanol is (0.4~0.7):1; In step (3), the molar ratio of compound ZD to pinacol 4-bromomethylbenzylboronic acid is 1.0:(1.0~1.4), and the molar ratio of anhydrous potassium carbonate to DMF is (0.3~0.5):

1.

7. The two types of fluorescent probes according to claim 1 or 4, characterized in that, It was used as a reagent to monitor the dynamic changes of hydrogen peroxide in different subcellular organelles.

8. The two types of fluorescent probes according to claim 7, characterized in that, The fluorescent probe PML was applied to the detection of hydrogen peroxide in lysosomal organelles. Its molecular structure contains a morpholine group, which is protonated in the acidic environment of lysosomes at pH 4.5 to 5.

0. Through lysosomal-specific enrichment under the ion trapping effect, lysosomal targeting was achieved, enabling the monitoring of dynamic changes in hydrogen peroxide in lysosomes during RSL3-induced ferroptosis.

9. The two types of fluorescent probes according to claim 7, characterized in that, Fluorescent probes PML or PZD are used in the detection of hydrogen peroxide in lysosomal organelles, specifically for locating lysosomal fluorescence imaging.

10. The two types of fluorescent probes according to claim 7, characterized in that, The fluorescent probe PZD was used to detect hydrogen peroxide in lipid droplets. By modifying long-chain hexyl groups, it was able to embed itself into the hydrophobic core of lipid droplets for targeted localization, thereby enabling the monitoring of dynamic changes in intracellular hydrogen peroxide during RSL3-induced ferroptosis.

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