Synthesis and application of a fluorescent probe for simultaneous discrimination of hydrogen peroxide and glutathione

Abstract

This invention discloses a fluorescent probe that simultaneously distinguishes between hydrogen peroxide and glutathione. The chemical structure of the probe is as follows: This fluorescent probe ingeniously combines a 1,8-naphthalenedimide fluorophore and an isophorone fluorophore, achieving for the first time a dual-channel simultaneous detection of hydrogen peroxide and glutathione. When reacting with glutathione, it emits 500 nm green fluorescence at an excitation wavelength of 380 nm; when reacting with hydrogen peroxide, it emits 680 nm near-infrared fluorescence at an excitation wavelength of 560 nm. The two emissions differ by 180 nm, and the spectra do not interfere with each other, allowing for the selective simultaneous detection of hydrogen peroxide and glutathione. This probe exhibits excellent selectivity for detecting hydrogen peroxide and glutathione, high fluorescence quantum yield, and a large Stokes shift, showing great promise for applications in analytical chemistry, life sciences, and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to the synthesis and application of a fluorescent probe that simultaneously distinguishes between hydrogen peroxide and glutathione. This probe novelly combines two fluorophores and enables rapid and selective detection of hydrogen peroxide and glutathione from various bioactive substances. The green fluorescence channel selectively detects glutathione, while the near-infrared fluorescence channel selectively detects hydrogen peroxide. It possesses advantages such as a large Stokes shift, high fluorescence quantum yield, high detection sensitivity, and visual detection. Background Technology

[0002] Hydrogen peroxide (H2O2) is a major member of the reactive oxygen species (ROS) family, involved in a wide range of physiological and pathological processes (Mol. Cell Biol. 2007, 8, 813-824.). H2O2 can act as a second messenger for normal cell growth and proliferation. Under normal H2O2 conditions, many life activities such as cell signal transduction, proliferation, and apoptosis can be effectively regulated (Physiol. Rev. 2007, 87, 245–313; Sens. Actuators B Chem. 2019, 288, 127–132; Chem. Sci. 2019, 10, 2025–2033; Chin. Chem. Lett. 2020, 31, 3149–3152). However, abnormal levels can cause various diseases, such as inflammation, cardiovascular disease, cancer, and diabetes (Sens. Actuators B Chem. 2020, 32, 128296; Biosens. Bioelectron. 2017, 94, 536–543; Eur. J. Med. Chem. 2021, 226, 113828). Glutathione (GSH) is the most abundant non-protein thiol (1–10 mM) in cells and has been shown to be useful in maintaining intracellular redox homeostasis, signal transduction, gene regulation, and xenobiotic metabolism (Nat. Methods 2008, 5, 553–559). The presence of GSH effectively prevents damage to cellular components from reactive oxygen species (ROS) (Free Radic. Biol. Med. 1999, 27, 916–921). H2O2 and GSH work together to maintain redox balance in living organisms. They are closely related and influence each other. Abnormal levels can lead to a variety of diseases. Therefore, it is of great significance to develop a method to simultaneously distinguish and detect H2O2 and GSH.

[0003] Currently, various methods have been developed for the detection of H2O2 and GSH, such as liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GCMS), and fluorescence spectroscopy. Among these methods, fluorescent probe analysis is generally favored due to its rapid response, high sensitivity, spatial resolution, and satisfactory biocompatibility (Angew. Chem. Int. Ed. 2017, 56, 16611–16615; Anal. Chem. 2016, 76, 166–181). Many fluorescent probes for the detection of hydrogen peroxide / glutathione have been reported, most of which detect only one of them (Sens. Actuators B Chem. 2018, 266, 528–533; J. Am. Chem. Soc. 2011, 133, 10629–10637). However, to date, only a very few reports have described fluorescent probes that simultaneously detect hydrogen peroxide and glutathione, and these are single-channel reversible detection methods; they cannot simultaneously image the dynamic changes of both through dual channels. This indicates that simultaneously distinguishing and imaging hydrogen peroxide and glutathione in organisms using dual channels remains a significant challenge. Summary of the Invention

[0004] In view of the above, and to overcome some shortcomings of existing technologies, the present invention aims to provide a fluorescent probe that can simultaneously distinguish between hydrogen peroxide and glutathione. This probe can rapidly and selectively detect hydrogen peroxide and glutathione from various bioactive substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the above-mentioned fluorescent probe that is simple to prepare, highly sensitive, has a low detection limit, and is low in cost.

[0006] The specific technical solution adopted by this invention to solve the problem is the synthesis and preparation of a fluorescent probe that simultaneously distinguishes hydrogen peroxide and glutathione, and the application of a device for quantitatively analyzing hydrogen peroxide and glutathione in the environment and simultaneously distinguishing and imaging hydrogen peroxide and glutathione in living cells. The chemical structural formula of the probe is as follows:

[0007]

[0008] A method for synthesizing a fluorescent probe that simultaneously distinguishes between hydrogen peroxide and glutathione, characterized in that the preparation method of the fluorescent probe includes the following steps:

[0009] Step 1. Synthesis of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester: 4-(3-(6-(methylthio)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of m-chloroperoxybenzoic acid. The mixture was stirred at 60°C for 6 hours. After the reaction was complete, the reaction system was evaporated to dryness, and the product was purified by column chromatography to obtain 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester;

[0010] Step 2. Synthesis of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate: 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester was added to anhydrous dichloromethane, followed by trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was evaporated to dryness, and the mixture was purified by column chromatography to obtain 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate;

[0011] Step 3. Synthesis of tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)

[0012] Tert-butyl acetate (E)-2-cyano-2-(3,5,5-trimethylcyclohexyl-2-en-1-yl) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzaldehyde were added to anhydrous ethanol, followed by a few drops of piperidine. The mixture was stirred at 95°C for 24 hours. After the reaction was complete, the reaction system was evaporated to dryness, and the mixture was purified by column chromatography to obtain tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohexyl-2-en-1-yl).

[0013] Step 4. Synthesis of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid

[0014] Tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl) was added to anhydrous dichloromethane, followed by the addition of trifluoroacetic acid. The mixture was stirred overnight at room temperature until the reaction was complete. The reaction system was then evaporated to dryness, and the mixture was purified by column chromatography to obtain (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid.

[0015] Step 5. Synthesize the fluorescent probe.

[0016] (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid was added to dry redistilled dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 30 min. Subsequently, 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate was added, and the mixture was stirred for 5 min. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction system was evaporated to dryness, and the fluorescent probe was purified by column chromatography.

[0017] A method for using a fluorescent probe that can simultaneously distinguish between hydrogen peroxide and glutathione according to the present invention: Unless otherwise specified, the probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature, and the analysis and detection are performed in an environment where the volume ratio of organic phase to aqueous phase is 5:5. The organic phase is dimethyl sulfoxide (DMSO), and the aqueous phase is phosphate buffer solution (PBS) with pH=7.4.

[0018] The specific characteristics of the fluorescent probe of this invention that simultaneously distinguishes between hydrogen peroxide and glutathione are as follows: The fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), with the probe molecules dissolved in an organic and aqueous (5:5, v / v) solution. After reacting with hydrogen peroxide for 30 minutes, it emits near-infrared fluorescence at an excitation wavelength of 680 nm at 560 nm; after reacting with glutathione at room temperature for 30 minutes, it emits strong green fluorescence at an excitation wavelength of 500 nm at 380 nm. Therefore, it enables the detection of specific analytes using specific excitation and fluorescence emission signals. When both substances are present, they can be well distinguished using different excitation and fluorescence emission signals. The above-mentioned fluorescent probe achieves simultaneous detection of hydrogen peroxide and glutathione under different detection conditions, and shows no significant response to other reactive oxygen species, reactive sulfur, common amino acids, metal ions, and reactive nitrogen. The detection limits for hydrogen peroxide and glutathione are as low as 15.6 μM and 840 μM, respectively. Therefore, the fluorescent probe disclosed in this invention can achieve highly sensitive differential and quantitative detection of both. Attached Figure Description

[0019] Figure 1 The proton NMR spectrum of the fluorescent probe described in this invention.

[0020] Figure 2 The fluorescent probe of this invention exhibits ultraviolet and fluorescence spectra in response to hydrogen peroxide and glutathione.

[0021] Figure 3 The fluorescence quantitative analysis diagram of the fluorescent probe of the present invention in response to hydrogen peroxide and glutathione. Detailed Implementation

[0022] The invention will be further explained in conjunction with the synthetic route below.

[0023] The synthetic route of the fluorescent probe described in this invention is as follows:

[0024]

[0025] Example 1. Synthesis of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester

[0026] 500.00 mg (1.03 mmol) of 4-(3-(6-(methylthio)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester was added to 30 mL of anhydrous dichloromethane, followed by 713.69 mg (4.14 mmol) of m-chloroperoxybenzoic acid. The mixture was stirred at 60 °C for 6 hours. After the reaction was complete, the reaction system was evaporated to dryness, and purified by column chromatography to obtain 422.00 mg of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester, with a yield of 81.31%.

[0027] Step 2. Synthesis of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate salt. 400.00 mg (800.67 μmol) of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1 12 mL of anhydrous dichloromethane was added to tert-butyl carboxylate, followed by 3 mL of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was evaporated to dryness, and the product was purified by column chromatography to obtain 370.00 mg of 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate, with a yield of 59.99%.

[0028] Step 3. Synthesis of tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)

[0029] 1.00 g (3.83 mmol) of tert-butyl acetate (E)-2-cyano-2-(3,5,5-trimethylcyclohexyl-2-en-1-yl) and 1.07 g (4.59 mmol) of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)benzaldehyde were added to 40 mL of anhydrous ethanol, followed by 0.2 mL of piperidine. The mixture was stirred at 95 °C for 24 hours. After the reaction was complete, the reaction mixture was evaporated to dryness, and the product was purified by column chromatography to obtain 584.00 mg of tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)styryl)cyclohexyl-2-en-1-yl), with a yield of 32.10%.

[0030] Step 4. Synthesis of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohexyl-2-en-1-yl)acetic acid. 400.00 mg (841.33 μmol) of tert-butyl acetate (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid was added to 8 mL of anhydrous dichloromethane, followed by 2 mL of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was completed, the reaction mixture was evaporated to dryness, and the product was purified by column chromatography to obtain 240.00 mg of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid, with a yield of 68.03%.

[0031] Step 5. Synthesize the fluorescent probe.

[0032] 100.00 mg (238.48 μmol) of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid was added to 8 mL of dry, redistilled dichloromethane, followed by 2.91 mg (23.85 μmol) of 4-dimethylaminopyridine (DMAP). The mixture was reacted at room temperature for 30 min, and then 122.46 mg (238.48 μmol) of acetic acid was added. 4-(3-(6-(methylsulfinyl)-1,3-dioxy-1H-benzo[de]isoquinoline-2(3H)-yl)propionyl)piperazine-1-onium 2,2,2-trifluoroacetate was added, and stirred for 5 min. Then, 68.58 mg (357.72 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was evaporated to dryness, and the fluorescent probe was purified by column chromatography to obtain 28.00 mg of the fluorescent probe, with a yield of 14.66%.

[0033] Example 9. Detection of hydrogen peroxide and glutathione by fluorescent probes in vitro.

[0034] The fluorescent probe spectral properties experiment of this invention: The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution, and 10 mM hydrogen peroxide and glutathione aqueous solutions were prepared respectively. The specific testing method was as follows: 20 μL of the 1 mM probe solution was taken, followed by 20 μL of the 10 mM analyte solution, and finally 980 μL of analytical grade DMSO and 980 μL of PBS were added. All tests maintained an organic phase to aqueous phase volume ratio of 5:5 (total volume of each test sample was 2 mL). For example, when testing the fluorescence intensity of hydrogen peroxide at a concentration of 100 μM, the sample preparation is as follows: Take 20 μL of 1 mM probe solution, 20 μL of 10 mM hydrogen peroxide aqueous solution, and then add 980 μL of analytical grade DMSO and 980 μL of PBS buffer solution to a 2 mL sample tube. After shaking and mixing at room temperature for 30 minutes, the fluorescence emission intensity can be measured using an excitation wavelength of 560 nm. Other testing procedures are similar to the above steps. This probe enables the differentiation and detection of hydrogen peroxide and glutathione, two bioactive substances, using different excitation wavelengths and fluorescence emission signals. It has high sensitivity, with detection limits as low as 15.6 μM and 840 μM, respectively, making it ideal for imaging / quantitative analysis of endogenous hydrogen peroxide and glutathione in live cells.

[0035] This invention provides a fluorescent probe that simultaneously distinguishes between hydrogen peroxide and glutathione. It connects a 1,8-naphthalenedimide fluorophore and an isophorone fluorophore via piperazine. Upon reaction with glutathione, it emits 500nm green light at an excitation wavelength of 380nm, and upon reaction with hydrogen peroxide, it emits 680nm near-infrared fluorescence at an excitation wavelength of 560nm, exhibiting significant fluorescence. Furthermore, the reaction product exhibits good water solubility, high fluorescence quantum yield, and a large Stokes shift. It has significant practical application value in biochemistry, analytical detection, and other fields. Although the invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the invention. Various modifications and substitutions to the invention will be obvious to those skilled in the art after reading the above content. Therefore, fluorescent probes with similar technical features as described herein fall within the protection scope of this patent.

Claims

1. A fluorescent probe for simultaneously distinguishing hydrogen peroxide and glutathione, characterized by, The chemical structure of the fluorescent probe is shown as (1):

2. The synthesis of fluorescent probe as claimed in claim 1, wherein, The synthesis method of the fluorescent probe comprises the following steps: Step 1. Synthesis of tert-butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate tert-Butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate is added into anhydrous dichloromethane, then m-chloroperbenzoic acid is added, and the reaction is stirred at 60 DEG C for 6 hours; after the reaction is completed, the reaction system is evaporated and dried, and column chromatography is used for separation and purification to obtain tert-butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate; Step 2. Synthesis of tert-butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate tert-Butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate is added into anhydrous dichloromethane, then m-chloroperbenzoic acid is added, and the reaction is stirred at 60 DEG C for 6 hours; after the reaction is completed, the reaction system is evaporated and dried, and column chromatography is used for separation and purification to obtain tert-butyl 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de] isoquinolin-2(3H)-yl) propanoyl)piperazine-1-carboxylate; Step 3. Synthesis of tert-butyl (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinan-2-yl)styryl)cyclohex-2-en-1-yl) acetate tert-Butyl (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinan-2-yl)styryl)cyclohex-2-en-1-yl) acetate is added into anhydrous ethanol, then a few drops of piperidine are added, and the reaction is stirred at 95 DEG C for 24 hours; after the reaction is completed, the reaction system is evaporated and dried, and column chromatography is used for separation and purification to obtain tert-butyl (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinan-2-yl)styryl)cyclohex-2-en-1-yl) acetate; Step 4. Synthesis of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborinan-2-yl)styryl)cyclohex-2-en-1-ylidene)acetic acid (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid was added into dry and distilled dichloromethane, then 4-dimethylaminopyridine (DMAP) was added, and the reaction was carried out at room temperature for 30 min, followed by the addition of 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazin-1-ium 2,2,2-trifluoroacetate, and after stirring for 5 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction system was dried, and column chromatography was used for purification and separation to obtain the fluorescent probe. Step 5. Synthesis of the fluorescent probe (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid was added into dry and distilled dichloromethane, then 4-dimethylaminopyridine (DMAP) was added, and the reaction was carried out at room temperature for 30 min, followed by the addition of 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazin-1-ium 2,2,2-trifluoroacetate, and after stirring for 5 min, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction system was dried, and column chromatography was used for purification and separation to obtain the fluorescent probe.

3. The method of claim 2, wherein the probe is synthesized by a method comprising: The molar ratio of (E)-2-cyano-2-(5,5-dimethyl-3-((E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)styryl)cyclohex-2-en-1-yl)acetic acid and 4-(3-(6-(methylsulfinyl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazin-1-ium 2,2,2-trifluoroacetate in step 5 is 1:

1.

4. The use of the fluorescent probe according to claim 1, wherein The fluorescent probe is prepared for quantitative analysis of hydrogen peroxide and glutathione in the environment, and is applied to the device for simultaneously distinguishing and imaging hydrogen peroxide and glutathione in living cells.

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