Synthesis and application of a bifunctional fluorescent probe for simultaneous detection of homocysteine ​​and hydrogen peroxide

Abstract

The present invention discloses a bifunctional fluorescent probe for the simultaneous detection of homocysteine ​​and hydrogen peroxide. The chemical structure of the bifunctional probe is as follows: #imgabs0#. This fluorescent probe is the first to simultaneously detect homocysteine ​​and hydrogen peroxide. It adopts a dual-position design for the selective detection of homocysteine, and after the reaction, it emits a red fluorescence of 620 nm at an excitation wavelength of 530 nm. Hydrogen peroxide is selectively identified by a borate sensitive unit, emitting a yellow fluorescence of 550 nm at an excitation wavelength of 440 nm. The probe has no obvious reaction with other substances such as active sulfur, active oxidizing agents, and metal ions. The reaction product has advantages such as good water solubility and a large Stokes shift. The probe can simultaneously detect homocysteine ​​and hydrogen peroxide in two channels and has great application prospects in technical fields such as analytical chemistry, life sciences, and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to the synthesis and application of a bifunctional fluorescent probe for the simultaneous detection of homocysteine ​​and hydrogen peroxide. This probe uses a dual-site pattern to highly selectively detect homocysteine, with a boronate-sensitive unit responsive to hydrogen peroxide and two fluorophores chemically linked. This probe enables rapid, selective imaging of hydrogen peroxide using the yellow fluorescence channel and homocysteine ​​using the red fluorescence channel from various reactive sulfur, reactive oxygen, and metal ion species. It exhibits advantages such as a large Stokes shift, high sensitivity, and visual detection. Background Art

[0002] Homocysteine ​​(Hcy), a sulfur-containing amino acid, is a key clinical diagnostic marker and is closely associated with the development and progression of many diseases. The kidneys play a crucial role in regulating Hcy metabolic homeostasis both locally and systemically. Chronic and acute kidney disease impairs the transsulfurization pathway and desulfurization reactions, leading to hyperhomocysteinemia (The Lancet Neurology 2003, 2, 425-428; Vascular Pharmacology 2016, 78, 1-9; Diabetes Care 2004, 27, s79-s83). Hydrogen peroxide (H2O2), a relatively stable intracellular second messenger, plays a variety of essential roles in cell signaling and homeostasis (Science 2006, 312, 1882-1883; Nature Communications 2015, 6, 6907-6915). Abnormally elevated levels of H2O2 can lead to a range of oxidative stress and inflammatory responses. In acute kidney injury, homocysteine ​​accumulates, potentially leading to hyperhomocysteinemia and elevated reactive oxygen species (H2O2) (Chemical Science 2018, 9, 7606-7613; Analytical Chemistry 2019, 91, 14019-14028; ACS Sensors 2020, 5, 2457-2466). Homocysteine ​​and hydrogen peroxide play crucial roles in acute kidney injury or chronic kidney disease, yet their simultaneous monitoring is rarely reported. Therefore, designing a system to simultaneously monitor intracellular homocysteine ​​and hydrogen peroxide levels is crucial for understanding acute kidney injury and other biological functions.

[0003] To date, some chemical sensors have been reported for detecting Hcy, which are used for the detection of Hcy in serum and cells. (Journal of the American Chemical Society 2005, 127, 15949-15958; Nature Protocols 2006, 1, 2759-2762) However, most small molecule probes cannot distinguish Hcy from other thiol compounds, there is a certain overlap between the fluorescence spectra, and the sensitivity is low, which cannot achieve selective fluorescence imaging analysis of Hcy in cells / tissues / living bodies (AngewandteChemie International Edition 2017, 56, 13188-13198; Biosensors and Bioelectronics 2016, 81, 341-348). In addition, there are also a large number of literature reports on probes for hydrogen peroxide, but there are still few dual-functional probes that can simultaneously detect changes in homocysteine ​​and hydrogen peroxide. Therefore, the simultaneous detection of homocysteine ​​and hydrogen peroxide by dual channels remains a huge challenge. Summary of the Invention

[0004] In view of the above situation, and to overcome some of the deficiencies of the prior art, the present invention aims to provide a dual-function fluorescent probe for the simultaneous detection of homocysteine ​​and hydrogen peroxide. This probe can rapidly and selectively detect homocysteine ​​and hydrogen peroxide from various substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the fluorescent molecular probe with simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize a dual-function fluorescent probe for simultaneous detection of homocysteine ​​and hydrogen peroxide and to prepare a device for quantitative analysis of homocysteine ​​and hydrogen peroxide in the environment and simultaneous differentiation and imaging of homocysteine ​​and hydrogen peroxide in living cells. The chemical structure of the dual-function probe is as follows:

[0007]

[0008] The synthesis of a dual-function fluorescent probe for simultaneous detection of homocysteine ​​and hydrogen peroxide is characterized in that the preparation method of the dual-function fluorescent probe comprises the following steps:

[0009] Step 1. Synthesis of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde

[0010] 1. Mix appropriate amounts of sulfuric acid and nitric acid and cool them in an ice-salt bath. Then add 4-(butylthio)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde to the mixed solution and stir for 1 hour. After the reaction is complete, slowly add water, adjust the pH of the solution to 6 with sodium hydroxide, extract with ethyl acetate, spin dry, and purify by column chromatography to obtain 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde;

[0011] Step 2. Synthesis of tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate

[0012] I. Add 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde to an appropriate amount of dichloromethane, then add appropriate amounts of tert-butyl cyanoacetate and triethylamine, and stir at room temperature overnight. After completion of the reaction, spin dry the mixture and perform column chromatography to obtain tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-2-cyanoacrylate.

[0013] Step 3. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid

[0014] i. tert-Butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate was added to an appropriate amount of dichloromethane, followed by an appropriate amount of trifluoroacetic acid. The reaction was allowed to proceed overnight at room temperature, filtered, and the solid was dried under vacuum to give (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate;

[0015] Step 4. Synthesis of tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2)(3H)-yl)propanoyl)piperazine-1-carboxylate

[0016] ⒈ Add an appropriate amount of tert-butyl 4-(3-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carboxylate to anhydrous DMF, then add an appropriate amount of tetrakistriphenylphosphine palladium, then add bipyraclostrobin, and stir at 105°C for 12 hours. After the reaction is completed, slowly introduce the reaction solution into water, extract with ethyl acetate, spin dry, and separate by column chromatography to obtain tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2)(3H)-yl)propionyl)piperazine-1-carboxylate;

[0017] Step 5. Synthesis of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

[0018] ⑴. The tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-2)(3H)-yl)propionyl)piperazine-1-carboxylate was added to an appropriate amount of dichloromethane, and then an appropriate amount of trifluoroacetic acid was added. The reaction was allowed to proceed overnight at room temperature, filtered, and the solid was dried under vacuum to give 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione;

[0019] Step 6. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carbonyl)acrylonitrile

[0020] ①. Add an appropriate amount of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid to anhydrous dichloromethane, then add an appropriate amount of 4-dimethylaminopyridine (DMAP) and react at room temperature for 30 minutes.

[0021] ②. An appropriate amount of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione was slowly added to the above solution, and then an appropriate amount of 1-ethyl-3(3-dimethylpropylamine)carbodiimide was added, reacted at room temperature for 24h, dried, and purified by column chromatography to obtain the A dual-functional fluorescent probe (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carbonyl)acrylonitrile.

[0022] The present invention discloses a method for using a dual-function fluorescent probe for the simultaneous detection of homocysteine ​​and hydrogen peroxide: unless otherwise specified, when detecting homocysteine ​​and hydrogen peroxide, the molecular fluorescent probe is usually dissolved in dimethyl sulfoxide (DMSO), and the probe molecules are dissolved in a solution of organic phase and aqueous phase (5:5, v / v). After reacting with homocysteine ​​at room temperature for 30 minutes, it emits red fluorescence of 620nm at an excitation wavelength of 530nm; after reacting with hydrogen peroxide at room temperature for 30 minutes, it emits strong green fluorescence of 550nm at an excitation wavelength of 450nm. Thus, specific excitation and fluorescence emission signals are used to detect specific analytes. When both substances are present, the different excitation and fluorescence emission signals can also be used to distinguish the two well. The above-mentioned fluorescent molecular probe realizes the simultaneous and differentiated detection of homocysteine ​​and hydrogen peroxide under different detection conditions. It has no obvious response to other reactive oxygen species, reactive sulfur species, common amino acids, metal ions, and reactive nitrogen species. The detection limits for homocysteine ​​and hydrogen peroxide are as low as 5μM and 454nM, respectively. Therefore, the bifunctional fluorescent molecular probe disclosed in the present invention can achieve high-sensitivity detection of both. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The dual-function fluorescent probe of the present invention detects fluorescence spectra of homocysteine ​​and hydrogen peroxide. DETAILED DESCRIPTION

[0024] The synthesis route of the dual-function fluorescent probe of the present invention is as follows:

[0025]

[0026] Example 1. Synthesis of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde

[0027] 1. Mix 1 mL of sulfuric acid and 3 mL of nitric acid and cool in an ice-salt bath. Then, 2.00 g (6.00 mmol) of 4-(butylthio)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde was added to the mixed solution and stirred for 1 hour. After the reaction, water was slowly added, and the pH of the solution was adjusted to 6 with sodium hydroxide. The solution was extracted with ethyl acetate, dried, and purified by column chromatography to give 400 mg of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde in a yield of 17.62%.

[0028] Example 2. Synthesis of tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate

[0029] I. 400 mg (1.06 mmol) of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde was added to 10 mL of dichloromethane, followed by 161.26 mg (1.27 mmol) of tert-butyl cyanoacetate and 160.44 mg (1.59 mmol) of triethylamine. The mixture was stirred and reacted overnight at room temperature. After completion of the reaction, the mixture was spin-dried and purified by column chromatography to obtain 345 mg of tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-2-cyanoacrylate in a yield of 65.07%.

[0030] Example 3. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid

[0031] i. 300 mg (598.09 μmol) of tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate was added to 6 mL of dichloromethane, followed by 2 mL of trifluoroacetic acid. The mixture was reacted overnight at room temperature, filtered, and the solid was dried under vacuum to give 210 mg of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate in a yield of 78.82%.

[0032] Example 4. Synthesis of tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2)(3H)-yl)propionyl)piperazine-1-carboxylate

[0033] ⒈ 4.00g (7.75mmol) of tert-butyl 4-(3-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carboxylate was added to 30mL of anhydrous DMF, followed by 895.12mg of tetrakis(triphenylphosphine)palladium and 3.93g (15.49mmol) of biboronic acid pinacol ester. The mixture was stirred at 105°C for 12 hours. After completion of the reaction, the reaction solution was slowly introduced into water, filtered, and dried to obtain 3.84g of tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2)(3H)-yl)propionyl)piperazine-1-carboxylate in a yield of 87.98%.

[0034] Example 5. Synthesis of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

[0035] ⑴. 2.00 g (3.55 mmol) of tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-2)(3H)-yl)propionyl)piperazine-1-carboxylate was added to 20 mL of dichloromethane, and 4 mL of trifluoroacetic acid was added. The reaction was allowed to proceed overnight at room temperature, filtered, washed with ethanol, and the solid was dried in vacuo to give 1.4 g of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione in a yield of 85.13%.

[0036] Example 6. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carbonyl)acrylonitrile

[0037] ①. Add 150 mg (336.71 μmol) of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid to 10 mL of anhydrous dichloromethane, then add 10 mg of 4-dimethylaminopyridine (DMAP) and react at room temperature for 30 minutes.

[0038] ②. Slowly add 156.01 mg (336.71 μmol) of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to the above solution, and then add 96.82 mg (505.06 μmol) of 1-ethyl-3(3-dimethylpropylamine)carbodiimide, react at room temperature for 24 hours, and rotate The mixture was dried and purified by column chromatography to obtain 40 mg of the bifunctional fluorescent probe (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carbonyl)acrylonitrile with a yield of 13.34%.

[0039] Example 7. Application of a Dual-Functional Fluorescent Probe for Detecting Homocysteine ​​and Hydrogen Peroxide in Vitro. The spectral properties of the dual-function fluorescent probe described herein for simultaneous detection of homocysteine ​​and hydrogen peroxide were tested: the probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution, and a 10 mM homocysteine / hydrogen peroxide aqueous solution was prepared. The specific testing method was as follows: 20 μL of the 1 mM probe solution was added to 20 μL of a 10 mM analyte solution, followed by 980 μL of analytical grade DMSO and 980 μL of PBS buffer solution in a 2 mL sample tube (the total volume of each test sample was 2 mL). For example, to measure the fluorescence intensity of the probe after the reaction with hydrogen peroxide at a hydrogen peroxide concentration of 100 μM, the sample preparation is as follows: 20 μL of a 1 mM probe solution, 20 μL of a 10 mM aqueous hydrogen peroxide solution, 980 μL of analytical grade DMSO, and 980 μL of PBS buffer are placed in a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured using an excitation wavelength of 440 nm. Other test procedures are similar to those described above. This dual-function probe enables the simultaneous detection of homocysteine ​​and hydrogen peroxide using different excitation wavelengths and fluorescence emission signals, with high sensitivity, achieving detection limits of as low as 5 μM for homocysteine ​​and 454 nM for hydrogen peroxide. It is ideally suited for imaging and quantitative analysis of endogenous homocysteine ​​and hydrogen peroxide in living cells.

[0040] The present invention provides a bifunctional fluorescent probe for the simultaneous detection of homocysteine ​​and hydrogen peroxide. A dual-position design is adopted for the selective detection of homocysteine, and after the reaction, a red fluorescence of about 620nm is emitted at an excitation wavelength of 530nm. Hydrogen peroxide is selectively identified by the sensitive unit of borate ester, and a yellow fluorescence of about 550nm is emitted at an excitation wavelength of 440nm. In addition, the probe has no obvious phenomenon with other active sulfur, active oxidizing, amino acids, metal ions and other substances, and the product after the reaction has good water solubility and large Stokes shift. Although the content of the present invention has been described in detail through the above embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be obvious to those skilled in the art for various modifications and substitutions of the present invention. Therefore, fluorescent mother nuclei similar to the technical features described herein all fall within the scope of protection of this patent.

Claims

1. A dual-function fluorescent probe for simultaneous detection of homocysteine ​​and hydrogen peroxide, characterized in that: The structural formula of the dual-function fluorescent probe is as follows:

2. The synthesis of the dual-function fluorescent probe according to claim 1, wherein The synthesis method of the dual-function fluorescent probe comprises the following steps: Step 1. Synthesis of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde: Appropriate amounts of sulfuric acid and nitric acid were mixed and cooled in an ice-salt bath. 4-(butylthio)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde was then added to the mixed solution and stirred for 1 hour. After the reaction was complete, the mixture was slowly added to water, and the pH of the solution was adjusted to 6 with sodium hydroxide. The solution was extracted with ethyl acetate, dried by spin drying, and purified by column chromatography to obtain 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde. Step 2. Synthesis of tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate 4-(Butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde was added to an appropriate amount of dichloromethane, followed by the addition of appropriate amounts of tert-butyl cyanoacetate and triethylamine. The mixture was stirred at room temperature and reacted overnight. After completion of the reaction, the mixture was spin-dried and subjected to column chromatography to obtain tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-2-cyanoacrylate. Step 3. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid Tert-butyl (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate was added to an appropriate amount of dichloromethane, followed by an appropriate amount of trifluoroacetic acid. The mixture was reacted overnight at room temperature, filtered, and the solid was dried under vacuum to obtain (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylate. Step 4. Synthesis of tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carboxylate An appropriate amount of tert-butyl 4-(3-(6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carboxylate was added to anhydrous DMF, followed by an appropriate amount of tetrakis(triphenylphosphine)palladium and an appropriate amount of pinacol diboron. The mixture was stirred at 105° C. for 12 hours. After completion of the reaction, the reaction solution was slowly introduced into water, extracted with ethyl acetate, and dried by spin drying. The mixture was separated by column chromatography to obtain tert-butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carboxylate. Step 5. Synthesis of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione tert-Butyl 4-(3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carboxylate was added to an appropriate amount of dichloromethane, followed by an appropriate amount of trifluoroacetic acid. The mixture was reacted overnight at room temperature, filtered, and the solid was dried under vacuum to obtain 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione; Step 6. Synthesis of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propanoyl)piperazine-1-carbonyl)acrylonitrile Add an appropriate amount of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid to anhydrous dichloromethane, then add an appropriate amount of DMAP, and react at room temperature for 30 minutes. Then slowly add an appropriate amount of 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione to the above solution. Then, an appropriate amount of 1-ethyl-3(3-dimethylpropylamine)carbodiimide was added, the reaction was carried out at room temperature for 24 hours, the mixture was dried by rotary evaporation, and the mixture was purified by column chromatography to obtain (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-(4-(3-(1,3-dioxo)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)propionyl)piperazine-1-carbonyl)acrylonitrile.

3. The synthesis of the bifunctional probe according to claim 2, wherein: The molar ratio of the appropriate amount of (E)-3-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-2-cyanoacrylic acid and 2-(3-oxo-3-(piperazin-1-yl)propyl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[de]isoquinoline-1,3(2H)-dione in step 6 is 1:

1.

4. The use of the dual-function fluorescent probe according to claim 1, wherein The dual-functional fluorescent probe can quantitatively analyze homocysteine ​​and hydrogen peroxide in the environment, and the application is for non-disease diagnosis and treatment purposes.

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