A curcumin-based fluorescent probe for detecting hydrogen peroxide and a preparation method and application thereof

Abstract

The application discloses a curcumin-based fluorescent probe for detecting hydrogen peroxide and a preparation method and application thereof. The fluorescent probe is DFCB. The application utilizes natural curcumin as raw material, and performs complexation reaction with boron trifluoride diethyl ether to obtain DFC; the DFC performs etherification reaction with 4-bromomethylphenylboronic acid pinacol ester to obtain compound DFCB. The compound DFCB can specifically react with hydrogen peroxide, the color of the solution changes from orange to light blue under sunlight, and the fluorescent color of the solution changes from orange to colorless under 365nm ultraviolet light, so that the compound can be used as a colorimetric probe and a quenching fluorescent probe for detecting hydrogen peroxide, and the compound can also sensitively detect the content of hydrogen peroxide in a solution, and the detection limit reaches 1.31muM, and the compound has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fine organic synthesis, and relates to a curcumin-based fluorescent probe for detecting hydrogen peroxide and a preparation method and application thereof. BACKGROUND

[0002] Hydrogen peroxide is an important component of reactive oxygen species and is mainly produced in mitochondria. As a necessary oxygen metabolism product of organisms, hydrogen peroxide plays a key role in activating immune cells and maintaining normal metabolism. However, abnormal proliferation of hydrogen peroxide can cause irreversible damage to the organism, can attack cell biological macromolecules such as proteins and DNA, affect cell metabolism and proliferation, and cause diseases such as cancer, abnormal amino acid modification, and Alzheimer's disease. Therefore, it is of great significance to study an efficient detection method for hydrogen peroxide for disease prevention and medical diagnosis.

[0003] Compared with traditional analysis and detection methods, fluorescent probes have been widely used in the fields of environmental science and life science due to the advantages of fast detection speed, high sensitivity, good selectivity, and simple operation. There is no related report on a novel fluorescent probe for detecting hydrogen peroxide synthesized by using curcumin as a raw material. SUMMARY

[0004] In view of the deficiencies in the prior art, the technical problems to be solved by the present application are to provide a curcumin-based fluorescent probe for detecting hydrogen peroxide, which can meet the use requirements. Another technical problem to be solved by the present application is to provide a preparation method of the curcumin-based fluorescent probe for detecting hydrogen peroxide. The present application also solves a technical problem of providing an application of the curcumin-based fluorescent probe for detecting hydrogen peroxide.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0006] The curcumin-based fluorescent probe is 2,2-difluoro-4,6-di(3-methoxy-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)styryl)-2H-1,3,2-dioxaborinane (abbreviated as DFCB), and the structural formula is as follows:

[0007]

[0008] The preparation method of the curcumin-based fluorescent probe for detecting hydrogen peroxide comprises the following steps:

[0009] 1) Complexing reaction of curcumin and boron trifluoride ether to obtain curcumin difluoroborane complex (abbreviated as DFC);

[0010] 2) etherification reaction of compound DFC with 4-bromomethylphenylboronic acid pinacol ester to obtain 2,2-difluoro-4,6-di(3-methoxy-4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)styryl)-2H-1,3,2-dioxaborinane (abbreviated as DFCB).

[0011] Step 1), complexation reaction of curcumin and boron trifluoride etherate in anhydrous dichloromethane solution to obtain compound DFC, and the specific preparation method comprises:

[0012] (1) 1 mmol of curcumin, 30-45 mL of dichloromethane were sequentially added to a three-necked flask equipped with a stirrer, 1.2-3.6 mmol of boron trifluoride etherate was added dropwise into the reaction bottle, and the reaction was carried out at room temperature for 5-10 h;

[0013] (2) After the reaction solution was distilled to remove half of the solvent, it was frozen and crystallized in a low-temperature refrigerator for 1-4 h;

[0014] (3) The crude product of compound DFC obtained by crystallization was filtered, washed with dichloromethane, and then dried to obtain red powdery solid DFC.

[0015] In step 2), etherification reaction of DFC with 4-bromomethylphenylboronic acid pinacol ester under the action of potassium carbonate to obtain compound DFCB, and the specific preparation method comprises:

[0016] (1) 1 mmol of compound DFC, 1.2-3.6 mmol of potassium carbonate, 1.2-3.6 mmol of 4-bromomethylphenylboronic acid pinacol ester, and 8-20 mL of anhydrous acetonitrile were sequentially added to a three-necked flask equipped with a stirrer, a thermometer and a reflux condenser, and the reaction was carried out at 80°C for 6 h;

[0017] (2) The reaction solution was placed in a low-temperature refrigerator overnight, and the brown precipitate precipitated was filtered to obtain the crude product of compound DFCB;

[0018] (3) The crude product of compound DFCB was dissolved in ethyl acetate, washed with water until neutral, then dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain brown solid DFCB.

[0019] The compound DFCB is applied to detect hydrogen peroxide.

[0020] The compound DFCB is applied to prepare a fluorescent probe for detecting hydrogen peroxide.

[0021] Beneficial effects: compared with the prior art, the application utilizes natural renewable curcumin as raw material, and complexation reaction with boron trifluoride diethyl ether to prepare compound DFC; the compound DFC is subjected to etherification reaction with 4-bromomethylphenylboronic acid pinacol ester to prepare compound DFCB, which can selectively recognize hydrogen peroxide, and the solution color changes from orange to light blue under sunlight, and the solution fluorescence color changes from orange to colorless under 365nm ultraviolet light, so that the compound can be used as a colorimetric probe and a quenching type fluorescent probe for hydrogen peroxide detection, and the detection limit reaches 1.31μM, and the compound has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is an ultraviolet absorption spectrum diagram of compound DFCB and different concentrations of hydrogen peroxide;

[0023] Figure 2 is a fluorescence emission spectrum diagram of compound DFCB and different concentrations of hydrogen peroxide;

[0024] Figure 3 is a fluorescence emission spectrum diagram of compound DFCB and different metal ions, anions and active oxygen. DETAILED DESCRIPTION

[0025] The application will be further described below in combination with specific examples.

[0026] Example 1

[0027] Preparation of compound DFCB, the reaction formula is as follows:

[0028]

[0029] The specific steps are as follows:

[0030] 1) Preparation of compound DFC:

[0031] 1mmol of curcumin, 25mL of dichloromethane were sequentially added into a three-necked flask provided with a stirrer, 2.4mmol of boron trifluoride diethyl ether was dropped into the reaction bottle, and the reaction was carried out at room temperature for 5h; after the reaction liquid was subjected to distillation to remove half of the solvent, it was placed in a low-temperature refrigerator for freezing crystallization for 2h; the DFC crude product obtained by crystallization was filtered, washed with dichloromethane again, and then dried to obtain red powdery solid DFC, with a yield of 98% and a purity of 99.3%. 1H NMR (600 MHz, DMSO-d6) δ: 10.09 (s, 2H), 7.92 (d, J = 15.5 Hz, 2H), 7.47 (s, 2H), 7.34 (d, J = 10.3 Hz, 2H), 7.01 (d, J = 15.6 Hz, 2H), 6.87 (s, 2H), 6.45 (s, 1H), 3.85 (s, 6H). 13 C NMR (151 MHz, DMSO) δ: 178.89, 151.52, 148.35, 147.14, 126.16, 125.44, 118.03, 116.13, 112.56, 101.29, 55.94. HRMS (m / z): [M+H] + calcd for C 21 H 19 BO6F2Na+H + , 439.1140; found, 439.1141.

[0032] 2) Preparation of compound DFCB:

[0033] Into a three-necked flask equipped with a stirrer, thermometer and reflux condenser, 1 mmol of compound DFC, 2.4 mmol of potassium carbonate, 2.4 mmol of 4-bromomethylphenylboronic acid pinacol ester and 8 mL of anhydrous acetonitrile were sequentially added, and the reaction was refluxed at 80°C for 6 h. The reaction solution was placed in a low-temperature refrigerator overnight, and the brown precipitate separated out was filtered to obtain the crude product DFCB. The crude product DFCB was dissolved in ethyl acetate, washed with water until neutral, then dried over anhydrous sodium sulfate, filtered and evaporated to obtain brown solid DFCB with a yield of 53% and a purity of 98.4%. 1 H NMR (600 MHz, DMSO-d6) δ: 10.09 (s, 2H), 7.92 (d, J = 15.5 Hz, 2H), 7.47 (s, 2H), 7.34 (d, J = 10.3 Hz, 2H), 7.01 (d, J = 15.6 Hz, 2H), 6.87 (s, 2H), 6.45 (s, 1H), 3.85 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ: 179.43, 158.87, 158.65, 158.42, 158.20, 151.59, 149.70, 147.12, 144.36, 140.22, 134.91, 127.77, 127.32, 125.34, 124.51, 119.42, 117.70, 115.75, 113.67, 111.96, 84.00, 70.03, 56.12, 24.95. HRMS (m / z): [M+H] + calcd for C 47 H 53 B3O 10 F2Na+H + ,871.3784; found, 871.3794.

[0034] Example 2

[0035] Compound DFCB was prepared into 1 x 10 -4 M PBS buffer solution (containing 50% acetonitrile), and hydrogen peroxide was dissolved in pure water to prepare a solution with a concentration of 0, 2.0 x 10 -5 , 4.0 x 10 -5 , 6.0 x 10 -5 , 8.0 x 10 -5 , 1.0 x 10 -4 , 1.2 x 10 -4 , 1.4 x 10 -4 , 1.6 x 10 -4 , 1.8 x 10 -4 , 2.0 x 10 -4 , 2.2 x 10 -4 , 2.4 x 10 -4 , 2.6 x 10 -4 , 2.8 x 10 -4 , 3.0 x 10 -4 M hydrogen peroxide solution. The UV absorption spectrum of compound DFCB in the presence of different concentrations of hydrogen peroxide was measured by UV spectrophotometric titration on a UV spectrophotometer, as shown in Figure 2. The results show that as the concentration of hydrogen peroxide in the solution gradually increases, the UV absorption intensity of compound DFCB at 503 nm gradually decreases, while the UV absorption intensity at 601 nm gradually increases, and the color of the solution changes from orange to light blue under sunlight, indicating that the compound can be used as a colorimetric probe for detecting hydrogen peroxide. Figure 1

[0036] Example 3

[0037] Compound DFCB was prepared into 1 x 10 -4 ​M's PBS buffer solution (containing 50% acetonitrile) was prepared by dissolving hydrogen peroxide in pure water to a concentration of 0.2.0 × 10⁻⁶. -5 4.0×10 -5 6.0×10 -5 8.0×10 -5 1.0×10 -4 1.2×10 -4 1.4×10 -4 1.6×10 -4 1.8×10 -4 2.0×10 -4 2.2×10 -4 2.4×10 -4 2.6×10 -4 2.8×10 -4 3.0×10 -4 M is a hydrogen peroxide solution. The fluorescence emission spectra of compound DFCB in the presence of different concentrations of hydrogen peroxide were measured using a fluorescence spectrophotometer by fluorescence spectrophotometric titration. For example... Figure 2 As shown in the figure, the results indicate that as the concentration of hydrogen peroxide in the solution gradually increases, the fluorescence emission intensity of compound DFCB at 601 nm gradually decreases. Simultaneously, under 365 nm ultraviolet light, the fluorescence color of the solution gradually changes from orange to colorless. This demonstrates that the compound can be used as a quenching fluorescent probe for detecting hydrogen peroxide, and the detection limit reaches 1.31 μM, indicating that the compound can sensitively detect the hydrogen peroxide content in the solution.

[0038] Example 4

[0039] Compound DFCB was formulated into 1×10 -4 M was prepared using a PBS buffer solution (containing 50% acetonitrile), while different metal ions, anions, and reactive oxygen species were dissolved in pure water to prepare a solution with a concentration of 3.0 × 10⁻⁶. -4 A solution of M. The fluorescence emission spectra of compound DFCB in the presence of different metal ions, anions, and reactive oxygen species were measured using a fluorescence spectrophotometer via fluorescence titration. For example... Figure 3 As shown. The results indicate that the addition of hydrogen peroxide significantly reduces the fluorescence emission intensity of the probe at 601 nm, while the addition of different metal ions (Ag) increases the fluorescence intensity. + ,K + Mg 2+ ,Mn 2+ Ca 2+ Na + ,Cs 2+ Hg 2+ Co 2+ Cu 2+ ,Ba 2+ ,Zn2+ Ni 2+ ), anion (I - , HSO3 - , HSO4 - , SO4 2- , ClO - , ClO4 - , Cl - , Br - , AcO - , NO3 - , H2PO4 - , CrO7 2- , NO2 - , ONOO - ) and active oxygen (·OH, TBHP, PAA, MCPBA) and so on, the fluorescence emission spectrum of the probe did not change obviously. Thus, the compound can be used as a specific fluorescence probe for detecting hydrogen peroxide.

Claims

1. A method for preparing curcumin-based fluorescent probe for detecting hydrogen peroxide, characterized in that, Comprising the following steps: 1) Preparation of compound DFC: 1 mmol of curcumin, 25 mL of dichloromethane were sequentially added into a three-necked flask equipped with a stirrer, 2.4 mmol of boron trifluoride etherate was added dropwise into the reaction flask, and the reaction was carried out at room temperature for 5 h; after removing half of the solvent from the reaction solution by distillation, the solution was frozen and crystallized in a low-temperature refrigerator for 2 h; the crude product DFC obtained by crystallization was filtered, washed with dichloromethane, and then dried to obtain red powdery solid DFC with a yield of 98% and a purity of 99.3%; 2) Preparation of compound DFCB: 1 mmol of compound DFC, 2.4 mmol of potassium carbonate, 2.4 mmol of 4-bromomethylphenylboronic acid pinacol ester, and 8 mL of anhydrous acetonitrile were sequentially added into a three-necked flask equipped with a stirrer, a thermometer, and a reflux condenser, and the reaction was carried out at 80°C for 6 h; the reaction solution was placed in a low-temperature refrigerator overnight, and the brown precipitate separated out was filtered to obtain crude product DFCB; the crude product DFCB was dissolved in ethyl acetate, washed with water until neutral, and then dried over anhydrous sodium sulfate, filtered, and evaporated to obtain brown solid DFCB with a yield of 53% and a purity of 98.4%; the reaction formula is as follows: 。 2. The application of the curcumin-based fluorescent probe DFCB prepared by the method of claim 1 in detecting hydrogen peroxide in a solution.

3. The application of DFCB prepared by the method of claim 1 in preparing a fluorescent probe for detecting hydrogen peroxide.

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