Fluorescent probe for detecting active oxygen as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting active oxygen as well as a preparation method and application of the fluorescent probe. Cyclohexanone and 3-ethyl-2-2-methylbenzothiazole iodide are used for preparing a probe TL, the structural formula of the probe TL is # imgabs0 #, after H2O2 is added into the probe TL, the fluorescence intensity is enhanced by about 7 times, the color is rapidly changed from purple to light green, after ONOO-is added into the probe TL, the fluorescence intensity is enhanced by about 3 times, the color is rapidly changed from purple to dark blue, selected interference ions have no response to the probe TL, and the probe TL can be used for detecting the fluorescence intensity of the probe TL. H2O2 and ONOO <-> can be rapidly and selectively detected by naked eyes, and the sensor can be used for detecting active oxygen and is expected to be applied to biological system and environment monitoring.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis, and particularly relates to a fluorescent probe for detecting reactive oxygen species, a preparation method thereof, and an application thereof. Background Art

[0002] Reactive oxygen species (ROS) are important active substances in cells, which are continuously generated and eliminated in the physiological environment and participate in regulating a variety of normal physiological functions and abnormal pathological processes. However, when their generation and elimination are out of balance, it will lead to cell damage and the occurrence of diseases. Tumor cells usually have a high level of ROS. The occurrence, growth, and metastasis of tumors, and even the apoptosis, necrosis, and autophagy of tumor cells are all closely related to ROS. ROS are used as tumor markers, which not only helps in the early diagnosis and prognosis evaluation of tumors, but also provides methods for tumor prevention and treatment strategies related to the ROS pathway. Therefore, achieving rapid, sensitive, and accurate detection of it has become an urgent need in biomedicine.

[0003] Currently, the methods for detecting reactive oxygen species mainly include fluorescent probe method, mass spectrometry analysis, electrochemistry method, spectrophotometry, and chemiluminescence. The fluorescent probe method is widely used in the fields of biology and medicine due to its advantages such as high sensitivity, good selectivity, non-invasive detection, and real-time imaging. Currently, the fluorescent probes for detecting reactive oxygen species have short emission wavelengths and are easily interfered by their own background fluorescence, which is not conducive to deep tissue and in vivo imaging detection. Therefore, constructing a fluorescent probe with a long emission wavelength, high sensitivity to reactive oxygen species, and rapid response is of great significance in improving tumor targeting and achieving early tumor diagnosis and treatment. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent probe for detecting reactive oxygen species, and also provide a preparation method and an application of the fluorescent probe.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A fluorescent probe TL for detecting reactive oxygen species, the structural formula is as follows:

[0007]

[0008] The preparation method of the above-mentioned fluorescent probe TL for detecting reactive oxygen species, the synthesis route of the fluorescent probe TL is as follows:

[0009]

[0010] Specifically, it includes the following steps:

[0011] 1) Cyclohexanone reacts with phosphorus tribromide to obtain compound 1, and the structural formula of compound 1 is as follows:

[0012]

[0013] 2) The reaction of compound 1 with 4-bromo-2-hydroxybenzaldehyde gives compound 2, and the structural formula of compound 2 is as follows:

[0014]

[0015] 3) The reaction of compound 2 with bis(pinacolato)diboron gives compound 3, and the structural formula of compound 3 is as follows:

[0016]

[0017] 4) The reaction of compound 3 with 3-ethyl-2-methylbenzothiazolium iodide gives probe TL, and the structural formula of probe TL is as follows:

[0018]

[0019] Preferably, the specific preparation method of compound 1 in step (1) is as follows: Under a protective atmosphere, phosphorus tribromide is added dropwise to N,N-dimethylformamide and chloroform. After stirring in an ice bath for 0.5 h to 1 h, cyclohexanone is added, and the reaction is stirred at room temperature until complete. Water is added to the reaction solution, and the solution is adjusted to neutral with sodium bicarbonate, then extracted with dichloromethane. The organic phase is dried, and the solvent is removed by rotary evaporation to obtain compound 1.

[0020] Preferably, the specific preparation process of compound 2 in step (2) is as follows: Under a protective atmosphere, N,N-dimethylformamide, 4-bromo-2-hydroxybenzaldehyde, cesium carbonate and compound 1 are placed in a reaction flask, protected from light, and stirred at room temperature until the reaction is complete. After the reaction, it is washed with ethyl acetate, the organic phase is dried, the remaining solvent is evaporated, and compound 2 is obtained by column chromatography purification. When eluting by column chromatography, the eluent is petroleum ether:ethyl acetate = 5 - 15:1 (volume ratio).

[0021] Preferably, the specific preparation process of compound 3 in step (3) is as follows: Under a protective atmosphere, compound 2, bis(pinacolato)diboron, palladium dichloride catalyst and 1,4-dioxane are successively added to a reaction flask, and stirred and refluxed under anhydrous and anaerobic conditions. After the reaction, the solution is rotary evaporated and then purified by column chromatography to obtain compound 3. When eluting by column chromatography, the eluent is petroleum ether:ethyl acetate = 8 - 20:1 (volume ratio).

[0022] In step (1), the molar ratio of PBr3, N,N-dimethylformamide to cyclohexanone is (2 - 3):(2 - 3):1; in step (2), the molar ratio of 4-bromo-2-hydroxybenzaldehyde, cesium carbonate and compound 1 is 1:(2 - 4):(2 - 3); in step (3), the molar ratio of compound 2, bis(pinacolato)diboron and palladium dichloride catalyst is 1:(1 - 3):(1.5 - 2).

[0023] Preferably, the specific preparation process of the fluorescent probe for detecting reactive oxygen species in step (4) is as follows: Under a protective atmosphere, compound 3, 3-ethyl-2-methylbenzothiazolium iodide, and ethanol are successively placed in a reaction flask, and refluxed. After the reaction, the solution is rotary evaporated and purified by column chromatography to obtain the probe. The mass ratio of compound 3 to 3-ethyl-2-methylbenzothiazolium iodide is 1:1 to 3. When eluting with column chromatography, the eluent is methanol:dichloromethane = 1:20 to 50 (volume ratio).

[0024] The application of the above fluorescent probe in the detection of reactive oxygen species for non-diagnostic purposes. The reactive oxygen species refer to H2O2 and ONOO - .

[0025] Furthermore, it is used for the fluorescence detection and visual qualitative detection of the contents of H2O2 and ONOO - .

[0026] All raw materials used in the present invention are ordinary commercially available products, or are obtained by methods known to those skilled in the art or methods disclosed in the prior art.

[0027] The present invention prepared a novel probe TL using cyclohexanone and 3-ethyl-2-methylbenzothiazolium iodide as raw materials, and characterized it by high performance liquid chromatography, ultraviolet-visible spectrophotometer, and fluorescence emission spectrometer; after adding H2O2 to probe TL, the fluorescence intensity increased by nearly 7 times and the color quickly changed from purple to light green. After adding ONOO - to probe TL, the fluorescence intensity increased by nearly 3 times and the color quickly changed from purple to dark blue. The selected interfering ions did not respond to probe TL, realizing the naked-eye rapid and selective detection of H2O2 and ONOO - , and it can be used for the detection of reactive oxygen species. Description of the Drawings

[0028] Figure 1 , Figure 2 is the 1H NMR and 13C NMR spectra of the fluorescent probe TL;

[0029] Figure 3 , Figure 4 is the fluorescence spectrum and ultraviolet absorption spectrum of the fluorescent probe TL in response to H2O2 over time;

[0030] Figure 5 is the fluorescence spectrum of the fluorescent probe TL at different H2O2 concentrations;

[0031] Figure 6 is the linear fitting diagram of the fluorescence emission intensity of the fluorescent probe TL varying with the H2O2 concentration;

[0032] Figure 7 is the ultraviolet absorption spectrum of the fluorescent probe TL at different H2O2 concentrations;

[0033] Figure 8 The fluorescence probe TL with different concentrations of ONOO - Fluorescence spectra change diagrams with different concentrations;

[0034] Figure 9 is the fluorescence probe TL with different concentrations of ONOO - Linear fitting diagrams of fluorescence emission intensities with different concentrations;

[0035] Figure 10 is the fluorescence probe TL with different concentrations of ONOO - Ultraviolet absorption spectra with different concentrations;

[0036] Figure 11 is the fluorescence spectrum diagram of the fluorescence probe TL for different ion selectivities;

[0037] Figure 12 is the bar chart of the fluorescence spectra of the fluorescence probe TL for different ion responses;

[0038] Figure 13 is the ultraviolet absorption spectrum of the fluorescence probe TL for different ion selectivities;

[0039] Figure 14 is the solution color photo taken under a fluorescent lamp after adding 17 different ions to the fluorescence probe TL;

[0040] Figure 15 is the solution color photo taken under a 365nm fluorescent lamp after adding 17 different ions to the fluorescence probe TL. Detailed implementation manners

[0041] The present invention will be further described in detail below through preferred embodiments, but the protection scope of the present invention is not limited thereto.

[0042] Example 1

[0043] (1) Preparation of Compound 1

[0044] The synthesis route is as follows:

[0045]

[0046] A constant pressure dropping funnel, a nitrogen balloon and a rubber stopper were respectively installed on a 250 mL three-necked flask. 11.7 mL (0.15 mol) of N,N-dimethylformamide / 50 mL of chloroform and 1 magnetic stir bar were added in sequence. 13.8 mL (0.147 mol) of phosphorus tribromide was added to the constant pressure dropping funnel, sealed, protected by nitrogen, and placed in an ice bath for stirring reaction for 1 h. After the reaction was completed, 5.3 mL of cyclohexanone (0.05 mol) was slowly added with a syringe, and stirred at room temperature overnight. The product was poured into a beaker containing 300 mL of water. After adjusting the reaction solution to neutral with solid sodium bicarbonate, the solution was poured into a separatory funnel, extracted twice with dichloromethane, washed three times with saturated sodium chloride solution, dried with anhydrous magnesium sulfate for half an hour, and the dried solution was poured into a 50 mL distillation flask. The remaining dichloromethane was removed by rotary evaporation, and the remaining DMF was removed under reduced pressure to obtain a dark brown oily liquid. 2.00 g of compound 1 was obtained with a yield of 78%.

[0047] (2) Preparation of compound 2

[0048] The synthetic route is as follows:

[0049]

[0050] 50.0 mL of N,N-dimethylformamide, 2.00 g (0.01 mol) of 4-bromo-2-hydroxybenzaldehyde, 9.50 g (0.03 mol) of cesium carbonate, 1.5 g of compound 1 (0.02 mol) and 1 magnetic stir bar were added in sequence to a 100 mL round-bottomed flask, protected by nitrogen, treated in the dark, and stirred at room temperature for 48 h. After the reaction was completed, the product was transferred to a separatory funnel, washed with ethyl acetate, the upper organic layer was separated, dried with anhydrous magnesium sulfate, and the remaining solvent was removed under reduced pressure to obtain the crude product of compound 2. The crude product of compound 2 was further separated by column chromatography with the eluent of petroleum ether:ethyl acetate = 10:1 (volume ratio). The solvent was removed by rotary evaporation to obtain 1.73 g of yellow granular solid compound 2 with a yield of 67%.

[0051] (3) Synthesis of compound 3

[0052] The synthetic route is as follows:

[0053]

[0054] In a dry two-necked flask, 0.06 g (0.2 mmol) of compound 2, 0.10 g (0.4 mmol) of bis(pinacolato)diboron, 0.06 g (0.34 mmol) of palladium dichloride catalyst, 8 mL of 1,4-dioxane and 1 magnetic stir bar were added successively. A nitrogen balloon was placed above the reflux condenser to ensure an anhydrous and anaerobic reaction. The mixture was heated under reflux for 3 h, and the solvent was removed by rotary evaporation to obtain the crude product compound 3. The crude product of compound 3 was further separated by column chromatography, and the eluent was petroleum ether:ethyl acetate = 10:1. The solvent was removed by rotary evaporation to obtain yellow solid compound 3 with a mass of 0.04 g and a yield of 54%. 5 mg of compound 3 was taken for 1H NMR scanning.

[0055] 1 H NMR(400MHz,CDCl3)δ10.30(s,1H),7.23(s,2H),7.13(d,J=7.5Hz,1H),6.66(s,1H),2.49(dt,J=12.1,5.8Hz,4H),1.51–1.35(m,1H),1.33–1.16(m,12H).

[0056] (4) Preparation of fluorescent probe TL

[0057] The synthetic route is as follows:

[0058]

[0059] 0.0523 g (0.15 mmol) of compound 3, 0.0651 g (0.2 mmol) of 3-ethyl-2-methylbenzothiazolium iodide and 6 mL of ethanol were weighed successively into a two-necked flask. A reflux condenser was installed, and nitrogen protection was applied. The mixture was heated and stirred under reflux for 3 h. The crude product was further separated by column chromatography, and the eluent was methanol:dichloromethane = 1:30 to obtain 0.03 g of pure probe TL with a yield of 61%. 10 mg of probe TL was taken and dissolved in deuterated chloroform for NMR scanning. The 1H NMR and 13C NMR spectra are as Figure 1 、 2 shown.

[0060] 11H NMR (400 MHz, CDCl3) δ 8.44 (t, J = 18.7 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.66 (s, 1H), 7.60 (t, J = 6.4 Hz, 2H), 7.28 (d, J = 4.5 Hz, 1H), 7.11 (d, J = 14.7 Hz, 1H), 6.93 (s, 1H), 5.10 (dd, J = 14.2, 6.9 Hz, 2H), 2.88 (t, J = 5.7 Hz, 2H), 2.69 (d, J = 5.5 Hz, 2H), 1.95 (d, J = 5.4 Hz, 2H), 1.61 (t, J = 6.9 Hz, 3H), 1.40 (s, 12H). 13 13C NMR (101 MHz, CDCl3) δ 169.76, 158.16, 152.17, 144.07, 141.13, 131.74, 130.88, 129.60, 129.51, 127.69, 126.89, 126.38, 124.09, 123.06, 121.32, 115.57, 114.29, 106.73, 84.45, 45.68, 31.93, 29.70, 25.71, 24.93, 24.88, 22.70, 20.24.

[0061] (5) Application test

[0062] 1) Preparation of stock solutions for detection

[0063] a. Preparation of the stock solution of probe TL: Weigh 0.0025 g of probe TL (M = 513.50 g·mol -1 ) into a 10 mL small brown bottle, and add 5.00 mL of acetonitrile with a pipette to prepare a stock solution of probe TL with a concentration of 1.0×10 -3 mol·L -1 .

[0064] b. Preparation of stock solutions of ions to be measured: Sodium acetate (CH3COO - ) solution, potassium bromide (Br - ) solution, sodium chloride (Cl - ) solution, hydrogen peroxide (H2O2) solution, potassium fluoride (F - ) solution, potassium dihydrogen phosphate (H2PO4 - ) solution, sodium hydrosulfide (HS - ) solution, sodium bisulfate (HSO4 - ) solution, sodium nitrate (NO3 - ) solution, potassium iodide (I- ) Solutions, sodium sulfide (S 2- ) solution, sodium sulfite (SO3 - ) solution, sodium sulfate solution, glutathione (GSH) solution, glycine (Gly) solution, leucine (Leu) solution, peroxynitrous acid (ONOO - ) and other substances were used to prepare an ionic stock solution of 1.0×10 -2 mol·L -1 .

[0065] c. Preparation of PBS buffer solution (0.01 mol·L -1 , pH = 7.4):

[0066] Weigh 4.3 g of sodium chloride, 1.1 g of disodium hydrogen phosphate, and 0.1 g of sodium dihydrogen phosphate, add them to a 500 mL volumetric flask, dissolve with deionized water and make up to the mark. Finally, a PBS buffer solution with pH = 7.4 was prepared.

[0067] 2) Detection and analysis

[0068] Add 3 mL of the system solution (ethanol:PBS = 4:6) and 30 μL of 1.00×10 -3 mol·L -1 probe TL buffer solution to the cuvette. First, scan the fluorescence curve of pure probe TL, then add 30 μL of 1.00×10 -2 mol·L -1 hydrogen peroxide ion solution, mix well, and detect the change of the fluorescence spectrum with time within 15 minutes. The excitation wavelength of fluorescence is 625 nm, the excitation slit width is 5 nm, and the emission slit width is 5 nm for scanning. The results are as Figure 3 shown. The addition of hydrogen peroxide to probe TL immediately caused a decrease in the absorption peak at 675 nm, an increase in the absorption peak at 730 nm, and the highest peak increase at 760 nm; after about 30 minutes, the fluorescence intensity of the reaction system reached the maximum, indicating that probe TL has high sensitivity for the detection of H2O2 ions.

[0069] Add 3 mL of the system solution (ethanol:PBS = 4:6) to the cuvette for baseline correction and zero adjustment, then add 30 μL of 1.00×10 -3 mol·L -1 probe TL stock solution, scan the absorption curve of pure probe TL, then add 30 μL of 1.00×10 -2 mol·L -1 hydrogen peroxide ion solution, stir evenly, scan until the peak no longer changes, and record the time required for the reaction process. Set the scanning range of the UV-visible spectrophotometer to 250 - 800 nm, with an interval of 1 nm, starting fromFigure 4 It can be seen that the ultraviolet absorption is at 710 nm. After adding the probe TL to H2O2, the absorbance of the system at 710 nm increases with time, the absorbance at 575 nm decreases with time, and reaches an equilibrium state at 30 min. The color of the solution changes from purple to light green visibly to the naked eye.

[0070] Take 3 mL of the system solution, add 30 μL of 1.00×10 -3 mol·L -1 probe TL, add different equivalents of aqueous H2O2 solution, and prepare solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, and 260 μL mol·L -1 , shake well, and detect the ultraviolet spectrum and fluorescence spectrum. The results are as Figure 5 、 6 、Figure 7 shows that in the fluorescence spectrum, the fluorescence intensity at 730 nm increases with the increase of H2O2 ion concentration. When the H2O2 ion concentration is 9 Equiv of the probe TL concentration, there is a good linear relationship between the solution fluorescence intensity and the H2O2 concentration. After linear fitting, the expression of the linear equation of one variable is: y = 129.48509 + 22.22485x, and the linear correlation coefficient is R 2 = 0.97346. According to the linear equation, the detection limit of the probe for H2O2 is 5.8 μM. In the ultraviolet spectrum, the absorption peak at 575 nm gradually decreases with the increase of H2O2 ion concentration, and the absorption peak at 710 nm gradually increases with the increase of H2O2 ion concentration;

[0071] Take 3 mL of the system solution, add 30 μL of 1.00×10 -3 mol·L -1 probe TL, add different equivalents of ONOO - aqueous solution, with concentrations of 0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, and 260 μLmol·L -1 , detect the ultraviolet spectrum absorbance and fluorescence spectrum. After adding ONOO - ions, the solution reacts rapidly, and the reaction time is fast. The results are as Figure 8 shown. In the fluorescence spectrum, the absorption peak at 751 nm gradually increases with the increase of ONOO - ion concentration, and the fluorescence intensity of the solution gradually increases. As Figure 9 can be seen, the fluorescence intensity of the solution is related to ONOO -There is a good linear relationship between the concentrations. After linear fitting, the expression of the linear equation of one variable is: y = 80.10714 + 3.110095x, and the linear correlation coefficient is R 2 = 0.9794. According to the linear equation, the detection limit of the probe for ONOO - ions is 1.8 μM. As Figure 10 shown, when the concentration of ONOO - ions gradually increases, the ultraviolet absorption of the solution at 600 nm and 650 nm gradually rises. It can be seen from the intuitive photos taken that when 0.42 Equiv ONOO - ions are added and the reaction is complete, the solution changes from purple to blue and the ultraviolet absorption no longer rises. Under the ultraviolet lamp at 625 nm, the fluorescence color of the solution changes from dark purple to deep purple, and the color change of the solution can be recognized with the naked eye.

[0072] Take 18 portions of 3 mL buffer solution, add 30 μL of the probe TL sample solution (1.00×10 -3 mol·L -1 ), and add 30 μL of 1.00×10 -2 mol·L -1 of ionic solution to 17 of them. (1) Probe, (2) CH3COO - , (3) Br - , (4) Cl - , (5) H2O2, (6) F - , (7) H2PO4 - , (8) HS - , (9) HSO4 - , (10) NO3 - , (11) I - , (12) S 2- , (13) SO3 - , (14) SO4 2- , (15) glutathione (GSH) solution, (16) glycine (Gly) solution, (17) leucine (Leu) solution, (18) ONOO - . Measure the ultraviolet absorption spectrum and fluorescence spectrum, and the results are as shown in Figure 11 , 12 , 13. As can be seen from Figure 11 and 12 , the probe TL has weak fluorescence emission at 763 nm. When H2O2 and ONOO - are added, the fluorescence emission of the probe TL solution is significantly enhanced, while the fluorescence spectrum does not change significantly when other anions are added. Therefore, the probe TL can be used as a highly selective fluorescence probe TL for detecting reactive oxygen species. As can be seen from Figure 13It can be seen from the ultraviolet absorption spectrum that only after adding H2O2 and ONOO - ions, the ultraviolet absorption changes significantly, while adding other ions makes little difference from the pure probe.

[0073] Take 18 portions of 3 mL buffer solution, add 30 μL of the probe TL sample solution (1.00×10 -3 mol·L -1 ), and add 30 μL of 1.00×10 -2 mol·L -1 ionic solution to 17 of them. Conduct color comparison under daylight and 365 fluorescence lamps. From left to right: (1) Probe, (2) 100 μM CH3COO - , (3) 100 μM Br - , (4) 100 μM Cl - , (5) 100 μM H2O2, (6) 100 μM F - , (7) 100 μM H2PO4 - , (8) 100 μM HS - , (9) 100 μM HSO4 - , (10) 100 μM NO3 - , (11) 100 μM I - , (12) 100 μM S 2- , (13) 100 μM SO3 - , (14) 100 μM SO4 2- , (15) 100 μM GSH, (16) 100 μM Gly, (17) 100 μM Leu, (18) 100 μM ONOO - . The results are as shown in Figure 14 、 15 . After adding H2O2 and ONOO - to the probe TL, the color visible to the naked eye and the color under the fluorescence lamp are significantly different from those of other anion solutions. Therefore, this probe TL can be used to detect the presence of reactive oxygen species at a certain concentration with the naked eye.

Claims

1. A fluorescent probe for detecting reactive oxygen species, characterized in that, The structural formula is as follows: 。 2. The preparation method of the fluorescence probe for detecting reactive oxygen species according to claim 1, characterized in that, It includes the following steps: React compound 3 with 3-ethyl-2-methylbenzothiazolium iodide to obtain the probe.

3. The preparation method of the fluorescent probe for detecting reactive oxygen species according to claim 2, characterized in that, The specific preparation process of the probe is as follows: Under a protective atmosphere, compound 3, 3-ethyl-2-methylbenzothiazolium iodide, and ethanol are successively placed in a reaction flask and stirred under reflux. After the reaction, the solution is rotary evaporated and then purified by column chromatography to obtain the probe.

4. The preparation method of the fluorescent probe for detecting reactive oxygen species according to claim 3, characterized in that, The molar ratio of compound 3 to 3-ethyl-2-methylbenzothiazolium iodide is 1:(1~3).

5. Application of the fluorescent probe according to claim 1 in the detection of reactive oxygen species for non-disease diagnosis purposes.

6. The application according to claim 5, characterized in that, The reactive oxygen species referred to herein are H2O2 and ONOO - .

7. The application according to claim 6, characterized in that, For the fluorescence detection and visual qualitative detection of H2O2 and ONOO - contents.

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