A fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide and its preparation method and application

Abstract

The invention discloses a fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide, a preparation method and an application thereof, wherein the fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-benz[e]indole-3-iodide, and its structural formula is The invention also specifically discloses a preparation method of the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide, and its application in simultaneously and rapidly and highly selectively identifying sodium bisulfite and hydrogen peroxide, and in preparing a dual-channel imaging detection drug for hydrogen peroxide and sodium bisulfite in cells. The invention realizes colorimetric detection of sodium bisulfite and hydrogen peroxide, realizes simultaneous detection of sodium bisulfite and hydrogen peroxide for the first time, and has fast response, low detection limit and high selectivity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes for detecting sodium bisulfite (NaHSO3) and hydrogen peroxide (H2O2), and specifically relates to a fluorescent probe for detecting sodium bisulfite (NaHSO3) and hydrogen peroxide (H2O2) with dual channels, and a preparation method and application thereof. Background Art

[0002] Sulfur dioxide (SO2) is both a major atmospheric pollutant and a valuable commercial reagent. Exposure to sulfur dioxide gas may not only cause respiratory reactions, but may also lead to lung cancer, cardiovascular disease, and some neurological diseases such as migraine and brain cancer. Sulfur dioxide dissolves in water to form bisulfite and sulfite (HSO3 - / SO3 2- According to the information recorded in the European Journal of Pharmacology ((1) Eur. J. Pharmacol. 2010, 645, 143, Sulfur dioxide upregulates the aortic nitric oxide pathway in rats; (2) Eur. J. Pharmacol. 2011, 670, 1, The biological effect of endogenous sulfur dioxide in the cardiovascular system.), HSO3 - / SO3 2- At low concentrations, it has an endothelial vasodilation effect, and hydrogen peroxide (H2O2) is an important member of reactive oxygen species (ROS) and is involved in the innate immune system. According to Analytical Chemistry (Anal. Chem. 2022, 94, 15, 5962, Target-Binding Accelerated Response for Sensitive Detection of Basal H2O2in Tumor Cells and Tissues via a Dual-Functional Fluorescence Probe), abnormal production of hydrogen peroxide is involved in various diseases such as cancer, diabetes, neurodegenerative diseases, inflammation, and cardiovascular diseases. The level of hydrogen peroxide in tumor cells (5μM to 1.0mM) is significantly higher than that in normal cells (less than 0.7μM). Recent studies have found that hydrogen peroxide plays an important role in the occurrence, growth, and apoptosis of tumors. Therefore, the detection of sodium bisulfite and hydrogen peroxide has attracted much attention.

[0003] So far, many probes that can be used to detect sodium bisulfite and hydrogen peroxide alone have been proposed, but these publicly available probes can only detect one of sodium bisulfite or hydrogen peroxide alone ((1) Sens. Actuat. B: Chem. 2022, 366, 131962, A reversible NIR fluorescent probe for monitoring of SO2 and formaldehyde in live cells and zebrafish; (2) Anal. Chim. Acta. 2022, 1214, 339939, Attractive benzothiazole-based fluorescence probe for the highly efficient detection of hydrogen peroxide.), and there are almost no probes that can detect both at the same time. Therefore, it is of practical research significance and application value to develop a fluorescent probe that can simultaneously and rapidly and highly selectively identify sodium bisulfite and hydrogen peroxide. Summary of the invention

[0004] The purpose of the present invention is to provide a dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide and a preparation method thereof. The fluorescent probe prepared by the method can be used to simultaneously and rapidly and highly selectively identify sodium bisulfite and hydrogen peroxide.

[0005] The present invention adopts the following technical scheme to achieve the above-mentioned purpose: a dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide, characterized in that: the fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide, whose structural formula is

[0006]

[0007] The preparation method of the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide of the present invention is characterized by the specific process of: taking acetic anhydride as a reaction medium, taking 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide, 4-formylphenylboronic acid pinacol ester and sodium acetate as reaction raw materials, reacting at 50-70°C under the protection of inert gas, filtering off acetic anhydride after the reaction is complete, and then recrystallizing in acetonitrile to obtain the target product, which is the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide.

[0008] It is further defined that the molar ratio of the 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide to 4-formylphenylboronic acid pinacol ester to sodium acetate is 1:1.05:1.

[0009] The synthesis route in the preparation method of the dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide of the present invention is:

[0010]

[0011] The invention discloses an application of a dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide in simultaneously and rapidly identifying sodium bisulfite and hydrogen peroxide with high selectivity. The dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide presents a light yellow color in a mixed solution of a phosphate buffer solution and dimethyl sulfoxide in a volume ratio of 9:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after sodium bisulfite is added to the mixed solution, the color of the solution changes to colorless, and exhibits blue fluorescence under a 365nm portable ultraviolet lamp; after hydrogen peroxide is added to the mixed solution, the solution changes from light yellow to plum red, and exhibits red fluorescence under a 365nm portable ultraviolet lamp.

[0012] The invention discloses an application of a fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide in the preparation of a dual-channel imaging detection drug for hydrogen peroxide and sodium bisulfite in cells.

[0013] Compared with the existing fluorescent probe technology for detecting sodium bisulfite and hydrogen peroxide, the dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide prepared by the present invention has a simple preparation process, can be obtained by only one-step reaction, has mild reaction conditions, is simple to operate, and does not require column chromatography. The dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide prepared by the present invention can realize colorimetric detection of sodium bisulfite and hydrogen peroxide, realizes simultaneous detection of sodium bisulfite and hydrogen peroxide for the first time, and has fast response, low detection limit and high selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the fluorescence emission spectrum of the fluorescent probe BI prepared in Example 1 reacting with sodium bisulfite and hydrogen peroxide in solution respectively.

[0015] Figure 2 These are the fluorescence emission spectra of the fluorescent probe BI prepared in Example 1 reacting with different equivalents of sodium bisulfite in a solution.

[0016] Figure 3 These are the fluorescence emission spectra of the fluorescent probe BI prepared in Example 1 reacting with different equivalents of hydrogen peroxide in a solution.

[0017] Figure 4It is a histogram of the fluorescence intensity of the fluorescent probe BI prepared in Example 1 reacting with different interfering ions in solution.

[0018] Figure 5 This is a cell imaging picture of the fluorescent probe BI prepared in Example 1 reacting with sodium bisulfite and hydrogen peroxide in cells. Specific implementation plan

[0019] The following is a further detailed description of the preparation method and specific application of the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide of the present invention through specific examples.

[0020] Example 1

[0021] Synthesis of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide

[0022] Fluorescent probe compound The probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide was reacted by the following synthetic route:

[0023]

[0024] Under nitrogen protection, 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide (236 mg, 0.65 mmol) and 4-formylphenylboronic acid pinacol ester (158 mg, 0.68 mmol) were added to a single-mouth bottle, and then sodium acetate (53 mg, 0.65 mmol) and 5 mL of acetic anhydride were added. The mixture was reacted at 60 ° C for 12 hours. After the reaction was completed, it was cooled to room temperature and filtered to obtain a crude product. It was then recrystallized from acetonitrile to obtain 308 mg of a brick-red solid product with a yield of 82%.

[0025] The brick-red solid product prepared in this example has a hydrogen nuclear magnetic resonance spectrum: 1HNMR (400MHz, CDCl3, TMS): δ = 8.28 (d, J = 16.3Hz, 1H, = CH), 8.22 (d, J = 8.4Hz, 1H, Ar-H) ,8.10-8.13(m,3H,Ar-H),8.06(d,J=8.1Hz,1H,Ar-H),7.96(d,J=7.9Hz,2H,Ar-H),7.8 0-7.86(m,2H,Ar-H),7.75(t,J=7.1Hz,1H,Ar-H),7.67(t,J=7.4Hz,1H,Ar-H),5.19(q, J=7.2Hz,2H,CH2),2.10(s,6H,CH3),1.68(t,J=7.2Hz,3H,CH3),1.35(s,12H,CH3)ppm.

[0026] Its carbon NMR spectrum is: 1 H NMR (400MHz, CDCl3, TMS): δ=182.0,152.9,139.0,137.8,135.8,135.7,135.2,134.0,132.0, 130.4,129.8,128.7,127.7,127.3,122.8,113.0,112.7,84.3,54.3,45.3,26.7,24.9,14.6.

[0027] Its high resolution mass spectrum is: HRMS (ESI) m / z: [MI] + calcd.For C 30 H 35 BNO2 + :452.2755;found:452.2750.

[0028] This proves that the brick-red solid product obtained is (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI), and its structural formula is

[0029] Example 2

[0030] Fluorescence response of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benz[e]indole-3-iodide (BI) to sodium bisulfite and hydrogen peroxide.

[0031] The fluorescent probe BI prepared in Example 1 was dissolved in a mixed solution of 0.1 M pH 7.4 phosphate buffer and DMSO in a volume ratio of 9:1 to obtain a test solution with a concentration of 10 μM fluorescent probe BI. Figure 1 As shown in the figure, after adding 2 times the equivalent of sodium bisulfite solution and 40 times the equivalent of hydrogen peroxide solution, respectively, the fluorescent probe was excited at 333nm and 452nm, and it was found that the fluorescence of the fluorescent probe was obviously turned on at 470nm and 574nm. Figure 2 As shown, 0-2 equivalents (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0) of sodium bisulfite were added to the test solution with a concentration of 10 μM fluorescent probe BI, and the fluorescence intensity of the test solution at 470 nm was observed to gradually increase under 33 nm excitation. Figure 3 It is the fluorescence spectrum after adding 0-40 equivalents (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50) of hydrogen peroxide solution to the test solution with a concentration of 10 μM fluorescent probe BI. The fluorescence intensity at 574 nm of the solution after adding different concentrations of hydrogen peroxide increases with the increase of hydrogen peroxide concentration under excitation at 452 nm. Figure 2 and Figure 3 It is shown that the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI) prepared by the present invention can realize dual-channel recognition of sodium bisulfite and hydrogen peroxide.

[0032] Example 3

[0033] Anti-interference detection of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI)

[0034] 400 μM of active oxygen compounds (·OH, NO·, ·O2) were added to the test solution with a concentration of 10 μM of the fluorescent probe BI prepared in the above embodiment of the present invention. - 、HClO、 1 O2, t-BuO·, ONOO - ), 50 μM of various metal cations (Mg 2+ , Ca 2+ , Fe 2+ 、Na + ) and various anions (SO4 2- , Cl- 、CO3 2- 、NO2 - ,AcO - , HS - ) and 50 μM biothiols (Cys, Hcy, GSH); hydroxyl radicals (·OH) were prepared from hydrogen peroxide and ferrous sulfate; nitric oxide (NO·) was obtained from sodium nitroprusside; superoxide anions (·O2 - ) is obtained by dissolving potassium superoxide in DMSO; hypochlorous acid (HClO) is prepared from sodium hypochlorite; singlet oxygen ( 1 O2) is obtained by the reaction of hydrogen peroxide and sodium hypochlorite; tert-butyloxy radical (t-BuO·) is obtained by the reaction of tert-butyl peroxide and ferrous sulfate; peroxynitrosyl anion (ONOO - ) is obtained by the reaction of sodium nitrite, hydrogen peroxide and hydrochloric acid; various metal cations and anions are prepared by magnesium chloride (MgCl2), calcium chloride (CaCl2), ferrous sulfate (FeSO4), sodium carbonate (NaCO3), sodium nitrite (NaNO2), sodium acetate (AcONa), sodium sulfide (Na2S), and biothiols are directly prepared with cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH). When the mixed solution is excited at 452nm, the fluorescent probe BI will have obvious fluorescence at 574nm only after reacting with hydrogen peroxide, while when the solution is excited at 333nm, it is found that the fluorescent probe BI will have obvious fluorescence at 470nm only after reacting with sodium bisulfite. The above data show that the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI) has a good selective detection effect on hydrogen peroxide and sodium bisulfite.

[0035] Example 4

[0036] Application of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI) in cell imaging

[0037] Hela cells were cultured in DMEM high glucose medium supplemented with 100 U / mL penicillin-streptomycin. The cell culture medium was placed in a humidified incubator at 37°C containing 95% air and 5% CO2. Hela cells were cultured at 5×10 4The cells were seeded on a 14 mm glass coverslip at a density of 10 cells / mL. After reaching 70% confluence, 10 μM fluorescent probe BI was added to the culture medium and cultured for 30 min, then washed three times with phosphate buffered saline (PBS, pH = 7.4). Thereafter, NaHSO3 and H2O2 diluted in PBS were added to the cells, respectively, and incubated at 37°C for 2 hours. The cells were then rinsed three times with PBS, sealed with antifade sealing agent, and imaged using a confocal microscope system.

[0038] Figure 5 The imaging results of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide (BI) prepared by the present invention on NaHSO3 and H2O2 are given. Figure 5 In the first row, we can see that the fluorescent probe BI has no obvious fluorescence in the blue channel and the red channel; in the second row after adding sodium bisulfite, it can be found that the fluorescent probe BI emits obvious blue fluorescence in the blue channel after being co-cultured with sodium bisulfite for 2 hours, while there is no obvious fluorescence in the red channel; in the third row after the fluorescent probe BI reacts with hydrogen peroxide, we can see that there is obvious red fluorescence in the red channel, while there is no obvious fluorescence in the blue channel. Therefore, the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benz[e]indole-3-iodide (BI) prepared by the present invention can well realize the imaging of sodium bisulfite and hydrogen peroxide in cells.

[0039] As can be seen from the above, compared with the existing probe technology in the field of hydrogen peroxide and sodium bisulfite detection, the target compound (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benz[e]indole-3-iodide (BI) of the present invention can well realize the characteristics of dual-channel detection of two detection objects by one molecule, and show good selectivity, realizing the dual-channel imaging of hydrogen peroxide and sodium bisulfite in cells for the first time.

[0040] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide, characterized in that: The fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylvinyl)-1H-benzo[e]indole-3-iodide, and its structural formula is 2. A method for preparing a dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide according to claim 1, characterized in that The specific process is: using acetic anhydride as the reaction medium, 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide, 4-formylphenylboronic acid pinacol ester and sodium acetate as reaction raw materials, reacting at 50-70°C under the protection of inert gas, and after the reaction is complete, filtering out acetic anhydride, and then recrystallizing in acetonitrile to obtain the target product, a dual-channel fluorescent probe for detecting sodium bisulfite and hydrogen peroxide.

3. The method for preparing the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide according to claim 2, characterized in that: The molar ratio of the 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide to 4-formylphenylboronic acid pinacol ester to sodium acetate is 1:1.05:

1.

4. The method for preparing a fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide according to claim 2, characterized in that The synthetic route is:

5. Use of the fluorescent probe for dual-channel detection of sodium bisulfite and hydrogen peroxide according to claim 1 in the preparation of drugs for dual-channel imaging detection of hydrogen peroxide and sodium bisulfite in cells.

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