A fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in living organisms, and its preparation method and application

Abstract

This invention discloses a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms, its preparation method, and its application. The fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide, with the following structural formula: The invention also specifically discloses the preparation method of this fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms and its application in the rapid and highly selective identification of sulfur dioxide and hydrogen peroxide, as well as in the preparation of imaging detection reagents for hydrogen peroxide and sulfur dioxide in cells and zebrafish. This invention achieves colorimetric detection of sulfur dioxide and hydrogen peroxide, enabling simultaneous detection of sulfur dioxide and hydrogen peroxide, with fast response, low detection limit, and high selectivity. It is also the first time this technology has been used for selective imaging detection of sulfur dioxide and hydrogen peroxide in zebrafish.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes for detecting sulfur dioxide (SO2) and hydrogen peroxide (H2O2), and specifically relates to a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body, and a preparation method and application thereof. Background Art

[0002] Sulfur dioxide (SO2) is a major air pollutant. The inorganic salts sodium bisulfite and sodium sulfite formed by it are also valuable chemical products. According to the information reported in the Journal of Analytical Chemistry (Anal. Chim. Acta, 2022, 1211, 339908, A quinoline-coumarin near-infrared ratiometric fluorescent probe for detection of sulfur dioxide derivatives), excessive inhalation of sulfur dioxide can cause physical discomfort, such as respiratory diseases, nervous system diseases and cardiovascular diseases. After sulfur dioxide dissolves in water, it will be in the form of bisulfite and sulfite (HSO3 - / SO3 2- ) exists in the form of endogenous SO2(HSO3 - , SO3 2- ) has a wide range of balancing effects in the body, regulating endocrine levels, lowering blood pressure, and regulating vasodilation. In contrast, hydrogen peroxide (H2O2) is an important member of reactive oxygen species (ROS) and is considered to be a ubiquitous intracellular messenger at the physiological level, participating in the innate immune system. It is also an important biomolecule involved in host defense, oxidative biosynthetic reactions, metabolism, oxidative stress, and signal transduction. Abnormal production of H2O2 is involved in a variety of diseases, such as cancer, diabetes, neurodegenerative diseases, inflammation, and cardiovascular diseases. It has been demonstrated that SO2 can enhance H2O2 signals by forming sulfoxide peroxide, leading to redox changes in cysteine ​​residues throughout the vascular smooth muscle cell proteome (Redox Biology, 2021, 41, 101898, Endogenous SO2-dependent Smad3 redox modification controls vascular remodeling). When the antioxidant properties of SO2 and the oxidative properties of H2O2 work together to maintain the redox balance in cells, they work synergistically. However, due to the lack of effective tools, the relationship between SO2 and H2O2 in living systems and their relationship with related diseases have not been confirmed.

[0003] So far, many probes that can be used to detect sulfur dioxide and hydrogen peroxide have been proposed, but these publicly available probes can only detect one of sulfur dioxide or hydrogen peroxide alone ((1) Chem. Commun., 202056, 10549, A turn-on TADF chemosensor for sulfite with a microsecond-scale luminescence lifetime, (2) Talanta, 2021, 235, 122719, A dual lock-and-key two photon fluorescence probe in response to hydrogen peroxide and viscosity: Application in cellular imaging and inflammation therapy.), 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 quickly and selectively identify sulfur dioxide and hydrogen peroxide. Summary of the invention

[0004] The purpose of the present invention is to provide a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms and a preparation method thereof. The fluorescent probe prepared by the method can be used for rapid and highly selective identification of sulfur dioxide and hydrogen peroxide and for biological imaging.

[0005] The present invention adopts the following technical scheme to achieve the above-mentioned purpose: a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body, characterized in that: the fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI), and its structural formula is

[0006]

[0007] The method for preparing a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body according to the present invention is characterized in that the specific process comprises: using acetic anhydride as a reaction medium, using 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide, 4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenoxy)benzaldehyde and sodium acetate as reaction raw materials, reacting at 60-80°C under the protection of an inert gas, removing acetic anhydride by suction filtration after the reaction is complete, and then recrystallizing in acetonitrile to obtain a target product, which is a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body.

[0008] It is further defined that the molar ratio of 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide, 4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenoxy)benzaldehyde and sodium acetate is 1:1:1.

[0009] The synthetic route in the method for preparing the fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms of the present invention is:

[0010]

[0011] The fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body of the present invention is used in the simultaneous rapid and highly selective identification of sulfur dioxide and hydrogen peroxide. The fluorescent probe is dark yellow in a mixed solution of phosphate buffer and dimethyl sulfoxide in a volume ratio of 9:1, and has no fluorescence under a 365nm portable ultraviolet lamp. After sulfur dioxide is added to the mixed system, the color of the mixed system changes to colorless, and exhibits bright blue fluorescence under a 365nm portable ultraviolet lamp. After hydrogen peroxide is added to the mixed system, the mixed system changes from light yellow to plum red, and exhibits red fluorescence under a 365nm portable ultraviolet lamp.

[0012] The invention discloses an application of the fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in an organism in the preparation of a detection reagent for imaging hydrogen peroxide and sulfur dioxide in cells or zebrafish.

[0013] Compared with the existing fluorescent probe technology for detecting sulfur dioxide and hydrogen peroxide, the fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body 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 fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body prepared by the present invention can realize colorimetric detection of sulfur dioxide and hydrogen peroxide, and realizes the simultaneous detection of sulfur dioxide and hydrogen peroxide by a single fluorescent probe for the first time, and has fast response, low detection limit and high selectivity. It is also the first time to realize selective imaging detection of sulfur dioxide and hydrogen peroxide in a living body. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 These are the fluorescence emission spectra of the fluorescent probe BBI prepared in Example 1 reacting with different equivalents of sulfur dioxide (NaHSO3 was added as a substitute) in a solution.

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

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

[0018] Figure 5 This is a cell imaging picture of the fluorescent probe BBI prepared in Example 1 reacting with sulfur dioxide and hydrogen peroxide in cells.

[0019] Figure 6 This is a zebrafish imaging picture of the fluorescent probe BBI prepared in Example 1 reacting with sulfur dioxide and hydrogen peroxide in zebrafish. Specific implementation plan

[0020] The following is a further detailed description of the preparation method and specific application of the fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms of the present invention through specific examples.

[0021] Example 1

[0022] Synthesis of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)phenylethylene)-1H-benzo[e]indole-3-iodide (BBI)

[0023] Fluorescent probe compound The probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) was reacted by the following synthetic route:

[0024]

[0025] Under argon protection, 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide (236 mg, 0.65 mmol) and 4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenoxy)benzaldehyde (219 mg, 0.65 mmol) were added to a single-necked bottle, and sodium acetate (53 mg, 0.65 mmol) and acetic anhydride 5 mL were added. The mixture was reacted at 70 ° 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 dark red solid product with a yield of 80%.

[0026] The brick-red solid product prepared in this example has a hydrogen nuclear magnetic resonance spectrum:1 H NMR (400MHz, CDCl3, 25℃, TMS): δ = 8.28-8.32 (m, 3H, Ar-H), 8.21 (d, J = 8.1Hz, 1H, Ar-H), 8.12 (d, J= 8.9Hz,1H,Ar-H),8.06(d,J=8.1Hz,1H,Ar-H),7.84(d,J=8.1Hz,2H,Ar-H),7.72-7.78(m,3H,Ar-H) ,7.64-7.68(m,1H,Ar-H),7.43(d,J=8.0Hz,2H,Ar-H),7.13(d,J=8.8Hz,2H,Ar-H),5.17(s,2H,CH 2), 5.12 (q, J = 7.2Hz, 2H, CH2), 2.10 (s, 6H, CH3), 1.67 (t, J = 7.2Hz, 3H, CH3), 1.35 (s, 12H, CH3) ppm.

[0027] Its carbon NMR spectrum is: 13 C NMR (100MHz, CDCl3, 25℃, TMS): δ=181.7,164.1,153.8,138.8,138.3,137.8,135.2,134.2,133.7,131.8,130.4,12 8.6,127.4,127.4,127.0,126.6,122.7,122.6,116.2,112.1,109.5,83.9,70.4,53.7,44.3,27.0,24.9,14.4ppm.

[0028] Its high resolution mass spectrum is: HRMS (ESI) m / z calcd for C 37 H 41 BNO3 + ,Exact Mass:558.3174,([MI - ]),found 558.3176.

[0029] This proved that the obtained dark red solid product was (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI), and its structural formula is

[0030] Example 2

[0031] Fluorescence response of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)phenylvinyl)-1H-benzo[e]indole-3-iodide (BBI) to sulfur dioxide and hydrogen peroxide

[0032] The fluorescent probe BBI prepared in Example 1 was dissolved in a mixed solution of 0.1 M pH 7.4 phosphate buffer and DMSO, wherein the volume ratio of the phosphate buffer to DMSO was 9:1, and finally a test solution with a concentration of 10 μM fluorescent probe BBI was obtained. Figure 1 As shown in the figure, after adding 2 times the equivalent of sodium bisulfite solution and 50 times the equivalent of hydrogen peroxide solution, respectively, the fluorescent probe was excited at 333nm and 470nm, 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 of sodium bisulfite were added to the test solution with a concentration of 10 μM fluorescent probe BBI, and the fluorescence intensity of the test solution at 470 nm was observed to gradually increase under 333 nm excitation. Figure 3 This is the fluorescence spectrum after adding 0-50 times equivalent hydrogen peroxide solution to the test solution with a concentration of 10 μM fluorescent probe BBI. The fluorescence intensity of the solution at 574 nm after adding different concentrations of hydrogen peroxide increases with the increase of hydrogen peroxide concentration when excited at 470 nm. Figure 2 and Figure 3 It is shown that the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) prepared by the present invention can realize the recognition of sulfur dioxide and hydrogen peroxide.

[0033] Example 3

[0034] Anti-interference detection of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)phenylvinyl)-1H-benzo[e]indole-3-iodide (BBI)

[0035] To the test solution of the fluorescent probe BBI prepared in the above embodiment of the present invention with a concentration of 10 μM, interfering reactive oxygen compounds (·OH, NO·, ·O2 - 、HClO、 1 O2, t-BuO·, ONOO - ) to a concentration of 400 μM, various metal cations (Mg2+ , Ca 2+ , Fe 2+ 、Na + ) and various nucleophilic anions (SO4 2- , Cl - 、CO3 2- 、NO2 - ,AcO - , HS - ) to a concentration of 50 μM and biothiols (Cys, Hcy, GSH) to a concentration of 50 μM; 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 from 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 using cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH). When the mixed solution was excited at 333 nm, it was found that the fluorescent probe BBI would have obvious fluorescence at 470 nm only after reacting with sodium bisulfite, while when the solution was excited at 470 nm, the fluorescent probe BBI would have obvious fluorescence at 574 nm only after reacting with hydrogen peroxide. The above data indicate that the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) has a good selective detection effect on sodium bisulfite and hydrogen peroxide.

[0036] Example 4

[0037] Application of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)phenylvinyl)-1H-benzo[e]indole-3-iodide (BBI) in cell imaging

[0038] 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. 4 The density of cells was seeded on a 14 mm glass coverslip. After reaching 70% confluence, the fluorescent probe BBI was added to the culture medium at a concentration of 10 μM and cultured for 30 min, and 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. Rinse with PBS three times, then seal the cells with a sealant, and image them on a confocal microscope system.

[0039] Figure 5 The imaging results of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) prepared by the present invention on sodium bisulfite or hydrogen peroxide are given. Figure 5 It can be seen in the first row that the fluorescent probe BBI has no obvious fluorescence in the blue channel and the red channel; in the second row where sulfur dioxide is added, it can be found that the fluorescent probe BBI emits obvious blue fluorescence in the blue channel after being co-cultured with sulfur dioxide for 2 hours, while there is no obvious fluorescence in the red channel; in the third row after the fluorescent probe BBI reacts with hydrogen peroxide, it can be seen 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-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benz[e]indole-3-iodide (BBI) prepared by the present invention can well realize the selective imaging detection of sulfur dioxide and hydrogen peroxide in cells.

[0040] Example 5

[0041] Imaging application of fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)phenylvinyl)-1H-benzo[e]indole-3-iodide (BBI) in zebrafish

[0042] Three-day-old zebrafish were cultured in fresh E3 medium containing the fluorescent probe BBI for 30 minutes, then washed three times with E3 medium, and then sodium bisulfite or hydrogen peroxide was added and cultured for another 2 hours before imaging.

[0043] Figure 6 The imaging results of the fluorescent probe compound (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) prepared by the present invention on sodium bisulfite or hydrogen peroxide are given. Figure 6 In the first row, it can be seen that the zebrafish cultured with the fluorescent probe BBI 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 BBI emits obvious blue fluorescence in the blue channel after being co-cultured with sulfur dioxide for 30 minutes, while there is no obvious fluorescence in the red channel; in the third row after the fluorescent probe BBI reacts with hydrogen peroxide, it can be seen 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-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide (BBI) prepared by the present invention can well realize the dual-channel imaging detection of sulfur dioxide and hydrogen peroxide in the organism zebrafish.

[0044] As can be seen from the above, compared with the existing probe technology in the field of hydrogen peroxide and sulfur dioxide detection, the target compound of the present invention (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benz[e]indole-3-iodide (BBI) can well realize the recognition and detection characteristics of two detection objects by one molecule, and shows good selectivity, realizing the dual-channel imaging detection of bisulfite and hydrogen peroxide in cells and zebrafish for the first time.

[0045] 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 fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms, characterized in that: The fluorescent probe is (E)-3-ethyl-1,1-dimethyl-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)benzyl)oxy)styrene)-1H-benzo[e]indole-3-iodide, and its structural formula is The fluorescent probe is used for selectively identifying sulfur dioxide and hydrogen peroxide at the same time, and the specific process is as follows: the fluorescent probe appears dark yellow in a mixed solution of phosphate buffer and dimethyl sulfoxide in a volume ratio of 9:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after sulfur dioxide is added to the mixed system, the color of the mixed system changes to colorless, and shows bright blue fluorescence under a 365nm portable ultraviolet lamp; and after hydrogen peroxide is added to the mixed system, the mixed system changes from light yellow to rose red, and shows red fluorescence under a 365nm portable ultraviolet lamp.

2. A method for preparing a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body 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-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenoxy)benzaldehyde and sodium acetate as reaction raw materials, reacting at 60-80°C under the protection of inert gas, and filtering out acetic anhydride after the reaction is complete, and then recrystallizing in acetonitrile to obtain the target product, which is a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in organisms.

3. The method for preparing a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body according to claim 2, characterized in that: The molar ratio of 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-iodide, 4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxolan-2-yl)phenoxy)benzaldehyde and sodium acetate is 1:1:

1.

4. The method for preparing a fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in a living body according to claim 2, characterized in that The synthetic route is:

5. Use of the fluorescent probe for imaging sulfur dioxide and hydrogen peroxide in an organism as claimed in claim 1 in preparing a detection reagent for imaging hydrogen peroxide and sulfur dioxide in cells or zebrafish.

Images