A novel bodipy-based near-infrared ratio fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid and a preparation method and application thereof

Abstract

The application discloses a novel BODIPY-based near-infrared ratio fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid and a preparation method and application thereof. The fluorescent probe is 4-((4-(3,7-di(2-(10-butyl-10H-phenothiazine-3-yl)vinyl)-1,9-dimethyl-5H-4-(dipyrromethene difluoroboron-10-yl)phenoxy)methyl)benzenesulfonamide, which is abbreviated as PTB-BSA. The probe PTB-BSA can specifically recognize hypochlorous acid, and the color of the probe PTB-BSA solution added with hypochlorous acid rapidly changes from green to dark blue under sunlight. In addition, the fluorescence emission spectrum of the probe PTB-BSA is obviously blue-shifted, and the fluorescence emission wavelength is shifted from 752 nm to 681 nm. The linear range of the probe for hypochlorous acid is 0-60 muM, the detection limit is as low as 35 nM, and the response time is within 10 s. The probe exhibits good selectivity for hypochlorous acid, and is not affected and interfered by other anions, biological thiols and active oxygen. More importantly, the probe can also target the endoplasmic reticulum, and can realize organelle-level detection of the content of hypochlorous acid. This has very important significance for in-depth study on the action process of hypochlorous acid in a biological body.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes and relates to a novel BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid, a preparation method thereof and an application thereof. Background Art

[0002] Reactive oxygen species, as important intracellular signaling molecules, play a vital role in the human body. Hypochlorous acid is produced by the reaction of chloride ions and hydrogen peroxide catalyzed by myeloperoxidase in white blood cells under certain physiological conditions. It is one of the most important reactive oxygen species in biological systems. In human physiological processes, hypochlorous acid can prevent pathogens and bacteria from invading the biological immune system, but excessive hypochlorous acid can cause oxidation of biological molecules such as proteins, nucleic acids, lipids and enzymes in the body, leading to a series of diseases such as tissue damage and inflammation, such as cardiovascular disease, kidney disease, neurodegenerative diseases, and even tumors. Therefore, it is of great significance to develop a method with high selectivity, high sensitivity, and real-time monitoring of hypochlorous acid in the human body and the environment.

[0003] Currently, the main methods for detecting hypochlorous acid include colorimetry, iodine titration, electrochemistry, and chemiluminescence. Compared to these detection methods, fluorescent probes offer advantages such as rapid detection, high sensitivity, good selectivity, and simple operation, making them widely used in fields such as life sciences, environmental science, and food science. However, the subcellular distribution of hypochlorous acid remains unclear, and its intracellular functions remain elusive. Therefore, it is necessary to develop fluorescent probes capable of detecting the subcellular distribution of hypochlorous acid. Although various fluorescent probes capable of imaging hypochlorous acid fluorescence in subcellular organelles have been reported, these organelle-targeted fluorescent probes mostly emit in the visible light region and produce only a single emission signal, which limits their application in biological systems. Therefore, the design of a near-infrared ratiometric fluorescent probe for the targeted detection of hypochlorous acid in the endoplasmic reticulum is particularly important. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a new BODIPY-based near-infrared ratiometric fluorescent probe for the targeted detection of hypochlorous acid in the endoplasmic reticulum. The fluorescent probe has a long wavelength (in the near-infrared region), high sensitivity and high selectivity for detecting hypochlorous acid.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A novel BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid, characterized in that the probe is 4-((4-(3,7-di(2-(10-butyl-10H-phenothiazine-3-yl)vinyl)-1,9-dimethyl-5H-4-(dipyrroledifluoroborane-10-yl)phenoxy) methyl)benzenesulfonamide, abbreviated as PTB-BSA, molecular formula: C 60 H 56 BF2N5O3S3, structural formula:

[0007]

[0008] The synthetic route of the novel BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid is as follows:

[0009]

[0010] Specifically includes the following steps:

[0011] 1) Dissolve 0.5-2.5 mmol of compound B-OH (0.17-0.85 g), 1.5-7.5 mmol of 10-butyl-10H-phenothiazine-3-formaldehyde (0.42-2.2 g), and 1.5-7.5 mmol of p-toluenesulfonic acid in 30-60 mL of anhydrous toluene, gradually add 0.5-2.0 mL of piperidine, and reflux the reaction mixture under stirring for 5-7 h (TLC monitoring). After washing with water, drying, and distillation, the obtained crude product is purified by silica gel column (petroleum ether: dichloromethane = 5:1, v / v) to obtain green solid compound PTB-OH.

[0012] 2) Dissolve 1.0 mmol of PTB-OH (0.87 g), 1.0-3.0 mmol of 4-(bromomethyl)benzenesulfonamide (0.25-0.75 g), and 2.0-4.0 mmol of anhydrous potassium carbonate (0.27-0.52 g) in 5-15 mL of DMF, and stir the reaction mixture at 80°C for 6 h (TLC monitoring). Add 30-40 mL of dichloromethane to the reaction solution, then wash with distilled water, dry, and distill the organic phase. The obtained crude product is purified by silica gel column (petroleum ether: dichloromethane = 1:1, v / v) to obtain compound PTB-BSA as a dark green solid.

[0013] The probe PTB-BSA can selectively react with hypochlorous acid. The color of the probe PTB-BSA solution added with hypochlorous acid rapidly changes from green to dark blue under sunlight. In addition, the fluorescence emission spectrum of the probe PTB-BSA obviously blue shifts, and the fluorescence emission wavelength shifts from 752 nm to 681 nm. The linear range for hypochlorous acid is 0-60 μM, the detection limit is as low as 35 nM, and the response time is within 10 s.

[0014] Beneficial effects: Compared with the prior art, the BODIPY-based endoplasmic reticulum-targeting near-infrared ratio fluorescent probe PTB-BSA of the present application can be used for high-sensitivity detection of hypochlorous acid. In the presence of hypochlorous acid, the color of the probe solution rapidly changes from green to dark blue, and the fluorescence emission spectrum obviously blue shifts. Meanwhile, the fluorescent probe responds to hypochlorous acid rapidly. The fluorescent probe exhibits good selectivity to hypochlorous acid and is not affected by other active oxygen, active sulfur and anions. PTB-BSA as a fluorescent probe for detecting hypochlorous acid has many advantages such as convenient synthesis, rapid response, high sensitivity, good selectivity, long emission wavelength and the like, and has good application value. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a fluorescence spectrum diagram of the fluorescence intensity of PTB-BSA after reacting with different concentrations of hypochlorous acid at 650-850 nm;

[0016] Figure 2 is a linear relationship diagram of the change of the fluorescence intensity ratio (fluorescence intensity at 681 nm / fluorescence intensity at 752 nm) of PTB-BSA after reacting with different concentrations of hypochlorous acid;

[0017] Figure 3 is a change of the ultraviolet absorption spectrum of PTB-BSA after reacting with different concentrations of hypochlorous acid;

[0018] Figure 4 is a fluorescence columnar diagram of the change of the fluorescence intensity ratio of PTB-BSA after reacting with other interference analytes (a: blank, b: HCO3 - , c: CO3 2- , HSO3 - , SO3 2- , SO4 2- , NO2 - , NO3 - , Cl - , Br - , I - , ClO4 - , AcO - , H2PO4 - , HS - , TBHP, N2H4, 1O2, ONOO-, O2 - H2O2, NO, Cys, Hcy, GSH, HClO);

[0019] Figure 5 is the relationship diagram of the fluorescence intensity at 681 nm and the fluorescence intensity at 752 nm over time after the PTB-BSA reacts with hypochlorous acid;

[0020] Figure 6 is the fluorescence confocal picture of the PTB-BSA and the commercial endoplasmic reticulum green dye co-localization staining experiment. Among them, (a-c) are the fluorescence imaging pictures of the endoplasmic reticulum green localization agent and the probe PTB-BSA co-staining experiment in HepG2 cells; (a) the excitation wavelength is 580 nm, and the emission wavelength scanning range is 650-850 nm; (b) the excitation wavelength is 405 nm, and the emission wavelength scanning range is 420-520 nm; (c) is a bright field picture; (d) is a superimposed picture of (a), (b) and (c); (e) is a fluorescence distribution scatter plot; (f) is a linear area red and green fluorescence position and fluorescence intensity relationship diagram. DETAILED DESCRIPTION

[0021] The application will be further described below in combination with specific examples.

[0022] Example 1

[0023] Preparation of the fluorescence probe PTB-BSA:

[0024] 1) Compound B-OH (0.5 mmol, 0.4 g), 10-butyl-10H-phenothiazine-3-formaldehyde (2.5 mmol, 0.38 g) and p-toluenesulfonic acid (2.5 mmol, 0.43 g) were placed in a three-necked flask with a Dean-Stark device together, and dissolved in 50 mL of anhydrous toluene. Then, piperidine (1 mL) was gradually added, and the reaction mixture was stirred at 120°C under reflux for 6 h. After the reaction, the reaction mixture was subjected to rotary evaporation, washing and drying treatment. The crude product was purified by silica gel column chromatography (PE:DCM=5:1) to obtain a green solid, i.e. the target compound PTB-OH (yield 30%). 1H NMR (600 MHz, DMSO-d6) δ: 9.83 (s, 1H), 7.48-7.43 (m, 4H), 7.39 (d, J = 16.2 Hz, 2H), 7.33 (d, J = 1.9 Hz, 2H), 7.24-7.20 (m, 2H), 7.18-7.13 (m, 4H), 7.07 (dd, J = 16.7, 8.4 Hz, 4H), 6.97 (t, J = 7.5 Hz, 2H), 6.94-6.89 (m, 4H), 3.90 (t, J = 7.1 Hz, 4H), 1.69 (p, J = 7.3 Hz, 4H), 1.48 (s, 6H), 1.42 (q, J = 7.5 Hz, 4H), 0.89 (t, J = 7.4 Hz, 6H); 13 C NMR (150 MHz, DMSO-d6) δ: 158.58, 152.17, 145.94, 144.31, 141.98, 139.23, 135.90, 133.65, 131.09, 129.97, 128.25, 127.64, 127.26, 126.03, 124.25, 123.34, 123.13, 118.54, 116.79, 116.52, 116.45, 46.87, 28.78, 19.85, 19.12, 14.87, 14.08, 14.01; [M+Na] + calcd for C 53 H 49 BF2N4OS2, 893.3307; found, 893.3297.

[0025] 2) PTB-B (1 mmol, 0.4 g), 4-(bromomethyl)benzenesulfonamide (2 mmol, 0.38 g) and anhydrous potassium carbonate (2.5 mmol, 0.34 g) were dissolved in 10 mL of anhydrous DMF, and then the mixture was stirred at 60 °C for 6 h. After the reaction, the reaction mixture was extracted, washed and dried. The crude product was purified by silica gel column chromatography (PE:DCM = 1:1) to obtain a dark green solid, which was the target probe PTB-BSA (yield 25%). 1 H NMR (600 MHz, CDCl3) δ: 1H NMR (600 MHz, DMSO-d6) δ 7.86 (d, J = 7.8 Hz, 2H), 7.69 (dd, J = 20.5, 7.9 Hz, 2H), 7.51 - 7.44 (m, 4H), 7.38 (t, J = 8.4 Hz, 4H), 7.36 - 7.31 (m, 4H), 7.27 - 7.20 (m, 4H), 7.19 - 7.15 (m, 2H), 7.08 (dd, J = 22.7, 8.4 Hz, 4H), 6.98 (t, J = 7.4 Hz, 2H), 6.92 (s, 2H), 5.29 (s, 2H), 3.92 (t, J = 7.1 Hz, 4H), 1.70 (p, J = 7.2 Hz, 4H), 1.48 - 1.38 (m, 10H), 0.90 (t, J = 7.4 Hz, 6H); 13 C NMR (150 MHz, CDC13) δ: 146.01, 144.32, 144.05, 143.47, 142.31, 141.91, 141.18, 137.02, 136.08, 133.51, 131.08, 129.76, 129.54, 128.42, 128.28, 128.07, 127.66, 127.32, 126.27, 126.08, 124.27, 123.38, 123.11, 116.74, 116.61, 116.51, 116.05, 55.38, 46.90, 28.79, 21.38, 19.85, 14.89, 14.10; [M+H]+calcd for C 60 H 56 BF2N5O3S3, 1040.3685; found, 1040.3602.

[0026] Example 2

[0027] PTB-BSA was prepared to 2 x 10 -5 M of PBS buffer solution (containing 30% THF), 10 M sodium hypochlorite solution was diluted with distilled water to concentrations of 0, 2 x 10 -5 , 4 x 10 -5 , 7 x 10 -5 , 10 x 10 -5 , 14 x 10 -5 , 19 x 10 -5 , 24 x 10 -5 , 29 x 10 -5 , 35 x 10 -5 , 41 x 10 -5 M, 50 x 10 -5 M, 60 x 10 -5M solution, add different concentrations of hypochlorous acid solution to the probe solution. Fluorescence emission spectrum of PTB-BSA in the presence of different concentrations of hypochlorous acid was measured on a fluorescence spectrophotometer using fluorescence spectrum titration method, with an excitation wavelength of 580nm and collecting the fluorescence emission peak at 650nm-850nm. Figure 1 It can be seen that with the addition of hypochlorous acid, the emission peak of PTB-BSA at 752nm is greatly weakened, and the emission peak at 681nm is rapidly enhanced. And with the increase of hypochlorous acid concentration, the fluorescence intensity ratio of the probe is also continuously enhanced. Then, the relationship between the fluorescence intensity ratio of the probe (fluorescence intensity at 681nm / fluorescence intensity at 752nm) and the concentration change of hypochlorous acid is linearly fitted to obtain the following: Figure 2 The linear relationship shown (y = 0.042x + 0.637, R 2 =0.991), and further calculation (LOD = 3Sb / m) yielded a detection limit of 35×10 -9 M. Meanwhile, under sunlight, the compound solution changes color from green to dark blue upon encountering hypochlorous acid. Therefore, this probe can be used as a colorimetric ratiometric fluorescent probe for sensitive detection of changes in hypochlorous acid concentration.

[0028] Example 3

[0029] PTB-BSA was prepared into 2×10 -5 M solution, and different active oxygen, active sulfur and other anions were dissolved in the aqueous solution to prepare a concentration of 1×10 -2 M solution. The fluorescence emission spectrum of the probe at 681nm and 752nm was measured in the presence of different active oxygen, active sulfur, cations and anions using a fluorescence spectrophotometer. The results are as follows Figure 4 As shown in Figure 2, only the addition of hypochlorous acid significantly increased the fluorescence intensity ratio of the probe, while the other analytes (blank, HCO3 - , CO3 2- , HSO3 - , S03 2- , SO4 2- , NO2 - , NO3 - , Cl - , Br - , I - , ClO4 - ,ACOO - , H2PO4 - , HS - ,TBHP,N2H4, 1O2, ONOO-, O2-, H2O2, NO, Cys, Hcy, GSH) were observed, the fluorescence intensity ratio of the probe did not change significantly. Thus, the compound can be used as a specific and rapid fluorescence probe for the detection of hypochlorous acid. With the addition of 60 x 10 -5 M hypochlorous acid solution, the fluorescence intensity ratio (681 nm fluorescence intensity / 752 nm fluorescence intensity) of the probe was measured by fluorescence spectroscopy. The results are shown in Figure 5 Figure 6, which shows that the addition of hypochlorous acid can rapidly reduce the fluorescence intensity of the probe at 752 nm within 13 s, and significantly increase the fluorescence intensity at 681 nm within 13 s and remain stable.

[0030] Example 4

[0031] We carried out fluorescence imaging studies of the co-localization of the probe PTB-BSA with the endoplasmic reticulum of HepG2 cells. In a confocal dish, 2 x 10 -5 M of the probe solution was incubated with HepG2 cells for 20 min, and then 0.2 x 10 -5 M of the commercial endoplasmic reticulum dye (ER-Tracker Green) was co-incubated for 20 min. After washing three times with PBS solution, fluorescence imaging was carried out under a confocal microscope, and the results are shown in Figure 6 Figure 7. The image f was obtained by superimposing the red channel image b, the green channel image a, and the bright field image c. The red channel and the green channel had good overlap, and the Pearson correlation coefficients under the two algorithms were both 0.98. Images d and e are scatter plots of the fluorescence distribution, and it can also be seen that the red fluorescence region and the green fluorescence region are highly overlapped. Image g is a position and intensity correlation analysis of the red and green fluorescence intensity of the linear region, and it was found that the change trends of the two were completely consistent. The above results show that PTB-BSA is a lysosome-targeting probe, and has potential practical application value.

Claims

1. A BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid, characterized in that: Its molecular formula is C 60 H 56 BF2N5O3S3, the structural formula is:

2. The method for preparing a BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid according to claim 1, characterized in that The 10-(4-hydroxyphenyl)-1,3,7,9-tetramethyldipyrroledifluoroborane complex is subjected to a condensation reaction with 10-butyl-10H-phenothiazine-3-carboxaldehyde to obtain 4-(3,7-bis(-2-(10-butyl-10H-phenothiazine-3-yl)vinyl)-1,9-dimethyl-5H-4-dipyrroledifluoroborane-10-yl)phenol; the 10-(4-hydroxyphenyl)-1,3,7,9-tetramethyldipyrroledifluoroborane complex is referred to as B-OH, the 4-(3,7-bis(-2-(10-butyl-10H-phenothiazine-3-yl)vinyl)-1,9-dimethyl-5H-4-dipyrroledifluoroborane-10-yl)phenol is obtained. PTB-OH is abbreviated as PTB-OH; PTB-OH then undergoes an etherification reaction with 4-(bromomethyl)benzenesulfonamide to obtain the probe 4-((4-(3,7-bis(2-(10-butyl-10H-phenothiazin-3-yl)vinyl)-1,9-dimethyl-5H-4-(dipyrrolodifluoroborane-10-yl)phenoxy)methyl)benzenesulfonamide, and the 4-((4-(3,7-bis(2-(10-butyl-10H-phenothiazin-3-yl)vinyl)-1,9-dimethyl-5H-4-(dipyrrolodifluoroborane-10-yl)phenoxy)methyl)benzenesulfonamide is abbreviated as PTB-BSA.

3. The method for preparing a BODIPY-based near-infrared ratiometric fluorescent probe for targeted detection of endoplasmic reticulum hypochlorous acid according to claim 2, characterized in that: The specific preparation method includes: 1) 0.5-2.5 mmol of compound B-OH, 1.5-7.5 mmol of 10-butyl-10H-phenothiazine-3-carboxaldehyde, and 1.5-7.5 mmol of p-toluenesulfonic acid were dissolved in 30-60 mL of anhydrous toluene, and 0.5-2.0 mL of piperidine were gradually added. The reaction mixture was refluxed with stirring for 5-7 hours, and the reaction was monitored by TLC. The reaction mixture was washed with water, dried, and distilled, and the resulting residue was purified on a silica gel column with an eluent of petroleum ether:dichloromethane = 5:1, v / v, to obtain a green solid compound PTB-OH. 2) 1.0 mmol PTB-OH, 1.0-3.0 mmol 4-(bromomethyl)benzenesulfonamide, and 2.0-4.0 mmol anhydrous potassium carbonate were dissolved in 5-15 mL DMF and stirred at 80°C for 6 h. The reaction was monitored by TLC. 30-40 mL of dichloromethane was added to the reaction solution, which was then washed with distilled water. The organic phase was dried and distilled, and the crude product was purified on a silica gel column with a 1:1 ratio of petroleum ether to dichloromethane (v / v) as the eluent to obtain PTB-BSA as a dark green solid.

4. Use of the BODIPY-based near-infrared ratiometric fluorescent probe PTB-BSA for targeted detection of endoplasmic reticulum hypochlorous acid according to claim 1 in the preparation of a reagent for detecting hypochlorous acid.

5. The use according to claim 4, characterized in that The probe PTB-BSA can selectively react with hypochlorous acid; when hypochlorous acid is added to the probe PTB-BSA solution, the color of the solution quickly changes from green to dark blue under sunlight; the fluorescence emission spectrum of the probe PTB-BSA undergoes a significant blue shift, with the fluorescence emission wavelength shifting from 752nm to 681nm. The linear range for hypochlorous acid is 0-60μM, the detection limit is as low as 35nM, and the response time is within 10s.

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