A lysosome-targeted hypochlorite fluorescent probe and preparation method and use method thereof
By constructing a photoinduced electron transfer system of fluoroboron dipyrrole and phenylselenoether, the problem of poor specificity and reactivity of existing lysosomal fluorescent probes in acidic environments was solved, achieving highly sensitive, specific detection and precise localization of hypochlorous acid.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG SCI-TECH UNIV
- Filing Date
- 2023-06-05
- Publication Date
- 2026-06-19
Smart Images
Figure CN116813652B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a fluoroboron dipyrrole derivative used as a hypochlorous acid fluorescent probe targeting lysosomes, and its preparation and usage methods. Background Technology
[0002] Hypochlorous acid (HOCl) is one of the important endogenous reactive oxygen species (ROS) in organisms, playing a crucial role in many physiological and pathological processes. Hypochlorous acid is typically produced by the catalysis of myeloperoxidase (MPO) by Cl-. − HOCl is produced through peroxidation. In the immune process, HOCl acts as a nemesis of microorganisms, eliminating invading bacteria and pathogens, playing a crucial role in immunity. Lysosomes, with a pH of 4.0–5.5, contain various hydrolytic enzymes and are important organelles for breaking down biological macromolecules such as proteins, nucleic acids, and polysaccharides. They play vital roles in cellular processes such as digestion, defense, oxidative stress, and autophagy. At the organelle level, HOCl is essential for maintaining lysosomal redox balance and lysosomal function. However, disordered or excessive HOCl can induce lysosomal rupture, leading to apoptosis and subsequently triggering various physiological or pathological processes, including neurodegenerative diseases, arthritis, and atherosclerosis. Therefore, targeted monitoring of HOCl levels in lysosomes is of great significance for analyzing related mechanisms and diagnosing diseases.
[0003] Compared with traditional detection methods, fluorescent probes have many advantages, including high sensitivity, high selectivity, real-time imaging, non-destructive testing, and ease of modification. Furthermore, by introducing targeting groups into organelles, probes can be enriched in the target organelles, making fluorescent probes an important tool for organelle imaging.
[0004] Currently developed small-molecule fluorescent probes for detecting hypochlorous acid are mainly designed based on the selective oxidation, deprotection, and oxidative cleavage reactions of hypochlorous acid. In the presence of hypochlorous acid, the detection group in the probe molecule undergoes a specific reaction with the acid, altering the molecule's original electronic or steric effects, leading to changes in the probe molecule's fluorescence properties, thereby achieving specific recognition of hypochlorous acid.
[0005] However, reactive hypochlorous acid fluorescent probes (see review Nahyun Kwon, Yahui Chen, Xiaoqiang Chen, Myung Hwa Kim, Juyoung Yoon, Dyes and Pigments(2022, 200, 110132.), including the oxidation of sulfur-containing groups to sulfoxides, the conversion of dithioacetal protecting groups to carbonyl groups, and the selective conversion of oximes or hydrazones to aldehydes or carboxylic acids, which have become commonly used selective recognition mechanisms for hypochlorous acid fluorescent probes. However, because the pH value in lysosomes is around 4.0~5.5, which is more acidic than the cytoplasm with a pH value of 7.0~7.3, its acidic conditions directly affect the occurrence of various biochemical processes in lysosomes. Using H... + Bifunctional probes for lysosome targeting can not only specifically locate lysosomes but also monitor ROS and related intracellular diseases within lysosomes. In recent years, groups used for lysosome localization have included morpholine groups, diethylamino derivatives, piperazine, and boric acid. While increasingly sophisticated fluorescent probes targeting lysosomes have been developed, they still have some limitations. For example, the lysosomal targeting region exhibits low specificity and may be affected by various microenvironments. Furthermore, most existing probes suffer from short fluorescence emission wavelengths and poor reactivity in acidic environments, limiting their application in organisms. Summary of the Invention
[0006] To overcome the aforementioned deficiencies in the prior art, this invention proposes a fluorescently activated probe (BpySe) for targeting lysosomes and detecting hypochlorous acid, along with its preparation and application methods. This invention enables the quantitative detection of trace amounts of hypochlorous acid within lysosomes in a sample.
[0007] The core of this invention lies in constructing a classic photo-electron transfer (PET) system using fluoroboron dipyrrole and phenylselenoether, and utilizing the basic pyridine-vinyl group bonded to the fluorescent parent group of fluoroboron dipyrrole. The probe itself exhibits excellent photo-electron transfer (PET) effect with almost no fluorescence emission. However, in the presence of hypochlorous acid, the hypochlorous acid reacts with the selenylphenyl portion of the probe molecule to form a selenium-oxygen double bond, thereby blocking the PET effect of the molecule. The probe molecule then emits strong orange-yellow fluorescence. Through this scheme, an "on" type fluorescence response is obtained, achieving highly sensitive and specific detection of hypochlorous acid. Furthermore, the introduction of the pyridine group enables the probe to accurately locate lysosomes.
[0008] The hypochlorous acid fluorescent probe described in this invention is named BpySe, and its structural formula is shown in formula (I):
[0009]
[0010] The preparation method of the above fluorescent probe is as follows: a certain amount of (E)-5,5-difluoro-1-methyl-3-(2-(pyridin-4-yl)vinyl)-5H-5λ is added. 4 ,6λ 4-pyrrole[1,2-c:2',1'-f][1,3,2]diazaborine, diphenyldiselenoether, and [bis(trifluoroacetoxy)iodide]benzene were dissolved in dichloromethane. After reacting for a period of time, the mixture was evaporated to dryness and then subjected to column chromatography to give compound (E)-5,5-difluoro-1-methyl-2-(phenylselenoyl)-3-(2-(pyridin-4-yl)vinyl)-5H-5λ 4 , 6λ 4 -pyrrole[1,2-c:2',1'-f][1,3,2]diazaborine (4), namely: BpySe.
[0011] The reaction formula for preparing the above probe is as follows:
[0012]
[0013] Preferably, the molar ratio of pyridine-dimethylbodipy, diphenyldiselenoether, and [bis(trifluoroacetyl)iodide]benzene is 1:1.5:1.5.
[0014] Preferably, the mass-to-volume ratio of pyridine-dimethylbodipy to dichloromethane is 1:500. Preferably, the reaction temperature is 15 °C.
[0015] Preferably, the reaction time is 1 hour.
[0016] The usage method of the above-mentioned hypochlorous acid fluorescent probe is as follows:
[0017] Step 1: Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions (10 mM, pH = 4.0) of hypochlorous acid at different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents.
[0018] The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM. ~ 10 μM;
[0019] The standard solution shown contains 0.1 nM hypochlorous acid. ~ 1 mM;
[0020] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 520 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 562 Establish a standard curve with the vertical axis as the ordinate;
[0021] I 562 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 562 nm;
[0022] Step 3: Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under excitation light with an excitation wavelength of 520 nm, and calculate the hypochlorous acid content of the sample to be tested based on the standard curve.
[0023] This invention has the following characteristics:
[0024] 1) The fluorescent probe provided by this invention is a purplish-black solid powder with a stable structure.
[0025] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of hypochlorous acid. As the concentration of hypochlorous acid increases, the fluorescence of its aqueous solution under ultraviolet light changes from no fluorescence to a bright orange-yellow.
[0026] 3) The fluorescent probe provided by this invention has an emission wavelength of 562 nm, which is a fluorescence "on" response, and the fluorescence intensity changes significantly before and after the reaction (about 30 times). This can greatly eliminate the influence of differences in detection conditions on the results and improve the sensitivity of detection.
[0027] 4) The fluorescent probe provided by this invention contains a pyridine group and can be used for precise localization of lysosomes.
[0028] 5) The fluorescent probe provided by this invention has a linear relationship with the concentration of hypochlorous acid and can be used for the accurate measurement of hypochlorous acid concentration.
[0029] The "open" hypochlorous acid probe based on dimethyl fluoroboron dipyrrole dye provided by this invention has a good response to hypochlorous acid solution, enabling sensitive quantitative detection of hypochlorous acid in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description
[0030] Figure 1 : The proton NMR spectrum of the fluorescent probe BpySe.
[0031] Figure 2 Color response of the fluorescent probe BpySe to hypochlorite-phosphate buffer solution (pH = 4.0).
[0032] Figure 3 Fluorescence response of the fluorescent probe BpySe to hypochlorite-phosphate buffer solution (pH = 4.0).
[0033] Figure 4 UV titration curve of fluorescent probe BpySe in hypochlorous acid in phosphate buffer solution (pH = 4.0), where the probe concentration is 10.0 μM.
[0034] Figure 5Fluorescence titration curve of fluorescent probe BpySe in hypochlorous acid in phosphate buffer solution (pH = 4.0), where the excitation wavelength is 520 nm and the probe concentration is 10.0 μM.
[0035] Figure 6 Selectivity experiment of fluorescent probe BpySe for various analytes in phosphate buffer solution (pH = 4.0), with excitation wavelength of 520 nm and probe concentration of 10.0 μM.
[0036] Figure 7 : Localization experiment of lysosomes by fluorescent probe BpySe.
[0037] Figure 8 Fluorescent imaging of HOCl in living cells by the fluorescent probe BpySe. Detailed Implementation
[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0040] The compound numbers in the examples correspond to the numbers in the compounds described above.
[0041] Example 1: Synthesis of compound BpySe.
[0042] Synthesis of compound 3-selenylphenyl-dimethylfluoroboron dipyrrole (4).
[0043] 100 mg of pyridine-fluoroboron dipyrrole chloride (1) (0.32 mmol) and 151.5 mg of diphenyldiselenoether (2) (0.485 mmol) were dissolved in 10 mL of dichloromethane, and then 208.7 mg of [bis(trifluoroacetyl)iodide]benzene (3) (0.485 mmol) were added. The mixture was reacted at room temperature for 1 hour, dried by rotary evaporation, and washed with water to give 75 mg of compound 5-selenophenyl-pyridine-dimethylfluoroboron dipyrrole (4), with a yield of 50%.
[0044] 1 H NMR (400 MHz, CDCl3) δ 8.65 – 8.59 (m, 3H), 8.14 (d, J = 16.6 Hz,1H), 7.90 (s, 1H), 7.83 (d, J = 16.6 Hz, 1H), 7.43 (s, 1H), 7.37 (d, J= 5.3Hz, 2H), 7.29 – 7.19 (m, 4H), 7.16 (d, J = 4.3 Hz, 1H), 6.65 – 6.58 (m, 1H), 2.40 (s, 3H); such as Figure 1 The image shows the 1H NMR spectrum of the fluorescent probe BpySe.
[0045] Example 2: Color response of compound BpySe to hypochlorous acid.
[0046] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe BpySe for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution and add it dropwise to a phosphate buffer solution of hypochlorous acid (pH = 4.0), and then dilute to 5 mL with the corresponding phosphate buffer solution, so that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 20.0 μM, for color response testing. Figure 2 and 3 As shown, after adding hypochlorous acid solution, the color of the solution changed from purple to pink, and the fluorescence of the solution also changed from no fluorescence to bright orange-yellow fluorescence, indicating that the probe BpySe has a direct colorimetric response to hypochlorous acid.
[0047] Example 3: UV response of hypochlorous acid to compound BpySe at pH = 4.
[0048] Prepare a 1 mM N,N-dimethylformamide stock solution of the fluorescent probe BpySe for detecting hypochlorous acid described in this invention. Measure 50 μL of this stock solution into a phosphate buffer solution at pH = 4.0, and then dilute to 5 mL with the corresponding phosphate buffer solution, so that the probe concentration in the test solution is 10.0 μM, and the hypochlorous acid concentration is 0 and 20.0 μM, respectively. Then perform absorption spectroscopy measurements. Figure 4 The UV absorption curve shown indicates that the absorbance at 525 nm increases after the addition of hypochlorous acid (A). 525 Meanwhile, the absorbance at 570 nm decreased (A 570 ).
[0049] Example 4: Fluorescent titration detection of compound BpySe with different concentrations of hypochlorous acid.
[0050] Prepare a 1 mM N,N-dimethylformamide solution of the fluorescent probe BpySe for detecting hypochlorous acid described in this invention. Take 50 μL of this stock solution and add it dropwise to phosphate buffer solutions of different concentrations of hypochlorous acid, then dilute to 5 mL with the corresponding phosphate buffer solution. This ensures that the probe concentration in the test solution is 10.0 μM and the hypochlorous acid concentration is 0-20.0 μM for fluorescence detection (λex = 520 nm, λem = 562 nm). Measure the fluorescence intensity in each system and establish a standard curve of fluorescence intensity versus hypochlorous acid concentration. Figure 5 As shown, the fluorescence intensity at 562 nm gradually increases with the increase of hypochlorous acid concentration.
[0051] Example 5: Selectivity of compound BpySe for different common reactive oxygen species.
[0052] Prepare a 1.0 mM N,N-dimethylformamide stock solution of the fluorescent probe BpySe for detecting hypochlorous acid described in this invention. Prepare 10 mM solutions of various small reactive oxygen species to be tested as backup. Measure 50 μL of this stock solution and add it dropwise to phosphate buffer solutions of different small molecule analytes, and then dilute to 5 mL with the corresponding phosphate buffer solutions, so that the concentration of the probe in the test solution is 10.0 μM and the concentration of the small molecule analyte is 100.0 μM for fluorescence detection (λex = 520 nm, λem = 562 nm). Measure the fluorescence intensity in each system and establish a fluorescence intensity (IL) measurement. 562 A bar chart showing the relationship between the analytes and the various test objects. (e.g.) Figure 6 As shown, other common reactive oxygen species (ROS) molecules have almost no effect on the fluorescence of the probe on BpySe.
[0053] Example 6: Localization of lysosomes by the fluorescent probe BpySe
[0054] A 1.0 mM stock solution of N,N-dimethylformamide (NDM) in which the fluorescent probe BpySe for detecting hypochlorous acid described in this invention was prepared and set aside for later use. 50 μL of the BpySe NDM stock solution was used to stain HeLa cells. The stained HeLa cells were then treated with HOCl. The probe BpySe and a commercial lysosomal tracker (Lyso Tracker Green) were co-incubated in the HeLa cells, and the cells were treated with NaOCl (10 μM) for 20 min. Imaging was then performed under a fluorescence microscope. The experimental results are as follows: Figure 7 As shown, the red channel is the imaging channel for the probe BpySe in the presence of HOCl. Figure 7 (a) The green channel is the Lyso Tracker Green imaging channel for lysosomes. Figure 7(b) The merged image is Figure 7 The c. BpySe staining showed good agreement with the lysosomal tracker staining, and the intensity distribution of the linear regions of interest in HeLa cells changed closely and synchronously in both channels, with a colocalization coefficient of 0.88. Figure 7 (d)
[0055] Example 7: Fluorescence imaging of HOCl in living cells by the fluorescent probe BpySe.
[0056] A 1.0 mM stock solution of BpySe, the fluorescent probe for detecting hypochlorous acid described in this invention, in N,N-dimethylformamide was prepared and set aside for later use. 50 μL of the BpySe N,N-dimethylformamide stock solution was used to stain HeLa cells. The stained HeLa cells were then treated with HOCl. Under a fluorescence microscope, HeLa cells showed bright fluorescence in the red channel, while untreated HeLa cells showed no fluorescence emission in the red channel. Figure 8 This confirms that the probe has good membrane permeability and can be used for fluorescence imaging of HOCl in live HeLa cells.
[0057] Example 8: Synthesis of compound BpySe.
[0058] Synthesis of compound 3-selenylphenyl-dimethylfluoroboron dipyrrole (4).
[0059] 100 mg of pyridine-fluoroboron dipyrrole chloride (1) (0.32 mmol) and 303 mg of diphenyldiselenoether (2) (0.97 mmol) were dissolved in 30 mL of dichloromethane, and then 417.4 mg of [bis(trifluoroacetyl)iodide]benzene (3) (0.97 mmol) were added. The mixture was reacted at 0 °C for 10 hours. After drying by rotary evaporation and washing with water, compound 5-selenophenyl-pyridine-dimethylfluoroboron dipyrrole (4) was obtained with a yield of 47%.
[0060] Example 9: Synthesis of compound BpySe.
[0061] Synthesis of compound 3-selenylphenyl-dimethylfluoroboron dipyrrole (4).
[0062] 100 mg of pyridine-fluoroboron dipyrrole chloride (1) (0.32 mmol) and 505 mg of diphenyldiselenoether (2) (1.61 mmol) were dissolved in 50 mL of dichloromethane, and then 695.7 mg of [bis(trifluoroacetyl)iodide]benzene (3) (1.61 mmol) were added. The mixture was reacted at 40 °C for 24 hours. After drying by rotary evaporation and washing with water, compound 5-selenophenyl-pyridine-dimethylfluoroboron dipyrrole (4) was obtained with a yield of 46%.
Claims
1. A hypochlorous acid fluorescent probe targeting lysosomes, characterized in that: Its molecular formula C 23 H 18 BF2N3Se, abbreviated as BpySe, has the structural formula (I). 。 2. The method for preparing a hypochlorous acid fluorescent probe targeting lysosomes according to claim 1, characterized in that, The synthesis steps are as follows: The compound shown in formula (1), diphenyl diselenide, was dissolved in dichloromethane. [bis(trifluoroacetoxy)iodide]benzene was added dropwise and reacted for a period of time to obtain the hypochlorous acid fluorescent probe targeting lysosomes shown in formula (4), namely: BpySe; The preparation reaction formula is as follows: 。 3. The method for preparing a fluorescent probe for hypochlorous acid targeting lysosomes according to claim 2, characterized in that: The compound shown in formula (1) has a molar ratio of diphenyldiselenoether to [bis(trifluoroacetoxy)iodide]benzene of 1:
1. ~ 5:1 ~ 5; The mass-to-volume ratio of the compound shown in formula (1) to dichloromethane is 1:
100. ~ 500; reaction temperature is 0 ~ 40 o C, the reaction time is 1~24 hours.
4. The method for preparing a fluorescent probe for hypochlorous acid targeting lysosomes according to claim 2, characterized in that: The compound shown in formula (1) has a molar ratio of diphenyl diselenyl ether to [bis(trifluoroacetoxy)iodo]benzene of 1:1.5:1.
5.
5. The method for preparing a fluorescent probe for hypochlorous acid targeting lysosomes according to claim 2, characterized in that: The reaction temperature is 15°C. o C.
6. The method for preparing a fluorescent probe for hypochlorous acid targeting lysosomes according to claim 2, characterized in that: The reaction time is 1 hour.
7. The method for preparing a fluorescent probe for hypochlorous acid targeting lysosomes according to claim 2, characterized in that: The mass-to-volume ratio of the compound shown in formula (1) to dichloromethane is 1:
500.
8. The method of using the fluorescent probe for hypochlorous acid targeting lysosomes according to claim 1; characterized in that: 1) Under the pH conditions of lysosomes, add the same concentration of the compound shown in formula (I) to phosphate buffer solutions of hypochlorous acid of different concentrations to prepare at least 5 standard solutions containing the compound shown in formula (I) with different hypochlorous acid contents. The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM. ~ 10 μM; The standard solution shown contains 0.1 nM hypochlorous acid. ~ 1 mM; 2) Measure the fluorescence emission spectra of the standard solutions respectively, with an excitation wavelength of 520 nm. Plot the hypochlorous acid concentration on the x-axis and Ig on the y-axis. 562 Establish a standard curve with the vertical axis as the ordinate; I 562 This indicates the fluorescence emission peak intensity value of the standard solution at a wavelength of 562 nm; 3) Add the compound shown in formula (I) to the sample to be tested, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; The fluorescence emission spectrum of the sample was measured under excitation light with an excitation wavelength of 520 nm, and the hypochlorous acid content of the sample was calculated based on the standard curve.