Ratio fluorescent probe based on FRET mechanism and preparation method and application thereof
A ratiometric fluorescent probe using a FRET mechanism with coumarin fluorophore and 2-(5-hydroxypiperidin-1-1,3,3H-indole-1-onium salt as a linking group, and piperidine-4-ol as a linking group; its chemical structure is shown in formula (I).
Patent Information
- Application Number
- CN202411242190.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2024-12-27
AI Technical Summary
Existing hypochlorous acid imaging techniques lack effective, real-time detection methods that can accurately measure spatiotemporal and concentration distribution while minimizing environmental interference, and there is a need for new FRET systems with balanced energy transfer efficiency and emission wavelengths.
A ratiometric fluorescent probe is developed using a coumarin fluorophore as the energy donor, 2-(2-(5-(dimethylamino)thiophene-2-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-onium salt as the energy acceptor, and piperidine-4-ol as a linking group, with a synthesis method involving condensation reactions and column chromatography to create a FRET mechanism for hypochlorous acid detection.
The probe selectively reacts with hypochlorous acid, exhibiting a linear fluorescence intensity ratio change with concentration and viscosity, enabling quantitative detection in living cells and zebrafish, with minimal interference from other analytes, and providing insights into viscosity-related diseases.
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Figure CN119192164A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, and specifically relates to a hypochlorous acid ratio fluorescent probe based on the FRET mechanism, its preparation method and application. Background Art
[0002] Hypochlorous acid is an important reactive oxygen species in living organisms, primarily produced by hydrogen peroxide and chloride ions in the presence of myeloperoxidase. Normally, hypochlorous acid protects the immune system from attacks by bacteria, viruses, chlamydia, and mycoplasma. However, abnormal hypochlorous acid levels can lead to oxidative stress and damage to internal systems. Furthermore, uncontrolled hypochlorous acid production is closely associated with certain diseases, such as arthritis, kidney disease, lung injury, arteriosclerosis, and tumors. Therefore, developing in vivo hypochlorous acid imaging techniques and real-time detection of the spatiotemporal and concentration distribution of intracellular hypochlorous acid are of great significance for studying the physiological and pathological effects of hypochlorous acid in vivo.
[0003] Hypochlorous acid fluorescent probes have attracted widespread attention from scientists due to their high selectivity, ultrasensitivity, and rapid response. Ratiometric fluorescent probes are particularly favored because they can effectively eliminate interference from the environment, probe concentration, and excitation light intensity. Among the many types of ratiometric fluorescent probes, ratiometric fluorescent probes based on the FRET mechanism consist of an energy donor fluorophore connected to an energy acceptor fluorophore via a linker unit. After absorbing energy, the energy donor transfers it to the energy acceptor, which then emits fluorescence. The fluorescence intensities of the donor and acceptor change according to the concentration of the analyte, thus the ratio of their fluorescence intensities is linearly related to the concentration of the analyte. To balance energy transfer efficiency and the distance between the two emission wavelengths, it is necessary to research and develop new energy donor and acceptor fluorophores to form a new FRET system. Therefore, developing new hypochlorous acid ratiometric fluorescent probes based on the FRET mechanism is of great significance. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the present invention aims to provide a ratiometric fluorescent probe based on the FRET mechanism, its preparation method, and its applications.
[0005] The first objective of this invention is to provide a ratiometric fluorescent probe based on the FRET mechanism, wherein the fluorescent probe comprises a coumarin fluorophore as an energy donor, 2-(2-(5-(dimethylamino)thiophene-2-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-onium salt as an energy acceptor, and piperidine-4-ol as a linking group; its chemical structure is shown in formula (I). A second objective of this invention is to provide a method for synthesizing the FRET-based ratiometric fluorescent probe, comprising the following steps: S1. Dissolve (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-onium (0.1 mmol) and triethylamine (0.3 mmol) in dichloromethane; S2. Dissolve 0.1 mmol of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyl chloride in dichloromethane and then add it to the solution from step S1. S3. The resulting mixed solution is subjected to a condensation reaction. After the reaction is complete, the solvent is removed by vacuum evaporation. The solid compound is further purified by column chromatography to obtain the target product.
[0006] Furthermore, the molar ratio of (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-onium II to (7-(diethylamino)-2-oxo-2Hchromene-3-carboxyl chloride) is 1:1; The molar ratio of (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indole-1-onium and triethylamine is 1:3.
[0007] Furthermore, the condensation reaction was carried out under nitrogen atmosphere with stirring in an ice bath for 12 hours.
[0008] Furthermore, the eluent for column chromatography is a mixed solution of dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of 35:1.
[0009] A third objective of this invention is to provide the application of the FRET-based ratiometric fluorescent probe in the detection of hypochlorous acid.
[0010] Furthermore, the FRET-based ratiometric fluorescent probe is used to detect endogenous hypochlorous acid in cells, zebrafish, or solutions containing hypochlorous acid.
[0011] Furthermore, the ratiometric fluorescent probe based on the FRET mechanism is used for the quantitative detection of hypochlorous acid.
[0012] A fourth objective of this invention is to provide the application of the FRET-based ratiometric fluorescent probe in viscosity detection.
[0013] Furthermore, the FRET-based ratiometric fluorescent probe is used to detect intracellular viscosity.
[0014] Beneficial technical effects of the present invention: The probe of this invention can selectively react with hypochlorous acid. As the concentration of hypochlorous acid increases, its fluorescence emission intensity gradually increases at 475 nm and gradually decreases at 580 nm; the ratio of these two intensity levels (I0...)2 475 / I 580 The fluorescence intensity of the probe exhibits a linear relationship with the concentration of hypochlorous acid within a certain range. Furthermore, as the viscosity of the test system increases, the fluorescence intensity of the probe at 580 nm gradually increases, and its fluorescence emission intensity shows a linear relationship with the viscosity of the test system within a certain range. The probe of this invention can realize the detection of hypochlorous acid and viscosity in living cells and zebrafish, and is expected to play a role in public health and clinical medicine detection, possessing broad application prospects. Attached Figure Description
[0015] Figure 1 This is a schematic diagram illustrating the selectivity of the fluorescent probe described in this invention in response to various analytes.
[0016] Figure 2 The graph shows the fluorescence intensity changes of the fluorescent probe described in this invention at 475 nm and 580 nm, and the linear relationship between the ratio and the concentration of hypochlorous acid.
[0017] Figure 3 The fluorescence intensity of the ratiometric fluorescent probe prepared for Example 1 at 580 nm as viscosity increases (b), the linear relationship between its value Log(I) and Logη (b), and photographs showing the change with viscosity under sunlight and ultraviolet light (c).
[0018] Figure 4 The image shows a fluorescence microscopy image (A) of the hypochlorous acid probe described in this invention in RAW264.7 cells, with the blue channel (435-550nm) and the red channel (560-630nm).
[0019] Wherein: (A) is a cell imaging image under the conditions shown; (B) is a bar chart of the ratio of blue channel fluorescence intensity to red channel fluorescence intensity under the conditions corresponding to (A).
[0020] Figure 5 This is a confocal fluorescence imaging image of HeLa cells with different viscosities using the fluorescent probe described in this invention. The excitation wavelength is 405 nm, and the red channel is 560-630 nm.
[0021] Wherein: (a) is a cell imaging image under the conditions shown; (b) is a bar chart of relative fluorescence intensity of the red channel under the conditions corresponding to (a).
[0022] Figure 6 This is a fluorescence microscopic image of the fluorescent probe described in this invention in zebrafish, showing the blue channel (435-550nm) and the red channel (560-630nm). Detailed Implementation
[0023] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.
[0024] Unless otherwise specified, the materials, reagents, strains, vectors, etc. used in the following examples are all commercially available.
[0025] Example 1 Synthesis of probe molecules (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-onium (0.1 mol, 49.4 mg) and triethylamine (0.3 mol) were dissolved in 10 mL of dichloromethane. Then, 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyl chloride (0.1 mol, 27.6 mg) was further dissolved in 12 mL of DCM and slowly added to the above reaction solution. These mixtures were stirred in an ice bath under nitrogen atmosphere for 12 hours. The solvent was removed by vacuum evaporation, and the solid compound was further purified by column chromatography (DCM / MeOH, V / V, 35 / 1) to give the target product in 31% yield.
[0026] Use HRMS 1 H NMR and 13 The molecular structure of the target product obtained in Example 1 was characterized by C NMR, and the results are as follows: HRMS: m / z, calculated for [C 36 H 40 N3O4S] + : 610.2734, found: 610.2787. 1H NMR: 1.140 (t, J=7.0 Hz, 6H), 1.675 (s, 6H), 1.950 (m, 2H), 2.113(m, 2H), 3.489 (q, J=14.0 Hz, 4H), 3.673 (s, 3H),3.811-3.922 (m, 4H), 5.248(s, 1H), 6.128 (d, J=13.6 Hz, 1H), 6.555 (s, 1H), 6.797 (d, J=9.2 Hz, 1H), 6.913 (s, 1H), 7.295 (t, J=7.6 Hz, 1H), 7.444 (q,J=12.4 Hz, 2H), 7.641 (q, J=7.2 Hz, 2H), 8.051 (s, 1H), 8.396 (d, J=23.6 Hz, 1H), 8.667 (s, 1H). 13 C NMR: 12.90, 27.27, 28.91, 30.13, 31.65, 43.98, 44.84, 48.61,49.71, 67.93, 74.44, 96.31, 105.61, 105.76,110.36, 111.94, 118.67, 122.17,122.82, 125.76, 127.97, 127.99, 132.31, 141.54, 143.03, 143.88, 150.08,153.45, 157.58, 158.64, 163.06. The preparation reaction of the above fluorescent probe is shown in the following formula: .
[0027] Example 2 Prepare a solution of the 5 μmol / L fluorescent probe prepared in Example 1 using PBS buffer (pH = 7.4). Add 20 μmol of aqueous solutions of reactive oxygen species, reactive nitrogen species, and metal ions (including Zn) to a 10 mL volumetric flask containing the 5 μmol / L ratiometric fluorescent probe solution using a microsyringe. 2+ Fe 2+ Fe 3+ Mg 2+ , Cl - , I - SO4 2- , F - CH3COO - C2O42- , Br - NO 2- , S 2- The results are as follows: H₂O₂, t-BuOOH, OH· and t-BuOO·. Figure 1 As shown, the results indicate that the probe described in this invention can specifically identify and detect hypochlorous acid, and the aforementioned ions have minimal interference with the probe, demonstrating excellent anti-interference capabilities.
[0028] Example 3 To a 10 mL volumetric flask containing a solution of the fluorescent probe prepared in Example 1 at a concentration of 5 μmol / L (solvent: pH 7.4 PBS buffer), sodium hypochlorite aqueous solutions of varying concentrations were added using a microsyringe. After mixing, fluorescence testing was performed, and the results are as follows. Figure 2 As shown.
[0029] The results show that as the concentration of hypochlorous acid in the system increases, the fluorescence intensity at 475 nm gradually increases and the fluorescence intensity at 580 nm gradually decreases. The ratio of the fluorescence intensity at 475 nm to that at 580 nm is linearly related to the concentration of hypochlorous acid within a certain range. Therefore, the fluorescent probe of the present invention can quantitatively detect the concentration of hypochlorous acid.
[0030] The detection mechanism is as follows: In the absence of hypochlorous acid, the fluorescent probe, upon excitation of the energy donor, transfers energy to the energy acceptor, which then emits fluorescence. In the presence of hypochlorous acid, the addition reaction of hypochlorous acid to the double bond disrupts the partially conjugated structure of the acceptor (see Equation 1), preventing the intramolecular FRET mechanism and thus blocking energy transfer. When the energy donor is excited, it emits fluorescence. The fluorescence intensity at the two emission wavelengths changes depending on the concentration of hypochlorous acid, thereby achieving ratiometric fluorescence detection of hypochlorous acid.
[0031] .
[0032] Example 4 Different volume ratios of PBS buffer (pH = 7.4) and glycerol were prepared to simulate a viscosity environment, with the glycerol ratio increasing from 0% to 99.5%. After thorough mixing, the fluorescence intensity was measured. The results showed that the fluorescence intensity of the fluorescent probe of this invention at 580 nm gradually increased with increasing glycerol content in the system. (See figure) Figure 3 Subsequently, when the viscosity increased from 0.95 cp to 683 cp, calculations based on the Förster-Hoffmann equation showed that Log(I 580 The value of ) and Logη(R) 2The values (=0.9910, K=0.3487) show a good linear relationship. This indicates that the fluorescent probe of the present invention can effectively detect viscosity levels, providing a promising method for the diagnosis and prevention of viscosity-related diseases.
[0033] Example 5: Intracellular Fluorescence Imaging Assay The laser scanning confocal microscope was set with an excitation wavelength of 405 nm, a blue fluorescence channel wavelength collection range of 435-550 nm, and a red fluorescence channel wavelength collection range of 560-630 nm.
[0034] After RAW264.7 cells were transferred to small confocal dishes and incubated for 24 hours, they were divided into groups for experiments.
[0035] Control group: RAW264.7 cells were treated with the fluorescent probe (2 μM) solution described in this invention (the probe was dissolved in DMSO to prepare a 10 μM solution). -2 A 2 μmol / L fluorescent probe solution was prepared by adding a mol / L solution to a culture dish containing culture medium and incubating for 1 hour. After washing three times with PBS, the cells were imaged and detected using a laser scanning confocal microscope. It can be seen that the control group cells showed stronger red fluorescence and weaker blue fluorescence. (See...) Figure 4 .
[0036] Experimental group: RAW264.7 cells were treated with 2 μg / mL -1 LPS (LPS: a stimulator of hypochlorous acid production in cells) (LPS dissolved in deionized water to prepare a corresponding solution) was incubated for 12 hours, followed by incubation for 2 hours with the fluorescent probe (2 μM) described in this invention. Then, the cells were washed three times with PBS for confocal cell imaging. LPS indirectly promoted hypochlorous acid production. The red fluorescence in this group was weaker than that in the control group, while the blue fluorescence was significantly stronger. The change in the ratio of blue fluorescence to red fluorescence intensity was very significant, as shown in [the diagram]. Figure 4 The results indicate that LPS incubation in the experimental group resulted in the production of endogenous hypochlorous acid within the cells. The red fluorescence of the fluorescent probe in the cells was weaker than that in the control group, while the blue fluorescence was significantly stronger, and the relative fluorescence intensity ratio was significantly increased. These experiments demonstrate that the fluorescent probe can effectively detect endogenous hypochlorous acid in living cells.
[0037] Example 6: Fluorescence imaging test of changes in cell viscosity.
[0038] HeLa cells were incubated with nystatin (10 μmol / L, 20 μmol / L, 40 μmol / L) for half an hour. Nystatin increased cell viscosity. Then, the cells were incubated with the fluorescent probe solution (2 μmol / L) prepared in Example 1 (the probe was dissolved in DMSO to prepare a 10 μmol / L solution). - 2A 2 μmol / L fluorescent probe solution was prepared by adding a mol / L solution to a culture dish containing culture medium and incubating for 1 hour. Confocal fluorescence imaging was then performed. The results showed that the fluorescence of the red channel increased with increasing viscosity. Figure 5 As the concentration of nystatin in cultured cells increases, the intracellular viscosity gradually increases, and the red fluorescence of the probe within the cell gradually intensifies, indicating that the fluorescent probe can effectively detect intracellular viscosity.
[0039] Example 7: Probe fluorescence imaging test in zebrafish.
[0040] Zebrafish aged 4-5 days were incubated in E3 medium and imaged. Zebrafish were incubated with LPS (0, 1, 2, 5 μg / mL) for 1 hour, followed by incubation with the fluorescent probe described in this invention (4 μM) for half an hour, and then confocal imaging was performed. Compared with the control group (incubated with the probe only), with increasing LPS levels, the blue fluorescence of the fluorescent probe described in this invention and the LPS-cultured zebrafish continuously increased, while the red fluorescence decreased (Figure 6). This indicates that the fluorescent probe can effectively detect hypochlorous acid in zebrafish.
[0041] In summary, the ratiometric fluorescent probe described in this invention can not only quantitatively detect low concentrations of hypochlorous acid and its viscosity, but also be used for ratio imaging within cells and zebrafish. It is a superior dual-sensitivity fluorescent probe for hypochlorite and viscosity based on the fluorescence resonance energy transfer mechanism. It is expected to play an important role in industrial production and clinical medicine, and has broad application prospects.
Claims
1. A ratiometric fluorescent probe based on the FRET mechanism, characterized in that: The fluorescent probe is composed of a coumarin fluorophore as an energy donor, 2-(2-(5-(dimethylamino)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium salt as an energy acceptor, and piperidine-4-ol as a linking group; its chemical structure is shown in formula (I), 。 2. A method for synthesizing a ratiometric fluorescent probe based on the FRET mechanism as claimed in claim 1, characterized in that: The steps include: S1. Dissolve (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium and triethylamine in dichloromethane; S2, dissolving 7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl chloride in dichloromethane, and then adding it to the solution of step S1; S3, subjecting the obtained mixed solution to a condensation reaction, removing the solvent by vacuum evaporation after the reaction is completed, and further purifying the solid compound by column chromatography to obtain the target product.
3. The method for synthesizing a ratiometric fluorescent probe based on the FRET mechanism according to claim 2, characterized in that: The molar ratio of the (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-ium II to (7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl chloride is 1:1; The molar ratio of the (E)-2-(2-(5-(4-hydroxypiperidin-1-yl)thiophen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium to triethylamine is 1:
3.
4. The method for synthesizing a ratiometric fluorescent probe based on the FRET mechanism according to claim 2, characterized in that: The condensation reaction was carried out under nitrogen atmosphere with stirring in an ice bath for 12 hours.
5. The method for synthesizing a ratiometric fluorescent probe based on the FRET mechanism according to claim 2, characterized in that: The eluent for column chromatography is a mixed solution of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the mixed solution is 35:
1.
6. Use of the ratiometric fluorescent probe based on the FRET mechanism as claimed in claim 1 in detecting hypochlorous acid.
7. The use of the ratiometric fluorescent probe based on the FRET mechanism in detecting hypochlorous acid according to claim 6, characterized in that: The ratiometric fluorescent probe based on the FRET mechanism is used for detecting endogenous hypochlorous acid in cells, zebrafish or a solution containing hypochlorous acid.
8. The use of the ratiometric fluorescent probe based on the FRET mechanism in detecting hypochlorous acid according to claim 6, characterized in that: The ratiometric fluorescent probe based on the FRET mechanism is used for quantitative detection of hypochlorous acid.
9. Use of the ratiometric fluorescent probe based on the FRET mechanism as claimed in claim 1 in viscosity detection.
10. The use of the ratiometric fluorescent probe based on the FRET mechanism in viscosity detection according to claim 9, characterized in that: The ratiometric fluorescent probe based on the FRET mechanism is used to detect intracellular viscosity.