CN113461706A - Fluorescent probe for rapidly identifying hydroxyl free radicals and preparation method and application thereof - Google Patents
Fluorescent probe for rapidly identifying hydroxyl free radicals and preparation method and application thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 title abstract description 4
- 239000000523 sample Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 8
- 238000001727 in vivo Methods 0.000 claims abstract 2
- -1 9-ethyl-9H-carbazole-3-yl Chemical group 0.000 claims description 15
- PZWWYAHWHHNCHO-FGHAYEPSSA-N (4r,7s,10s,13r,16s,19r)-n-[(2s,3r)-1-amino-3-hydroxy-1-oxobutan-2-yl]-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-10-(3-aminopropyl)-7-[(1r)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-3,3-dimethyl-6,9,12,15,18-pentaoxo-1,2-dithia Chemical compound C([C@H]1C(=O)N[C@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCN)C(=O)N[C@H](C(=O)N[C@@H](C(SSC[C@@H](C(=O)N1)NC(=O)[C@H](N)CC=1C=CC=CC=1)(C)C)C(=O)N[C@@H]([C@H](O)C)C(N)=O)[C@@H](C)O)C1=CC=C(O)C=C1 PZWWYAHWHHNCHO-FGHAYEPSSA-N 0.000 claims description 12
- 108700040302 phenylalanyl-cyclo(cysteinyltyrosyl-tryptophyl-ornithyl-threonyl-penicillamine)threoninamide Proteins 0.000 claims description 12
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- 239000002904 solvent Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- 238000006555 catalytic reaction Methods 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- PQMFVUNERGGBPG-UHFFFAOYSA-N (6-bromopyridin-2-yl)hydrazine Chemical compound NNC1=CC=CC(Br)=N1 PQMFVUNERGGBPG-UHFFFAOYSA-N 0.000 claims description 2
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- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 2
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- GMZFKUXFTJMSDM-UHFFFAOYSA-N CCCCN1C=CC(C)=CC1.Br Chemical compound CCCCN1C=CC(C)=CC1.Br GMZFKUXFTJMSDM-UHFFFAOYSA-N 0.000 claims 1
- 206010003246 arthritis Diseases 0.000 claims 1
- 239000000376 reactant Substances 0.000 claims 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 abstract description 7
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- 238000001514 detection method Methods 0.000 description 8
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- 238000006243 chemical reaction Methods 0.000 description 4
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- NZCHHEFOTMKOJX-UHFFFAOYSA-K [6-[[3-carboxy-4-(3-oxido-6-oxoxanthen-9-yl)phenyl]carbamothioylamino]hexoxy-oxidophosphoryl] [5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound O1C(COP([O-])(=O)OP([O-])(=O)OCCCCCCNC(=S)NC=2C=C(C(=CC=2)C2=C3C=CC(=O)C=C3OC3=CC([O-])=CC=C32)C(O)=O)C(O)C(O)C1N1C=CC(=O)NC1=O NZCHHEFOTMKOJX-UHFFFAOYSA-K 0.000 description 3
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- UWVZAZVPOZTKNM-UHFFFAOYSA-M 1-butyl-4-methylpyridin-1-ium;bromide Chemical compound [Br-].CCCC[N+]1=CC=C(C)C=C1 UWVZAZVPOZTKNM-UHFFFAOYSA-M 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
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- JUBKLYCRFZTXBA-UHFFFAOYSA-N (9-ethylcarbazol-3-yl)boronic acid Chemical compound OB(O)C1=CC=C2N(CC)C3=CC=CC=C3C2=C1 JUBKLYCRFZTXBA-UHFFFAOYSA-N 0.000 description 1
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- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
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- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- CUILPNURFADTPE-UHFFFAOYSA-N hypobromous acid Chemical compound BrO CUILPNURFADTPE-UHFFFAOYSA-N 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
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- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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Abstract
The invention discloses a fluorescent probe for rapidly identifying hydroxyl radicals and a preparation method and application thereof. The structural formula of the fluorescent probe is as follows:
the synthesis of the probe is relatively simple and easy to operate. In vitro experiments prove that the hydroxyl radical fluorescent probe can rapidly identify the hydroxyl radical, and the process belongs to a fluorescence closing process. The fluorescent probe has outstanding advantages for detecting hydroxyl free radicals of organisms and is applied to in vivo experiments.
Description
Technical Field
The invention relates to a fluorescent probe for rapidly identifying hydroxyl radicals and a preparation method and application thereof, belonging to the field of organic small-molecule fluorescent probes.
Background
Appropriate amount of biologically active species (ROS, including O)2·、H2O2、HOCl，1O2OH, HOBr, etc.) are important for maintaining cellular homeostasis and play a crucial role in many physiological processes, such as pathogen defense, signal transduction, and cell differentiation. However, excessive production of ROS can lead to the development of oxidative stress, leading to a decline in organ system function and even a range of diseases.
Hydroxyl free radical is one of the most toxic and harmful free radicals for organisms in active oxygen, and can cause excessive oxidation of molecules such as DNA, lipid, carbohydrate, protein and the like in cells to cause cell damage. These lesions have been shown to further contribute to the development of a variety of degenerative diseases, aging, mutations and cancers.
The hydroxyl radical has short service life and low content, and the detection of the hydroxyl radical is one of the most difficult problems in the fields of chemical analysis and life science analysis. The traditional method for detecting the hydroxyl free radical mainly comprises the following steps: spin trapping-electron spin magnetic resonance, high performance liquid chromatography, electrochemical detection, spectrophotometry, and fluorometry. In recent years, with the development of small organic molecule fluorescent probes, specific properties such as: the method has the advantages of high sensitivity, simplicity and convenience in operation, good reproducibility, good membrane permeability, in-situ detection, good selectivity and the like, so that the organic small-molecule fluorescent probe is increasingly applied to hydroxyl radical detection and imaging analysis in various complex biological and environmental samples. Therefore, it is of great importance to design and develop a method that is efficient and can be used for the detection of hydroxyl radicals in biological systems.
Disclosure of Invention
One of the purposes of the invention is to provide a fluorescent probe which has good permeability to cells and small toxic and side effects and is suitable for detecting hydroxyl radicals in organisms.
The invention also aims to provide a synthetic method of the fluorescent probe with simple process.
The invention also aims to provide the application of the fluorescent probe.
The invention adopts the following technical scheme:
a fluorescent probe for rapidly identifying hydroxyl radicals is characterized in that the chemical structural formula of the probe is shown as the formula (I):
the preparation method of the fluorescent probe for rapidly identifying the hydroxyl radicals is characterized by comprising the following steps:
under the nitrogen environment, (9-ethyl-9H-carbazole-3-yl) boric acid, 7-bromo-2, 3-dihydrothieno [3, 4-b)][1,4]dioxane-5-Formaldehyde, Pd (PPH)3)4And K2CO3Dissolved in organic solvent and water and the reaction refluxed overnight. Cooling to room temperature, pouring the reacted product into water, and adding CH2Cl2Extraction and drying with anhydrous magnesium sulfate, column chromatography separation to obtain yellow intermediate 1.
Intermediate 1 and 1-butyl-4-methylpyridinium bromide were added to dry ethanol under argon and refluxed overnight under the catalysis of a few drops of piperidine. After cooling to room temperature, the solvent was evaporated under reduced pressure. And performing column chromatography separation to obtain a red-black product CTOP.
The application of the fluorescent probe for rapidly identifying the hydroxyl free radicals comprises the following steps: the fluorescent probe can be applied to the content sensing detection of hydroxyl radicals in organisms and water environments; the sensing detection is mainly fluorescence detection.
The invention has the advantages that: (1) the synthesis of the probe only needs two steps, and the post-treatment process is relatively simple and easy to operate; (2) the emission wavelength of the invention is in the near infrared region, which can effectively reduce the interference of autofluorescence of biological tissues; (3) the invention realizes the rapid detection of the hydroxyl free radical. Under a common ultraviolet lamp (365nm), the obvious color change (from red to clear) before and after the action of the reagent and hydroxyl radicals can be observed, the reagent can realize the in vivo imaging of arthritic mice, and has wide application prospect in the field of biomolecule detection.
Drawings
In order to more clearly illustrate the technical solution in the embodiment of the present invention, the drawings required in the description of the embodiment will be briefly introduced as follows:
FIG. 1 is a synthesis scheme of a fluorescent probe 1 obtained in example of the present invention.
FIG. 2 is a synthesis scheme of a fluorescent probe CTOP obtained in the example of the present invention.
FIG. 3 shows a fluorescent probe 1 obtained in example of the present invention1H-NMR spectrum.
FIG. 4 shows the fluorescence probe CTOP obtained in the example of the present invention1H-NMR spectrum.
FIG. 5 is a spectrum before and after the interaction of the fluorescent probe CTOP obtained in the example of the present invention with a hydroxyl radical, wherein the line of the peak height represents the hydroxyl radical (0 equivalent).
FIG. 6 shows that the fluorescence probe CTOP obtained in the embodiment of the present invention is applied to a biological system to test the biological toxicity.
Detailed Description
The following detailed description of the embodiments of the present invention will be provided with reference to the drawings and examples, so that how to apply the technical means to solve the technical problems and achieve the technical effects can be fully understood and implemented. It should be noted that, as long as there is no conflict, the features in the embodiments of the present invention may be combined with each other, and the formed technical solutions are within the scope of the present invention.
The invention discloses a fluorescent probe for rapidly identifying hydroxyl radicals, which is characterized in that the chemical structural formula of the probe is shown as the formula (I):
example 1: synthesis of fluorescent Probe 1
The synthetic route is as follows:
to (9-ethyl-9H-carbazol-3-yl) boronic acid (478mg, 2mmol), 7-bromo-2, 3-dihydrothieno [3,4-b ] in a nitrogen atmosphere][1,4]Dioxane-5-carbaldehyde (498mg, 2mmol), Pd (PPH)3)4To a mixture of (84mg, 0.073mmol) and toluene (20ml) was added K2CO3(2.0M, 1.5ml) in water and the reaction was refluxed overnight. After the reaction is finished and the temperature is cooled to room temperature, the product after the reaction is poured into water and CH is used2Cl2Extracted and dried over anhydrous magnesium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether/DCM ═ 1/2) to give yellow intermediate 1 powder (493mg, 68% yield), which was designated 7- (9-ethyl-9H-carbazol-3-yl) -2, 3-dihydrothieno [3,4-b ] as a yellow intermediate][1,4]Dioxin-5-formaldehyde.1H NMR(600MHz,DMSO-d6)δ9.88(s,1H),8.59(d,J＝1.8Hz,1H),8.24(d,J＝7.7Hz,1H),7.91(dd,J＝8.6,1.8Hz,1H),7.71(d,J＝8.6Hz,1H),7.65(d,J＝8.2Hz,1H),7.52–7.48(m,1H),7.25(t,J＝7.4Hz,1H),4.55–4.45(m,6H),1.33(t,J＝7.1Hz,3H)。
Intermediate 1(363mg,1mmol) and 1-butyl-4-methylpyridinium bromide (230mg,1mmol)) were added to dry ethanol under an argon atmosphere and refluxed overnight under the catalysis of a few drops of piperidine. After cooling to room temperature, the solvent was evaporated under reduced pressure. With Al2O3The residue was purified by column chromatography using a mixture of DCM and methanol (90:1v/v) as the eluting solvent to give a red-black CTOP powder (333mg, 58% yield).1H NMR(600MHz,DMSO-d6)δ8.81(d,J＝6.7Hz,2H),8.52(d,J＝1.8Hz,1H),8.19(dd,J＝13.7,7.3Hz,3H),8.03(d,J＝15.8Hz,1H),7.87(dd,J＝8.6,1.9Hz,1H),7.71(d,J＝8.7Hz,1H),7.66(d,J＝8.2Hz,1H),7.51(ddd,J＝8.2,7.0,1.2Hz,1H),7.25(t,J＝7.4Hz,1H),7.02(d,J＝15.8Hz,1H),4.52–4.48(m,4H),4.43(t,J＝7.4Hz,2H),1.87(tt,J＝9.1,6.6Hz,2H),1.36–1.31(m,4H),1.26–1.22(m,3H),0.92(t,J＝7.4Hz,3H)。
CTOP is collectively referred to as: (E) -1-butyl-4- (2- (7- (9-ethyl-9H-carbazol-3-yl) -2, 3-dihydrothiophene [3,4-b ] [1,4] dioxin-5-yl) vinyl) pyridin-1-ambroxide salt.
Example 2: change of fluorescence spectrum of probe CTOP with addition of OH
The CTOP fluorescent probe prepared in example 1 was dissolved in DMF to prepare a 1mmol/L stock solution. mu.L of the stock solution was taken out and put into a 5mL centrifuge tube, 100 equivalents OH standard solution was added, and the solution was diluted to 3mL (10. mu.M) with DMF/PBS (1:1, v/v) to measure the fluorescence property. The fluorescence spectrum is shown in FIG. 5, and it can be seen from FIG. 5 that the fluorescence intensity of CTOP decreases with the addition of OH, because the active oxygen five is generated to bleach the probe.
Example 3: biological compatibility test of CTOP Probe
The cells are cultured for 24h and 48h by using cell culture solution containing probes with different concentrations at 37 ℃, the percentage of the living cells is measured by using a microplate reader, and the obtained experimental result is shown in figure 6.
The above-listed values are exemplary, and other suitable ratios may be selected by those skilled in the art in light of the teachings of the present application and are within the scope of the present invention.
Claims (3)
1. A fluorescent probe for rapidly identifying hydroxyl radicals is characterized in that the chemical structural formula of the probe is shown as the formula (I):
2. the method for preparing the fluorescent probe capable of rapidly identifying the hydroxyl radicals as claimed in claim 1, which comprises the following steps:
(1) under the nitrogen environment, (9-ethyl-9H-carbazole-3-yl) boric acid, 7-bromo-2, 3-dihydrothieno [3, 4-b)][1,4]dioxane-5-Formaldehyde, Pd (PPH)3)4And K2CO3Dissolving in organic solvent and water, and refluxing the reactant overnight; cooling to room temperature, pouring the reacted product into water, and adding CH2Cl2Extracting, drying by anhydrous magnesium sulfate, and performing column chromatography separation to obtain a yellow intermediate 1;
(2) adding the intermediate 1 and 1-butyl-4-methylpyridine bromide into dry ethanol in an argon environment, and refluxing overnight under the catalysis of piperidine; after cooling to room temperature, the solvent was evaporated under reduced pressure; and (3) performing column chromatography separation to obtain a red-black product CTOP:
3. the use of the fluorescent probe for rapidly identifying hydroxyl radicals as claimed in claim 1, wherein the fluorescent probe is used for detecting hydroxyl radicals in an organism, and the in vivo model is arthritis.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114621248A (en) * | 2022-03-11 | 2022-06-14 | 华南理工大学 | Fluorescent probe for identifying RNA and having photodynamic effect and preparation method thereof |
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| JP5124780B2 (en) * | 2006-03-03 | 2013-01-23 | 国立大学法人 東京大学 | Fluorescent probe |
| CN103342720A (en) * | 2013-07-01 | 2013-10-09 | 南京大学 | Near infrared BODIPY (Boron Dipyrromethene Compounds) hydroxyl radical probes and synthesis method and usage thereof |
| CN105154065A (en) * | 2015-08-19 | 2015-12-16 | 济南大学 | Fluorescence probe for identifying hydroxyl radicals rapidly and specifically as well as preparation method and application of fluorescence probe |
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| JP5124780B2 (en) * | 2006-03-03 | 2013-01-23 | 国立大学法人 東京大学 | Fluorescent probe |
| CN103342720A (en) * | 2013-07-01 | 2013-10-09 | 南京大学 | Near infrared BODIPY (Boron Dipyrromethene Compounds) hydroxyl radical probes and synthesis method and usage thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114621248A (en) * | 2022-03-11 | 2022-06-14 | 华南理工大学 | Fluorescent probe for identifying RNA and having photodynamic effect and preparation method thereof |
| CN114621248B (en) * | 2022-03-11 | 2023-11-17 | 华南理工大学 | Fluorescent probe for recognizing RNA and having photodynamic and preparation method thereof |
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