CN117586245A - DNA fluorescent probe and preparation method and application thereof - Google Patents
DNA fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN117586245A CN117586245A CN202311546764.6A CN202311546764A CN117586245A CN 117586245 A CN117586245 A CN 117586245A CN 202311546764 A CN202311546764 A CN 202311546764A CN 117586245 A CN117586245 A CN 117586245A
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 51
- 238000000034 method Methods 0.000 claims abstract description 12
- 210000004027 cell Anatomy 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 16
- 239000003960 organic solvent Substances 0.000 claims description 16
- 238000003384 imaging method Methods 0.000 claims description 15
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- NTHMTYNJFSUBMF-UHFFFAOYSA-N 5-(5-bromothiophen-2-yl)thiophene-2-carbaldehyde Chemical compound S1C(Br)=CC=C1C1=CC=C(C=O)S1 NTHMTYNJFSUBMF-UHFFFAOYSA-N 0.000 claims description 9
- KFDVPJUYSDEJTH-UHFFFAOYSA-N 4-ethenylpyridine Chemical compound C=CC1=CC=NC=C1 KFDVPJUYSDEJTH-UHFFFAOYSA-N 0.000 claims description 7
- 238000007341 Heck reaction Methods 0.000 claims description 7
- YJVFFLUZDVXJQI-UHFFFAOYSA-L palladium(ii) acetate Chemical compound [Pd+2].CC([O-])=O.CC([O-])=O YJVFFLUZDVXJQI-UHFFFAOYSA-L 0.000 claims description 7
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- COIOYMYWGDAQPM-UHFFFAOYSA-N tris(2-methylphenyl)phosphane Chemical compound CC1=CC=CC=C1P(C=1C(=CC=CC=1)C)C1=CC=CC=C1C COIOYMYWGDAQPM-UHFFFAOYSA-N 0.000 claims description 6
- XXCFMHCLWHXDAV-UHFFFAOYSA-M 1,2-dimethylquinolin-1-ium;iodide Chemical compound [I-].C1=CC=CC2=[N+](C)C(C)=CC=C21 XXCFMHCLWHXDAV-UHFFFAOYSA-M 0.000 claims description 4
- 210000004556 brain Anatomy 0.000 claims description 4
- KEOLYURDXNRJEY-UHFFFAOYSA-N 1,2-dimethylquinolin-1-ium Chemical class C1=CC=CC2=[N+](C)C(C)=CC=C21 KEOLYURDXNRJEY-UHFFFAOYSA-N 0.000 claims description 3
- 210000004958 brain cell Anatomy 0.000 claims description 3
- 230000036647 reaction Effects 0.000 claims description 3
- 239000000523 sample Substances 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 5
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 3
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 3
- 239000001257 hydrogen Substances 0.000 abstract description 3
- 238000012984 biological imaging Methods 0.000 abstract description 2
- 238000001917 fluorescence detection Methods 0.000 abstract description 2
- 230000003993 interaction Effects 0.000 abstract description 2
- 238000009830 intercalation Methods 0.000 abstract description 2
- 230000002687 intercalation Effects 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 150000003577 thiophenes Chemical class 0.000 abstract description 2
- 102000053602 DNA Human genes 0.000 description 33
- 108020004414 DNA Proteins 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 11
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- 239000012452 mother liquor Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
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- 238000010438 heat treatment Methods 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
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- 230000035484 reaction time Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000007614 solvation Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- -1 50 mL) Chemical compound 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
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- 238000011534 incubation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000011369 resultant mixture Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
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- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 229940121863 DNA inhibitor Drugs 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention provides a DNA fluorescent probe and a preparation method and application thereof, belonging to the technical field of biochemical research. The DNA fluorescent probe (called compound BDSK for short) provided by the invention is a novel probe synthesized based on thiophene compounds, and can be combined with DNA through hydrogen bond interaction, TICT mechanism, intercalation and the like by virtue of the structural characteristics of the probe, so that biological imaging can be carried out on the DNA by a fluorescence detection method, and especially detection and fluorescence imaging of the DNA in cells can be realized. The compound BDSK provided by the invention is used for detecting DNA, and the operation method is simple and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biochemical research, in particular to a DNA fluorescent probe and a preparation method and application thereof.
Background
Deoxyribonucleic acid (DNA) is not only widely present in cells, but also has important implications for human health in studying the nature of DNA present in cells and humans. The detection of DNA in biological cells by the fluorescent probe BDSK is helpful for comprehensively knowing the characteristics of DNA distribution in the cells, and further, the deep research on the DNA is realized. Imaging of DNA within tumor cells via BDSK would provide an important reference for exploring the pathogenesis of genes causing major diseases.
However, the current techniques for DNA imaging in cells have shortcomings in terms of fluorescence intensity and staining time.
Disclosure of Invention
Aiming at the defects in the prior art, the invention develops a novel probe BDSK for the first time, which is used for detecting DNA in living cells and fluorescent imaging of DNA in cells. The DNA fluorescent probe BDSK provided by the invention not only has the tracking and imaging capabilities, but also has the advantages of near infrared fluorescent signals, strong fluorescent signals, short dyeing time and the like.
The invention also aims at providing a preparation method of the DNA fluorescent probe.
The invention also provides application of the DNA fluorescent probe in cell imaging.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a fluorescent probe, which has a structure shown in a formula II:
。
in the synthesis of formula II, the invention provides a synthesis method of an essential intermediate compound, which has a structure shown in formula I:
。
the invention also provides a preparation method of the necessary intermediate compound, which comprises the following specific operation steps: mixing 5-bromo-2, 2 '-bithiophene-5' -formaldehyde, 4-vinyl pyridine, palladium acetate, tri (o-methylphenyl) phosphorus and potassium carbonate with an organic solvent, and performing Heck reaction to obtain an intermediate with a structure shown in formula I.
Preferably, the molar ratio of the compound 5-bromo-2, 2 '-bithiophene-5' -formaldehyde, 4-vinylpyridine, palladium acetate, tris (o-methylphenyl) phosphorus and potassium carbonate is 1: (1.5-2.0): (0.1-0.3): (0.2-0.6): (2-5).
Preferably, the organic solvent includes, but is not limited to, DMF or acetonitrile, and the ratio of the organic solvent to the 5-bromo-2, 2 '-bithiophene-5' -formaldehyde is (2-6 mL): 1 mmol.
The invention provides a preparation method of an essential intermediate compound, wherein the Heck reaction is carried out under a heating condition, and the reaction time is 70-75 h. The heating temperature time of the reaction was 128 ℃.
The invention also provides a preparation method of the DNA fluorescent probe shown in the formula II, which specifically comprises the following steps: and mixing an intermediate compound shown in a formula I, iodinated 1, 2-dimethylquinolinium and tetrahydropyrrole with an organic solvent, and performing a brain cell reaction to obtain the DNA fluorescent probe with a structure shown in a formula II.
Preferably, the molar ratio of the intermediate compound to the 1, 2-dimethylquinolinium iodide to the tetrahydropyrrole is 1: (1-1.5): (1-5).
Preferably, the organic solvent includes, but is not limited to, methanol, ethanol or acetonitrile, and the ratio of the organic reagent to the intermediate compound of formula I is 300 mL:1 mmol.
According to the preparation method of the DNA fluorescent probe, the brain Wen Ge reaction is carried out at room temperature, and the reaction time is 8-24 h.
The invention also provides application of the DNA fluorescent probe in DNA imaging in living cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a fluorescent probe with a structure shown in a formula II, and the fluorescent probe (BDSK) is a novel probe synthesized based on thiophene compounds, and can be combined with DNA through hydrogen bond interaction, TICT mechanism, intercalation and the like by virtue of the structural characteristics of the probe, so that the DNA can be subjected to biological imaging by a fluorescent detection method. The compound BDSK provided by the invention is used for detecting DNA, and the operation method is simple and has wide application prospect.
Drawings
FIG. 1 is a Nuclear Magnetic Resonance (NMR) spectrum of a compound of formula I;
FIG. 2 is a high resolution mass spectrum of the compound of formula I;
FIG. 3 is a Nuclear Magnetic Resonance (NMR) spectrum of compound BDSK;
FIG. 4 is a high resolution mass spectrum of compound BDSK;
FIG. 5 is a graph showing the results of a solvation test of compound BDSK;
FIG. 6 is a graph showing the effect of compound BDSK on DNA imaging in cells;
FIG. 7 is a graph showing the effect of compound BDSK on DNA imaging in cells under different staining conditions;
Detailed Description
In the present invention, the raw materials used are commercially available products well known to those skilled in the art unless specified otherwise.
The invention provides a fluorescent probe, which has a structure shown in a formula II:
。
the DNA is a biological molecular compound, polymerized by a plurality of nucleotide monomers and in a spiral double-stranded structure, and the compound with the structure shown in the formula II provided by the invention is used as a fluorescent probe, and can realize detection of DNA in cells or environment through hydrogen bond self-assembly with a nucleic acid groove.
The invention provides a preparation method of the fluorescent probe, which comprises the following steps:
mixing 5-bromo-2, 2 '-bithiophene-5' -formaldehyde, 4-vinyl pyridine, palladium acetate, tri (o-methylphenyl) phosphorus and potassium carbonate with an organic solvent, and performing Heck reaction to obtain an intermediate compound with a structure shown in a formula I; and then mixing an intermediate compound shown in a formula I, iodinated 1, 2-dimethylquinolinium and tetrahydropyrrole with an organic solvent to perform a brain cell reaction, so as to obtain the fluorescent probe with a structure shown in a formula II.
In some embodiments, the compound 5-bromo-2, 2 '-bithiophene-5' -carbaldehyde, 4-vinylpyridine, palladium acetate, tris (o-methylphenyl) phosphorus, potassium carbonate is present in a molar ratio of 1: (1.5-2.0): (0.1-0.3): (0.2-0.6): (2-5). The organic solvent comprises DMF or acetonitrile, and the dosage ratio of the organic reagent to the 5-bromo-2, 2 '-bithiophene-5' -formaldehyde is (2-6 mL): 1 mmol. The Heck reaction is carried out under the heating condition, and the reaction time is 70-75 h. The heating temperature time of the reaction was 128 ℃.
The molar ratio of the compound shown in the structural formula I to the 1, 2-dimethylquinolinium iodide to the tetrahydropyrrole in the reaction of the brain Wen Ge is 1: (1-1.5): (1-5). The organic solvent comprises methanol, ethanol or acetonitrile, and the dosage ratio of the organic solvent to the compound shown in the structural formula I is 300 mL:1 mmol. The reaction is carried out at room temperature for a period of 8 to 24 h.
The synthesis steps of the fluorescent probe are as follows:
after the Heck reaction, the obtained mixture is preferably extracted by water and methylene dichloride, so that the solvent of the organic phase is evaporated to dryness, the obtained mixture is subjected to column chromatography, and finally the obtained red solid is an intermediate with the structure shown in the formula I. In the invention, the reagent used for the column chromatography separation is preferably petroleum ether: ethyl acetate = 1:1.
after the brain Wen Ge substitution reaction, the obtained mixture is preferably distilled in vacuum to evaporate the reaction solvent, and the obtained mixture is subjected to column chromatography separation, so that the finally obtained black solid is the fluorescent probe with the structure shown in the formula II. In the invention, the reagent used for the column chromatography separation is preferably dichloroethane: methanol=10: 1.
the invention provides application of the fluorescent probe in detecting DNA. In the present invention, the use of the fluorescent probe in detecting DNA may be, for example, the use of the fluorescent probe in preparing a DNA inhibitor. In the present invention, the DNA is preferably in a cell.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
5-bromo-2, 2 '-dithiophene-5' -carbaldehyde (2.73 g, 10 mmol) was dissolved in N, N-dimethylformamide (DMF, 50 mL), and palladium acetate (448 mg, 2.0 mmol), triorthophenylphosphine (912 mg, 3.0 mmol), and potassium carbonate (1.65 g, 12 mmol) were sequentially added to the resultant mixture with stirring. The mixture was stirred under nitrogen at 45 ℃ for 0.5 h. 4-vinylpyridine was then added to the reaction flask via syringe and the temperature was raised to 128 degrees celsius for 72 hours. The reaction system changes from yellow to black; after the reaction was completed, the mixture was extracted with water and methylene chloride. Evaporating the extracted organic phase in vacuum, and separating the mixture by column chromatography, wherein the reagent used for the column chromatography is petroleum ether: ethyl acetate = 1:1, the final red solid obtained is a compound of formula I in 35% yield.
FIG. 1 shows Nuclear Magnetic Resonance (NMR) spectra of a compound represented by formula I, and FIG. 2 shows a high resolution mass spectrum of a compound represented by formula I. As can be seen from FIGS. 1 and 2, the target product prepared in this example is indeed a compound of formula I.
Example 2
The compound shown in formula I (0.297 g, 1 mmol) was dissolved in methanol (MeOH, 200 mL), and 1, 2-dimethylquinolinium iodide (0.427 g, 1.5 mmol) was added to the resultant mixture with stirring, then tetrahydropyrrole 0.05 Ml was added dropwise, after the addition was completed, the reaction was performed at room temperature under nitrogen protection, 16 h was performed, and the reaction system was changed from yellow to black; after the reaction is finished, evaporating the obtained product system in vacuum, and separating the obtained mixture by column chromatography, wherein the reagent used for separating by column chromatography is dichloroethane: methanol=10: 1, the black solid finally obtained is compound BDSK, with a yield of 55%.
Fig. 3 is a Nuclear Magnetic Resonance (NMR) spectrum of compound BDSK, and fig. 4 is a high resolution mass spectrum of compound BDSK. As can be seen from fig. 1 and 2, the target product prepared in this example is indeed compound BDSK.
Test example 1
The solvation effect of compound BDSK was determined as follows:
dissolving compound BDSK in DMSO to obtain BDSK mother liquor with concentration of 8 mL of 1 mM; and respectively taking 25 mu L of BDSK mother liquor, adding the BDSK mother liquor into 5 identical 5mL volumetric flasks, respectively diluting and fixing the volume by using acetonitrile, tetrahydrofuran, dioxane, methanol, DMF, dimethyl sulfoxide, ethanol, PBS buffer solution (pH value is 7.2-7.4) and pure water, and then carrying out fluorescence detection under the excitation wavelength 510 and nm.
Fig. 5 is a graph showing the results of the solvation effect test of compound BDSK, and as can be seen from fig. 5, the maximum emission peak of compound BDSK in the organic phase and the maximum emission peak in the aqueous phase both appear around 690 nm.
Test example 2
Imaging application of analytical compound BDSK in living cells is specifically as follows:
dissolving compound BDSK in DMSO to obtain BDSK mother liquor with 10 mL concentration of 1 mM;
SW620 cells were cultured in dishes for 3 days using medium consisting of 0.9 DMEM and 0.1 foetal calf serum, respectively. During cell culture and culture, the cell culture dish and imaging dish were placed in a constant temperature incubator at 37 ℃ and 5% carbon dioxide. These cells were processed on a sterile super clean bench.
All cells were then incubated with probe BDSK (1. Mu.M) and commercial dye DAPI (5. Mu.M) for 30 min, washed 2 times with PBS and fluorescence imaged with confocal laser microscopy at excitation wavelength 561-nm and emission 600-700 nm.
Fig. 6 is a fluorescence imaging diagram of compounds BDSK and DAPI in SW620 cells, and as can be seen from fig. 6, the compound BDSK provided by the present invention can realize DNA imaging in SW620 cells.
Test example 3
Imaging application of analytical compound BDSK in living cells is specifically as follows:
dissolving compound BDSK in DMSO to obtain BDSK mother liquor with 10 mL concentration of 1 mM;
SW620 cells were cultured in dishes for 3 days using medium consisting of 0.9 DMEM and 0.1 foetal calf serum, respectively. During cell culture and culture, the cell culture dish and imaging dish were placed in a constant temperature incubator at 37 ℃ and 5% carbon dioxide. These cells were processed on a sterile super clean bench.
Cells were then incubated with probe BDSK under different conditions, including 1,2 μm and 20 min,10 min,5 min conditions, including treatment modes that were not used after incubation.
Fig. 7 is a fluorescence imaging diagram of compound BDSK in SW620 cells under different staining conditions, and as can be seen from fig. 7, the compound BDSK provided by the present invention can be incubated in SW620 cells for DNA imaging with short incubation time and low concentration. And the cells may not be washed prior to imaging.
As can be seen from the above test examples, the fluorescent probe provided by the invention can realize the detection of DNA in cells; the operation method is simple and has wide application prospect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An intermediate compound having the structure of formula i:
formula I.
2. A DNA fluorescent probe is characterized by having a structure shown in a formula II:
formula II.
3. A process for the preparation of an intermediate compound according to claim 1, comprising the steps of: mixing 5-bromo-2, 2 '-bithiophene-5' -formaldehyde, 4-vinyl pyridine, palladium acetate, tri (o-methylphenyl) phosphorus and potassium carbonate with an organic solvent, and performing Heck reaction to obtain an intermediate compound with a structure shown in a formula I.
4. The method according to claim 3, wherein the molar ratio of the 5-bromo-2, 2 '-bithiophene-5' -carbaldehyde, 4-vinylpyridine, palladium acetate, tris (o-methylphenyl) phosphorus and potassium carbonate is 1: (1.5-2.0): (0.1-0.3): (0.2-0.6): (2-5); the dosage ratio of the organic solvent to the 5-bromo-2, 2 '-bithiophene-5' -formaldehyde is (2-6 mL): 1 mmol.
5. The method of preparation according to claim 3 or 4, wherein the organic solvent comprises, but is not limited to DMF or acetonitrile.
6. The process according to any one of claims 3 to 5, wherein the Heck reaction is carried out at a temperature of 128 ℃ for 70 to 75 hours.
7. A method for preparing the DNA fluorescent probe as set forth in claim 2, comprising the steps of: and mixing an intermediate compound shown in a formula 1, iodinated 1, 2-dimethylquinolinium and tetrahydropyrrole with an organic solvent, and performing a brain cell reaction to obtain the fluorescent probe with the structure shown in a formula II.
8. The preparation method according to claim 7, wherein the molar ratio of the intermediate compound, 1, 2-dimethylquinolinium iodide and tetrahydropyrrole is 1: (1-1.5): (1-5); the dosage ratio of the organic solvent to the intermediate compound is (300 mL): 1 mmol.
9. The method of preparation according to claim 7 or 8, wherein the organic solvent includes, but is not limited to, methanol, ethanol or acetonitrile; the brain Wen Ge reaction is 8-24 h reaction at room temperature.
10. Use of a DNA fluorescent probe according to claim 2 or prepared by a method according to any one of claims 7 to 9 for detecting DNA and imaging living cells.
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