CN114014848B - RNA fluorescent probe and preparation method and application thereof - Google Patents
RNA fluorescent probe and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- 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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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
Abstract
The invention relates to the technical field of probe detection, in particular to an RNA fluorescent probe and a preparation method and application thereof. The RNA fluorescent probe is a novel probe synthesized on the basis of thiophene compounds, can be combined with RNA through hydrogen bond interaction, intercalation action and the like by virtue of the characteristic that a self V-shaped conjugated structure is connected with a rigid plane in a single bond form, is limited in free rotation of a single bond of the probe after the RNA is embedded to generate a Torsion Intramolecular Charge Transfer (TICT) mechanism effect, can carry out biological imaging on the RNA through a fluorescence detection method, and particularly can realize the detection of the RNA in a cell or water environment. According to the description of the embodiment, the RNA fluorescent probe not only has high stability, low background fluorescence and high sensitivity, but also has the tracking and imaging capabilities.
Description
Technical Field
The invention relates to the technical field of probe detection, in particular to an RNA fluorescent probe and a preparation method and application thereof.
Background
Ribonucleic acid (RNA) is not only present in living cells, but is also distributed in environmental microorganisms. Therefore, it is important to study the existence characteristics of RNA in microorganisms and human bodies to evaluate the ecological environment and human health.
At present, the number of commercialized RNA probes is small, and SYTO RNAselect Green fluorescence Cell Stain produced in the United states is more commonly used, but the probe has poor light stability and poor detection effect.
Disclosure of Invention
The invention aims to provide an RNA fluorescent probe, a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an RNA fluorescent probe, which has a structure shown in a formula I:
the invention also provides a preparation method of the RNA fluorescent probe in the technical scheme, which comprises the following steps:
carrying out first mixing on a compound with a structure shown in a formula II, iodoethane and an organic solvent, and then carrying out nucleophilic substitution reaction to obtain the RNA fluorescent probe;
preferably, the molar ratio of the compound having the structure shown in the formula II to the iodoethane is 1: (3.0-3.5).
Preferably, the compound having the structure shown in the formula II and the first organic solvent are used in a ratio of 1 mmol: (90-120) mL.
Preferably, the first mixing comprises mixing the compound with the structure shown in the formula II and the first organic solvent, and then dropwise adding iodoethane.
Preferably, the nucleophilic substitution reaction is performed under reflux conditions;
the temperature of the nucleophilic substitution reaction is 75-95 ℃, and the time is 70-75 h.
Preferably, the preparation method of the compound with the structure shown in the formula II comprises the following steps:
and (3) carrying out second mixing on bithiophene dibromide, potassium carbonate, palladium acetate, 3-o-tolylphosphorus and a second organic solvent, then dropwise adding 4-vinylpyridine, and carrying out coupling reaction to obtain the compound with the structure shown in the formula II.
Preferably, the mol ratio of bithiophene dibromide to 4-vinylpyridine is (0.8-1.2): 4.
preferably, the coupling reaction is carried out under a protective atmosphere and under reflux conditions;
the temperature of the coupling reaction is 110-140 ℃, and the time is 65-80 h.
The invention also provides the application of the RNA fluorescent probe in the technical scheme or the RNA fluorescent probe prepared by the preparation method in the technical scheme in the detection of RNA.
The invention provides an RNA fluorescent probe, which has a structure shown in a formula I:
the RNA fluorescent probe is a novel probe synthesized on the basis of thiophene compounds, can be combined with RNA through hydrogen bond interaction, intercalation action and the like by virtue of the characteristic that a self V-shaped conjugated structure is connected with a rigid plane in a single bond form, and can generate a Torsion Intramolecular Charge Transfer (TICT) mechanism effect by limited free rotation of the single bond of the RNA fluorescent probe after the RNA is embedded, so that the RNA can be subjected to biological imaging by a fluorescence detection method, and especially the detection of the RNA in a cell or water environment can be realized. According to the description of the embodiment, the RNA fluorescent probe not only has high stability, low background fluorescence and high sensitivity, but also has the tracking and imaging capabilities. Therefore, the RNA fluorescent probe can realize the detection of RNA in cells or water environment, and the RNA in various tumor cells is imaged by the RNA fluorescent probe, so that important reference is provided for exploring pathogenesis of gene-induced major diseases.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of Bptp-R1 prepared in example 1;
FIG. 2 is a high resolution mass spectrum of Bptp-R1 prepared in example 1;
FIG. 3 is a graph showing the results of the solvation effect test of Bptp-R1 prepared in example 1;
FIG. 4 is a graph of the fluorescence spectrum of Bptp-R1 in example 1 under different RNA concentrations in an aqueous environment;
FIG. 5 is a graph showing the variation of the highest emission peak under different RNA concentrations in FIG. 4;
FIG. 6 is a photograph of the fluorescence images of Bptp-R1 in different cell types according to example 1.
Detailed Description
The invention provides an RNA fluorescent probe, which has a structure shown in a formula I:
the invention also provides a preparation method of the RNA fluorescent probe in the technical scheme, which comprises the following steps:
carrying out first mixing on a compound with a structure shown in a formula II, iodoethane and a first organic solvent, and then carrying out nucleophilic substitution reaction to obtain the RNA fluorescent probe;
in the present invention, all the starting materials for the preparation are commercially available products known to those skilled in the art unless otherwise specified.
In the present invention, the compound having the structure shown in formula II is named as 5,5 '-bis (2- (4-pyridyl) ethenyl) -2,2' -bithiophene in Chinese, and abbreviated as PPVB in English.
In the present invention, the preparation method of the compound having the structure shown in formula ii preferably comprises the following steps:
and (3) carrying out second mixing on bithiophene dibromide, potassium carbonate, palladium acetate, 3-o-tolylphosphorus and a second organic solvent, then dropwise adding 4-vinylpyridine, and carrying out coupling reaction to obtain the compound with the structure shown in the formula II.
In the present invention, the molar ratio of bithiophene dibromide, potassium carbonate, palladium acetate and 3-o-tolylphosphorus is preferably 3.1:10.0:0.3: 0.9.
In the present invention, the second organic solvent is preferably N, N-dimethylformamide; the usage ratio of the bithiophene dibromide to the second organic solvent is preferably 3.1 mmol: 30 mL.
In the present invention, the second mixing is preferably performed under stirring, and the stirring time is preferably 0.5 h; the stirring speed is not limited in any way, and the stirring can be carried out by adopting a process well known to a person skilled in the art.
In the present invention, the 4-vinylpyridine is preferably added dropwise.
In the invention, the molar ratio of bithiophene dibromide to 4-vinylpyridine is preferably (0.8-1.2): 4, more preferably 3.1: 12.
In the present invention, the coupling reaction is preferably carried out under a protective atmosphere and under reflux; the protective atmosphere is preferably a nitrogen atmosphere. In the invention, the temperature of the coupling reaction is preferably 110-140 ℃, more preferably 120-130 ℃, most preferably 120 ℃, and the time is preferably 65-80 h, more preferably 68-75 h, most preferably 72 h.
After the coupling reaction is finished, the invention also preferably comprises the steps of mixing the obtained product system with an ice-water mixture, and then sequentially filtering, extracting and separating by column chromatography. In the present invention, the mixing of the product system obtained with an ice-water mixture is carried out in order to rapidly lower the temperature of the product system obtained and to wash out the poorly soluble impurities from the system. The filtration process is not particularly limited in the present invention, and may be performed by a process known to those skilled in the art. In the present invention, the extractant used for the extraction is preferably a mixed solution of dichloromethane and water in a volume ratio of 1: 5. After the extraction is completed, the present invention preferably performs column chromatography separation on the obtained organic phase. In the invention, the reagent used for column chromatography separation is preferably a mixed solution of dichloroethane and methanol in a volume ratio of 50: 1.
In the present invention, the first organic solvent is preferably acetonitrile and/or ethanol, and when the first organic solvent is acetonitrile and ethanol, the ratio of acetonitrile to ethanol is not particularly limited, and the first organic solvent and the ethanol may be mixed in any ratio.
In the present invention, the molar ratio of PPVB to iodoethane is preferably 1: (3.0 to 3.5), more preferably 1: (3.1 to 3.4), most preferably 1: (3.2-3.3).
In the present invention, the ratio of the amounts of PPVB and the first organic solvent is preferably 1 mmol: (90-120) mL, more preferably 1 mmol: (100-110) mL.
In the present invention, the first mixing preferably includes mixing the compound having the structure represented by formula ii with the first organic solvent, and then dropping ethyl iodide.
In the present invention, the nucleophilic substitution reaction is preferably performed under reflux conditions; the temperature of the nucleophilic substitution reaction is preferably 75-95 ℃, more preferably 80-90 ℃, and most preferably 85 ℃; the time is preferably 70-75 h, and more preferably 75 h. In the present invention, the initiation time of the nucleophilic substitution reaction is preferably based on the time after completion of the dropwise addition of iodoethane.
After the nucleophilic substitution reaction is completed, the method also preferably comprises vacuum distillation and column chromatography separation which are sequentially carried out; the vacuum distillation process is not particularly limited in the present invention, and may be performed by a process known to those skilled in the art. In the invention, the reagent used for column chromatography separation is preferably a mixed solution of dichloroethane and methanol in a volume ratio of 10: 1.
The invention also provides the application of the RNA fluorescent probe in the technical scheme or the RNA fluorescent probe prepared by the preparation method in the technical scheme in the detection of RNA. In the present invention, the application is preferably detection of aqueous environment or detection of endogenous RNA in imaging of different kinds of cancer cells.
The following will describe the RNA fluorescent probe provided by the present invention, its preparation method and application in detail with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Adding bithiophene dibromide (1.0g, 3.1mmol) and K2CO3Dissolving (1.38g, 10.0mmol), palladium acetate (0.07g, 0.3mmol) and 3-o-tolylphosphorus (0.9mmol) in N, N-dimethylformamide (DMF, 30mL), stirring and mixing for 0.5h, dropwise adding 4-vinylpyridine (1.3g, 12.0mmol) to the obtained mixture, and after the dropwise addition is finished, refluxing and reacting at 120 ℃ for 72h under the protection of nitrogen, wherein the reaction system is changed from light yellow to black; after the reaction is finished, mixing the obtained product system with an ice water mixture (0 ℃) to rapidly reduce the temperature of the product system, precipitating impurities with poor solubility in the system, filtering, and extracting the obtained filtrate by adopting dichloromethane and water, wherein the volume ratio of the dichloromethane and the water used for extraction is 1: 5; and (3) carrying out column chromatography separation on the organic phase obtained by extraction, wherein the reagents used in the column chromatography separation are dichloroethane: methanol 50:1, the yellow solid finally obtained is a compound PPVB, and the yield is 35%;
dissolving the PPVB (0.372g and 1.0mmol) in acetonitrile (MeCN and 100mL), stirring and mixing, dropwise adding iodoethane (0.468g and 3.0mmol) into the obtained mixture, after the dropwise adding is finished, carrying out reflux reaction at 85 ℃ for 75 hours, changing the reaction system from yellow to black, after the reaction is finished, carrying out vacuum evaporation on the obtained product system, and carrying out column chromatography separation by taking a mixed solution of dichloroethane and methanol with a volume ratio of 10:1 as a separation reagent to obtain an RNA fluorescent probe (recorded as Bptp-R1), wherein the yield is 35%;
performing a nuclear magnetic resonance test on the Bptp-R1, wherein the test result is shown in FIG. 1; the Bptp-R1 is subjected to high-resolution mass spectrometry, the test result is shown in FIG. 2, and the Bptp-R1 is shown in FIGS. 1-2.
Test example 1
The solvation effect of the Bptp-R1 was determined by the following test procedure:
dissolving the Bptp-R1 in N, N-Dimethylformamide (DMF) to obtain 8mL of 1mM Bptp-R1 mother liquor;
adding 25 mu L of Bptp-R1 mother liquor into 5 identical volumetric flasks of 5mL, diluting the volumetric flasks with DMF, acetonitrile, PBS buffer solution (pH value is 7.2-7.4), methanol and pure water to constant volume, and performing fluorescence detection at an excitation wavelength of 488 nm.
FIG. 3 is a graph showing the results of the solvation effect test of the Bptp-R1, wherein A is an absorption spectrum and B is a fluorescence spectrum; as can be seen from FIG. 3, the maximum emission peak of Bptp-R1 in the organic phase and the maximum emission peak in the aqueous phase both appear around 550 nm.
Test example 2
Dissolving the Bptp-R1 in DMF to obtain 8mL of 1mM Bptp-R1 mother liquor;
dissolving RNA in Tris-HCl buffer solution (10mM Tris-HCl,100mM KCl, pH 7.2) to obtain RNA mother liquor with higher concentration;
determining the concentration by Lambert beer's law, then diluting to different concentrations (0-0.5 mM), respectively taking 1.99mL of RNA diluent with different concentrations to 20 identical 2mL volumetric flasks, adding 10 muL of Bptp-R1 mother liquor into each volumetric flask to obtain a series of solutions to be detected with different RNA concentrations, and carrying out fluorescence detection on each solution to be detected under the condition of an excitation wavelength of 550 nm;
FIG. 4 is a fluorescence spectrum of the Bptp-R1 under different RNA concentrations in an aqueous environment, and it can be seen from FIG. 4 that an emission peak appears at 635nm, and the fluorescence intensity gradually increases with the increase of the RNA concentration in the solution to be detected, which indicates that the Bptp-R1 provided by the invention can realize the detection of RNA in the aqueous environment;
FIG. 5 is a graph showing the variation trend of the highest emission peak under different RNA concentrations in FIG. 4. from FIG. 5, it can be seen that the emission peak shows a trend of increasing gradually to equilibrium at 635nm under the condition of the excitation wavelength of 550nm, which indicates that the Bptp-R1 interacts with RNA to change the fluorescence spectrum of the Bptp-R1, specifically, the fluorescence intensity gradually increases with the increase of the RNA concentration.
Test example 3
Analyzing the imaging application of the Bptp-R1 in the living cells, and concretely, the following steps are carried out:
dissolving the Bptp-R1 in DMF to obtain 10mL of 1mM Bptp-R1 mother liquor;
HeLa, A549 and HepG2 cells were cultured in a petri dish for 3 days using a culture medium consisting of 0.9DMEM and 0.1 fetal bovine serum, respectively. During cell culture and growth, the cell culture dishes and imaging dishes were placed in an incubator at 37 ℃ and 5% carbon dioxide. These cells were processed on a sterile clean bench.
In cell digestion experiments, cells were fixed on the imaging dish surface with 4% formalin fixation solution, and then 0.4 wt% Triton X-100 was added to perforate the cell membrane. After washing the cells with PBS, each cell was divided into two groups. One group was treated with RNase-containing PBS (30. mu.g/mL) for 2h, and the other group was treated with clean PBS as a control. Then all cells are incubated with a probe Bptp-R1(10 mu M) for 30min, washed for 2 times by PBS, and then fluorescence imaging is carried out by a confocal laser microscope, wherein the excitation wavelength is 488nm, and the emission is 570-620 nm.
FIG. 6 is a graph of fluorescence images of the Bptp-R1 in different cell types, wherein (A1, B1 and C1) are fluorescence image contrast before and after RNase digestion of the probe in HeLa (A1), HepG2(B1) and A549(C1) cells respectively, (A2, B2 and C2) are fluorescence intensity contrast in cytoplasm and nucleus of HeLa (A2), HepG2(B2) and A549(C2) cells before and after digestion respectively; as can be seen from FIG. 6, the Bptp-R1 provided by the present invention can realize the imaging of RNA in different kinds of cells.
Therefore, the fluorescent probe provided by the invention not only can detect RNA in a water phase, but also can detect the RNA in cells; the operation method is simple and has wide application prospect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An RNA fluorescent probe, which is characterized by having a structure shown in a formula I:
2. The method for preparing the RNA fluorescent probe of claim 1, which comprises the following steps:
carrying out first mixing on a compound with a structure shown in a formula II, iodoethane and an organic solvent, and then carrying out nucleophilic substitution reaction to obtain the RNA fluorescent probe;
3. The method of claim 2, wherein the compound having the structure of formula ii and the iodoethane are present in a molar ratio of 1: (3.0-3.5).
4. The method according to claim 2, wherein the compound having the structure represented by formula ii and the first organic solvent are used in a ratio of 1 mmol: (90-120) mL.
5. The method of claim 2, wherein the first mixing comprises mixing the compound having the structure of formula ii with the first organic solvent, and then adding iodoethane dropwise.
6. A production method according to any one of claims 2 to 5, wherein the nucleophilic substitution reaction is carried out under reflux conditions;
the temperature of the nucleophilic substitution reaction is 75-95 ℃, and the time is 70-75 h.
7. The method of any one of claims 2 to 5, wherein the method for preparing the compound having the structure represented by formula II comprises the following steps:
and (3) carrying out second mixing on bithiophene dibromide, potassium carbonate, palladium acetate, tri (o-methylphenyl) phosphorus and a second organic solvent, then dropwise adding 4-vinylpyridine, and carrying out coupling reaction to obtain the compound with the structure shown in the formula II.
8. The method according to claim 7, wherein the bithiophene bisbromide and 4-vinylpyridine are present in a molar ratio of (0.8 to 1.2): 4.
9. the method of claim 7, wherein the coupling reaction is carried out under a protective atmosphere and under reflux;
the temperature of the coupling reaction is 110-140 ℃, and the time is 65-80 h.
10. Use of the RNA fluorescent probe of claim 1 or the RNA fluorescent probe prepared by the preparation method of any one of claims 2 to 9 in preparation of an RNA detection probe.
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