CN111100151A - Preparation method of probe for specifically detecting parallel configuration G-quadruplex DNA - Google Patents

Preparation method of probe for specifically detecting parallel configuration G-quadruplex DNA Download PDF

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CN111100151A
CN111100151A CN201911318427.5A CN201911318427A CN111100151A CN 111100151 A CN111100151 A CN 111100151A CN 201911318427 A CN201911318427 A CN 201911318427A CN 111100151 A CN111100151 A CN 111100151A
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王明齐
马瑞
于全琦
刘洪孛
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Abstract

The invention discloses a preparation method of a probe for specifically detecting a parallel configuration G-quadruplex DNA, belonging to the technical field of biological probes. The specific structural formula of the related probe is as follows:
Figure DDA0002326489980000011
the probe has the advantages of simple preparation steps, easily obtained raw materials and low fluorescence background. The rapid dual-function specificity detection of the parallel configuration G-quadruplex DNA can be realized by an ultraviolet spectrophotometer and a fluorescence spectrophotometer. Overcomes the defects of single and difficult differentiation of G-quadruplex DNA with different configurations of the traditional detection method, and has the advantages of simple structure, low cost, high specificityHas wide application prospect.

Description

Preparation method of probe for specifically detecting parallel configuration G-quadruplex DNA
Technical Field
The invention relates to construction of a novel bifunctional probe, a preparation method and application thereof, belonging to the technical field of biological probes.
Background
The G-quadruplex DNA is a special DNA structure, and is a high-level DNA secondary structure formed by self-assembling four guanine rings of a guanine-rich DNA sequence into a G-tetrad through Hoogsteen hydrogen bonds under a certain condition and forming two or more G-tetrads through pi-pi stacking action. The G-quadruplexes can be divided into three different structures, i.e., parallel, antiparallel and mixed, according to the orientation of the four chains in the G-quadruplexes. Recent bioinformatics studies have shown that approximately 37 million guanine-rich gene sequences, which may form a G-quadruplex structure in humans, are common, particularly in the telomere ends and oncogene promoter regions of humans (e.g., c-myc, ckit, bcl-2, Pu27, kRAS, VEGFR, TERT, etc.). This "new" DNA structure is widely distributed in eukaryotic genomes, such as telomere ends, rDNA, and gene promoter regions. Biological function research shows that the structure relates to the processes of DNA replication, telomere maintenance, gene expression and regulation, and the like, and is closely related to the proliferation and apoptosis of cells and the occurrence and development of tumors. However, only a few G-quadruplex DNAs have been studied in detail so far. Therefore, the construction and related action research of the probe molecule with the specific recognition G-quadruplex DNA structure lays a foundation for understanding the distribution, the function and the mechanism of the structure in the gene, and provides a new opportunity for specifically regulating and controlling the cell biological behavior.
Fluorescence imaging is widely applied to molecular detection as an intuitive and in-situ visual observation technology. The molecular fluorescent probe based on the organic fluorophore has the advantages of high sensitivity, simplicity and convenience in operation, good reproducibility and the like, can be conveniently used for in-situ real-time nondestructive detection of target molecules in a biological system, is used for monitoring biomolecules and biological processes of living cells and living bodies, and increasingly becomes an indispensable molecular tool in the fields of modern life science, disease diagnosis and the like. To date, the detection of many small molecule probes targeting the G-quadruplex DNA structure in vitro and even in vivo has made a number of important research advances. But more problems need to be solved in practical application due to the complex in vivo environment. The G-quadruplex DNA structure is very small in the whole genome and almost submerged in the sea of double helix DNA. Furthermore, the secondary structure of G-quadruplex DNA is complex and diverse, including parallel, antiparallel and mixed configurations. Thus, there are few reports on the ability of probe molecules to selectively detect G-quadruplex DNA structures, even one type of configuration. Most reported probes have single means for detecting G-quadruplex DNA conformation research and have larger interference.
In recent years, BODIPY (BODIPY) is an ideal bioluminescent probe which is widely researched and applied and has excellent optical properties such as low biotoxicity, adjustable fluorescence characteristics, insensitivity to environment, strong photo-thermal stability and the like. The applicant synthesizes a series of aryl ethylene compounds with conjugated functional side chains in the early stage, and the aryl ethylene compounds have better binding capacity to G-quadruplex DNA. The applicant combines a functional side chain into a BODIPY skeleton structure to obtain a novel G-quadruplex DNA probe molecule with a bifunctional detection parallel configuration.
Disclosure of Invention
The invention aims to provide a synthetic method of a G-quadruplex DNA probe with a double-function detection parallel configuration and application thereof aiming at the defects of the prior art.
The invention provides a G-quadruplex DNA probe with a bifunctional detection parallel configuration, which has the following structural formula:
Figure BDA0002326489960000021
the invention relates to a preparation method of a G-quadruplex DNA probe with a bifunctional detection parallel configuration, which has the following reaction equation:
Figure BDA0002326489960000022
the invention relates to a preparation method of a G-quadruplex DNA probe with a double-function detection parallel configuration, which comprises the following steps:
(1) under the protection of nitrogen, using anhydrous dichloromethane as a solvent, reacting ether oxygen chain modified p-hydroxybenzaldehyde (compound 1) with 2, 4-dimethylpyrrole, 2, 3-dichloro-5, 6-dicyano-p-benzoquinone, boron trifluoride diethyl ether and triethylamine in a molar ratio of 1:2:1:30:35 under stirring at room temperature, and separating and purifying a product to obtain a compound 3. The structural formula of compound 3 is:
Figure BDA0002326489960000023
(2) dissolving the compound 4, 4-fluorobenzaldehyde and potassium carbonate in anhydrous acetonitrile according to the molar ratio of 1:1:3, performing reflux reaction, and separating and purifying a product to obtain a compound 5. The structural formula of compound 4 is:
Figure BDA0002326489960000024
the structural formula of compound 5 is:
Figure BDA0002326489960000031
(3) dissolving the compound 3, the compound 5, piperidine and glacial acetic acid in toluene according to the molar ratio of 1:1:10:10, reacting at 130 ℃, and separating and purifying the product to obtain the final probe compound. The structural formula of the probe compound is:
Figure BDA0002326489960000032
the invention also provides application of the fluorescent probe in selective recognition of G-quadruplex DNA with parallel configuration.
The method comprises the following steps:
1) dissolving a G-quadruplex DNA probe with a dual-function detection parallel configuration by using DMSO, and diluting by using a buffer solution with pH7.4 to obtain a solution B; the DNA sample to be tested was dissolved in a buffer solution of pH 7.4.
2) And mixing the solution B and the solution A to ensure that the molar ratio of the DNA sample to be detected to the probe in the mixed solution is 2-1, analyzing the spectral change of the mixed solution by using a fluorescence spectrophotometer or an ultraviolet spectrophotometer to judge whether the DNA sample to be detected is a G-quadruplex DNA structure with a parallel configuration. The judgment method comprises the following steps:
(a) when the mixed solution is analyzed by a fluorescence spectrophotometer, compared with the solution B, if the fluorescence intensity of the fluorescence spectrum of the mixed solution at 606nm is obviously enhanced (the enhancement range is 150-250 times), the DNA sample to be detected can be judged to be a G-quadruplex DNA structure with parallel configuration; if the fluorescence intensity of the mixed solution is not obviously enhanced (less than 150 times) relative to that of the solution B, the detected DNA sample can be judged to be a G-quadruplex DNA structure with a non-parallel configuration.
(b) When the mixed solution is analyzed by an ultraviolet spectrophotometer, compared with the solution B, if the absorption spectrum of the mixed solution has an obvious new peak at 584nm (A: A)0>1.05) and the peak width is narrowed, the G-quadruplex DNA structure of the parallel configuration of the DNA sample to be detected can be judged; if the absorbance spectrum of the mixed solution does not change or decreases with respect to that of solution B (A: A)0<1.05), the detected DNA sample can be judged to be a G-quadruplex DNA structure with a non-parallel configuration.
Compared with the prior art, the invention has the following advantages:
the probe preparation synthetic route has strong operability, easily obtained raw materials and low preparation cost. And the parallel G-quadruplex DNA structure can be detected with dual-function specificity through ultraviolet spectrum or fluorescence spectrum, so that the parallel G-quadruplex DNA structure can be distinguished from other antiparallel G-quadruplex, single-strand and double-strand DNA structures. Overcomes the defects of single and difficult differentiation of G-quadruplex DNA with different configurations in the traditional detection method
Drawings
FIG. 1 is a UV spectrum of probe-titrated parallel configuration G-quadruplex DNA (c-myc);
FIG. 2 is a fluorescence spectrum of probe-titrated parallel configuration G-quadruplex DNA (c-myc);
FIG. 3 is an ultraviolet spectrum of probe-titrated antiparallel configuration G-quadruplex DNA (Oxy 28);
FIG. 4 is a fluorescence spectrum of probe-titrated antiparallel configuration G-quadruplex DNA (Oxy 28);
FIG. 5 is a UV spectrum of probe-titrated double-stranded DNA (ds 26);
FIG. 6 is a fluorescence spectrum of probe-titrated double-stranded DNA (ds 26);
FIG. 7 shows UV spectra of probe-titrated single-stranded DNA (ss 26);
FIG. 8 shows fluorescence spectra of probe-titrated single-stranded DNA (ss 26);
FIG. 9 shows the ultraviolet (A/A) of a probe for DNAs of different structures0) A dot plot of responses;
FIG. 10 shows fluorescence (F/F) of different structural DNAs with a probe0) Histogram of the response.
Detailed Description
The present invention will be further described with reference to the following detailed description and accompanying drawings so that those skilled in the art can better understand the technical solution of the present invention.
Example 1: synthesis of Compound 3
Under the protection of nitrogen, 1.0g (3.7mmol) of ether oxygen chain modified p-hydroxybenzaldehyde (compound 1) and 0.7g (7.4mmol) of 2, 4-dimethylpyrrole are dissolved in 100mL of anhydrous dichloromethane, 3-5 drops of a catalytic amount of trifluoroacetic acid are added dropwise, and the mixture is stirred at room temperature for 12 hours, then 0.84g (3.7mmol) of 2, 3-dichloro-5, 6-dicyano-p-benzoquinone is added, and the mixture is stirred for 12 hours, 18mL (129mmol) of triethylamine is added, the mixture is stirred for 30 minutes, 15mL (118.0mmol) of boron trifluoride diethyl ether is added, and the mixture is stirred for 8 hours. Washing with ultrapure water (25mL × 2 times) and saturated NaCl aqueous solution (25mL × 2 times) in this order, drying the obtained organic phase with anhydrous sodium sulfate, and distilling off dichloromethane under reduced pressure to obtain a crude product, which is subjected to column chromatography purification using a mixed solvent of ethyl acetate and petroleum ether at a volume ratio of 1:1 as a mobile phase and silica gel as a stationary phase, to obtain 0.45g of red solid product 3 with a yield of 24.8%.1H NMR(400MHz，CDCl3)δ：7.16(m，2H)，7.02(m，2H)，5.97(s，2H)，4.18(t，J＝4.6Hz，2H)，3.91(t，J＝4.88Hz，2H)，3.78-3.75(m，2H),3.72-3.65(m，4H)，3.57-3.55(m，2H)，3.38(s，3H)，2.54(s，6H)，1.42(s，6H)。
Example 2: synthesis of Compound 5
0.65g (5.0mmol) of N-hydroxyethylpiperazine was dissolved in 50mL of DMSO, 0.62g (5.0mmol) of 4-fluorobenzaldehyde and 2.0g (15.0mmol) of K were added2CO3The reaction was heated in an oil bath at 100 ℃ for 24 hours. After the reaction, 100mL of water was added, extraction was performed with dichloromethane (50 mL. times.3 times), the resulting organic phase was dried over anhydrous sodium sulfate, dichloromethane was distilled off under reduced pressure, and the crude product was obtained in a volume ratio of ethyl acetate to petroleum etherAnd (3) performing column chromatography purification by using a mixed solvent of 2:1 as a mobile phase and silica gel as a stationary phase to obtain 0.80g of a yellow solid product 5, wherein the yield is 68.3%.
Example 3: synthesis of Probe
0.4g (0.82mmol) of Compound 3, 0.19g (0.82mmol) of Compound 5, 0.8mL of glacial acetic acid, and 1mL of piperidine were dissolved in 50mL of toluene, and heated to 120 ℃ for reaction for 8 hours. After the reaction, the toluene was distilled off under reduced pressure, and the obtained crude product was purified by column chromatography using a mixed solvent of dichloromethane and methanol in a volume ratio of 20:1 as a mobile phase and silica gel as a stationary phase to obtain 0.21g of a dark blue solid product probe, with a yield of 36.8%.1H NMR(400MHz，CDCl3)δ：7.54-7.49(m，3H),7.19-7.15(m，3H)，7.02(d，J＝8.24Hz,2H)，6.88(d，J＝8.56Hz,2H)，6.57(s，1H)，5.98(s，1H)，4.19(t，J＝4.28Hz，2H)，3.91(t，J＝4.64Hz，2H),3.78-3.76(m，6H)，3.70-3.66(m，4H)，3.57-3.55(m，2H)，3.38(m，5H)，2.86(br，4H)，2.76(m，2H)，2.57(s，3H)，1.46(s，3H)，1.42(s，3H)；19FNMR(376MHz，CDCl3) δ: -139.66, -139.73, -139.84, -139.92; HRMS (ESI) m/z: theoretical value C39H50BF2N4O5[M+H]+703.3842; experimental value, 703.3831.
Example 4: application of the Probe of the present invention
(1) Preparation of DNA samples. DNA samples were purchased from Shanghai Producers, Inc. The DNA sample was dissolved in an appropriate amount of buffer solution (10mM Tris-HCl, containing 60mM KCl) at pH 7.4.
The parallel configuration G-quadruplex DNA sequence used in this experiment is:
sequence number CM 22: 5'-TGAGGGTGGGTAGGGTGGGTAA-3'
Sequence number c-myc: 5'-TTGAGGGTGGGTAGGGTGGGTAAA-3'
Sequence number Ckit 1: 5'-AGGGAGGGCGCTGGGAGGAGGG-3'
Sequence number TRF 2: 5'-CGGGAGGGCGGGGAGGGC-3'
Sequence number Chl 1: 5'-CGGGCGGGGAAGGGGTGGGA-3'
Sequence number VAV 1: 5'-GGGCAGGGAGGGAACTGGG-3'
Sequence number Bcl 2: 5'-GGGCGGGCGCGGGAGGAAGGGGGCGGG-3'
Sequence number EAD: 5'-CTGGGTGGGTGGGTGGGA-3'
Sequence number Pu 27: 5'-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3'
The antiparallel G-quadruplex DNA sequence used in this experiment was:
sequence number Ckit: 5'-GGCGAGGAGGGGCGTGGCCGGC-3'
Sequence number HRAS: 5'-TCGGGTTGCGGGCGCAGGGCACGGGCG-3'
Sequence number Hum 24: 5'-TTAGGGTTAGGGTTAGGGTTAGGG-3'
Sequence number ASC 20: 5'-GGCTTAGGCTTAGGCTTAGG-3'
Sequence number ODN: 5'-GGGATGGGACACAGGGGACGGG-3'
Sequence number Oxy 28: 5'-GGGGTTTTGGGGTTTTGGGGTTTTGGGG-3'
The double-stranded DNA sequences used in this experiment were:
sequence number ds 26: 5'-CAATCGGATCGAATTCGATCCGATTG-3'
Sequence number d (A-T)2：5’-GCATGCGCGCGCGCATGC-3’
Sequence number d (A-T)5：5’-GCGCATATATATATGCGC-3’
Sequence number d (A-T)9：5’-ATATATATATATATATAT-3’
Sequence number d (G-C)9：5’-GCGCGCGCGCGCGCGCGC-3’
The single-stranded DNA sequences used in this experiment were:
sequence number ss 26: 5'-CCGCGAACGCCTAAGCTGCTAACCGC-3'
(2) And (3) preparing a probe solution. The probes were formulated as stock solutions in DMSO solvent and diluted with 10mM Tris-HCl buffer (pH 7.4, containing 60mM KCl) for testing.
(3) And (5) detecting by ultraviolet spectrum. The concentration of the fixed fluorescent probe solution is 2 mu M, different DNA samples are respectively dripped into the probe solution, the probe solution is stabilized for 1 minute after being homogenized, and the absorption spectrum of the system is measured by an ultraviolet spectrophotometer. The titration experiment result is shown in figures 1, 3, 5, 7 and 9, if a new absorption peak is generated at 584nm of the test sample, and the absorbance increase multiple at 584nm is more than 1.05 times, the DNA sample to be detected can be judged to be a G-quadruplex DNA structure with parallel configuration; if the absorbance of the mixture is not changed or decreased and the absorbance at 584nm is increased by a factor of less than 1.05, the detected DNA sample can be determined to be a G-quadruplex DNA structure with a non-parallel configuration.
(4) And (4) detecting fluorescence spectrum. The concentration of the fixed fluorescent probe solution is 1 mu M, different DNA samples are respectively dripped into the probe solution, the probe solution is stabilized for 1 minute after being homogenized, the fluorescence emission of the system is measured by using a fluorescence spectrum, and 575nm is set as an excitation wavelength. The titration experiment results are shown in FIGS. 2,4, 6, 8 and 10, if the fluorescence intensity of the system at 605nm is enhanced by more than 150 times, the DNA sample to be detected can be judged to be a parallel configuration G-quadruplex DNA structure; if the fluorescence intensity of the system is less than 150 times, the DNA sample to be detected can be judged to be in a non-parallel configuration G-quadruplex structure.

Claims (3)

1. A G-quadruplex DNA probe with a bifunctional detection parallel configuration is characterized in that the structural formula is as follows:
Figure FDA0002326489950000011
2. the method for preparing the G-quadruplex DNA probe with the bifunctional detection parallel configuration according to claim 1, which is characterized by comprising the following steps:
(1) under the protection of nitrogen, taking anhydrous dichloromethane as a solvent, reacting ether oxygen chain modified p-hydroxybenzaldehyde (compound 1) with 2, 4-dimethylpyrrole, 2, 3-dichloro-5, 6-dicyano-p-benzoquinone, boron trifluoride diethyl ether and triethylamine in a molar ratio of 1:2:1:30:35 by stirring at room temperature, and separating and purifying a product to obtain a compound 3; the structural formula of compound 3 is:
Figure FDA0002326489950000012
(2) dissolving the compound 4, 4-fluorobenzaldehyde and potassium carbonate in anhydrous acetonitrile according to the molar ratio of 1:1:3, performing reflux reaction, and separating and purifying a product to obtain a compound 5; the structural formula of compound 4 is:
Figure FDA0002326489950000013
the structural formula of compound 5 is:
Figure FDA0002326489950000014
(3) dissolving the compound 3, the compound 5, piperidine and glacial acetic acid in toluene according to the molar ratio of 1:1:10:10, reacting at 130 ℃, and separating and purifying a product to obtain a final probe compound; the structural formula of the probe compound is:
Figure FDA0002326489950000015
3. the use of the bifunctional G-quadruplex DNA detection probe with the parallel configuration as claimed in claim 1 for detecting G-quadruplex DNA, which is carried out according to the following steps:
1) dissolving a G-quadruplex DNA probe with a dual-function detection parallel configuration by using DMSO, and diluting by using a buffer solution with pH7.4 to obtain a solution B; dissolving a DNA sample to be detected by using a buffer solution with pH 7.4;
2) mixing the solution B and the solution A to enable the molar ratio of the DNA sample to be detected to the probe in the mixed solution to be 2-1, analyzing the spectral change of the mixed solution by using a fluorescence spectrophotometer or an ultraviolet spectrophotometer to judge whether the DNA sample to be detected is a G-quadruplex DNA structure with a parallel configuration; the judgment method comprises the following steps:
(a) when the mixed solution is analyzed by a fluorescence spectrophotometer, compared with the solution B, if the fluorescence intensity of the fluorescence spectrum of the mixed solution at 606nm is obviously enhanced (the enhancement range is 150-250 times), the DNA sample to be detected can be judged to be a G-quadruplex DNA structure with parallel configuration; if the fluorescence intensity of the mixed solution is not obviously enhanced (less than 150 times) relative to that of the solution B, the detected DNA sample can be judged to be a G-quadruplex DNA structure with a non-parallel configuration;
(b) when the mixed solution is analyzed by an ultraviolet spectrophotometer, compared with the solution B, if the absorption spectrum of the mixed solution has an obvious new peak at 584nm (A: A)0>1.05) and the peak width is narrowed, the G-quadruplex DNA structure of the parallel configuration of the DNA sample to be detected can be judged; if the absorbance spectrum of the mixed solution does not change or decreases with respect to that of solution B (A: A)0<1.05), the detected DNA sample can be judged to be a G-quadruplex DNA structure with a non-parallel configuration.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN109796483A (en) * 2019-03-11 2019-05-24 福州大学 A kind of water-soluble cationic photosensitizer and its preparation and application

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109796483A (en) * 2019-03-11 2019-05-24 福州大学 A kind of water-soluble cationic photosensitizer and its preparation and application

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Title
ZHANG, JINGTUO ET AL: "Near-infrared fluorescent probes based on piperazine-functionalized BODIPY dyes for sensitive detection of lysosomal pH", 《JOURNAL OF MATERIALS CHEMISTRY B: MATERIALS FOR BIOLOGY AND MEDICINE》 *

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