CN107417598B - Fluorescent probe for detecting G-quadruplex DNAs (deoxyribonucleic acids) and preparation method thereof - Google Patents
Fluorescent probe for detecting G-quadruplex DNAs (deoxyribonucleic acids) and preparation method thereof Download PDFInfo
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Abstract
The invention provides a fluorescent probe for detecting G-quadruplex DNAs (deoxyribonucleic acids), which is named as 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodide salt. The preparation method comprises the following steps: (1) 4-pyridine acetonitrile hydrochloride reacts with triethylamine to prepare 4-pyridine acetonitrile; (2) 4-pyridine acetonitrile reacts with methyl iodide to obtain 4-pyridine acetonitrile methylated iodized salt; (3) 4-pyridine acetonitrile methylated iodized salt, 4-dimethylaminobenzaldehyde and potassium tert-butoxide react to obtain a target product 3- (4-dimethylamino phenyl) -2- (N-methylpyridine-4-yl) acrylonitrile iodized salt. The binding capacity of the prepared fluorescent probe and the G-quadruplex DNAs is far greater than that of the prepared fluorescent probe and the double-helix DNA, and the fluorescence intensity after the fluorescent probe and the G-quadruplex DNAs are combined is also obviously greater than that after the fluorescent probe and the double-helix DNA are combined, so that the fluorescent probe can be used for specific detection of the G-quadruplex DNAs.
Description
Technical Field
The invention relates to a fluorescent probe, in particular to a fluorescent probe for detecting G-quadruplex DNAs (deoxyribonucleic acids), and a preparation method and application thereof.
Background
Human telomere terminal DNA consists of guanine (G) -rich repeats in monovalent cations (e.g., K)+And Na+) Can form planar G-tetrads through Hoogsteen hydrogen bonding among G bases under the induction of the (G) and further stack to form a G-quadruplex structure. The G-quadruplex structure is also present in promoter regions of protooncogenes such as c-myc, c-kit and bcl-2, etc. c-myc, c-kit and bcl-2 are overexpressed in cancer cells, and their associated proteins affect the proliferation and apoptosis of cancer cells. Human telomere G-rich single-stranded DNA is a substrate for telomerase, and telomerase does not express activity or has low activity in normal somatic cells, but has high activity in 85-90% of cancer cells. The organic small molecular compound can induce the formation of a G-quadruplex structure and stabilize the G-quadruplex structure, so that the activity of telomerase can be inhibited or the transcription expression of oncogenes can be reduced to achieve the aim of resisting tumors, and the organic small molecular compound can be used as a fluorescent probe of the G-quadruplex DNAs to assist the research on the biological functions of the G-quadruplex DNAs and the diagnosis of related diseases. However, a large amount of double-helix DNA exists in human cell nuclei, so that the development of probe molecules capable of selectively recognizing DNAs with a G-quadruplex structure has important theoretical and practical significance. Therefore, the structure of G-quadruplex DNAs with good selectivity and high sensitivityThe design of fluorescent probes is one of the important leading areas of chemical biology in recent years.
Disclosure of Invention
The invention aims to provide a fluorescent probe for detecting G-quadruplex DNAs, a preparation method thereof and application of the probe in detecting structures of the G-quadruplex DNAs.
The invention provides a fluorescent probe for detecting G-quadruplex DNAs (deoxyribonucleic acids), which is 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodide salt (DPBAI), and has the following structural formula:
the synthetic route of the fluorescent probe for detecting G-quadruplex DNAs provided by the invention is as follows:
the invention provides a preparation method of a fluorescent probe for detecting G-quadruplex DNAs, which comprises the following steps:
(1) synthesis of 4-pyridine acetonitrile: completely dissolving 4-pyridine acetonitrile hydrochloride in N, N-dimethylformamide at room temperature, dropwise adding triethylamine, continuously stirring for 5-10 hours after dropwise adding, performing suction filtration, washing a filter cake with N, N-dimethylformamide for three times, performing rotary evaporation on filtrate to remove a solvent to obtain a mixture of black oily liquid and triethylamine hydrochloride, performing gradient elution on the mixture to perform column chromatography, separating residual N, N-dimethylformamide with petroleum ether, and separating and purifying a product with a mixed solution of ethyl acetate and dichloromethane in a volume ratio of 1: 6-10 to obtain yellow oily liquid 4-pyridine acetonitrile;
(2) 4-Synthesis of pyridine acetonitrile methylated iodide salt: under the cooling of an ice-water bath, completely dissolving 4-pyridine acetonitrile in N, N-dimethylformamide, rapidly and dropwise adding iodomethane under continuous stirring according to the molar ratio of 4-pyridine acetonitrile to iodomethane being 1: 5-7, sealing, continuously stirring for 5-10 h, and then placing the obtained reaction liquid in a refrigerator for cooling overnight to separate out a solid; performing suction filtration, washing the filter cake with ethanol and diethyl ether in sequence, and recrystallizing the obtained filter cake with a mixed solution of ethanol and water in a volume ratio of 1: 5-10 to obtain a gray black solid product, namely 4-pyridine acetonitrile methylated iodide;
(3) synthesis of 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodide salt: at room temperature, completely dissolving 4-pyridine acetonitrile methyl iodide salt and 4-dimethylaminobenzaldehyde by using anhydrous methanol, dropwise adding an anhydrous methanol solution of potassium tert-butoxide according to the molar ratio of 4-pyridine acetonitrile methyl iodide salt to 4-dimethylaminobenzaldehyde and potassium tert-butoxide: 1-2, continuously stirring at room temperature for 5-10 h after dropwise adding, carrying out vacuum filtration, washing the filter cake twice by using the anhydrous methanol, and then carrying out column chromatography separation by using ethyl acetate and N, N-dimethylformamide as eluent to obtain the target product.
The binding capacity of DPBAI and G-quadruplex DNAs is far larger than that of the DPBAI and the double-helix DNA, and the fluorescence intensity after the DPBAI and the G-quadruplex DNAs are combined is obviously stronger than that after the DPBAI and the double-helix DNA are combined, so that the DPBAI can be used for detecting the structure of the G-quadruplex DNAs.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the 3- (4-dimethylamino phenyl) -2- (N-methylpyridine-4-yl) acrylonitrile iodide salt (DPBAI) provided by the invention is simple, the reaction conditions are easy to control, and the mass preparation is easy; the binding capacity of the DPBAI compound and the G-quadruplex DNAs is far larger than that of the double-helix DNA, and the fluorescence intensity after the DPBAI compound is combined with the G-quadruplex DNAs is also obviously larger than that after the DPBAI compound is combined with the double-helix DNA, so that the DPBAI can be used for the specific detection of the G-quadruplex DNAs.
Drawings
FIG. 1 is a UV-VIS absorbance titration spectrum of DPBAI (10mM) interacting with DNAs of different secondary structures, in which: arrows indicate increasing DNA concentration; (A) + c-myc; (B) + c-kit; (C) + bcl-2; (D) + HTG (K buffer system, denoted HTG-K); (E) + HTG (Na buffer system, denoted HTG-Na); (F) + calf thymus DNA.
FIG. 2 is a graph of fluorescence enhancement fold for the effect of DPBAI (5mM) on G-quadruplex DNAs at various concentrations and calf thymus DNA, in which: (A) fluorescence enhancement fold plots of DPBAI (5mM) versus G-quadruplex DNAs at various concentrations, where + c-myc (. cndot.), + c-kit (. cndot.), + bcl-2 ()▲)，+HTG-K(▼)，+HTG-Na
(B) Fluorescence enhancement fold number plot of DPBAI (5mM) versus calf thymus DNA at different concentrations.
FIG. 3 bar graph of fluorescence enhancement at reaction equilibrium of DPBAI (5mM) with G-quadruplex DNAs and calf thymus DNA.
Detailed Description
Example 1 preparation of a fluorescent probe DPBAI that can be used for the detection of G-quadruplex DNAs:
(1) synthesis of 4-pyridine acetonitrile
100mL of N, N-dimethylformamide and 3.60g (23.46mmol) of 4-pyridineacetonitrile hydrochloride are sequentially added into a 250mL three-neck flask at room temperature, the 4-pyridineacetonitrile hydrochloride is completely dissolved under continuous stirring, 12mL of triethylamine is dropwise added by using a constant-pressure dropping funnel, stirring is continued for 5 hours after the dropwise addition, suction filtration is carried out, the filter cake is washed three times by using the N, N-dimethylformamide, the solvent is removed by rotary evaporation of the filtrate, a mixture of a black oily liquid and the triethylamine hydrochloride is obtained, column chromatography is carried out on the mixture by a gradient elution method, the residual N, N-dimethylformamide is separated by using petroleum ether, and then the product is separated and purified by using a mixed solution of ethyl acetate and dichloromethane in a volume ratio of 1: 8, so that 2.68g (22.08mmol) of the yellow oily liquid 4-pyridineacetonitrile is obtained, and the yield is 94.1%.
(2) Synthesis of 4-pyridine acetonitrile methylated iodized salt
Under the cooling of an ice-water bath, 2.68g (22.08mmol) of 4-pyridine acetonitrile and 30mL of N, N-dimethylformamide are sequentially added into a 100mL single-neck flask, 8mL of methyl iodide is rapidly added dropwise under continuous stirring, a hollow plug is tightly plugged after the dropwise addition is finished, the stirring is continued for 5 hours, and the reaction flask is placed in a refrigerator to be cooled overnight, so that a solid is precipitated. The mixture was filtered, washed with ethanol and diethyl ether, and the resulting cake was recrystallized from a mixture of ethanol and water at a volume ratio of 1: 5 to give 3.96g (14.84mmol) of 4-pyridineacetonitrile methyl iodide salt as a dark gray solid in 67.2% yield.
(3) Synthesis of 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodonium salt DPBAI
To a 100mL single-neck flask, 50mL of anhydrous methanol, 0.98g (3.68mmol) of 4-pyridineacetonitrile methylated iodide salt, and 0.55g (3.69mmol) of 4-dimethylaminobenzaldehyde were added in this order at room temperature, and after heating to dissolve completely, 15mL of an anhydrous methanol solution containing 0.61g (5.44mmol) of potassium tert-butoxide was added dropwise, and after the addition, stirring was continued at room temperature for 5 hours, whereby the solution gradually turned into a purple color and a purple precipitate was formed. Vacuum filtration is carried out, the filter cake is washed twice by absolute methanol, and then column chromatography separation is carried out on the obtained filter cake by using ethyl acetate and N, N-dimethylformamide as eluent in sequence, so that the target product DPBAI0.42g (1.07mmol) is obtained, and the yield is 29.1%.
The structural formula of DPBAI is:
the physical constants are as follows:
purple powder with molecular formula C17H18N3I；
ESI(+)-MS(m/z):[M-I]+Found 264.15 (calculated 264.34).
1H NMRδ/ppm(600MHz,DMSO)：8.848～8.837(d,2H,J＝6.6Hz),8.489(s,1H),8.219～8.209(d,2H,J＝6.0Hz),8.079～8.065(d,2H,J＝8.4Hz),6.945～6.931(d,2H,J＝8.4Hz),4.252(s,3H),3.144(s,6H)。
13C NMRδ/ppm(150MHz,DMSO)：154.37,151.11,145.37,134.47,121.45,120.02,118.45,112.57,95.24,47.12,40.54,0.58。
Example 2 investigation of the binding Capacity of DPBAI and DNAs of different Secondary structures
The interaction of DPBAI with different secondary structure DNAs was tested using uv-vis absorbance titration experiments, and the results are shown in fig. 1 and table 1. In the figure: arrow indicates increasing graduallyAdding the concentration of DNA; (A) + c-myc; (B) + c-kit; (C) + bcl-2; (D) + HTG (K buffer system, denoted HTG-K); (E) + HTG (Na buffer system, denoted HTG-Na); (F) calf thymus DNA. Table 1 lists the binding constants (K) for the interaction of cyanovinyl acrylonitrile methylated iodide salt DPBAI with DNAs of different secondary structuresb) The degree of discoloration (%) and the red shift value (nm) of the maximum absorption band. The results showed that DPBAI acted both with the G-quadruplex DNAs and with the calf thymus DNA, but the binding constant of DPBAI and G-quadruplex DNAs was much greater than that of the calf thymus DNA.
The double helix DNA used in this experiment was calf thymus DNA.
The sequences of the G-quadruplex DNAs used in this experiment were:
bcl-2(5'-GGGCGCGGGAGGAAGGGGGCGGG-3')；
c-kit(5'-CGGGCGGGCGCGAGGGAGGGG-3')；
c-myc(5'-TGAGGGTGGGGAGGGTGGGGAA-3')；
HTG(5'-GGGTTAGGGTTAGGGTTAGGG-3')。
the buffer system used in the experiment is as follows: k buffer System (10mM K)2HPO4/KH2PO4100mM KCl, pH 7.4) and Na buffer system (10mM Na)2HPO4/NaH2PO4,100mM NaCl,pH 7.4)。
In this experiment, calf thymus DNA was used as the duplex DNA, and the concentration thereof was represented by the base pair, and the concentration of G-quadruplex DNAs was represented by the G-tetrad.
TABLE 1 binding constants (K) for interaction of DPBAI with different secondary structure DNAsb) Color reduction ratio (%) and red shift value (nm) of maximum absorption band
Example 3 fluorescence Change study after interaction of DPBAI and DNAs of different Secondary structures
The fluorescence intensity changes of DPBAI (5uM) and DNAs with different secondary structures after reaction were investigated by fluorescence titration experiments, and the results are shown in FIG. 2 and FIG. 3, respectively. Fluorescence titration experiments show that after DNAs with different secondary structures are dropwise added into a DPBAI solution, the fluorescence enhancement times of the five G-quadruplex DNAs and the DPBAI are obviously greater than that of calf thymus DNA and the DPBAI, wherein the fluorescence enhancement times of the c-myc G-quadruplex and the DPBAI are the largest and almost reach 107 times. Whereas the fluorescence intensity after DPBAI and calf thymus DNA was only increased by about 16 times, the fluorescence intensity after the other G-quadruplex DNAs c-kit, bcl-2, HTG (K buffer), HTG (Na buffer) and DPBAI were increased by about 87, 89, 71 and 56 times, respectively.
The binding constants of the prepared fluorescent probe (DPBAI) and the G-quadruplex DNAs and the double-helix DNA are greatly different, wherein the binding constant difference between the DPBAI and the c-myc G-quadruplex DNA and the double-helix DNA is the largest, and the former is 85.3 times that of the latter. The fluorescence enhancement fold of the DPBAI after the action with the G-quadruplex DNAs is obviously larger than that of the double-helix DNA, wherein the difference of the fluorescence enhancement fold of the DPBAI after the action with the c-myc G-quadruplex DNA and the double-helix DNA is also largest, and the former is 6.6 times that of the latter. The difference of the affinity size and the fluorescence enhancement multiple after the probe DPBAI reacts with the G-quadruplex DNAs and the double helix DNAs can be used for detecting the structures of the G-quadruplex DNAs.
Claims (2)
1. A fluorescent probe capable of being used for detecting G-quadruplex DNAs, which is characterized by being 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodide salt and having a structural formula as follows:
2. the method of claim 1, wherein the method comprises the steps of:
(1) synthesis of 4-pyridine acetonitrile: completely dissolving 4-pyridine acetonitrile hydrochloride in N, N-dimethylformamide at room temperature, dropwise adding triethylamine, continuously stirring for 5-10 hours after dropwise adding, performing suction filtration, washing a filter cake with N, N-dimethylformamide for three times, performing rotary evaporation on filtrate to remove a solvent to obtain a mixture of black oily liquid and triethylamine hydrochloride, performing gradient elution on the mixture to perform column chromatography, separating residual N, N-dimethylformamide with petroleum ether, and separating and purifying a product with a mixed solution of ethyl acetate and dichloromethane in a volume ratio of 1: 6-10 to obtain yellow oily liquid 4-pyridine acetonitrile;
(2) 4-Synthesis of pyridine acetonitrile methylated iodide salt: under the cooling of an ice-water bath, completely dissolving 4-pyridine acetonitrile in N, N-dimethylformamide, rapidly and dropwise adding iodomethane under continuous stirring according to the molar ratio of 4-pyridine acetonitrile to iodomethane being 1: 5-7, sealing, continuously stirring for 5-10 h, and then placing the obtained reaction liquid in a refrigerator for cooling overnight to separate out a solid; performing suction filtration, washing the filter cake with ethanol and diethyl ether in sequence, and recrystallizing the obtained filter cake with a mixed solution of ethanol and water in a volume ratio of 1: 5-10 to obtain a gray black solid product, namely 4-pyridine acetonitrile methylated iodide;
(3) synthesis of 3- (4-dimethylaminophenyl) -2- (N-methylpyridin-4-yl) acrylonitrile iodide salt: at room temperature, completely dissolving 4-pyridine acetonitrile methyl iodide salt and 4-dimethylaminobenzaldehyde by using anhydrous methanol, dropwise adding an anhydrous methanol solution of potassium tert-butoxide according to the molar ratio of 4-pyridine acetonitrile methyl iodide salt to 4-dimethylaminobenzaldehyde and potassium tert-butoxide: 1-2, continuously stirring at room temperature for 5-10 h after dropwise adding, carrying out vacuum filtration, washing the filter cake twice by using the anhydrous methanol, and then carrying out column chromatography separation by using ethyl acetate and N, N-dimethylformamide as eluent to obtain the target product.
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| N,N-Dialkylaminostyryl dyes:specific and highly fluorescent substrates of peroxidase and their application in histochemistry;R. Krieg,等;《J Mol Hist》;20071129;第39卷;第169-191页 * |
| Oxidative Dehydrogenation on the r-Carbon of 4-Pyridylacetonitrile Complexes of Pentaammineruthenium(II);Scot Liu,等;《Inorganic Chemistry》;20011112;第40卷(第24期);第6139-6143页尤其是第6139页右栏第二段 * |
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