A fluorescent probe for identifying G-quadruplex DNA and a preparation method thereof

Abstract

The invention discloses a fluorescent probe for identifying G-quadruplex DNA and a preparation method thereof, and relates to the technical field of fluorescent probes. The compound TDFG provided by the invention contains an N positive ion alkaline group in its molecular structure, which can enhance the binding efficiency of the fluorescent probe with DNA, and forms a hydrogen bond with the G-quadruplex DNA to restrict intramolecular rotation, emit yellow fluorescence, and specifically identify the G-quadruplex DNA; and the compound TDFG provided by the invention can emit fluorescence responses of different magnifications after binding to G-quadruplex DNAs of different topological structures, which provides another method for quantitatively detecting G-quadruplex DNAs of different topological structures.

Description

Technical field:

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for identifying G-quadruplex DNA and a preparation method thereof. Background technology:

[0002] Nucleic acid is a double helix structure used to store genetic information in the cell nucleus. The nucleobases can form Hoogsteen hydrogen bonds and have unique base pairing and chain folding, which enables them to weave into other three-dimensional secondary structures. The self-assembly of guanylate in nucleic acids leads to the formation of guanine quadruplexes (G-quadruplexes, G4s). Four guanines are bound to each other through hydrogen bonding to form G-quartets planes. Several such G-quartets planes are stacked into a G4. G-quadruplexes are mainly formed by hydrogen bonding between guanines within the G-quadruplex, quadruple π-π stacking interactions, and monovalent cations (such as K + 、Na + NH 4 + ) is stabilized by the binding of the lone pair of electrons of O6 of guanine. In addition, G4s can be formed by the intramolecular folding of a single strand of DNA or RNA, or by the intermolecular association of two or four different strands of guanine. Therefore, G4s is a polymorphic dynamic structure that can be folded into different topological structures. For the G4s structure formed within the molecule, according to the orientation of the DNA sequence from 5' to 3', the G-quadruplex can be divided into a parallel G-quadruplex configuration, an antiparallel G-quadruplex configuration, and a hybrid G-quadruplex configuration.

[0003] Studies have found that in the human body, G-quadruplexes are mainly concentrated in telomeric DNA and oncogenes, especially in the promoter and untranslated regions of telomeric DNA, and most of them are intramolecular G-quadruplex configurations. However, the potential G-quadruplex formation region is not unique, it exists not only in nuclear DNA sequences, but also in mitochondrial DNA sequences. Therefore, specific detection of the presence of G-quadruplexes and identification of different G-quadruplex topological structures are of great significance for studying the biological functions of G-quadruplexes and developing anticancer drugs targeting G-quadruplexes.

[0004] At present, the main research methods for detecting and analyzing G-quadruplex structures include X-ray crystal diffraction, nuclear magnetic resonance, mass spectrometry, circular dichroism, UV-visible absorption spectroscopy, fluorescence analysis imaging, etc. Among them, fluorescence analysis imaging is an intuitive visualization detection technology using fluorescent probes. It has the advantages of easy synthesis of probes, high adjustability, simple operation, excellent spatiotemporal resolution, and can be used for living cell detection. It is widely used in G-quadruplex detection. Most of the fluorescent probes reported for detecting G-quadruplexes cannot specifically identify G-quadruplexes with different topological structures. Therefore, there is a lack of a fluorescent probe that can specifically identify G-quadruplexes with different topological structures. Summary of the invention:

[0005] The technical problem to be solved by the present invention is to provide a fluorescent probe which can specifically identify G-quadruplex DNA with different topological structures in vitro.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a styryl indolium iodide, referred to as compound TDFG, whose structural formula is as follows:

[0008]

[0009] The second object of the present invention is to provide a method for preparing the compound TDFG, which uses 2,3,3-trimethylindole as a starting material, reacts 2,3,3-trimethylindole with methyl iodide to obtain an intermediate, and the intermediate is reacted with N-methyl-N-chloroethyl-4-aminobenzaldehyde to obtain the compound TDFG through a condensation reaction.

[0010] The synthetic route is as follows:

[0011]

[0012] The third object of the present invention is to provide the use of the compound TDFG as a fluorescent probe.

[0013] The fourth object of the present invention is to provide use of the fluorescent probe in detecting G-quadruplex DNA in vitro.

[0014] The beneficial effects of the present invention are:

[0015] 1. The compound TDFG provided by the present invention contains N positive ion basic groups in its molecular structure, which can enhance the binding efficiency of the fluorescent probe with DNA, and form hydrogen bonds with G-quadruplex DNA, restricting intramolecular rotation, emitting yellow fluorescence, and can specifically identify G-quadruplex DNA.

[0016] 2. The compound TDFG provided by the present invention can emit fluorescence responses of different magnifications after binding to G-quadruplex DNAs of different topological structures, which provides another method for quantitatively detecting G-quadruplex DNAs of different topological structures (hereinafter referred to as G4DNA). Description of the drawings:

[0017] Figure 1 For compound TDFG 1 H NMR, 13 C NMR and HR-MS structural characterization results;

[0018] Figure 2 The UV absorption spectrum and fluorescence spectrum of compound TDFG: (a) UV absorption spectrum in PBS buffer; (b) fluorescence spectrum at an excitation wavelength of 520 nm in PBS buffer;

[0019] Figure 3 The selectivity of compound TDFG for various analytical substrates contained in cells;

[0020] Figure 4 For different topological structures of G4DNA, K is added + Circular dichroism spectra before and after ions: (a) antiparallel G4DNA; (b) hybrid G4DNA; (c) parallel G4DNA;

[0021] Figure 5 This is the fluorescence response spectrum of compound TDFG (10 μM) to DNA and RNA with different structures.

[0022] Figure 6 Fluorescence titration spectra and fitting patterns of compound TDFG (10 μM) on G4DNA with different topological structures: (a) hybrid G4DNA; (b) antiparallel G4DNA; (c) parallel G4DNA;

[0023] Figure 7 The fluorescence response spectra of compound TDFG (10 μM) to G4DNA with different topological structures before and after treatment with universal nuclease;

[0024] Figure 8 The fluorescence response spectra of compound TDFG (10 μM) to G4 DNA of different sequences. Specific implementation method:

[0025] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0026] The present invention provides a styryl indolium iodide, referred to as compound TDFG, whose structural formula is as follows:

[0027]

[0028] The invention provides a preparation method of the compound TDFG, which comprises taking 2,3,3-trimethylindole as a starting material, performing a salt-forming reaction between 2,3,3-trimethylindole and methyl iodide to obtain an intermediate, and performing a condensation reaction between the intermediate and N-methyl-N-chloroethyl-4-aminobenzaldehyde to obtain the compound TDFG.

[0029] The synthetic route is as follows:

[0030]

[0031] Preferably, the molar ratio of 2,3,3-trimethylindole to methyl iodide is 1:(2-5). Excessive methyl iodide can improve the utilization rate of 2,3,3-trimethylindole.

[0032] Preferably, the molar ratio of the intermediate to N-methyl-N-chloroethyl-4-aminobenzaldehyde is 1:(1.1-1.5). Excessive N-methyl-N-chloroethyl-4-aminobenzaldehyde can improve the utilization rate of the intermediate.

[0033] Preferably, the catalyst for the condensation reaction is selected from at least one of piperidine, tetramethylammonium hydroxide, tetraethylammonium hydroxide and tetrabutylammonium hydroxide. In the presence of the catalyst, the reaction rate is increased.

[0034] The present invention also provides the use of the compound TDFG as a fluorescent probe. The compound TDFG prepared by the present invention is used as a fluorescent probe.

[0035] The present invention also provides the use of the fluorescent probe in detecting G-quadruplex DNA in vitro. The fluorescent probe of the present invention is used to detect G-quadruplex DNA in vitro to specifically identify G-quadruplex DNA with different topological structures.

[0036] The technical solution of the present invention is described in detail below through specific embodiments:

[0037] Example 1: Synthesis of intermediates

[0038] First, 2,3,3-trimethylindole (1.00 g, 0.0063 mol) was dissolved in 50 mL of acetonitrile, and then (2.68 g, 0.0189 mol) of iodomethane was added dropwise, and the mixture was heated to 85°C and refluxed with stirring for 12 h. After the reaction was completed, a large amount of solvent was dried by spin drying, and the remaining small amount of liquid was filtered to obtain a large amount of dark red solid, which was washed and dried to obtain an intermediate, which could be directly used in the next step without subsequent treatment, with a yield of 95%. 1H NMR (400 MHz, Methanol-d 4 ,δ)7.89-7.78(m,1H),7.78-7.67(m,1H),7.67-7.51(m,2H),4.81-4.80(m,3H),4.05-4.03(m,3H),1.62-1.52(m,6H). 13 C NMR (400MHz, Methanol-d 4 ,δ)142.24,141.77,129.76,129.05,123.07,114.82,54.44,34.12,21.21.HR-MS (m / z, ESI): Cal.for[C 12 H 16 N] + ,m / z=174.1277;[M] + found, m / z = 174.1279.

[0039] Example 2: Synthesis of compound TDFG

[0040] The intermediate (1.00 g, 0.0033 mmol) was dissolved in 70 mL of ethanol, and then N-methyl-N-chloroethyl-4-aminobenzaldehyde (0.79 g, 0.0040 mol) was added, heated and stirred, and a small amount of piperidine was added dropwise as a catalyst, and heated to 80°C and refluxed and stirred for 12 hours. After the reaction was completed, a large amount of solvent was dried, and the remaining small amount of liquid was separated and purified by column chromatography, and the eluent used was dichloromethane and methanol in a volume ratio of 10:1. The obtained red-black solid was compound TDFG, and the yield was 90%. 1 HNMR (400MHz, Methanol-d 4 ,δ)8.29(d,J=15.6Hz,1H),7.94(d,J=8.6Hz,2H),7.70-7.41(m,4H),7.25(d,J=15.7Hz,1H),6.94(d,J=8.7Hz ,2H),3.98(s,3H),3.91(t,J=6.2Hz,2H),3.78(t,J=6.4Hz,2H),3.21(d,J=1.4Hz,3H),1.79(d,J=1.5Hz,6H). 13 C NMR (400MHz, Methanol-d 4,δ)155.07,154.26,142.68,142.14,133.82,128.89,127.99,123.11,122.36,113.23, 112.40,105.08,53.27,51.37,40.53,38.29,32.26,25.78.HR-MS(m / z, ESI): Cal.for[C 22 H 26 C1N 2 ] + ,m / z=353.1779;[M] + found, m / z = 353.1768.

[0041] Example 3: Determination of UV absorption spectrum and fluorescence spectrum

[0042] Compound TDFG was added as a fluorescent probe to 2 mL of PBS buffer (0.01 M, pH 7.4) to a concentration of 20 μM, and changes in the UV absorption spectrum and the fluorescence spectrum were recorded.

[0043] Depend on Figure 2 It can be seen that the fluorescent probe molecule has a very strong absorption peak at 520nm and a very strong fluorescence emission peak at 600nm.

[0044] Example 4: Selective exploration of fluorescent probes

[0045] Compound TDFG was added as a fluorescent probe to 2 mL PBS buffer (0.01 M, pH 7.4) to make its concentration reach 10 μM, and then common biological molecules in cells were added, such as singlet oxygen, calcium ions, copper ions, cysteine, calf thymus DNA, iron ions, glutathione, hydrogen peroxide, hypochlorous acid, potassium ions, lysine, magnesium ions, sodium ions, peroxynitrite ions, yeast RNA, sulfite ions, sulfate ions, zinc ions, arginine, glucose, histidine, antiparallel G4DNA, hybrid G4DNA, and parallel G4DNA, and the fluorescence changes after the reaction of these biological molecules with the probe were measured.

[0046] Depend on Figure 3 It can be seen that only when G4DNA is added, the fluorescence intensity of the fluorescent probe molecule at 600nm will change significantly, indicating that the fluorescent probe can respond to G4DNA.

[0047] Example 5: Circular dichroism analysis of G4DNA with different topological structures

[0048] To test whether the G4 DNA sequence can +Different G-quadruplex topological structures were formed under the action of K. + Changes in circular dichroism spectra before and after addition.

[0049] Depend on Figure 4 It can be seen that a) is an antiparallel structure. + After that, the spectral peaks at 266nm and 294nm appeared, proving that it formed an antiparallel G4 structure; b) is a hybrid structure. + After that, the spectral peaks at 237nm, 254nm and 283nm appeared, proving that it formed a hybrid G4 structure; c) a parallel structure, after adding K + Afterwards, spectral peaks at 245 nm and 264 nm appeared, proving that a parallel G4 structure was formed.

[0050] Example 6: Selective study of fluorescent probes for different DNA and RNA structures

[0051] In order to test the selectivity of fluorescent probe molecules for different DNA and RNA structures, the compound TDFG was added as a fluorescent probe to PBS buffer (0.01M, pH7.4) to make its concentration reach 10μM, and then single-stranded DNA, single-stranded RNA, double-stranded DNA and G4DNA with different topological structures were added respectively, and the fluorescence changes after reaction with the probe molecules were measured.

[0052] Depend on Figure 5 It can be seen that the fluorescence intensity of the fluorescent probe molecule will change significantly only when G4DNA is added, indicating that the fluorescent probe is selective for G4DNA.

[0053] Example 7: Changes in fluorescence intensity of fluorescent probe at different G4DNA concentrations

[0054] In order to test the change of fluorescence intensity of fluorescent probe molecules under different G4DNA concentrations, compound TDFG was added as a fluorescent probe to 200μL PBS buffer (0.01M, pH7.4) to make its concentration reach 10μM, and then parallel, antiparallel and hybridized G4DNA (2-50μM) were added respectively to study the change of fluorescence intensity.

[0055] Depend on Figure 6 It can be seen that as the concentration of G4DNA increases, the fluorescence intensity gradually increases. When the final concentration of parallel G4DNA is 50μM, the growth multiple of the fluorescent probe molecule can reach 17 times. When the final concentration of antiparallel G4DNA is 50μM, the growth multiple of the fluorescent probe molecule can reach 3 times. When the final concentration of hybrid G4DNA is 50μM, the growth multiple of the fluorescent probe molecule can reach 13 times.

[0056] Example 8: Changes in fluorescence intensity of fluorescent probe before and after G4 DNA treatment with universal nuclease

[0057] In order to test the change in fluorescence intensity of the fluorescent probe molecule before and after G4DNA was treated with omnipotent nuclease, compound TDFG was added as a fluorescent probe to 200 μL PBS buffer (0.01 M, pH 7.4) to make its concentration reach 10 μM, and then parallel, antiparallel and hybridized G4DNA (10 μM) were added respectively to test their fluorescence intensity, and then omnipotent nuclease was added, and after incubation at 37°C for 1 hour, its fluorescence intensity was tested.

[0058] Depend on Figure 7 It can be seen that after the addition of omnipotent nuclease treatment, the fluorescence intensity of the fluorescent probe molecule decreased significantly and dropped to the same level as the blank group, indicating that the fluorescent probe molecule is selective for the G-quadruplex structure formed by G4DNA.

[0059] Example 9: Changes in the fluorescence intensity of fluorescent probes in G4 DNA with different sequences

[0060] In order to test the change of fluorescence intensity of fluorescent probe molecules under different sequences of G4DNA, compound TDFG was added as a fluorescent probe to 200μL PBS buffer (0.01M, pH7.4) to make its concentration reach 10μM, and then various G4DNA sequences (10μM) were added respectively to test the change of their fluorescence intensity.

[0061] Depend on Figure 8 It can be seen that the fluorescent probe molecule has a high fluorescence response to the G4DNA sequence with parallel and hybrid structures, and a low response to the G4DNA sequence with antiparallel structure. This shows that the fluorescent probe molecule can identify G4DNA with different topological structures through fluorescence response.

[0062] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A styrylindolium iodide, referred to as compound TDFG, characterized in that: The structural formula of the compound TDFG is as follows:

2. The method for preparing the compound TDFG according to claim 1, characterized in that: Using 2,3,3-trimethylindole as a starting material, 2,3,3-trimethylindole and iodomethane undergo a salt-forming reaction to obtain an intermediate, and the intermediate undergoes a condensation reaction with N-methyl-N-chloroethyl-4-aminobenzaldehyde to obtain the compound TDFG; The synthetic route is as follows:

3. The preparation method according to claim 2, characterized in that: The molar ratio of the 2,3,3-trimethylindole to methyl iodide is 1:(2-5).

4. The preparation method according to claim 2, characterized in that: The molar ratio of the intermediate to N-methyl-N-chloroethyl-4-aminobenzaldehyde is 1:(1.1-1.5).

5. The preparation method according to claim 2, characterized in that: The catalyst for the condensation reaction is selected from at least one of piperidine, tetramethylammonium hydroxide, tetraethylammonium hydroxide and tetrabutylammonium hydroxide.

6. Use of the compound TDFG according to claim 1 or the compound TDFG obtained by the preparation method according to any one of claims 2 to 5 in preparing a fluorescent probe for detecting G-quadruplex DNA.

7. Use of the compound TDFG according to claim 1 or the compound TDFG obtained by the preparation method according to any one of claims 2 to 5 in preparing an in vitro reagent for detecting G-quadruplex DNA.

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