CN106147753B - Thiazole orange styrene compound as G-quadruplex nucleic acid fluorescent probe - Google Patents

Abstract

The invention discloses a fluorescent probe, a preparation method thereof and application thereof in detecting a nucleic acid G-quadruplex structure. The probe has a structure of a general formula (I), and is simple and stable in structure and easy to prepare. The invention also discloses that the probe can be used for specifically detecting the G-quadruplex secondary structure of nucleic acid, and the G-quadruplex secondary structure in a solution can be quickly detected by a fluorescence spectrophotometer or directly by observing the fluorescent lamp irradiation by naked eyes; the probe can be used for detecting a nucleic acid G-quadruplex structure in agarose gel or polyacrylamide gel; it can also be used to detect, label or display the presence and distribution of G-quadruplex structures in living cells. The fluorescent material has high-efficiency and specific recognition capability on a nucleic acid G-quadruplex structure, has the advantages of good cell membrane permeability, low phototoxicity, biotoxicity, photobleaching property and the like, and overcomes the defects of high price, high equipment requirement, relatively complex technical operation and the like of other detection methods.

Description

Thiazole Orange Styrenics as Fluorescent Probes for G-quadruplex Nucleic Acids

technical field

The present invention relates to a fluorescent probe and its preparation method, as well as its use in detecting the secondary structure of nucleic acid G-quadruplex in aqueous solution, gel and cell.

Background technique

Nucleic acids are not only the basic components of all biological cells, but also play a dominant role in major life phenomena such as growth, development, reproduction, inheritance and variation of organisms. Nucleic acid macromolecules are divided into two categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which play the role of storing and transmitting genetic information in the replication and synthesis of proteins.

G-quadruplex (G-quadruplex) is a special nucleic acid secondary structure. Many guanine-rich regions in the human genome have the ability to form this structure, including telomeric terminal guanine repeats, and the promoter regions of various genes, such as c-kit, c-myc, c-myb, bcl-2 , PDGF, kRAS, VEGF, Rb and insulin genes. The G-quadruplex structure is polymorphic. The number and orientation of the chains, the connection method of loops, the glycoside torsion angle of guanine, and the metal ions coordinated to the negatively charged center of the carbonyl group determine the type of G-quadruplex. and conformation, these differences also provide multiple recognition sites for proteins and small-molecule compounds. Depending on the orientation of the chains, G-quadruplexes are divided into three conformations: normal parallel, antiparallel and mixed.

The formation of G-quadruplex structure has a regulatory effect on a series of physiological processes in vivo. Studies have shown that the G-quadruplex structure in some promoter regions can significantly affect the transcription and translation levels of genes, so the G-quadruplex structure is considered to function as a molecular switch, and its formation and disassembly may involve signaling A series of important physiological processes in vivo, such as conduction, apoptosis and cell proliferation. Therefore, in vivo or in vitro experiments, the existence or formation of the G-quadruplex structure can be specifically detected, which is useful for studying the related biological functions of the G-quadruplex structure and developing the G-quadruplex structure as the target. Anticancer drugs and other aspects of the point have a very important role.

With the development of biotechnology, the requirements for nucleic acid labeling are getting higher and higher, and the previous method of DNA molecular sequencing by isotope effect has been unable to meet the demand. The labeling technology with the advantages of less amount and no radiation has been widely valued and has developed rapidly. The dyes that have been found are porphyrins, cyanines, styrenes and the like. Among them, thiazole orange (TO) is a classical non-selective cyanine nucleic acid fluorescent probe. In addition to generating fluorescence after binding to other nucleic acids, TO can also bind to G-quadruplex to generate strong fluorescence. However, TO cannot efficiently recognize G-quadruplex secondary structure from other forms of nucleic acid molecules, thus the poor selectivity limits the application of TO. In order to improve the selectivity of TO, we introduced a styrene group into the structure of TO, and obtained a kind of fluorescent probe with novel structure and strong specificity for nucleic acid G-quadruplex structure.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a fluorescent probe aiming at the deficiencies of the prior art.

Another object of the present invention is to provide a method for preparing the above probe.

Another object of the present invention is to provide the application of the above probe in detecting G-quadruplex structure in aqueous solution, gel and cell.

The present invention realizes above-mentioned purpose through following technical scheme:

The present invention provides a fluorescent probe whose structural formula is shown in the following formula I:

或

wherein R ₁ is selected from

or

R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ are respectively selected from H, F, Cl, Br, OH, OCH ₃ , N(CH ₃ ) ₂ , C _1-6 alkyl or C _{3 -6} cycloalkyl;

X is C or N.

The present invention also provides the preparation method of the above probe, which is expressed as follows:

The specific steps are:

将2-甲基苯并噻唑与碘甲烷反应，得到

然后将

和

反应，得到

最后将

和不同取代基的芳香醛反应，得到最终探针化合物

4-Chloro-2-methylquinoline is reacted with iodomethane to give the compound

2-methylbenzothiazole is reacted with iodomethane to give

followed by

and

react, get

will finally

React with aromatic aldehydes of different substituents to obtain the final probe compound

The present invention also provides the application of the above probe in detecting G-quadruplex structure in aqueous solution, gel and cell.

Since the probe provided by the present invention has a large electron conjugated system and plane, the intensity of the charge transfer effect in the probe molecule can affect the fluorescence emission intensity of the molecule. After the specific interaction with the G-quadruplex structure, the flexibility of the rotatable double bond in the molecule is limited, so that the intramolecular charge transfer effect is enhanced, and the fluorescence is also significantly enhanced. At the same time, the flexible conjugated plane of the molecular structure of this type of probe has a rotatable bond, so that it can be easily stacked on the plane of the G-quadruplex, and then has a strong interaction with the G-quadruplex. , and weakly interacts with nucleic acids of other secondary structures. Therefore, when the probe is mixed with nucleic acids with different secondary structures, if the nucleic acid has a G-quadruplex structure, the specific interaction between the probe and the probe molecule will result in a change in the fluorescence spectrum. When the secondary structure of the nucleic acid is other structures, there will be no obvious signal changes.

Compared with the prior art, the present invention has the following advantages:

(1) This kind of probe is simple to prepare, easy to obtain, and has a stable structure, which is convenient for storage.

(2) The probe provided by the present invention has lower biological toxicity, phototoxicity and photobleaching, and has good photostability.

(3) The probe provided by the present invention has good water solubility and good cell membrane permeability.

(4) The spectral range of the probe provided by the present invention is sufficiently different from the spectral range of the biological sample, and has a lower fluorescence background in solutions and cells where non-G-quadruplexes exist.

(5) The probe provided by the present invention can specifically detect and recognize the G-quadruplex structure, realize the distinction between the G-quadruplex structure and other secondary structures, and use a simple fluorescence spectrometer, or even an ordinary ultraviolet lamp. Under irradiation, the secondary structure of the nucleic acid sample can be identified by naked eye observation, which is fast, easy to operate, low cost, and can realize on-site detection.

Description of drawings

Figure 1 shows the fluorescence spectra of probe 6a and six nucleic acids AT, DA21, LQ1, Oxy28, random and RNA at 1:1 concentration.

Figure 2 shows the fluorescence spectrum of probe 6a titrated with four-stranded DNA (Oxy28).

Figure 3 shows the curve fitting of C and (FF ₀ )/F ₀ in the fluorescence spectrum of probe 6a titrating four-stranded DNA (Oxy28).

Figure 4 shows the polyacrylamide gel electrophoresis of probe 6a with double-stranded DNA (ds26) and G-quadruplex (Oxy28).

Figure 5 is a cell imaging image of PC3 cells stained with the dye DAPI.

Figure 6 is a cell imaging image of PC3 cells stained with probe 6a.

Figure 7 is a cell imaging image of PC3 cells counterstained with probe 6a and the dye DAPI.

Detailed ways

The technical solutions of the present invention are further described below through specific examples. It is proved by fluorescence spectrum experiments that the compound 6a involved in the present invention has a large electron conjugation system and plane, and after specific stacking with the G-quadruplex structure, the fluorescence spectrum changes significantly, and the fluorescence intensity increases by a hundred times. Even under the irradiation of ordinary ultraviolet light, it can be observed with the naked eye. At the same time, the interaction with nucleic acids of other secondary structures is weak, and there is no obvious fluorescence signal response, so that this type of probe has a good specific recognition effect. Therefore, when we mix the probe with DNA with different secondary structures, when the DNA is in a G-quadruplex structure, the specific interaction between it and the probe molecule results in a change in the fluorescence spectrum. When the secondary structure of DNA is other structures, there will be no obvious signal changes. Taking compound 6a as an example to illustrate the application of the fluorescent probe of the present invention in detecting the secondary structure of G-quadruplex nucleic acid in aqueous solution, gel and cell by fluorescence method (including fluorescence microscope and fluorescent gel imager).

Example 1: Synthesis of Compound 2

Weigh 0.2 g (1.1236 mmol) of 4-chloro-quinalidine into a 25 ml round-bottomed flask, add methyl iodide and solvent sulfolane, heat the mixture to 40-60 ° C, react for 18 hours, cool, add anhydrous ether After shaking, suction filtration, the solid was washed several times, dried in vacuo, and weighed. Thin-layer chromatography initially showed that there were no by-products, and 0.345 g of pure product 2 was obtained with a yield of 95.8%: ¹ H NMR (400 MHz, DMSO) δ8. 56(d, J=8.4Hz, 1H), 8.46(d, J=8.3Hz, 1H), 8.22(t, J=8.1Hz, 1H), 8.01(t, J=7.9Hz, 1H), 7.55( s, J=7.4Hz, 1H), 4.20 (s, 3H), 3.74 (s, 1H), 2.68 (s, 3H).

Example 2: Synthesis of Compound 4

Weigh 0.25g (1.68mmol) of 2-methyl-benzothiazole into a 25ml round-bottomed flask, add methyl iodide and solvent dehydrated ethanol, react at 80°C for 15 hours, and cool the reacted solution to room temperature , and then add a mixture of absolute ethanol and chloroform, oscillate and filter with suction, and wash the precipitate with a small amount of reagents. After vacuum drying, 0.448 g of a white powdery solid is obtained with a yield of 91.7%: ¹ H NMR (400 MHz, DMSO) δ8 .44 (d, J=8.1Hz, 1H), 8.30 (d, J=8.4Hz, 1H), 7.90 (t, J=7.8Hz, 1H), 7.81 (t, J=7.7Hz, 1H), 4.20 (s, 3H), 3.54 (s, 1H), 3.17 (s, 3H).

Example 3: Synthesis of Compound 5

Weigh 0.50 g of each of compounds 2 and 3 into a round-bottomed flask containing 10 ml of methanol, stir at room temperature, add a small amount of 0.5 mol/L aqueous sodium bicarbonate solution, and stir openly for about 1 hour. 4 ml of saturated KI solution was added to the reacted solution, stirred for about 5 minutes, filtered with suction, washed with water, and washed with acetone to finally obtain a brick-red solid, which was dried, filtered, concentrated, and purified by column chromatography to obtain 0.98 g of compound 4. Rate 81.7%: ¹ H NMR (400 MHz, DMSO) δ 8.77 (d, J=8.3 Hz, 1H), 8.18 (d, J=8.7 Hz, 1H), 8.02-7.96 (m, 2H), 7.74 ( d, J=8.2Hz, 2H), 7.59(t, J=7.7Hz, 1H), 7.39(t, J=7.5Hz, 1H), 7.34(s, 1H), 6.85(s, 1H), 4.07( s, 3H), 3.98 (s, 3H), 2.87 (s, 3H).

Example 4: Synthesis of Compound 6a

Weigh 0.0756g ( _0.170mmol ) of M3 in a 25ml round-bottomed flask, add 2 times the molar amount of 4,4-dimethylamino-benzaldehyde 0.0505g, solvent n-butanol, 4-methylpiperidine A few drops, reacted at 120-150°C for 3 hours, filtered with suction after cooling, washed the solid with a mixed solution of n-butanol and ice water, dried under vacuum and weighed to obtain 72 mg, yield 73.4%: ¹ H NMR (400MHz) , DMSO)δ8.70(d, J=8.3Hz, 1H), 8.13(d, J=8.8Hz, 1H), 8.03(d, J=7.7Hz, 1H), 7.97-7.92(m, 1H), 7.79(d, J=8.9Hz, 2H), 7.71(d, J=7.8Hz, 1H), 7.69-7.64(m, 2H), 7.63(s, 1H), 7.56(t, J=7.8Hz, 1H) ), 7.43(d, J=15.7Hz, 1H), 7.36(t, J=7.6Hz, 1H), 6.79(d, J=9.1Hz, 3H), 4.13(s, 3H), 3.94(s, 3H ), 3.05 (s, 6H).

Example 5: Synthesis of Compound 6b

The synthesis method was the same as that of 6a, and 70 mg of black solid 6b was obtained with a yield of 76.5%: ¹ H NMR: (400 MHz, DMSO-d ₆ )δ12.00(s, 1H), 8.72(d, J=8.0H _Z , 1H), 8.18(d, J=16.0H _Z , 3H), 8.00(m, 3H), 7.69(m, 3H), 7.58(d, J=8.0H _Z , 2H), 7.47(d, J=12.0H _Z , 1H), 7.35 (t, J= _8.0HZ , 1H), 7.27 (s, 2H), 6.82 (s, 1H), 4.19 (s, 3H), 4.94 (s, 3H).

Example 6: Synthesis of compound 6c

The synthesis method was the same as that of 6a, and 79 mg of purple solid 6c was obtained with a yield of 83.5%: ¹ H NMR (400 MHz, DMSO) δ 8.70 (d, J=8.5 Hz, 1H), 8.12 (d, J=8.9 Hz, 1H), 8.07(d, J=7.9Hz, 1H), 7.95(t, J=7.8Hz, 1H), 7.73-7.68(m, 2H), 7.59(dd, J=13.5, 7.0Hz, 3H), 7.49(d , J=8.7Hz, 2H), 7.39 (t, J=7.6Hz, 1H), 7.17 (dd, J=24.7, 13.8Hz, 3H), 6.83-6.76 (m, 3H), 4.09 (d, J= 8.5Hz, 3H), 3.96(s, 3H), 3.00(d, J=11.7Hz, 6H)

Example 7: Synthesis of compound 6d

The synthesis method was the same as that of 6a, and 76 mg of red solid 6d was obtained with a yield of 77.4%: ¹ H NMR (400 MHz, DMSO) δ 8.69 (d, J=8.4 Hz, 1H), 8.01 (ddd, J=13.8, 10.1, 7.4 Hz) , 4H), 7.94-7.89 (m, 1H), 7.69 (t, J=7.4Hz, 1H), 7.64 (d, J=4.5Hz, 1H), 7.60 (d, J=9.5Hz, 1H), 7.58 -7.49(m, 2H), 7.44(s, 1H), 7.34(dd, J=19.2, 8.3Hz, 3H), 6.79(s, 1H), 4.07(s, 3H), 3.92(s, 3H).

Example 8: Synthesis of compound 6e

The synthesis method was the same as that of 6a, and 82 mg of brown solid 6e was obtained in 85.6% yield: ¹ H NMR (400 MHz, DMSO) δ 9.10 (s, 1H), 8.77 (s, 1H), 8.66 (d, J=3.7 Hz, 1H) ), 8.39(d, J=7.3Hz, 1H), 8.17(s, 1H), 8.06(d, J=7.5Hz, 1H), 7.99(s, 1H), 7.91(d, J=15.9Hz, 1H) ), 7.74(s, 2H), 7.67(d, J=18.3Hz, 1H), 7.65-7.52(m, 3H), 7.41(s, 1H), 6.91(s, 1H), 4.16(s, 3H) , 4.00(s, 3H).

Example 9: Synthesis of compound 6f

The synthesis method was the same as that of 6a, and 80 mg of purple-black solid 6e was obtained in 83.2% yield: ¹ H NMR (400 MHz, DMSO) δ 8.73 (t, J=10.7 Hz, 1H), 8.10 (dd, J=18.2, 9.6 Hz, 2H), 7.97-7.92 (m, 1H), 7.74-7.68 (m, 2H), 7.67-7.61 (m, 2H), 7.60-7.51 (m, 3H), 7.45 (dd, J=13.6, 5.9Hz, 2H), 7.42-7.35 (m, 3H), 7.31 (d, J=15.2Hz, 2H), 6.83 (d, J=9.2Hz, 1H), 4.06 (d, J=14.2Hz, 3H), 3.97 ( d, J=7.1Hz, 3H).

Example 10: Nucleic acid selectivity

DNA configuration: DNA samples were purchased from Yingjun Biotechnology Co., Ltd. Dissolve an appropriate amount of DNA in Tris-HCl buffer (pH 7.4, 100mM Tris, 60mM KCl) or Tris-acetate buffer (pH 5.5, 100mM Tris, 60mM KCl), and heat at 95°C for 5min. After slow cooling and annealing to room temperature as a storage solution, stored at 4 °C.

The 5mM compound stock solution was diluted to a concentration of 5uM, and then different kinds of nucleic acids were added to measure their respective fluorescence intensities with a spectrofluorometer (slit width = 10, scan speed = 200, Ex = 475 nm), and found this. The fluorescence intensity was the strongest after the combination of these compounds with G-quadruplex DNA.

DNA sequence

Example 11: Determination of detection limit

Dilute the 5mM compound stock solution to a concentration of 5uM, then scan in a spectrofluorometer (slit width=10, scan speed=200, Ex=475nm), and then slowly add Oxy28 DNA to it to saturate it .Calculation formula of detection limit

LOD=K×S _b /m

LOD (limit of detection for a compound), m is the slope of the straight line drawn between concentration C and (FF ₀ )/F ₀ , S _b is the standard deviation of multiple measurements with instrument blanks, and K values are according to the International Union of Pure and Applied Chemistry It is recommended to usually take 3, and the measured LOD of 6a is 0.87nM.

Example 12: Nucleic acid gel electrophoresis experiment

First prepare 5×TBE running buffer, ammonium persulfate 10% m/V, 6X loading buffer, methylenebispropionamide (29:1) (%, m/V), then install the electrophoresis device and configure Gel solution, pour the gel, take out the comb and the separator after the gel is cooled, put it into the electrophoresis tank, the buffer submerges the gel for 1-2mm, and the DNA sample is made into 5μM (the loading buffer in the mixture is 1 ×), add 10 μL to the gel spotting well, turn on the entire electrophoresis apparatus, run at 45V for 1 hour, then run at 100V for 3 hours, take out the gel block, and then put the gel block into the dye and compound to soak in the dye. , dried after dyeing, and placed on a gel electrophoresis apparatus for observation.

Example Thirteen: Cell Imaging Experiment

The cells were first seeded in a 6-well plate so that the density of the cells was about 2×10 ³ cells/mL, and then cultured at 37° C. and 5% CO ₂ for 24 h. Then discard the cell culture medium in the 6-well plate in the previous step, wash 3 times with pre-cooled 1×PBS, then add 1.5 mL of pre-cooled pure methanol and place in the dark at room temperature for 1 min. Finally, discard the pure methanol and use pre-cooled pure methanol again. Washed 3 times with cold 1×PBS, added 1 mL of 5 μM compound and left for 15 min. Discard the compound solution in the 6-well plate in the previous step, wash 3 times with pre-cooled 1×PBS, add 1 mL of 1 μM DAPI solution to the above 6-well plate and place it at 37°C for 2min, and then use pre-cooled 1×PBS. Wash 6 times, soak for 5min each time. Cell staining was observed under an inverted fluorescence microscope.

Claims (5)

1. a G-quadruplex fluorescent probe is characterized in that chemical structural formula is as shown in I:

wherein R ₁ is selected from

One of R ₂ , R ₃ or R ₄ is N(CH ₃ ) ₂ , and the rest are H. 2. a preparation method of G-quadruplex fluorescent probe as claimed in claim 1 is characterized in that comprising the steps: 4-Chloro-2-methylquinoline is reacted with iodomethane to give the compound

2-methylbenzothiazole is reacted with iodomethane to give

followed by

react, get

will finally

Reaction with 4,4-dimethylamino-benzaldehyde to obtain the final probe compound

R ₁ is selected from

One of R ₂ , R ₃ or R ₄ is N(CH ₃ ) ₂ , and the rest are H. 3. The application of the fluorescent probe according to claim 1 in the preparation and detection of a nucleic acid G-quadruplex structure product in an aqueous solution. 4. The application of the fluorescent probe according to claim 1 in the preparation and detection of nucleic acid G-quadruplex structure products in agarose gel or polyacrylamide gel. 5. The application of the fluorescent probe according to claim 1 in the preparation and detection of nucleic acid G-quadruplex structure products in cells.

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