A neutral dye probe based on g-quadruplex targeting, synthesis method and application

Abstract

The application discloses a neutral dye probe based on G-quadruplex targeting, a synthesis method and application, and belongs to the field of fluorescent probe detection. The chemical structural formula of the neutral dye probe is shown in the following, and the synthesis method of the neutral dye probe comprises the following steps: 5-dimethylamino thiophene-2-formaldehyde is synthesized by using 5-bromothiophene-2-formaldehyde and dimethylamine; and 5-dimethylamino thiophene-2-formaldehyde is reacted with 2,9-dimethyl-1,10-phenanthroline to be dissolved in an acetic acid solution to obtain the neutral dye probe. The synthesized neutral dye probe can efficiently combine with DNA and RNA G-quadruplex, can sensitively detect and image G-quadruplex in living cells, and can provide raw materials and a theoretical basis for the research on the physiological and pathological processes of G-quadruplex in cancers.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent probe detection, in particular to a neutral dye probe based on G-quadruplex targeting, a synthesis method and application. BACKGROUND

[0002] G-quadruplexes (G4s) are a classic secondary structure of DNA or RNA formed by guanine-rich sequences. Computational analysis shows that there are about 500,000 repetitive guanine-rich sequences in the human genome, and G4s are widely distributed in the human genome, including telomeres, promoters and genomes. They play an important regulatory role in genome stability, DNA replication and gene expression. Recent studies have shown that the number of G4s in cancer tissues is abnormally high. In recent years, G4s have become an important target for tumor treatment. Therefore, it is extremely important to study the role of G4s in physiological and pathological processes.

[0003] So far, the methods for studying G4s are mainly biophysical techniques, including nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography and circular dichroism (CD) spectroscopy. These methods play a very important role in studying the structural information of G4s, however, they are mainly used to elucidate the synthesis of short-chain G4s in vitro. Small molecule fluorescent probes have become an effective method for detecting and imaging G4s in living cells due to their high cell permeability, no-washing and adjustable optical physical properties. Currently, G4s fluorescent probes based on perylene, cyanine, thiazole and carbazole derivatives have been developed for detecting and imaging G4s in living cells. However, most of the probes are cationic, and cationic G4s-targeting probes are greatly interfered by the mitochondrial microenvironment in cells, thus easily leading to false positive results. Therefore, it is still urgent to develop neutral G4s indicators with high sensitivity. SUMMARY

[0004] The purpose of the present application is to provide a neutral dye probe based on G-quadruplex targeting, a synthesis method and application, to solve the problems existing in the prior art, which can specifically bind to DNA or RNA G-quadruplex, and provide a theoretical basis for detecting and imaging G-quadruplex in living cells.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides a neutral dye probe based on G-quadruplex targeting, and the chemical structure formula of the neutral dye probe is as follows:

[0007]

[0008] The present application also provides a synthesis method of the neutral dye probe based on G-quadruplex targeting, comprising the following steps:

[0009] 5-dimethylaminothiophene-2-carboxaldehyde is reacted with 2,9-dimethyl-1,10-phenanthroline in acetic acid solution to obtain;

[0010] The synthetic route is shown as follows:

[0011]

[0012] Preferably, the 5-dimethylaminothiophene-2-carboxaldehyde is synthesized by using 5-bromothiophene-2-carboxaldehyde and dimethylamine;

[0013] The synthetic route is shown as follows:

[0014]

[0015] Preferably, the 5-dimethylaminothiophene-2-carboxaldehyde, the 2,9-dimethyl-1,10-phenanthroline and the acetic acid solution are used in a ratio of 1 mol:(1-2) mol:(1-2) mL.

[0016] Preferably, the reaction is carried out at 110 DEG C for 6-8 h.

[0017] Preferably, the 5-bromothiophene-2-carboxaldehyde and the dimethylamine are used in a ratio of (1-2) mg:2 mL.

[0018] Preferably, the synthesis is carried out at 90 DEG C for 4-6 h.

[0019] The application further provides application of the neutral dye probe based on G-quadruplex targeting in preparation of a fluorescent probe for detecting G-quadruplex DNA or RNA.

[0020] The application further provides application of the neutral dye probe based on G-quadruplex targeting in preparation of a fluorescent probe for detecting G-quadruplex in imaging living cells.

[0021] The application discloses the following technical effects:

[0022] The application discloses a neutral dye probe PMT, and experiments find that the probe PMT has high affinity with G-quadruplexes, is a fluorescent probe with superior performance, high stability and high selectivity. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0024] Figure 1 For detecting the UV spectrum of the probe PMT under the PBS buffer condition with or without G-quadruplex;

[0025] Figure 2 For the quantitative analysis result of the probe PMT on G-quadruplex; A: Influence of different concentrations of DNA G-quadruplex Bcl2 on the fluorescence intensity of the probe PMT; B: Linear relationship diagram of the probe PMT on the detection of DNA G-quadruplex Bcl2; C: Titration experiment analysis result;

[0026] Figure 3 For analyzing the response performance of RNA G-quadruplex Bcl2 on the probe PMT; A: Influence of different concentrations of RNA G-quadruplex Bcl2 on the fluorescence intensity of the probe PMT; B: Linear relationship diagram of the probe PMT on the detection of RNA G-quadruplex Bcl2; C: Titration experiment analysis result;

[0027] Figure 4 For detecting the viscosity response performance of the probe PMT;

[0028] Figure 5 For detecting the influence of pH value on the probe PMT;

[0029] Figure 6 For analyzing the selectivity of the probe PMT on different nucleic acid structure G-quadruplex;

[0030] Figure 7 For the cytotoxicity evaluation result of the probe PMT;

[0031] Figure 8 For analyzing the ability of the probe PMT on G-quadruplex structure fluorescence imaging in living cells. DETAILED DESCRIPTION

[0032] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentration, intensity, and time, an intermediate value of the range is specifically contemplated. Each of these intermediate values is also specifically contemplated. These smaller ranges are also specifically contemplated: the upper and lower limits of the ranges are independently includable and excludable from the ranges. In addition, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically contemplated.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0035] Various modifications and changes in the specific embodiments described herein can be made without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0036] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0037] The following examples relate to the sequences of the DNA strands or RNA strands of G4 structures, which were synthesized by BioSynthesis, Inc. The sequences are shown in Table 1.

[0038] Table 1 Sequences

[0039]

[0040] Example 1 Synthesis of a G-quadruplex-targeting neutral dye probe

[0041] The synthetic route is shown below:

[0042]

[0043] The synthetic method is:

[0044] (1) 190 mg of 5-bromothiophene-2-carboxaldehyde was added to 2.0 mL of 40% dimethylamine aqueous solution under stirring, and two drops of piperidine catalyst were added dropwise, and stirred at 90°C for 4-6 h. After the reaction was completed, extraction was performed twice with dichloromethane, and the organic phase was collected and further evaporated; column chromatography was performed using ethyl acetate (EA) and petroleum ether (PE) (1:10) as eluent to obtain a beige solid (yield about 70%), which was 5-dimethylaminothiophene-2-carboxaldehyde.

[0045] The prepared 5-dimethylaminothiophene-2-carboxaldehyde was detected by nuclear magnetic resonance, and the results were as follows: 1 H NMR (400 MHz, CDCl3) δ (ppm): 9.486 (1H, s), 7.486 (2H, d, J = 2.2 Hz), 5.938 (2H, d, J = 2.2 Hz), 3.098 (1H, s) 13 C NMR (100 MHz, CDCl3) δ (ppm): 180.11, 168.10, 140.68, 126.59, 103.19, 42.07.

[0046] (2) 155 mg of 5-dimethylaminothiophene-2-carboxaldehyde and 208.1 mg of 2,9-dimethyl-1,10-phenanthroline were dissolved in 2.0 mL of acetic acid solution, and the reaction was carried out at 110°C for 6-8 h under nitrogen protection. After the reaction was completed, the acetic acid was removed, and column chromatography was performed using methanol (MeOH) and dichloromethane (DCM) (1:10) as eluent to obtain a blue solid (yield about 20%), which was a neutral dye probe PMT targeting G-quadruplex targets.

[0047] The prepared probe PMT was detected by nuclear magnetic resonance, and the results were as follows: 1 H NMR (500 MHz, CD3OD) δ (ppm): 7.960 (2H, s), 7.705 (3H, d, J = 2.5 Hz), 7.44 (1H, s), 6.605 (4H, d, J = 2.5 Hz), 3.360 (3H, s), 3.310 (6H, s). MS (ESI) m / z: for C 21 H 19 N3S [M] + m / z 345.13, measured [M+H] + 346.30.

[0048] Example 2

[0049] 2.0 μM of PMT was detected for ultraviolet response with and without DNA G4s or RNA G4s.

[0050] The results are as follows:Figure 1 As shown, the UV absorption peak of the probe PMT in PBS buffer is strong at 580 nm. When the probe is added to solutions containing different G-quadruplexes, a red-shift in the absorption peak is observed, with the strongest absorption peak shifting to around 590 nm. This red-shift in the probe's UV absorption occurs because the probe PMT binds to the G-quadruplex, inhibiting its intramolecular rotation.

[0051] After testing the UV absorption spectra of the probe PMT before and after binding to the G-quadruplex, the excitation wavelength of the probe PMT is obtained. Then, the fluorescence response performance of the probe PMT before and after binding to different G-quadruplexes is studied. The specific steps are as follows:

[0052] The probe PMT was mixed with PBS buffer to prepare a 2.0 μM probe PMT solution, and the fluorescence intensity of the PMT itself was detected as a control.

[0053] After adding G-quadruplex Bcl2 to 2.0 μM probe PMT solution (10 mM, pH 7.4, 20 mM K + ), and the fluorescence intensity at 620 nm was detected.

[0054] like Figure 2 As shown in Figure A, when the probe PMT was tested alone in PBS buffer, the fluorescence intensity of the probe PMT itself was very weak. However, when G-quadruplex Bcl was added to the probe PMT solution, a strong fluorescence emission peak at around 620 nm was observed. As the G-quadruplex concentration increased, the fluorescence emission of the probe PMT at around 620 nm increased by approximately 70-fold. This indicates that the fluorescence signal of the probe PMT is activated by the G-quadruplex, likely due to the interaction between the probe PMT and the G-quadruplex structure, which limits the rotational energy dissipation pathway of the probe PMT itself. This result suggests that the probe PMT can serve as a potential activated G-quadruplex fluorescent probe.

[0055] Example 3

[0056] This example tests the response of the PMT probe to different concentrations of DNA G-quadruplexes. The specific steps are as follows:

[0057] First, PBS solutions of DNA G-quadruplex Bcl2 with different concentrations were prepared in a total volume of 200 μL, and then the probe PMT (final concentration 2.0 μmol / L) was added and mixed evenly. The mixture was incubated at 37°C for 2 minutes, and then the fluorescence emission spectrum was tested.

[0058] The results are as follows Figure 2As shown in Figure A, when the concentration of DNA G-quadruplex Bcl2 is 0 nmol / L, the fluorescence intensity of the probe PMT is very weak. However, as the concentration of DNA G-quadruplex Bcl2 increases, the fluorescence intensity of the probe PMT at 620 nm also increases. When the concentration of DNA G-quadruplex Bcl2 increases to 2.0 μmol / L, the fluorescence intensity of the probe PMT at 620 nm no longer increases with the increase in DNA G-quadruplex Bcl2 concentration, indicating that the saturation binding concentration of the probe PMT for DNA G-quadruplex Bcl2 is approximately 2.0 μmol / L. As can be seen from the figure, when the probe PMT reaches saturation response, the maximum fluorescence response of the probe PMT is approximately 70 times that of the control without G-quadruplex addition, indicating that the probe PMT is very sensitive to DNA G-quadruplex Bcl2.

[0059] The present invention uses different concentrations of DNA G-quadruplex Bcl2 as the horizontal axis and the strongest emission intensity of the probe PMT at 620nm as the vertical axis to draw a linear relationship diagram of the detection of DNA G-quadruplex Bcl2 by probe PMT, as shown in FIG. Figure 2 As shown in B, it can be found that the probe PMT has a very good linear response to the G-quadruplex concentration of 0-1000nmol / L, and its R 2 =0.992. Further titration experiments showed that the binding ratio of probe PMT to DNAG-quadruplex Bcl2 was 1:1 (e.g. Figure 2 (as shown in C), indicating that the probe PMT has a high affinity for DNAG-quadruplex Bcl2.

[0060] From the above experimental results, it can be seen that the probe PMT is a G-quadruplex fluorescent probe with excellent performance.

[0061] In order to further confirm that the probe PMT has a wide range of responses to G-quadruplexes, the present invention also studied the response performance of RNA G-quadruplex NRSA to the probe PMT. The experimental operation and steps are the same as those of DNA G-quadruplex NRSA. The results are as follows Figure 3 As shown in Figure A, when the concentration of RNAG-quadruplex NRSA is 0 nmol / L, the fluorescence intensity of the probe PMT is very weak. However, as the concentration of RNAG-quadruplex NRSA increases, the fluorescence intensity of the probe PMT at 620 nm also increases with the concentration of RNAG-quadruplex NRSA. At the same time, the present invention plots a linear relationship diagram of the detection of RNAG-quadruplex NRSA by the probe PMT using different concentrations of RNAG-quadruplex NRSA as the horizontal axis and the strongest emission intensity of PMT at 620 nm as the vertical axis, as shown in Figure 1. Figure 3As shown in B, it can be found that the probe PMT has a very good linear response to RNAG-quadruplex NRSA at a concentration of 50-1000 nmol / L, where R 2 =0.987. Further titration experiments showed that the binding ratio of probe PMT to RNAG-quadruplex NRSA was 1:1. Figure 3 As shown in Figure C, the PMT probe has a high affinity for RNA G-quadruplex NRSA. The above experiments indicate that the PMT probe is an excellent fluorescent probe for RNA G-quadruplex NRSA.

[0062] Example 4

[0063] This example tests the response of the probe PMT to viscosity. The response of the probe PMT is tested at different viscosities of a water / glycerol binary system. The specific steps are as follows:

[0064] 2 μM probe PMT was mixed with a water / glycerol binary system, wherein the glycerol component in the water / glycerol binary system increased from 0% to 90%, and the fluorescence intensity at 600 nm was detected.

[0065] The results are as follows Figure 4 As shown, the fluorescence intensity of the probe PMT at 600 nm gradually increases as the glycerol fraction increases from 0% to 90%. The enhanced fluorescence of the probe PMT with increasing glycerol fraction is due to viscosity restricting the intramolecular rotation of PMT. The experimental results indicate that the probe PMT is sensitive to viscosity and can be used as a potential indicator for quantitative detection of G-quadruplex structure.

[0066] Example 5

[0067] This example examines the effect of pH on the probe PMT. Due to the complex intracellular environment, pH values ​​vary significantly within different subcellular organelles. Therefore, we next investigated the effect of pH on the probe PMT and its fluorescence emission when bound to the DNA G-quadruplex Bcl2. Specifically, the fluorescence intensity of 2 μM probe PMT was measured at pH values ​​between 4.0 and 7.4 as a control. Furthermore, DNA G-quadruplex Bcl2 was added to the probe PMT at different pH values ​​and the fluorescence intensity was measured.

[0068] The results are as follows Figure 5As shown in the figure, the activation of the probe PMT is unaffected by pH between 4.0 and 7.4, as the probe PMT exhibits almost no fluorescence in this pH range. However, the fluorescence intensity of the probe PMT after binding to the DNA G-quadruplex Bcl2 remains constant within the pH range of 5.6 to 7.4, indicating that physiological pH has little effect on the response of the probe PMT to the DNA G-quadruplex Bcl2. The decreased fluorescence of the probe PMT at acidic pH may be related to the reduced stability of the DNA G-quadruplex Bcl2. In summary, these results demonstrate that the probe PMT can serve as a specific G-quadruplex fluorescent probe, and the influence of pH changes is negligible.

[0069] Example 6

[0070] This example studies the response of probe PMT to different nucleic acid structures (including DNA / RNA G-quadruplex structures and DNA double-stranded structures) to confirm the selectivity of probe PMT for G-quadruplexes. Specifically, 2.0 μM probe PMT was mixed with different nucleic acids at 20 mM K + Fluorescence detection was performed after incubation for 5 minutes under the same conditions.

[0071] The probe PMT showed good response to DNAG4 at typical telomere ends (Tel26 and 22AG) and oncogene promoter DNAG4 (Kit, Bcl2, VEGF and C-Myc, etc.). Figure 6 As shown, the experimental results show that the fluorescence enhancement observed after the probe PMT binds to DNA G4s is high, and the fluorescence intensity of the probe PMT also increases significantly in the presence of the typical RNA G4 structure NRSA. In contrast, the fluorescence enhancement of the probe PMT after responding to the double-stranded DNA structure (ds-15GC) is very weak. This result shows that the probe PMT can widely and specifically interact with DNA / RNA G4 structures and activate fluorescence, and its specific and selective response to DNA / RNA G4 structures is very high.

[0072] Example 7

[0073] This example is a cytotoxicity evaluation. The WST-8 reagent was used to evaluate the cytotoxicity of the probe PMT against HeLa cells. The specific steps are as follows:

[0074] Different concentrations of PMT (0 μM-40 μM) were added to different wells with cells at a density of about 30% for 24 h, washed three times with PBS, and then WST-8 reagent was added. After incubation for about 3 h, the absorbance at 490 nm was recorded with a microplate reader to explore cell viability.

[0075] The results are as follows Figure 7As shown, the results show that the survival rate of HeLa cells is more than 95% after incubation with probe PMT at different concentrations for 24h, which indicates that probe PMT has good biocompatibility.

[0076] Example 8

[0077] Based on the superior performance of probe PMT in vitro, the ability of probe PMT to image G-quadruplex structure fluorescence in living cells was also studied. The specific operation steps are as follows:

[0078] 2.0 μM probe PMT was used to culture HeLa cells with a density of about 40%, and then confocal fluorescence imaging was performed to obtain confocal fluorescence images.

[0079] The results are shown in Figure 8 The cytoplasm and nucleolus in the cells have strong fluorescence, which indicates that probe PMT can effectively penetrate the cell membrane and nuclear membrane and target G-quadruplex in the cells.

[0080] The above-described examples are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A neutral dye probe based on G-quadruplex targeting, characterized in that The chemical structural formula of the neutral dye probe is shown below:

2. The method for synthesizing a neutral dye probe based on G-quadruplex targeting according to claim 1, wherein: The following steps are involved: 5-dimethylaminothiophene-2-carboxaldehyde and 2,9-dimethyl-1,10-phenanthroline are dissolved in acetic acid solution and reacted to obtain the product; The synthetic route is as follows:

3. The synthesis method according to claim 2, wherein The 5-dimethylaminothiophene-2-carboxaldehyde is synthesized from 5-bromothiophene-2-carboxaldehyde and dimethylamine; The synthetic route is as follows:

4. The synthesis method according to claim 2, wherein The usage ratio of the 5-dimethylaminothiophene-2-carboxaldehyde, the 2,9-dimethyl-1,10-phenanthroline and the acetic acid solution is 1 mol:(1-2) mol:(1-2) mL.

5. The synthesis method according to claim 2, wherein The reaction conditions are: 110° C. for 6-8 hours.

6. The synthesis method according to claim 3, wherein The usage ratio of the 5-bromothiophene-2-carboxaldehyde to the dimethylamine is (1-2) mg:2 mL.

7. The synthesis method according to claim 3, wherein The synthesis conditions are stirring at 90° C. for 4-6 hours.

8. Use of the neutral dye probe based on G-quadruplex targeting according to claim 1 in preparing a fluorescent probe for detecting G-quadruplex DNA or RNA.

9. Use of the neutral dye probe based on G-quadruplex targeting according to claim 1 in preparing a fluorescent probe for detecting and imaging G-quadruplexes in living cells.

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