Compound, synthetic method thereof and application of compound in preparation of zinc ion fluorescent probe

Abstract

The invention discloses a compound, a synthesis method thereof and application of the compound in preparation of a zinc ion fluorescent probe. The chemical structural formula of the compound is as shown in formula I in the specification. The synthesis method of the compound comprises the following step: taking 4-diphenylaminobenzaldehyde and 2, 9-dimethyl-1, 10-phenanthroline as raw materials to react, thereby obtaining the compound. The compound has unique response to zinc ions and copper ions; therefore, the probe can be used as a probe for detecting zinc ions and copper ions; in addition, when the compound disclosed by the invention is used as a probe, the compound is stable when the pH value is in a range of 4.0-9.0; the cell toxicity is avoided, and the biocompatibility is good; meanwhile, the compound also can effectively penetrate through cell membranes and cell nuclear membranes and target Zn < 2 + > and Cu < 2 + > in cells; therefore, the fluorescent probe has important application value when being further used as the fluorescent probe for detecting the zinc ions and the copper ions in the cells. Raw materials and theoretical basis are provided for deep understanding of action mechanism research of zinc ions, copper ions and silver ions in physiological and pathological processes.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a compound and a synthesis method thereof and application thereof in preparing zinc ion fluorescent probes. Background Art

[0002] Zinc ion (Zn 2+ ), copper ions (Cu 2+ ) are important metal ions in cells. They play a key role in biological processes such as cell metabolism, signal transduction and DNA repair. The dynamic balance between zinc ions and copper ions is essential for maintaining cell function and genome stability. Zinc ions, as cofactors of many enzymes, are involved in the regulation of DNA synthesis, repair and transcription, while copper ions play an important role in oxidative stress response and are also involved in the structural stability of DNA and the regulation of gene expression.

[0003] Computational analysis shows that the concentration and distribution of zinc and copper ions in cells are crucial to maintaining the integrity of the genome. In the human genome, the promoter regions and important gene regions of many genes and regulatory elements contain specific DNA sequences that can bind to zinc, copper and silver ions. The structure of these sequences is similar to that of Zn 2+ , forming a stable secondary structure, known as an M-quadruplex (M represents a metal ion).

[0004] The latest research shows that in cancer cells, abnormal changes in the concentration of zinc and copper ions may lead to genomic instability and accumulation of DNA damage. The levels of zinc and copper ions in cancer cells significantly affect the proliferation ability and treatment resistance of cells. Therefore, a deep understanding of the mechanism of action of zinc and copper ions in physiological and pathological processes, especially their relationship with DNA structural stability and gene expression regulation, is of great significance for the development of new cancer treatment strategies.

[0005] So far, Zn 2+ There are many methods for detection, such as flame atomic absorption spectrometry in photochemistry and gel electrophoresis in electrochemistry, which have been widely used in various fields. These methods have the advantages of high sensitivity and fast response speed. However, the limitations and disadvantages of these methods are also obvious, including complex sample pretreatment steps, poor sample stability and other problems. At present, optical sensors have become the most suitable candidates due to their suitable detection environment and excellent sample stability. Among them, fluorescent probes are the most effective method, and fluorescent dyes are synthesized by selecting fluorophores. Therefore, the development of a fluorescent probe that is sensitive to zinc ions has important application value for the detection of zinc ions in cells. Summary of the invention

[0006] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a compound and a synthesis method thereof and an application thereof in the preparation of a zinc ion fluorescent probe.

[0007] The above technical problem to be solved by the present invention is achieved by adopting the following technical solution:

[0008] The present invention first provides a compound, whose chemical structure is shown in Formula I:

[0009]

[0010] The present invention also provides a method for synthesizing the above compound, which is obtained by reacting 4-diphenylaminobenzaldehyde and 2,9-dimethyl-1,10-phenanthroline as raw materials.

[0011] Preferably, the synthesis method specifically comprises the following steps: dissolving 4-diphenylaminobenzaldehyde and 2,9-dimethyl-1,10-phenanthroline in acetic acid solution and reacting them to obtain the product.

[0012] Preferably, the usage ratio of 4-diphenylaminobenzaldehyde, 2,9-dimethyl-1,10-phenanthroline and acetic acid is 1-2 mol:1-2 mol:1-5 mL.

[0013] More preferably, the usage ratio of 4-diphenylaminobenzaldehyde, 2,9-dimethyl-1,10-phenanthroline and acetic acid is 1 mol:1-2 mol:1-2 mL.

[0014] Preferably, the acetic acid is glacial acetic acid.

[0015] Preferably, the specific conditions of the reaction are: reaction at 100-120° C. for 6-10 h.

[0016] More preferably, the specific conditions of the reaction are: reacting at 110° C. for 6-8 hours.

[0017] The invention also provides an application of the compound in detecting metal ions.

[0018] Preferably, the metal ion is a zinc ion.

[0019] Preferably, the metal ions are zinc ions and / or copper ions.

[0020] The present invention also provides an application of the compound in preparing a fluorescent probe.

[0021] Preferably, the fluorescent probe is a fluorescent probe for detecting copper ions.

[0022] Preferably, the fluorescent probe for detecting zinc ions is specifically a fluorescent probe for detecting zinc ions in cells.

[0023] Preferably, the fluorescent probe for detecting zinc ions is specifically a fluorescent probe for detecting copper ions in cells.

[0024] Beneficial effects: The present invention provides a new compound; studies have shown that the compound has a unique response to zinc ions and copper ions; therefore, it can be used as a probe to detect zinc ions and copper ions; in particular, after the compound of the present invention is used as a probe to bind to zinc ions, the fluorescence emission at 620nm / 520nm is enhanced by about 16 times, which indicates that the compound of the present invention is used as a probe to detect Zn 2+ The response is very sensitive; at the same time, the compound described in the present invention is used as a probe with Zn 2+ It also has a very strong affinity.

[0025] In addition, the compound of the present invention is used as a probe, which is stable in the pH range of 4.0 to 9.0; has no cytotoxicity and good biocompatibility; and can effectively penetrate the cell membrane and the cell nuclear membrane and target the Zn in the cell. 2+ and Cu 2+ Therefore, further using it as a fluorescent probe to detect zinc ions and copper ions in cells has important application value; it provides raw materials and theoretical basis for in-depth understanding of the mechanism of action of zinc ions and copper ions in physiological and pathological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 UV spectra of probe ZNN in the presence or absence of zinc ions and copper ions in PBS buffer.

[0027] Figure 2 The quantitative analysis results of the probe ZNN on zinc ions, copper ions and silver ions; A: The effect of different concentrations of zinc ions, copper ions and silver ions on the fluorescence intensity of the probe ZNN; B: The effect of different concentrations of copper ions on the fluorescence intensity of the probe ZNN; C: Zn 2+ Linear relationship diagram of ion detection; D: Cu 2+ Linearity plot for ion detection.

[0028] Figure 3 The effect of different viscosities of water / glycerol binary system on the response performance of probe ZNN.

[0029] Figure 4 To detect the effect of pH on probe ZNN;

[0030] Figure 5 To analyze the selectivity of probe ZNN to different metal ions;

[0031] Figure 6 This is the result of the cytotoxicity evaluation of the probe ZNN;

[0032] Figure 7 To analyze the ability of probe ZNN to perform fluorescence imaging of zinc and copper ions in living cells. DETAILED DESCRIPTION

[0033] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0034] The reagents involved in the following examples were purchased from manufacturers, and the reagents are detailed in Table 1; the remaining raw materials whose sources are not indicated are conventional raw materials that can be obtained by those skilled in the art through conventional purchasing channels.

[0035] Table 1 Reagents

[0036]

[0037]

[0038] Example 1 Synthesis of Compounds

[0039] 273.1 mg of 4-diphenylaminobenzaldehyde and 208.2 mg of 2,9-dimethyl-1,10-phenanthroline were dissolved in 10.0 mL of glacial acetic acid (17.5 mol / L), and reacted at 110°C for 6-8 h under nitrogen protection. After the reaction, acetic acid was removed, and methanol (MeOH) and dichloromethane (DCM) (1:10) were used as eluents. Column chromatography was used to separate the yellow solid (yield of about 40%), thereby obtaining the compound shown in Formula I (hereinafter referred to as probe ZNN).

[0040] The prepared probe ZNN was subjected to nuclear magnetic resonance detection, and the results were: 1 H NMR (520MHz, CDCl3) δ8.20(d,J=8.4Hz,1H),8.15(d,J=8.1Hz,1H),7.92(d,J=8.4Hz,1H),7.73(s,2H),7.69(s,1H),7. 62(d,J=16.4Hz,1H),7.58–7.49(m,3H),7.29(t,J=7.8Hz,4H),7.16(d,J=7.9Hz,4H),7.12–7.03(m,4H),2.99(s,3H). 13C NMR (126 MHz, CDCl3) δ 159.50 (s), 156.52 (s), 148.27 (s), 147.36 (s), 136.42 (t, J = 6.9 Hz), 130.36 (d, J = 7.4 Hz), 129.33 (d, J = 7.1 Hz), 128.29 (s), 127.50 (s), 127.05 (s), 125.69 (s), 125.45 (s), 124.95 (s), 123.71 (s), 123.38 (s), 122.80 (s), 120.16 (s), 25.86 (s)..MS (ESI) m / z: for C 33 H 25 N3[M] + m / z 463.5, found [M+H] + 464.5.

[0041] Example 2

[0042] The UV response of 10.0 μM ZNN in PBS buffer with and without zinc ions and copper ions.

[0043] The results are as follows Figure 1 As shown in the figure, the UV absorption peak of the probe ZNN in PBS buffer has a strong absorption peak at 420nm. 2+ After being added to the solution, it can be seen that the absorption peak has a certain red shift process, and the strongest absorption peak has red shifted to around 450nm. 2+ The binding occurs, inhibiting the intramolecular rotation of the probe ZNN, causing the probe's ultraviolet absorption to red shift. 2+ After being put into the solution, it can be seen that the absorption peak has no red shift process.

[0044] Test probe ZNN and Zn 2+ and Cu 2+ After combining the UV absorption spectra before and after, the excitation wavelength of the probe ZNN was obtained. 2+ and Cu 2+ The specific steps are as follows (with Zn 2+ For example):

[0045] The probe ZNN was mixed with PBS buffer to prepare a 10.0 μM probe ZNN solution, which was used as a control to detect the fluorescence intensity of ZNN itself.

[0046] Add Zn to the 10.0 μM probe ZNN solution 2+ After addition of 5% paraformaldehyde (10 mM, pH 7.4, 20 mM), the fluorescence intensity at 520 nm was detected.

[0047] like Figure 2 As shown in A, when the probe ZNN was tested alone in PBS buffer, the fluorescence intensity of the probe ZNN itself was very weak. 2+ After that, we can see that the probe ZNN has a strong fluorescence emission peak at around 520nm. 2+ As the concentration increases, the fluorescence emission of probe ZNN at 620nm / 520nm increases by about 16 times. This indicates that the fluorescence signal of probe ZNN is stimulated by Zn 2+ Activation may be due to the interaction of the probe ZNN with Zn 2+ The results showed that the probe ZNN can be used as a potential activating Zn 2+ Fluorescent probes.

[0048] Example 3

[0049] This example is to detect the effect of probe ZNN on different concentrations of Zn 2+ and Cu 2+ The specific steps are as follows:

[0050] First, prepare different concentrations of Zn 2+ The PBS solution was added with probe ZNN (final concentration 10.0 μmol / L) and mixed evenly. The mixture was incubated at 37°C for 2 min and then the fluorescence emission spectrum was tested.

[0051] The results are as follows Figure 2 As shown in A, when Zn 2+ and Cu 2+ When the concentration is 0 nmol / L, the fluorescence intensity of the probe ZNN at 620 nm is very weak, but as Zn 2+ As the concentration of Zn increases, the fluorescence intensity of the probe ZNN at 620 nm also increases. 2+ The concentration of Zn increases. 2+ When the concentration increased to 130.0 μmol / L, the fluorescence intensity of the probe ZNN at 620 nm basically no longer changed with the Zn 2 + The concentration of Zn increased, indicating that the probe ZNN 2+ The saturation binding concentration of ZNN is about 100 μmol / L. As can be seen from the figure, when the probe ZNN reaches saturation response, the fluorescence emission of the probe at 620nm / 520nm is enhanced by about 16 times. The results show that the probe ZNN has a strong affinity for Zn 2+ Very responsive.

[0052] The present invention uses Zn 2+The different concentrations of Zn are plotted as the horizontal axis, and the strongest emission intensity of probe ZNN at 620nm is plotted as the vertical axis against the strongest emission intensity of Zn at 520nm. 2+ Linear relationship diagram of detection, Cu 2+ Follow the same method. Figure 2 As shown in D, it can be found that the probe ZNN is sensitive to Zn in the concentration range of 0-13eq. 2+ The response linear relationship is very good, and its R 2 =0.9941. Further titration experiments showed that the probe ZNN and Zn 2+ The combination ratio is 4:1 (such as Figure 2 (as shown in F), indicating that the probe ZNN is effective for Zn 2+ Has higher affinity.

[0053] From the above experimental results, it can be seen that the probe ZNN is a Zn 2+ and Cu 2+ Fluorescent probes.

[0054] Example 4

[0055] This embodiment is to detect the response performance of probe ZNN to viscosity. The response performance of probe ZNN is detected by using different viscosities of water / glycerol binary system. The specific steps are as follows:

[0056] 10 μM probe ZNN 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 520 nm was detected.

[0057] The results are as follows Figure 3 As shown in Figure 2, as the glycerol content increases from 0% to 90%, the fluorescence intensity of the probe ZNN at 520nm gradually decreases. As the glycerol content increases, the fluorescence of the probe ZNN increases because the viscosity restricts the intramolecular rotation of ZNN. The experimental results show that the probe ZNN is sensitive to viscosity. This result shows that ZNN can be used as a quantitative detection method for Zn 2+ and Cu 2+ Potential indicators of structure.

[0058] Example 5

[0059] This example is to detect the effect of pH value on probe ZNN. Due to the complex intracellular environment, the pH values ​​in different subcellular organelles vary greatly. Therefore, the following is to explore the effect of pH value on probe ZNN and probe ZNN and Zn 2+ Specifically, the fluorescence intensity of 10 μM probe ZNN was measured in the pH range of 3.0 to 9.0 as a control; Zn was added to the probe ZNN at different pH values. 2+, detect the fluorescence intensity.

[0060] The results are as follows Figure 4 As shown in Figure 2, when the pH value is 4.0-9.0, the activation of probe ZNN is not affected by pH, because the fluorescence of probe ZNN changes little in this pH range. 2+ The fluorescence intensity after binding remained unchanged in the pH range of 6.0 to 8.0, indicating that physiological pH has an important effect on the binding of probe ZNN and Zn 2+ The fluorescence reduction of probe ZNN at acidic pH may be related to the 2+ In summary, the above results show that the probe ZNN can be used as a specific Zn 2+ Fluorescent probes, and the effect of pH changes can be neglected.

[0061] Example 6

[0062] This example studies the reaction of probe ZNN to different metal ions (including DNA / RNA Zn2+ structure and DNA double-stranded structure) to confirm the effect of probe ZNN on Zn 2+ Specifically, 10.0 μM probe ZNN was incubated with different metal ions in 20 mM PBS for 5 minutes and then fluorescence test was performed.

[0063] The specificity of the probe ZNN was tested using a separate solution method, with a series of common metal ions and the probe undergoing separate fluorescence response tests. + , Cu 2+ 、Na 2+ 、Ag + 、Zn 2+ 、Ni 2+ , Hg 2+ Mg 2+ , Ca 2+ 、Al 3+ , Fe 3+ 、Co 3+ 、Cd 2 + Cr 3+ The free probe ZNN exhibits a typical absorption peak at 450nm in the UV-visible absorption spectrum ( Figure 5 ).like Figure 5 As shown, adding Zn 2+ After that, ZNN showed obvious fluorescence enhancement at 620nm, which means that 2+ After adding ZNN, a new red shift was observed at 620nm, and the fluorescence enhancement was relatively strong, which indicated that the probe was highly selective for Zn 2+In addition, ZNN has a separate response when Cu is added 2+ After that, the fluorescence at 520 nm was obviously weakened, which indicated that the probe 2+ There were also separate responses, and other metal ions did not show fluorescence reduction. These results demonstrate that ZNN can detect Zn 2 + ability, and confirmed that the probe has good selectivity and obvious visual detection ability.

[0064] Example 7

[0065] This example is a cytotoxicity evaluation. The WST-8 reagent was used to evaluate the cytotoxicity of the probe ZNN to HeLa cells. The specific operation steps are as follows:

[0066] Different concentrations of ZNN (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 450 nm was recorded with a microplate reader to explore the viability of the cells.

[0067] The results are as follows Figure 6 As shown, the results showed that after incubation with different concentrations of probe ZNN for 24 h, the viability of HeLa cells exceeded 95%, indicating that the probe ZNN has good biocompatibility.

[0068] Example 8

[0069] Based on the excellent performance of the probe ZNN in vitro, the present invention also studies the activity of the probe ZNN in Zn 2+ The ability of structural fluorescence imaging. The specific operation steps are as follows:

[0070] HeLa cells with a density of about 40% were cultured with 10.0 μM probe ZNN, and then confocal fluorescence imaging was performed, and confocal fluorescence images were obtained.

[0071] The results are as follows Figure 7 As shown in Figure 2, there is strong fluorescence enhancement and quenching in the cytoplasm and nucleolus of the cells, which indicates that the probe ZNN can effectively penetrate the cell membrane and nuclear membrane and target the Zn 2+ and Cu 2+ .

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound, characterized in that Its chemical structure is shown in Formula I:

2. The method for synthesizing the compound according to claim 1, characterized in that: It is obtained by reacting 4-diphenylaminobenzaldehyde and 2,9-dimethyl-1,10-phenanthroline as raw materials.

3. The synthesis method according to claim 2, characterized in that The method specifically comprises the following steps: dissolving 4-diphenylaminobenzaldehyde and 2,9-dimethyl-1,10-phenanthroline in acetic acid and reacting them to obtain the obtained product.

4. The synthesis method according to claim 3, characterized in that The dosage ratio of 4-diphenylaminobenzaldehyde, 2,9-dimethyl-1,10-phenanthroline and acetic acid is 1-2 mol:1-2 mol:1-5 mL. More preferably, the usage ratio of 4-diphenylaminobenzaldehyde, 2,9-dimethyl-1,10-phenanthroline and glacial acetic acid (17.5 mol / L) is 1 mol:1-2 mol:1-2 mL.

5. The synthesis method according to claim 3, characterized in that The specific conditions of the reaction are: reacting at 100-120° C. for 6-10 hours. More preferably, the specific conditions of the reaction are: reacting at 110° C. for 6-8 hours.

6. Use of the compound according to claim 1 in detecting metal ions.

7. The use according to claim 6, characterized in that: The metal ions are zinc ions and / or copper ions.

8. The use according to claim 6, characterized in that: The metal ions are metal ions in cells.

9. Use of the compound according to claim 1 in preparing a fluorescent probe.

10. The use according to claim 9, characterized in that: The fluorescent probe is a fluorescent probe for detecting zinc ions.

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