A compound and its synthesis method and application in preparing zinc ion fluorescent probe

Abstract

The present invention discloses a compound, a synthesis method thereof, and an application thereof in the preparation of a zinc ion fluorescent probe. The compound has a chemical structural formula as shown in Formula I. The synthesis method of the compound is as follows: 4-diphenylaminobenzaldehyde and 2,9-dimethyl-1,10-phenanthroline are reacted as raw materials to obtain the compound. 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 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 has good biocompatibility; at the same time, it can also 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, copper ions and silver ions in physiological and pathological processes.

Description

Technical Field

[0001] The present 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, playing key roles in biological processes such as cell metabolism, signal transduction, and DNA repair. The dynamic balance between zinc and copper ions is crucial for maintaining cell function and genomic stability. Zinc ions, as cofactors for many enzymes, are involved in the regulation of DNA synthesis, repair, and transcription, while copper ions play an important role in oxidative stress responses 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 for 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] Recent studies have shown that abnormal changes in zinc and copper ion concentrations in cancer cells can lead to genomic instability and accumulation of DNA damage. Zinc and copper ion levels in cancer cells significantly affect cell proliferation and treatment resistance. Therefore, a deeper understanding of the mechanisms of action of zinc and copper ions in physiological and pathological processes, particularly their relationship to DNA structural stability and gene expression regulation, is crucial for developing new cancer treatment strategies.

[0005] So far, Zn 2+ There are various detection methods, 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, these methods are also subject to obvious limitations and disadvantages, including complex sample pretreatment steps and poor sample stability. Currently, 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, which synthesize fluorescent dyes by selecting fluorophores. Therefore, the development of a fluorescent probe that is sensitive to zinc ions has important application value for the detection of intracellular zinc ions. 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 problems to be solved by the present invention are achieved by adopting the following technical solutions:

[0008] The present invention first provides a compound, the chemical structure of which 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: reaction at 110° C. for 6-8 hours.

[0017] The present 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 for Zn 2+ The response is very sensitive; at the same time, the compound of 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 nuclear membrane and target 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 results of the quantitative analysis of ZNN probe 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 ZNN probe; B: The effect of different concentrations of copper ions on the fluorescence intensity of ZNN probe; C: Zn 2+ Linear relationship diagram of ion detection; D: Cu 2+ Linearity plot of 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 towards different metal ions;

[0031] Figure 6 This is the result of cytotoxicity evaluation of 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 with reference to 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 without specified sources 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 under nitrogen protection for 6-8 hours. After the reaction, acetic acid was removed and column chromatography was used to separate the yellow solid (yield of about 40%) using methanol (MeOH) and dichloromethane (DCM) (1:10) as eluents to obtain the compound represented by 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, observed [M+H] + 464.5.

[0041] Example 2

[0042] The UV response of 10.0 μM ZNN in PBS buffer with or 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 solution has a strong absorption peak at 420nm. 2+ After the probe ZNN is 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 added to 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. Then, the probe ZNN was subjected to the UV absorption spectra of different Zn 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+ This result indicates that the probe ZNN can be used as a potential activating Zn 2+ Fluorescent probe.

[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 prepared, and the probe ZNN (final concentration 10.0 μmol / L) was added 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 the concentration of Zn 2+ As the concentration of Zn increases, the fluorescence intensity of the probe ZNN at 620 nm also increases with the 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 change of Zn 2 + The effect of ZNN on Zn 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 ZNN are plotted as the horizontal axis, and the strongest emission intensity of ZNN at 620nm is plotted as the vertical axis against Zn 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 with a concentration 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 sensitive to Zn 2+ Has higher affinity.

[0053] From the above experimental results, we can see that the probe ZNN is a Zn 2+ and Cu 2+ Fluorescent probe.

[0054] Example 4

[0055] This example tests the response of probe ZNN to viscosity. The response of probe ZNN was tested using different viscosities of a 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 the figure, 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 limits 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 test the effect of pH on probe ZNN. Due to the complex intracellular environment, the pH values ​​in different subcellular organelles vary greatly. Therefore, the following study explores the effect of pH on probe ZNN and probe ZNN and Zn 2+ Specifically: 10 μM probe ZNN was added to the pH range of 3.0 to 9.0, and the fluorescence intensity was measured 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 very 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 had a significant effect on the binding of probe ZNN to Zn 2+ The fluorescence of the probe ZNN at acidic pH may be affected by the presence of Zn 2+ In summary, the above results indicate 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 detection was performed.

[0063] The specificity of the probe ZNN was tested using a separate solution method, where a series of common metal ions were tested for their individual fluorescence responses to the probe. + 、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 shows 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 the Zn 2+ It is highly selective. After adding ZNN, a new red shift can be observed at 620nm, and the fluorescence enhancement is relatively large, which indicates that the probe is highly selective for Zn 2+There are separate responses. In addition, ZNN 2+ The fluorescence at 520 nm was obviously weakened, which indicated that the probe was sensitive to Cu 2+ There were also separate responses, and other metal ions did not show fluorescence reduction. These results demonstrated that ZNN could 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 ZNN probe against HeLa cells. The specific steps are as follows:

[0066] Different concentrations of ZNN (0 μM-40 μM) were added to different wells of cells with 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 survival rate 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 Zn 2+ The ability to perform structural fluorescence imaging. The specific 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 to obtain confocal fluorescence images.

[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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

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

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.

5. The synthesis method according to claim 4, characterized in that The usage ratio of 4-diphenylaminobenzaldehyde, 2,9-dimethyl-1,10-phenanthroline and glacial acetic acid with a concentration of 17.5 mol / L is 1 mol:1-2 mol:1-2 mL.

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

7. The synthesis method according to claim 6, characterized in that The specific conditions of the reaction are: reaction at 110° C. for 6-8 h.

8. Use of the compound according to claim 1 in the preparation of a reagent for detecting metal ions; the metal ions are zinc ions and / or copper ions.

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

10. Use of the compound according to claim 1 in the preparation of a fluorescent probe; the fluorescent probe is a fluorescent probe for detecting zinc ions.

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