Beta-diketone/diamino phenanthroline europium complex and synthesis method and application thereof
阅读说明：本技术 一种β-双酮/双氨基邻菲啰啉铕配合物及合成方法和用途 (Beta-diketone/diamino phenanthroline europium complex and synthesis method and application thereof ) 是由 田玉鹏 徐忠伟 王飞 苏文卿 张琼 李丹丹 李胜利 吴杰颖 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种β-双酮/双氨基邻菲啰啉铕配合物及合成方法和用途,其中β-双酮/双氨基邻菲啰啉铕配合物简记为Eu(TTA)-(3)·PN,其结构式如下所示：本发明β-双酮/双氨基邻菲啰啉铕配合物具有良好多光子吸收性质,可用于识别检测RNA和靶向溶酶体的荧光探针。(The invention discloses a beta-diketone/diamino phenanthroline europium complex and a synthesis method and application thereof, wherein the beta-diketone/diamino phenanthroline europium complex is abbreviated as Eu (TTA) 3 PN, the formula of which is shown below: the beta-diketone/diamino phenanthroline europium complex has good multi-photon absorption property, and can be used for identifying and detecting RNA and a fluorescent probe for targeting lysosome.)
1. Beta-diketone/diamino phenanthroline europium complex, abbreviated as Eu (TTA)3PN, characterized by the following structural formula:
2. a method for synthesizing β -diketone/diamino phenanthroline europium complex as claimed in claim 1, characterized by comprising the steps of:
weighing Eu (TTA)3·2H2O, in an appropriate amount of CHCl3Dissolving, adding CHCl into the reaction solution3Heating and refluxing the dissolved ligand PN solution for 12h, cooling and standing, separating out a light yellow solid, performing suction filtration, washing and drying to obtain a target product Eu (TTA)3·PN；
The synthetic route is as follows:
3. use of the β -diketone/diamino phenanthroline europium complex of claim 1, characterized in that:
the beta-diketone/diamino phenanthroline europium complex is used for preparing a fluorescent probe material; the fluorescent probe can specifically recognize RNA and target lysosomes of organelles.
Technical Field
The invention relates to a beta-diketone/diamino phenanthroline europium complex and a synthesis method and application thereof.
Background
Ribonucleic acid (RNA) is ubiquitous in biological cells, and its main physiological function is a genetic information carrier. RNA functions differently in cells in the form of rRNA, mRNA and microRNA. These RNA species vary significantly in their sequence, abundance and function. Since the function of RNA is determined by its sequence, specific fluorescent probes are required to specifically detect RNA, such as gene expression and developmental biology. The fluorescent probe is used for accurately detecting RNA of cells in organisms, and has obvious practical value in the aspects of disease diagnosis and treatment. At present, most researched RNA fluorescent probes are mainly concentrated on organic small molecules, and the probes have the defects of high toxicity, autofluorescence interference, poor light stability and the like, so that the clinical application of the probes is limited.
Lysosomes are the primary site for intracellular waste treatment, contain a variety of acid lytic enzymes, and have the primary physiological functions of digesting and clearing endocytic and endogenous intracellular materials. Research shows that the lysosome dysfunction can cause a series of genetic metabolic diseases of cell tissue and organ dysfunction, such as gaucher disease, mucopolysaccharidosis and the like. Therefore, biological studies on lysosomes are increasingly receiving attention.
The applicant has conducted the following literature search on the subject matter of the present application:
1. xueshu.baidu.com net search results: (2021/7/7)
2. Chinese knowledge network retrieval results:
the first retrieval method comprises the following steps:
discourse-multiphoton absorbing europium complexes with RNA recognition and lysosomal targeting functions: there is no relevant literature.
The discourse is a multi-photon absorption europium complex with the functions of RNA recognition and lysosome targeting and a preparation method thereof: there is no relevant literature.
And a second retrieval mode:
full text-multiphoton absorbing europium complexes with RNA recognition and lysosomal targeting functions: there is no relevant literature.
Full text-a multi-photon absorption europium complex with RNA recognition function and a preparation method thereof: there is no relevant literature.
Disclosure of Invention
The invention aims to provide a beta-diketone/diamino phenanthroline europium complex and a synthesis method and application thereof.
Rare earth ion (Ln)III) As a biological fluorescent probe, the fluorescent probe has the advantages of narrow emission band, large Stokes displacement, strong photobleaching resistance, long fluorescence life and the like, and is widely researched, developed and applied by researchers as a biological imaging material. The multi-photon absorption material is a luminescent material with low energy excitation and high energy emission, has the advantages of strong penetrating power to cells and biological tissues, small light damage, weak autofluorescence interference and the like, and has important application value in the field of biomedicine. Based on the consideration, the europium complex fluorescent probe designed and synthesized by the invention has the advantages of easily obtained raw materials, simple synthesis, capability of specifically identifying RNA and target organelle lysosome, low toxicity and high nonlinear optical activity.
The invention relates to a beta-diketone/diamino phenanthroline europium complex, which is abbreviated as Eu (TTA)3PN, the formula of which is shown below:
the invention relates to a synthesis method of beta-diketone/diamino phenanthroline europium complex, which comprises the following steps:
0.25g (0.50mmol) of Eu (TTA) are weighed out3·2H2O, put into a 100mL flask, and an appropriate amount of CHCl3Dissolving, adding appropriate amount of CHCl into the reaction solution3Heating and refluxing the dissolved 0.50mol of ligand PN solution for 12h, cooling and standing, precipitating a light yellow solid, filtering, washing and drying. Obtaining the complex Eu (TTA)3PN 0.26 g. Yield: 86 percent.
The synthetic route is as follows:
the application of the beta-diketone/diamino phenanthroline europium complex is used for preparing a fluorescent probe material; the fluorescent probe can specifically recognize RNA and target lysosomes of organelles.
The invention has the beneficial effects that:
1、Eu(TTA)3PN has a large two-photon absorption cross-section (42.9GM) under 750nm excitation, and exhibits a large three-photon absorption cross-section (30.2X 10) under 1300nm excitation-82cm6s2 photon-2) It is a multiphoton absorption material having excellent nonlinear optical properties, as shown in FIG. 1.
2、Eu(TTA)3Introduction of phenanthroline and amino groups in PN enables the europium complex to specifically recognize RNA, and after binding with RNA, the fluorescence emission intensity is significantly enhanced, as shown in fig. 2.
3. Hydrophilic group amino (-NH)2) Can reduce the cytotoxicity of the complex. Experiments show that the europium complex has low cytotoxicity to HepG2, the cell survival rate is still as high as 80% when the concentration reaches 25 mu M, and the requirement of biological application can be completely met, as shown in figure 3.
4. Complex Eu (TTA)3PN is well taken up by cells and targets lysosomes in cells with high specificity, as shown in figure 4.
5. The raw materials are easy to obtain and the synthesis is simple. Similar RNA recognition and lysosome targeting fluorescent probes do not exist, and the method has higher commercial value.
Drawings
FIG. 1(a) shows Eu (TTA)3The effective two-photon absorption cross section of the PN complex (42.9GM), (b) is Eu (TTA)3Effective three-photon absorption cross section of PN complex (30.2X 10)-82cm6s2 photon-2). Indicating Eu (TTA)3PN has excellent multiphoton absorption properties.
FIG. 2 shows the reaction of Eu (TTA)3When RNA was added to the PN solution to 50mM, the emission intensity increased 31-fold, and the solution was excited by 365nm wavelength light, the fluorescence emission turned into red light. Indicating the specific recognition of the europium complex to RNA.
FIG. 3 shows Eu (TTA)3Survival of HepG2 cells after 24h incubation of PN in medium at a concentration ranging from 5 to 25. mu.M. When the concentration of the europium complex reaches 25 mu M, the cell survival rate is still as high as 80 percent, which indicates that the europium complex has lower cytotoxicity to HepG2 and can completely meet the requirements of biological application.
FIG. 4 shows Eu (TTA)3Co-localization experiments of PN. Indicating that the europium complex can be specifically targeted to lysosomes in cells.
Detailed Description
Example 1: compound Eu (TTA)3Synthesis of PN
0.25g (0.50mmol) of Eu (TTA) are weighed out3·2H2O, put into a 100mL flask, and an appropriate amount of CHCl3Dissolving, adding appropriate amount of CHCl into the reaction solution3Heating and refluxing the dissolved 0.50mol of ligand PN solution for 12h, cooling and standing, precipitating a light yellow solid, filtering, washing and drying. Obtaining the complex Eu (TTA)3PN 0.26 g. Yield: 86 percent.1H-NMR(d6-DMSO，400MHz,ppm)δ：7.97(d,J＝27.7Hz,2H),7.62–7.14(m,6H),6.74–6.06(m,11H),4.50(s,3H),3.72(s,3H).13C-NMR(d6-DMSO,100MHz,ppm)δ：181.31,148.11,143.21,140.78,135.42,134.05,131.09,127.97,123.05,120.91,98.10,42.05.
Example 2: biological investigation of target molecules
1. MTT method test Eu (TTA)3Cytotoxicity of PN in europium complexesHepG2 cells were incubated for 24h in a medium at a concentration ranging from 5 to 25. mu.M, and cell viability was observed. Eu (TTA)3PN has low cytotoxicity to HepG2, and when the concentration reaches 25 mu M, the cell survival rate is still as high as 80 percent, thus completely meeting the requirements of biological application.
2. HepG2 cells were used as a model of the cells to be visualized, using 20. mu.M Eu (TTA)3PN staining and the test cells are incubated in a cell incubator for 30min and washed 3 times with PBS solution. To confirm the Complex Eu (TTA)3PN targeting to the cell site, lysosome-stained Lyso-tracker deep red selected for co-localization, and results showed that the complex Eu (TTA)3The coloring sites and phenomena of PN and lysosome are consistent, indicating that the complex Eu (TTA)3PN can target lysosomes within cells.