CN107880076B - Preparation method and application of phosphorescent metal complex - Google Patents
Preparation method and application of phosphorescent metal complex Download PDFInfo
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Abstract
The invention belongs to the technical field of photoelectric functional organic materials, and particularly relates to a preparation method and application of a phosphorescent metal complex; the phosphorescent metal complex comprises a metal center and a cyclometalated ligand; the preparation method obtains the phosphorescent metal complex by the coordination reaction of the N ^ N ligand and the C ^ N ligand with the amine chain; according to the invention, the oxygen content in organelles can be more effectively judged through the change of the service life, and then different organelles can be well separated by using the FLIM technology, so that the phosphorescent metal complex has an important significance in oxygen detection in cells, cell membranes and mitochondria can be effectively separated by means of the FILM technology, and the application of the complex in aspects of biological imaging, cell labeling, oxygen detection and the like is provided.
Description
Technical Field
The invention belongs to the technical field of photoelectric functional organic materials, and particularly relates to a preparation method and application of a phosphorescent metal complex.
Background
With the continuous development of cell biology, the resolution requirements of scientific research on biological imaging technology are increasing. In recent years, many biological imaging techniques have been developed, and the research performance in the field of cell imaging is remarkable, and at present, the main biological imaging techniques cover two major categories, namely, imaging diagnosis techniques and optical imaging techniques, wherein the imaging diagnosis techniques include ultrasonic imaging, X-ray tomography, nuclear magnetic resonance imaging and the like, and the optical imaging techniques include chemiluminescence imaging technique, fluorescence microscopy and the like. The optical imaging is combined with the luminescent material to carry out biological imaging on cells or tissues and the like by taking optical detection as a main mode, and the optical imaging is widely applied to basic research of biology and medicine because the optical imaging has the advantages of high sensitivity, high resolution, more visual imaging, no detection loss and the like, and is simple to operate and low in cost. At present, organic luminescent materials have become the main guide in luminescence research, and can be divided into fluorescent materials and phosphorescent materials, the former can only emit light by virtue of singlet exciton radiation, and the latter can emit light by virtue of the mixed radiation of singlet and triplet excitons due to the existence of intersystem crossing, so that the phosphorescent transition metal complex has higher quantum efficiency and better light stability, and is widely researched and applied by researchers by virtue of the excellent characteristics of abundant excited state characteristics at room temperature, larger Stokes shift, photochemical stability, easy wavelength adjustment, higher luminous efficiency and the like.
The cell membrane is a barrier for preventing extracellular substances from freely entering cells, and ensures the relative stability of the intracellular environment, so that various biochemical reactions can be orderly operated. However, the cells must exchange information, substances and energy with the surrounding environment to perform a specific physiological function, and therefore, the cells must have a substance transport system for obtaining desired substances and discharging metabolic wastes. It is estimated that the proteins on the cell membrane involved in substance transport account for 15-30% of the proteins encoded by nuclear genes, and the energy used by the cells in substance transport amounts to two thirds of the total energy consumed by the cells. One of the important morphological features obtained by the evolution of primitive life towards cells is the appearance of a layer of membranous structure, i.e. "cell membrane", outside the living material. The cell membrane is located on the surface of the cell, is usually 7-8 nm thick, and consists of lipid and protein. The most important characteristic of the cell is semi-permeability or selective permeability, which has strong selective permeability to substances entering and exiting cells. Cell membranes and intracellular membrane systems, collectively referred to as biofilms, share the same basic structural features.
The mitochondria are internalThe membrane is bent inwards and folded into a double-layer membrane structure with ridge and smooth outer membrane, so that the inner membrane and the outer membrane have different environments, and a plurality of ions form ion gradients such as H due to different concentrations of the inner membrane and the outer membrane+、Mg2+And a certain potential difference exists between the inner film and the outer film. Intracellular ATP is mainly provided by mitochondria, has the functions of free radical production, regulation of dynamic balance of ions and the like, and influences the aerobic respiration process of eukaryotic cells. Mitochondria are dynamically distributed in cells in a nearly elliptical and linear structure, and change in cell metabolism or internal environment can also affect the shape, size and distribution of mitochondria, so that the mitochondrial skeleton structure and distribution change possibly occurring in the earliest stage of cell death are found through research. With the gradual and deep research on the structure and function of mitochondria, the research and development of a specific mitochondrial probe has great significance for observing and researching mitochondria. Mitochondrial dyes have been proposed for a long time, and the localization mechanism of mitochondrial probes has been the mitochondrial localization hypothesis, the cationic lipophilic probe targeting theory, and the like. The membrane potential of mitochondria is negative so that a cationic probe with both lipophilicity can be developed to target mitochondria. And it is also considered that the phospholipid with strong lipophilicity on the mitochondrial inner membrane forms insoluble salt with the lipophilic cation probe, so researchers have proposed the hypothesis of the non-cationic mitochondrial targeting probe. According to different mitochondrial targeting theories, a novel mitochondrial probe with excellent performance is hopefully designed.
Lysosomes are intracellular digestive organs that affect cellular autolysis, defense, elimination of invading microorganisms and associated pathological processes, such as hypoxia caused by diseases such as myocardial infarction, which can cause rapid release of lysosomes, thereby causing rapid increase in blood levels of the associated enzymes. Protein degradation recycling, endocytosis, apoptosis, autophagy and the like are completed in lysosomes (pH 4.5-5.5) and endosomes (pH 4.5-6.8). Autophagy is a lysosome degradation pathway, and during autophagy, autophagosomes capture intracellular waste components and transfer the intracellular waste components to lysosomes for degradation and recycling, so that the autophagosomes play an important role in stabilizing the internal environment of cells and tissues and preventing and treating various diseases.
In organisms, the oxygen content of different organelles has difference, and the oxygen content is not very accurate according to the luminous intensity of the complex.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method and application of a phosphorescent metal complex, wherein the complex has the characteristics of high stability, good water solubility and high sensitivity, the oxygen content in organelles can be more effectively judged through the change of the service life, then different organelles can be well separated by using FLIM technology, so that the phosphorescent metal complex which targets different organelles and can detect oxygen can effectively separate cell membranes and mitochondria in cells by means of FILM technology, the preparation methods of the phosphorescent metal complex and the cell membrane can be provided, and the application of the complex in aspects of biological imaging, cell marking, oxygen detection and the like can be provided.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a phosphorescent metal complex having the structure:
a method for preparing a phosphorescent metal complex, comprising the steps of: the phosphorescent metal complex is obtained by the coordination reaction of N ^ N ligand and C ^ N ligand with an amine chain.
Preferably, the preparation method of the C ^ N ligand with the amine chain comprises the following steps:
1) the first step of condensation reaction: taking methanol as a solvent, and reacting for 12h at 65 ℃;
2) and a second step of reduction reaction: methanol is used as solvent, NaBH4As a reducing agent, the reaction is carried out for 12 hours at the temperature of 25 ℃.
Preferably, the coordination reaction takes dichloromethane and methanol as a mixed solvent, the reflux reaction is carried out for 8 hours under the protection of nitrogen, and then the ion exchange is carried out at room temperature.
A phosphorescent metal complex is applied to labeling and tracking of organelles.
A phosphorescent metal complex is applied to simultaneously targeting mitochondria and cell membranes.
A phosphorescent metal complex is applied to intracellular oxygen detection.
A phosphorescent metal complex is used for life imaging.
A phosphorescent metal complex is applied to biological imaging, biological detection and simultaneous targeting of different organelles.
A phosphorescent metal complex is applied to FLIM imaging.
The phosphorescent metal complex has the following beneficial effects: 1) has the functions of marking and tracing different organelles; 2) has the functions of simultaneously targeting mitochondria and cell membranes; 3) the complex is applied to intracellular oxygen detection; 4) in the future research process, cell imaging can be utilized to achieve a more sensitive and reliable detection result; 5) cell membranes and mitochondria can be successfully distinguished through lifetime imaging; 6) the method is applied to biological imaging, biological detection and simultaneous targeting of different organelles; 7) the method is applied to FLIM imaging, and can detect the oxygen content of different organelles. These results show that the phosphorescent complex has a wide application prospect in the fields of cell imaging, cell targeting labeling and biological detection.
Drawings
FIG. 1 is an absorption spectrum of complex 5 of example 5 and complex 6 of example 6 in MeOH;
FIG. 2 shows the emission spectra of complex 5 from example 5 in different solvents;
FIG. 3 is the emission spectrum of complex 5 of example 5 at different oxygen concentrations;
FIG. 4 shows the co-staining of Hela cells with the complex 6 of example 6;
FIG. 5 is an image of complex 6 of example 5 at the life of Hela cells.
Detailed Description
Embodiments of the present invention are described in further detail below with reference to the accompanying drawings.
The starting materials used in this embodiment are all commercially available compounds, and were synthesized by methods known in the art without any purification treatment prior to use. However, these examples do not limit the present invention.
Example 1
Synthesis of complex 1: weighing NH2Adding 1mmol of-C4 dichloro bridge and 2.2mmol of 4-methyl 4 '-carboxyl-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol of the product is reacted for 2h, dried by spinning, extracted by dichloromethane (3X 150mL), the organic phase is taken, dried by spinning, loaded into a silica gel column, separated and purified, and the yield is 59%.
Example 2
Synthesis of complex 2: weighing NH2Adding 1mmol of-C8 dichloro bridge and 2.2mmol of 4-methyl 4 '-carboxyl-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol of the product is reacted for 2h, the product is dried by spinning, dichloromethane (3X 150mL) is used for extraction, the organic phase is taken out, silica gel powder column is filled by spinning, and separation and purification are carried out, and the yield is 64%.
Example 3
Synthesis of complex 3: weighing NH2Adding 1mmol of-C8 dichloro bridge and 2.2mmol of 4,4 '-dicarboxy-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol reacted for 2h, spin-dried, using dichloromethane (3X 150 m)L) extracting, taking an organic phase, spin-drying, filling the organic phase into a silica gel powder column, separating and purifying, wherein the yield is 45%.
Example 4
Synthesis of complex 4: weighing NH2Adding 1mmol of-C6 dichloro bridge and 2.2mmol of 4,4 '-dimethyl-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol of the product is reacted for 2h, the product is dried by spinning, dichloromethane (3X 150mL) is used for extraction, the organic phase is taken out, silica gel powder column is filled by spinning, and the separation and purification are carried out, wherein the yield is 55%.
Example 5
Synthesis of complex 5: weighing NH2Adding 1mmol of-C8 dichloro bridge and 2.22mmol of 4,4 '-dimethyl-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol of the product is reacted for 2h, the product is dried by spinning, dichloromethane (3X 150mL) is used for extraction, an organic phase is taken, silica gel powder column is filled by spinning, and separation and purification are carried out, wherein the yield is 40%.
Example 6
Synthesis of complex 6: weighing NH2Adding 1mmol of-C10 dichloro bridge and 2.2mmol of 4,4 '-dimethyl-2, 2' -bipyridine into a reaction bottle, vacuumizing, reacting at 40 ℃ for 10h under the protection of nitrogen, and adding KPF630mmol of the reaction product is reacted for 2h, the reaction product is dried in a spinning mode, dichloromethane (3X 150mL) is used for extraction, an organic phase is taken out, silica gel powder columns are filled in the spinning mode, separation and purification are carried out, and the yield is 35%.
The absorption spectra of complex 5 and complex 6 in MeOH were determined and the results are shown in fig. 1: by the position of the absorption peak in the figureIt is clear that the complexes have strong spin allowability in the range of 250-300nm1IL (π → π) absorption peak. Characteristic peaks in the UV range and in the visible range of relatively weak absorption intensity are generally associated with spin-allowed metal-to-ligand interactions1MLCT charge transfer is of great concern.
The emission spectra of complex 5 in different solvents were determined and the results are shown in FIG. 2: the complex 5 has different emission intensities in different solvents, because the maximum emission intensity of the complex varies due to the different polarities of the solvents.
The emission spectra of complex 5 at different oxygen concentrations were determined and the results are shown in FIG. 3: in the case of the complex 5, under the condition of passing through nitrogen, the emission intensity of the complex is greatly reduced along with the increase of the oxygen concentration, and the increase of the nitrogen concentration can increase the emission intensity of the complex, because the phosphorescent complex is sensitive to oxygen, and the oxygen easily quenches the luminescence of the complex, so that the intensity is strong, and the increase of the nitrogen concentration can increase the emission intensity of the complex.
The co-staining experiment of the complex 6 on Hela cells shows that: the staining ratio of the cell membrane was 60% and increased with increasing amine chain length.
The complex 5 was imaged on the life of Hela cells, and the results are shown in fig. 5: under the condition of hypoxia, the complex can be found to be capable of simultaneously targeting mitochondria and cell membranes by analysis, the service life of the cell membranes is 270ns, and the service life of the mitochondria is 145ns, so that the oxygen concentration in the mitochondria can be judged to be larger than the oxygen concentration in the cell membranes, and based on the judgment, the positions of the cell membranes and the mitochondria and the oxygen content can be judged by the service life analysis, so that the purposes of detecting oxygen and distinguishing different organelles are achieved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are intended to be within the scope of the invention.
Claims (4)
1. A phosphorescent metal complex is applied to simultaneously targeting mitochondria and cell membranes, judges the positions of the cell membranes and the mitochondria and the oxygen content through fluorescence lifetime imaging analysis, and achieves the purposes of detecting oxygen and distinguishing different organelles, and has the following structure:
2. use according to claim 1, characterized in that the preparation method of the phosphorescent metal complex comprises the following steps:
the phosphorescent metal complex is obtained by the coordination reaction of N ^ N ligand and C ^ N ligand compound with an amine chain.
3. Use according to claim 2, characterized in that the C ^ N ligand compounds with amine chains are prepared by the following steps:
wherein n is 7;
1) the first step of condensation reaction: methanol is taken as a solvent, and the reaction is carried out for 12 hours under the condition of triethylamine at 65 ℃;
2) and a second step of reduction reaction: methanol is used as solvent, NaBH4As a reducing agent, the reaction is carried out for 12 hours at the temperature of 25 ℃.
4. The use according to claim 2, wherein the coordination reaction uses dichloromethane and methanol as mixed solvent, the reflux reaction is carried out for 8 hours under the protection of nitrogen, and then the ion exchange is carried out at room temperature.
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| Application Number | Priority Date | Filing Date | Title |
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| Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents;Kenneth Yin Zhang et al.;《Inorg. Chem.》;20150618;第54卷;摘要，chart 1, 6584页左栏第1段，6590页右栏第1段，第6586页右栏 * |
| Kenneth Yin Zhang et al..Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents.《Inorg. Chem.》.2015,第54卷 * |
| 铱配合物磷光探针的设计、合成及其在检测和生物成像中的应用;于海霞;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20150515;B014-114页 * |
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