CN108948093B - Phosphorescent metal iridium complex with singlet oxygen detection effect and preparation method and application thereof - Google Patents

Abstract

The invention provides a phosphorescent metal iridium complex with a singlet oxygen detection effect, and a preparation method and application thereof. The complex consists of a ring metal ligand, a metal center and an auxiliary ligand of a singlet oxygen detection functional group, and has the following structural general formula

Description

Phosphorescent metal iridium complex with singlet oxygen detection effect and preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic photoelectric functional materials, and particularly relates to a phosphorescent metal iridium complex capable of targeting mitochondria and having a singlet oxygen detection effect, a preparation method thereof, and applications thereof in cell imaging, mitochondrial marking and singlet oxygen detection.

Background

The phosphorescent transition metal complex has excellent photoelectric properties and is widely applied to the research of biological detection and sensing. Compared with a fluorescent probe, the phosphorescent transition metal complex has a series of advantages of good photostability, low toxicity, large Stokes shift and the like.

Some reactive oxygen species are produced in the tissue during biological metabolism, and the levels of reactive oxygen species are normally in a dynamic balance between production and clearance, but too highThe active oxygen substances in the amount can cause damage to a biological membrane system and intracellular oxidative phosphorylation disorder, and are directly participated in human diseases, aging and death, such as tumors, senile dementia, heart diseases, Parkinson's disease and the like. In addition, air pollution, sunlight radiation, smoking, pesticides and the like in the external environment can promote the human body to generate active oxygen free radicals, so that intracellular nucleic acid is mutated, and the process is the root of human diseases and aging. Singlet oxygen (¹O₂) Is one of the major active oxygen species, which is usually generated by energy transfer between triplet excitation of phosphorescent photosensitizers and the ground state of oxygen molecules, and low levels of singlet oxygen in the body are normally harmless to cells, are continuously generated and quenched in the body, and play a good or bad role in a variety of physiological and pathological processes. For example, under the conditions of dye photosensitization oxidation, various biological components (proteins, amino acids, nucleic acids, etc.) are easily reacted with oxygen to damage the organism, such as causing protein photooxidation diseases in animals and humans. When the equilibrium state of singlet oxygen in cells is destroyed, the singlet oxygen can have an injurious effect on the cells, so that the singlet oxygen is widely researched and applied in tumor treatment at present.

The iridium complex phosphorescent probe Ir1 designed and synthesized by the invention can capture singlet oxygen, so that the iridium complex phosphorescent probe Ir1 can be used for detecting the singlet oxygen in cells, can reduce the concentration of the singlet oxygen in the cells by combining with the singlet oxygen, reduces the damage to living cells, and has important research value in the field of biological application. In addition, the probe has good solvent effect and has targeting property to mitochondria. The low toxicity of the material per se also enables the material to capture and detect singlet oxygen in cells through confocal imaging and other technologies

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a phosphorescent metal iridium complex with a singlet oxygen detection effect, and a preparation method and application thereof.

The purpose of the invention is realized by the following technical scheme:

the phosphorescent metal iridium complex with the singlet oxygen detection effect has a structural formula shown in the specification,

wherein the C ^ N ligand is any one of the following:

r on the ancillary ligand₁The substituent is H or

A preparation method of a phosphorescent metal iridium complex with a singlet oxygen detection effect comprises the following steps:

s1, weighing 1 and SeO₂Adding into a reaction bottle, adding 1, 4-dioxane, vacuumizing, reacting for 24h under the protection of nitrogen at 102 ℃, and then weighing for heat filtration; spin-drying the filtrate, dissolving, filtering and extracting to obtain a white solid 2;

s2, weighing 2 prepared in S1, adding 95% ethanol solution, then adding silver nitrate solution, quickly stirring to obtain yellow suspension, slowly adding NaOH solution within 20min, reacting in a dark place, cooling and filtering, re-extracting the filtrate, adjusting the pH of the aqueous phase to acidity, and drying to obtain solid white powder 3;

s3, preparing a compound 4, 4-aminobenzene boric acid and tetrakis (triphenylphosphine) palladium, adding the mixture into a reaction bottle, vacuumizing the reaction bottle, adding deoxygenated ethanol, methylbenzene and a saturated potassium carbonate solution under the protection of nitrogen, carrying out light-shielding treatment, reacting for 24 hours at 85 ℃, cooling to room temperature after the reaction is finished, filtering, extracting, spin-drying the filtrate, and carrying out column chromatography separation to obtain a yellow solid 5;

s4, adding the compound 3, EDC and DMAP into a reaction bottle, vacuumizing, heating to remove water and simultaneously performing nitrogen protection, adding redistilled dichloromethane, adding the compound 5 dissolved in dichloromethane solution after 2min, adding dichloromethane for extraction after stirring and reacting at room temperature, performing rotary evaporation to remove an organic solvent, and performing recrystallization and purification to obtain a light yellow solid 6;

s5, stirring and refluxing the light yellow solid 6 prepared in the S4 and the phenylquinoline dichloro bridge in a mixed solvent of dichloromethane and MeOH at 50 ℃ under a nitrogen atmosphere for one night, cooling the reaction solution to room temperature, and adding KPF (Ketone peroxidase)₆Continuously stirring, removing the organic solvent by rotary evaporation after the reaction is finished, purifying the obtained solid by a column, and recrystallizing to obtain solid Ir 1;

the reaction formula of the above preparation method is as follows:

preferably, the S1 includes the following steps:

s11, weighing 1 and SeO₂Adding into a reaction bottle, adding 260mL of 1, 4-dioxane, vacuumizing, reacting for 24h under the protection of nitrogen at 102 ℃, weighing, and filtering;

s12, the filtrate in S11 was spin dried and dissolved in ethyl acetate, and the insoluble solid was removed by filtration again to give a yellow solution using 1.0M Na₂CO₃Extracting twice, and taking an organic phase;

s13, using 0.3M Na₂S₂O₅Taking inorganic phase, adding Na₂CO₃Adjusting the pH of the inorganic phase to 10, then adding CH₂Cl₂Extraction and spin-drying to obtain white solid 2.

Preferably, the S2 includes the following steps:

s21, weighing 2, adding 95% ethanol solution and silver nitrate solution, quickly stirring to obtain yellow suspension, slowly adding NaOH solution within 20min to obtain dark reaction mixed solution, and covering with tinfoil paper to react at 25 ℃ in a dark place.

S22, cooling and filtering after the reaction is finished, and removing Ag and Ag₂O, the filtrate was extracted with dichloromethane.

S23, taking the water phase extracted by the dichloromethane, adjusting the pH to 3.5 by using 1M HCl solution, placing the water phase in a refrigerator for freezing overnight, filtering the solution to obtain solid, and drying the solid to obtain solid white powder 3.

Preferably, the S3 includes the following steps:

s31, adding the compound 4, 4-aminophenylboronic acid and tetrakis (triphenylphosphine) palladium into a double-mouth reaction bottle, vacuumizing, adding deoxygenated ethanol, toluene and saturated potassium carbonate solution under the protection of nitrogen, carrying out light-shielding treatment, sealing a reaction device by using tinfoil paper, and reacting for 24 hours at 85 ℃;

s32, after the reaction is finished, cooling to room temperature, filtering, washing with saline solution, extracting with dichloromethane, spin-drying the filtrate, and performing column chromatography separation to obtain a yellow solid 5;

preferably, the S4 includes the following steps:

s41, adding the compound 3, EDC and DMAP into a reaction bottle, vacuumizing, heating to remove water and simultaneously performing nitrogen protection, adding redistilled dichloromethane, and adding the compound 5 dissolved in a dichloromethane solution after 2 min;

and S42, stirring at room temperature for 24 hours, repeatedly washing the reaction solution by using water and dilute hydrochloric acid after the reaction is finished, adding dichloromethane for extraction, removing the organic solvent by rotary evaporation, and recrystallizing and purifying to obtain a light yellow solid 6.

Preferably, the S5 includes the following steps:

s51, stirring and refluxing the compound 6 prepared in the S4 and the phenylquinoline dichloro bridge in a mixed solvent of dichloromethane and MeOH at 50 ℃ under a nitrogen atmosphere overnight;

s52, cooling the reaction solution to room temperature, and adding KPF into the reaction solution₆Stirring was continued for 4 hours, after the reaction was completed, the organic solvent was removed by rotary evaporation, and the obtained solid was purified by column chromatography and recrystallized to obtain Ir1 as a solid.

Preferably, the complex is used for capturing singlet oxygen and reducing damage of the singlet oxygen to organisms.

Preferably, the complexes are applied in the fields of cell imaging, biomarkers and singlet oxygen detection.

Preferably, the complex can be targeted to mitochondria and applied to mitochondrial marking of living cells.

The invention has the beneficial effects that: the phosphorescent iridium complex can be excited by visible light, and has emission in a red light region of 620nm after being combined with singlet oxygen, so that the damage of an excitation light source to a biological sample can be reduced. Meanwhile, the compound has low toxicity and the function of capturing singlet oxygen, can be used for various experiments in organisms, and reduces the damage of the singlet oxygen to cells. And after being combined with singlet oxygen, the fluorescent probe can target mitochondria to realize the labeling of the mitochondria. When applied to biological imaging, the probe has a simple chemical mechanism and good biocompatibility, and is a good biological cell probe for detecting singlet oxygen.

Drawings

FIG. 1: the invention is based on a histogram of MTT cytotoxicity experimental results of iridium complex Ir1(pqu) on the basis of HeLa cells.

FIG. 2: the invention is based on a histogram of MTT cytotoxicity experimental results of iridium complex Ir1(pqu) on the basis of 3T3 cells.

FIG. 3: the invention is based on a histogram of MTT cytotoxicity experimental results of iridium complex Ir1(pqu) on the basis of A549 cells.

FIG. 4: the iridium complex Ir1(pqu) of the invention is an imaging phosphorescence luminous intensity diagram of a confocal imaging experiment in living cells, and the cells are HeLa cells.

FIG. 5: the trend chart of phosphorescence luminous intensity of the iridium complex Ir1(pqu) in living cells in confocal imaging experiments along with time changes is shown, and the cells are HeLa cells.

FIG. 6: the image phosphorescence luminous intensity of the confocal imaging experiment of the iridium complex Ir1(pqu) in the living cells is shown, and the cells are 3T3 cells.

FIG. 7: the trend chart of phosphorescence luminous intensity of the iridium complex Ir1(pqu) in living cells in confocal imaging experiments along with time changes is shown, and the cells are 3T3 cells.

FIG. 8: the phosphorescence imaging picture of the confocal imaging experiment of the iridium complex Ir1(pqu) blended with the active oxygen indicator DCFH-DA in the living cells is shown, and the cells are HeLa cells.

FIG. 9: the phosphorescence imaging picture of the confocal imaging experiment of the iridium complex Ir1(pqu) blended with the active oxygen indicator DCFH-DA in the living cells is 3T3 cells.

FIG. 10: results of co-localization experiments of the iridium complex Ir1(pqu) of the present invention with the commercial dye MitoTracker Deep RedFM in living cells are shown schematically.

FIG. 11: the intracellular luminous intensity distribution diagram of the iridium complex Ir1(pqu) in the living cell under different illumination times in the flow cytometry experiment.

FIG. 12: comparison graph of intracellular luminescence intensity of the iridium complex Ir1(pqu) in living cells under different illumination time in flow cytometry experiment.

FIG. 13: comparison graph of flow cell apoptosis test of iridium complex Ir1(pqu) of the present invention.

Detailed Description

The invention discloses a phosphorescent metal iridium complex with a singlet oxygen detection effect, and a preparation method and application thereof, which are specifically described by combining the following embodiments:

preparation of ancillary ligands:

preparation of compound 2: 1(5.27g, 28mmol), SeO, was weighed₂(3.84g, 34.6mmol), adding 260mL of 1, 4-dioxane, vacuumizing, reacting for 24h at 102 ℃ under the protection of nitrogen, and weighing for heat filtration; the filtrate was spin dried and dissolved in 500mL of ethyl acetate, then filtered again to remove insoluble solids to give a yellow solution using 1.0M Na₂CO₃(2 × 100mL) was extracted 2 times, the organic phase was taken, and then 0.3M (3 × 100mL) Na was used₂S₂O₅Taking inorganic phase, adding Na₂CO₃Adjusting the pH of the inorganic phase to 10, and then usingCH₂C1₂Extraction (4 × 100mL) gave 2.5g of a white solid in 45% yield by rotary drying.

Hydrogen spectrum analysis by nuclear magnetic resonance:¹H NMR(400MHz，CDCl3，6)：10.1(1H，s)， 8.80(1H，d)，8.73(1H，s)，8.49(1H，d)，8.18(1H，s)，7.63(1H，d)，

7.11 (1H，d)，2.37(3H，s)。

preparation of compound 3: weighing 2(198.2mg, 1mmol), adding 20mL of 95% ethanol solution, adding 8mL of silver nitrate solution, rapidly stirring to obtain yellow suspension, slowly adding NaOH solution within 20min to obtain dark black reaction mixed solution, coating with tinfoil paper, reacting at 25 deg.C in the dark for 15h, cooling, and vacuum filtering (removing Ag and Ag after reaction is finished)₂O), the filtrate was extracted with dichloromethane (3 × 100mL), the aqueous phase was taken and the pH was adjusted to 3.5 using 1M HCl solution, frozen in the refrigerator overnight, filtered to give a solid which was then dried to give a solid white powder with a yield of 80%.

Hydrogen spectrum analysis by nuclear magnetic resonance:¹H NMR(400MHz，D₂O，)：8.89(1H，d)， 8.60(1H，s)，8.58(1H，s)，8.44(1H，s)，8.04(1H，s)，7.83(1H，d)，2.6(3H， s)。

preparation of compound 5: weighing the compound 4(500mg, 1.94mmol), 4-aminophenylboronic acid (266mg, 1.94mmol) and tetrakis (triphenylphosphine) palladium (76mg), vacuumizing, adding 5mL of deoxygenated ethanol, 10mL of toluene and 5mL of saturated potassium carbonate solution under the protection of nitrogen, carrying out light-resistant treatment, sealing a reaction device by using tinfoil paper, reacting for 24 hours at 85 ℃, cooling to room temperature after the reaction is finished, filtering, washing by using saline solution, extracting by using dichloromethane, spin-drying the filtrate, and carrying out column chromatography separation to obtain a yellow solid 5. The yield was 49%.

Hydrogen spectrum analysis by nuclear magnetic resonance:¹H NMR(CDCl₃)8.27(s，1H)，7.91(m， 4H)，7.54(d，2H)，7.39(m，4H)，6.58(d，2H)，6.27(s，2H)。

preparation of compound 6: adding the compound 3(401mg, 2.02mmol), EDC (485mg, 2.53mmol) and DMAP (309mg, 2.53mmol) into a reaction bottle, vacuumizing, heating to remove water and simultaneously protecting with nitrogen, adding redistilled dichloromethane (10mL), adding the compound 5(500mg, 1.69mmol) dissolved in a dichloromethane solution after 2min, stirring for 24h at room temperature and 25 ℃, repeatedly washing the reaction solution with water and diluted hydrochloric acid (3X 15mL) after the reaction is finished, adding dichloromethane (2X 50mL) for extraction, removing the organic solvent by rotary evaporation, and recrystallizing and purifying to obtain a light yellow solid 6. The yield was 54%.

Hydrogen spectrum analysis by nuclear magnetic resonance:¹H NMR(CDCl₃)9.15(s，1H)，8.90(m， 2H)，8.66(s，1H)，8.42(m，1H)，8.27(s，1H)，7.90(m，6H)，7.77(d，2H)， 7.38(m，5H)，7.19(m，1H)，2.36(s，3H)。

preparation of iridium complex Ir1(pqu) containing singlet oxygen detecting functional group:

preparation of compound Ir1 (pqu): after 6(47mg, 0.10mmol) and a phenylquinoline dichloro bridge (60mg, 0.045mmol) were added to a mixed solvent of dichloromethane (15mL) and MeOH (5mL) and stirred at 50 ℃ under a nitrogen atmosphere for reflux overnight, the reaction solution was cooled to room temperature, and KPF was added thereto₆(1.2mmol) is continuously stirred for 4 hours, after the reaction is finished, the organic solvent is removed by rotary evaporation, and the obtained solid is purified by a column and recrystallized to obtain an orange-red solid. The yield was 35%.

Hydrogen spectrum analysis by nuclear magnetic resonance:¹H NMR(400MHz，CDCl₃)9.20(s，1H)， 9.05(d，1H)，8.90(s，1H)，8.66(s，1H)，8.60(s，1H)，8.30(d，2H)，8.07 (m，4H)，7.98(d，2H)，7.78(d，2H)，7.54(m，10H)，7.36(m，5H)，7.18(s， 1H)，5.90(m，4H)，5.80(m，4H)，3.23(s，1H)，2.61(m，2H)，2.36(s，3 H)，2.23(m，2H)。

and (3) performance testing: MTT cytotoxicity assay of Iridium Complex Ir1(pqu)

(1) Dark toxicity test

Three cells, HeLa, 3T3 and A549 were used for the experiment. Cells were seeded at 104/well density in two 96-well plates, continued at 37 ℃ and 5% CO₂Incubating in an incubator whileWhen the cells can grow adherently and the number of the cells is appropriate, DMEM solutions of complex Ir1(pqu) with different concentrations of 5 mu M, 10 mu M, 20 mu M, 50 mu M, 75 mu M and 100 mu M are added into different wells, two 96-well plates are wrapped by tinfoil paper and put in a dark place at 37 ℃ and 5% CO₂After 18h, one of the 96-well plates was purged with oxygen and irradiated with a xenon lamp (P ═ 50mW) with a light source of 475 soil at 20nm for 2h, and after completion of the irradiation, the incubation in the incubator was continued for 2 h. Then 15. mu.L of MTT (5mg/mL) was added to each of the two 96-well plates and incubation was continued for 4 h. The solution in each well was aspirated and 150. mu.L of DMSO solution was added to each well, and the 96-well plate was placed on a shaker and shaken at a constant speed (120r/min) for 10min to dissolve the precipitated formazan crystals sufficiently. Finally, an enzyme-linked immunoassay tester is used for testing the absorption value of each hole in the 96-hole plate at 570nm, and the cell survival rate is calculated through a formula.

(2) Phototoxicity test in an oxygen-passing environment

Firstly, inoculating appropriate cell concentration of HeLa, 3T3 and A549 cells into a confocal culture dish, putting the confocal culture dish into an incubator to incubate so that the cells grow adherent to the wall for 24 hours, sucking old culture solution in the dish, and carefully washing the cells growing adherent to the wall in the confocal culture dish for 2 times by using fresh DMEM culture solution. To each of the confocal dishes for incubating different cell types, 1mL of DMEM medium containing the complex Ir1 (10. mu.M) was added, and the mixture was further incubated at 37 ℃ and 5% CO₂Into cells by incubation for 2 h. Then introducing oxygen, respectively illuminating for 0min, 30min, 60min and 120min by using a xenon lamp (P ═ 50mW), continuously placing the sample into an incubator for incubation for 30min after illumination is finished, flushing the cells for 2 times by using fresh DMEM medium after incubation is finished, respectively adding 1mL of fresh DMEM medium into a culture dish, and then carrying out cell imaging. Wherein λ is_ex＝405nm，λ_em＝550-650nm。

The results of MTT cytotoxicity experiments are shown in FIGS. 1-3, and when the concentration of the complex is lower than 100 μ M, the cell survival rate is greater than 70% when the complex is cultured under the illumination condition, which indicates that the material captures oxygen in cells after illumination and reduces the toxicity of generated singlet oxygen to the cells. The complex is proved to have low cytotoxicity and can be used for cell imaging.

Imaging experiment: confocal imaging experiments of iridium complex Ir1(pqu) in living cells

And (3) imaging the complex entering the Hela cells by using a laser confocal microscope under different time illumination conditions after oxygen is introduced. Adding 10 μ M of complex Ir1(pqu), incubating in an incubator for 2h, exciting with a 405nm laser, collecting the wave band set to 550-650 nm, selecting a cell on the image graph under different conditions, analyzing the luminous intensity of the intracellular phosphorescent material as shown in FIGS. 4-7,

when the light is not applied, no fluorescence appears in the cells, when the light is applied for 30min, weak light appears in the cells, and when the light is applied for 120min, stronger fluorescence can be seen in an imaging picture.

Observing the difference of the effect of the probe for detecting the singlet oxygen in the cancer cells and the normal cells: the normal cell 3T3 is selected to be compared with the experimental result in the cancer cell during the experiment, the experimental condition is the same as the test in the Hela cell, the cell imaging is respectively carried out under the condition of different illumination time, the material is found to have the same effect in the normal cell and the cancer cell, the material has the capability of capturing and detecting the singlet oxygen in the cell, the damage of the singlet oxygen to the cell can be reduced, the biological application prospect is wide, and the experimental result is shown in figures 8-9.

In order to investigate whether singlet oxygen is captured by the complex Ir1(pqu), an active oxygen indicator is selected for detecting singlet oxygen, the active oxygen indicator (DCFH-DA) used can permeate cells and has no fluorescence in the cells, when singlet oxygen exists in the cells, the indicator DCFH-DA is oxidized into DCF emitting green fluorescence, and the color change of the indicator is observed to detect whether singlet oxygen exists in the cells. When the light is not used, the green fluorescence intensity of the indicator is very weak, when the light is used for 120min, the green fluorescence intensity of the indicator is obviously enhanced, when the light is used for 130min, the green fluorescence of the indicator is slightly enhanced, and the result shows that when the light is used for 120min, the complex Ir1(pqu) captures singlet oxygen and completely converts the singlet oxygen into the complex Ir2(pqu) under the light condition.

Co-localization experiments: iridium complex Ir1(pqu) and commercial mitochondrial dye MitoTracerDeep Red FM co-localization experiment in living cells

In order to research the targeting position of the complex in cytoplasm of cells, 10 mu M of complex Ir1(pqu) is added into three cells of HeLa, A549 and 3T3 respectively, after the cells are illuminated by a 475 +/-20 nM xenon lamp for 120min, a MitoTracker Deep FM of a commercial dye 200nM targeting mitochondria and the complex are added for a co-localization experiment, the experimental result is shown in figure 10, in the three cells of HeLa, A549 and 3T3, the co-staining coefficients of the complex and the MitoTracker Deep FM are all above 0.85, and the complex can well target mitochondria after capturing singlet oxygen and can be used for marking living cell mitochondria.

Flow cytometry experiment: flow cytometry experiment of Iridium Complex Ir1(pqu)

The detection effect of the ligand probe on singlet oxygen is verified and analyzed through a flow cytometry experiment, as shown in fig. 11-12, the complex Ir1 with the concentration of 10 μ M is added into Hela cells, the incubation is continued for 2h, the incubation is continued for 30min after the light irradiation is respectively carried out for 0min, 30min, 60min and 120min, and the digestion, centrifugation and heavy suspension are carried out after the incubation is finished. And a 405nm laser is selected on a flow cytometer for intracellular information collection. The results of the analysis of the luminescence intensities in the cells under different illumination times are shown in fig. 11, and as the illumination time increases, the luminescence intensity curve represented by the abscissa in the image moves to the right, which indicates that the illumination time increases, and more complexes Ir1(pqu) absorb singlet oxygen in the cells and convert the singlet oxygen into complexes Ir2(pqu) capable of emitting light. This luminescence conversion process can be used to detect singlet oxygen in cells.

The apoptosis of a large number of cells by the transition metal complex selected under different illumination time is further explored by flow cytometry. In the flow cell apoptosis test, 3 kinds of cells of HeLa, A549 and 3T3 are selected for testing, 10 mu M of complex Ir1(pqu) is respectively added into HeLa, A549 and 3T3 cell dishes with proper cell number, the cells are put into an incubator for continuous incubation for 2 hours, and then the cells are washed twice by PBS. Respectively illuminating a cell confocal dish for incubating the probe for 0min, 30min, 60min and 120min, then continuously incubating for 30min, digesting and centrifuging after incubation is finished, removing supernatant, adding 195 mu L of Annexin binding solution into a centrifuge tube, then sequentially adding 5 mu L of Annexin-FITC and 10 mu L of PI dye, re-suspending cells, transferring into a 1mL centrifuge tube, carrying out testing on a flow cytometer after ice bath for 10min, selecting a 488nm laser for excitation, and carrying out cell collection on the flow cytometer. The flow analysis software was then used for analysis, and the results are shown in fig. 13, in which the proportion of living cells in the blank control was over 90%, and the number of living cells did not change much with the increase of the light irradiation time, which is consistent with the trend of the phototoxicity results obtained by the MTT toxicity test method. The complex captures partial singlet oxygen in cells and reduces damage to the cells.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (7)

1. The phosphorescent metal iridium complex with the singlet oxygen detection effect is characterized in that: the structural formula of the phosphorescent metal iridium complex is shown as follows,

2. a preparation method of a phosphorescent metal iridium complex with a singlet oxygen detection effect is characterized by comprising the following steps: the method comprises the following steps:

s1, weighing 1 and SeO₂Adding into a reaction bottle, adding 1, 4-dioxane, vacuumizing, reacting for 24h under the protection of nitrogen at 102 ℃, and then weighing for heat filtration; spin-drying the filtrate, dissolving, filtering, and extractingA white solid 2;

s2, weighing 2 prepared in S1, adding 95% ethanol solution, then adding silver nitrate solution, stirring quickly to obtain yellow suspension, slowly adding NaOH solution within 20min, reacting at room temperature in the dark place, cooling, filtering, re-extracting the filtrate, adjusting the pH of the aqueous phase to acidity, and drying to obtain solid white powder 3;

s3, preparing a compound 4, 4-aminobenzene boric acid and tetrakis (triphenylphosphine) palladium, adding the mixture into a reaction bottle, vacuumizing the reaction bottle, adding deoxygenated ethanol, methylbenzene and a saturated potassium carbonate solution under the protection of nitrogen, carrying out light-shielding treatment, reacting for 24 hours at 85 ℃, cooling to room temperature after the reaction is finished, filtering, extracting, spin-drying the filtrate, and carrying out column chromatography separation to obtain a yellow solid 5;

s4, adding the compound 3, EDC & HCl and DMAP into a reaction bottle, vacuumizing, heating to remove water and simultaneously performing nitrogen protection, adding redistilled dichloromethane, adding the compound 5 dissolved in dichloromethane solution after 2min, adding dichloromethane for extraction after stirring reaction at room temperature, performing rotary evaporation to remove an organic solvent, and performing recrystallization and purification to obtain a light yellow solid 6;

s5, putting the light yellow solid 6 prepared in the S4 and iridium dichloro bridge into a mixed solvent of dichloromethane and MeOH, wherein the volume ratio of dichloromethane to MeOH is 3:1, stirring and refluxing the mixture at 50 ℃ under a nitrogen atmosphere overnight, cooling the reaction solution to room temperature, and adding KPF (Ketone fluoride) into the reaction solution₆Continuously stirring, removing the organic solvent by rotary evaporation after the reaction is finished, purifying the obtained solid by a column, and recrystallizing to obtain solid Ir 1;

the reaction formula of the above preparation method is as follows:

the structural formula of the iridium dichloro bridge is as follows:

3. the method for preparing the phosphorescent metallic iridium complex with the singlet oxygen detection effect according to claim 2, wherein the method comprises the following steps: the S1 includes the following steps:

s11, weighing 1 and SeO₂Adding into a reaction bottle, adding 260mL of 1, 4-dioxane, vacuumizing, reacting for 24h under the protection of nitrogen at 102 ℃, weighing, and filtering;

s12, the filtrate in S11 was spin dried and dissolved in ethyl acetate, and the insoluble solid was removed by filtration again to give a yellow solution using 1.0M Na₂CO₃Extracting twice, and taking an organic phase;

s13, using 0.3M Na₂S₂O₅Taking inorganic phase, adding Na₂CO₃Adjusting the pH of the inorganic phase to 10, then adding CH₂Cl₂Extraction and spin-drying to obtain white solid 2.

4. The method for preparing the phosphorescent metallic iridium complex with the singlet oxygen detection effect according to claim 2, wherein the method comprises the following steps: the S2 includes the following steps:

s21, weighing 2, adding 95% ethanol solution and silver nitrate solution, quickly stirring to obtain yellow suspension, slowly adding NaOH solution within 20min to obtain dark reaction mixed solution, and covering with tinfoil paper to avoid light and react at 25 ℃;

s22, cooling and filtering after the reaction is finished, and removing Ag and Ag₂O, extracting the filtrate by using dichloromethane;

s23, taking the water phase extracted by the dichloromethane, adjusting the pH to 3.5 by using 1M HCl solution, placing the water phase in a refrigerator for freezing overnight, filtering the solution to obtain solid, and drying the solid to obtain solid white powder 3.

5. The method for preparing the phosphorescent metallic iridium complex with the singlet oxygen detection effect according to claim 2, wherein the method comprises the following steps: the S3 includes the following steps:

s31, adding the compound 4, 4-aminophenylboronic acid and tetrakis (triphenylphosphine) palladium into a double-mouth reaction bottle, vacuumizing, adding deoxygenated ethanol, toluene and saturated potassium carbonate solution under the protection of nitrogen, carrying out light-shielding treatment, sealing a reaction device by using tinfoil paper, and reacting for 24 hours at 85 ℃;

and S32, after the reaction is finished, cooling to room temperature, filtering, washing with saline, extracting with dichloromethane, spin-drying the filtrate, and performing column chromatography to obtain a yellow solid 5.

6. The method for preparing the phosphorescent metallic iridium complex with the singlet oxygen detection effect according to claim 2, wherein the method comprises the following steps: the S4 includes the following steps:

s41, adding the compound 3, EDC & HCl and DMAP into a reaction bottle, vacuumizing, heating to remove water and simultaneously protecting nitrogen, adding redistilled dichloromethane, and adding the compound 5 dissolved in dichloromethane solution after 2 min;

and S42, stirring at room temperature for 24 hours, repeatedly washing the reaction solution by using water and dilute hydrochloric acid after the reaction is finished, adding dichloromethane for extraction, removing the organic solvent by rotary evaporation, and recrystallizing and purifying to obtain a light yellow solid 6.

7. The method for preparing the phosphorescent metallic iridium complex with the singlet oxygen detection effect according to claim 2, wherein the method comprises the following steps: the S5 includes the following steps:

s51, stirring and refluxing the compound 6 prepared in the S4 and an iridium dichloro bridge in a mixed solvent of dichloromethane and MeOH at 50 ℃ under a nitrogen atmosphere overnight;

s52, cooling the reaction solution to room temperature, and adding KPF into the reaction solution₆Stirring was continued for 4 hours, after the reaction was completed, the organic solvent was removed by rotary evaporation, and the obtained solid was purified by column chromatography and recrystallized to obtain Ir1 as a solid.

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