Iridium complex as well as preparation method and application thereof

Abstract

The invention discloses an iridium complex as well as a preparation method and application thereof, and relates to the technical field of optogenetics. The iridium complex has the following structural formula, X is an anion, Y is selected from an alkane derivative, an ether derivative or an amine derivative, the iridium complex provided by the invention is regular in structure, the central metal atom iridium has excellent fluorescent property, and fluorine atoms and trifluoromethyl groups introduced into the ligand have excellent fluorescent property, so that the fluorescence intensity of the iridium complex is greatly improved. The electron structure and the energy level of the complex are obviously regulated, the photophysical performance of the complex is enhanced, and excellent two-photon absorption capacity is shown, so that the complex has huge application potential in the fields of biological imaging, photodynamic therapy and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of optogenetics, in particular to an iridium complex and a preparation method and application thereof. BACKGROUND

[0002] Optogenetics technology realizes the spatiotemporal precise regulation of cell behavior by introducing photosensitive proteins into target cells and activating or inhibiting ion channels with specific wavelengths of light, and is widely used in the fields of neuroscience, cell biology and biomedical engineering. However, the probe tools for two-photon optogenetic imaging are still very limited at present, and the existing mainstream tools such as optical fiber probes and upconversion nanomaterials have problems such as shallow penetration depth, strong phototoxicity, difficult synthesis or poor biocompatibility, which are difficult to meet the needs of high-resolution and low-damage imaging of deep tissues.

[0003] At present, metal iridium complexes have the advantages of high luminescent efficiency, long phosphorescent lifetime, strong light stability, large Stokes shift and adjustable emission wavelength, and are particularly suitable for high-resolution deep imaging. However, there is no public literature or patent reporting the use of metal iridium complexes for two-photon excitation optogenetic imaging probes, so there is an urgent need to develop new metal iridium probes with two-photon absorption capacity, biocompatibility and targetability to expand the toolbox of optogenetic imaging. SUMMARY

[0004] The main purpose of the present application is to provide an iridium complex and a preparation method and application thereof, aiming to provide a new metal iridium probe with two-photon absorption capacity, biocompatibility and targetability to expand the toolbox of optogenetic imaging.

[0005] To achieve the above-mentioned purpose, the present application provides an iridium complex, which has the following structural formula: wherein X is an anion, and Y is selected from alkane derivatives, ether derivatives or amine derivatives.

[0006] In an embodiment, the alkane derivatives include tert-butyl, methyl or isopropyl; and / or, the ether derivatives include methoxy, ethoxy or tert-butoxy; and / or, the amine derivatives include amino, dimethylamino or diethylamino; and / or, the anion includes hexafluorophosphate ion or chloride ion.

[0007] The present application provides a preparation method of an iridium complex, comprising the following steps: S10, reacting an iridium compound and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine in a first solvent to obtain an iridium dimer precursor; S20, performing a coordination substitution reaction on the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent, washing and purifying to obtain an iridium complex.

[0008] In an embodiment, in the step S10, the mass ratio of the iridium compound and the 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine is 1:(1~3).

[0009] In an embodiment, the iridium compound is hydrated iridium trichloride; and / or, The first solvent comprises a mixed solvent of water and 2-methoxyethanol; and / or, The second solvent comprises a mixed solvent of methanol and chloroform.

[0010] In an embodiment, in the step S20, the 2,2'-bipyridine compound is selected from 4,4'-bis(tert-butyl)-2,2'-bipyridine, 4,4'-bis(methoxy)-2,2'-bipyridine, 4,4'-bis(n,n-dimethyl)-2,2'-bipyridine or 4,4'-bis(amino)-2,2'-bipyridine.

[0011] In an embodiment, the step S20 comprises: S201, performing a coordination substitution reaction on the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent to obtain a reaction mixture; S202, extracting and washing the reaction mixture multiple times to remove impurities, then adding an aqueous solution of hexafluorophosphate, and purifying by a filter precipitation method to obtain an iridium complex.

[0012] The present application provides a fluorescent probe comprising the iridium complex as described above or the iridium complex prepared by the preparation method as described above.

[0013] The present application provides the use of the fluorescent probe as described above in the field of optogenetic imaging.

[0014] The present application provides the use of the fluorescent probe as described above in the field of cell biology research.

[0015] In the technical solution of the present invention, the iridium complex provided by the present invention has a regular structure, the central metal atom iridium has excellent luminescent properties, and the fluorine atoms and trifluoromethyl groups introduced in the ligands not only significantly regulate the electronic structure and energy level of the complex, but also enhance its photophysical properties. More importantly, the introduction of these strong electron-withdrawing groups is conducive to improving the two-photon absorption cross section of the complex, and the polyfluorinated bipyridine ligands and phenylpyridine ligands provide a good conjugated skeleton for the complex, which is conducive to the delocalization of electrons, thereby promoting the two-photon absorption process. In addition, the experimental results of the present invention predict that the iridium (III) complex provided by the present invention exhibits excellent two-photon absorption ability, which makes it have huge application potential in the fields of bioimaging and photodynamic therapy. By two-photon excitation, deeper tissue penetration, higher spatial resolution and lower background fluorescence interference can be achieved. This shows that the above-mentioned iridium complex has potential deep tissue imaging and precise therapeutic activity, and is expected to provide a new strategy for disease diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0017] Figure 1 This is a flow chart of the iridium complex prepared in an embodiment of the present invention; Figure 2 The structural formula of the iridium complex prepared in Examples 1 to 4 of the present invention; Figure 3 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of the iridium complex Ir1 prepared in Example 1 of the present invention; Figure 4 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of the iridium complex Ir2 prepared in Example 2; Figure 5 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of the iridium complex Ir3 prepared in Example 3; Figure 6 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of the iridium complex Ir4 prepared in Example 4; Figure 7 The iridium complexes prepared in Examples 1 to 4 of the present invention, namely, the UV absorption-emission spectra of Ir1-Ir4 in different solvents and the emission spectra in different solvents; Figure 8 Schematic diagram of overlapping luminescence lifetime curves of iridium complexes prepared in Examples 1 to 4 of the present invention; Figure 9 Two-photon absorption cross-sectional area diagrams of the iridium complexes prepared in Examples 1 to 4 of the present invention; Figure 10 This is a cytotoxicity test chart of the iridium complexes prepared in Examples 1 to 4 of the present invention; Figure 11 The iridium complexes prepared in Examples 1 to 4 of the present invention were tested in 293T cells to obtain cell imaging effects of the iridium complexes Ir1-Ir4; Figure 12 This is a test image of the iridium complex prepared in Example 1 of the present invention opening a calcium ion channel under irradiation of a two-photon light source.

[0018] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the various components under a certain specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0021] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of such features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0022] Currently, iridium complexes offer advantages such as high luminescence efficiency, long phosphorescence lifetime, strong photostability, large Stokes shift, and tunable emission wavelength, making them particularly suitable for high-resolution deep-layer imaging. However, there are no published literature or patent reports on the use of iridium complexes as two-photon excitation optogenetic imaging probes. Therefore, there is an urgent need to develop novel iridium probes with two-photon absorption capability, biocompatibility, and targetability to expand the optogenetic imaging toolbox.

[0023] In view of this, the present invention proposes an iridium complex having the following structural formula: , wherein X is an anion and Y is selected from alkane derivatives, ether derivatives or amine derivatives.

[0024] In the technical solution of the present invention, the iridium complex provided by the present invention has a regular structure, the central metal atom iridium has excellent fluorescence properties, and the fluorine atoms and trifluoromethyl groups introduced in the ligands not only significantly regulate the electronic structure and energy level of the complex, but also enhance its photophysical properties. More importantly, the introduction of these strong electron-withdrawing groups is conducive to improving the two-photon absorption cross section of the complex. The polyfluorinated bipyridine ligands and phenylpyridine ligands provide a good conjugated skeleton for the complex, which is conducive to the delocalization of electrons, thereby promoting the two-photon absorption process. In addition, the experimental results of the present invention predict that the iridium (III) complex provided by the present invention is expected to show excellent two-photon absorption ability, which makes it have huge application potential in the fields of bioimaging and photodynamic therapy. By two-photon excitation, deeper tissue penetration, higher spatial resolution and lower background fluorescence interference can be achieved. This shows that the above-mentioned iridium complex has potential deep tissue imaging and precise therapeutic activity, and is expected to provide new strategies for disease diagnosis and treatment.

[0025] Furthermore, the iridium complex introduces a D-π-Metal-π-A structure, creating a molecule with a large π-conjugated system at the iridium metal center, endowing it with excellent two-photon absorption properties. This design can significantly increase the two-photon absorption cross-section of the substance, as the large π-conjugated system helps enhance the probability of electron transitions between different energy levels, thereby improving the absorption efficiency of light of specific wavelengths. This is crucial for achieving effective two-photon excitation. Furthermore, using different types of ligands to coordinate with the metal center can produce complexes with different luminescence properties. By precisely controlling the ratio and type of ligands, the luminescence color can be continuously adjusted to a certain extent, achieving emission light output at different wavelengths, such as 470 nm to 632 nm. This allows for compatibility with a variety of photosensitive proteins, and the iridium complex probe can function in actual biological environments.

[0026] In some embodiments, the alkane derivative includes a tert-butyl group, a methyl group, or an isopropyl group.

[0027] In some embodiments, the ether derivative comprises a methoxy group, an ethoxy group or a tert-butoxy group; and / or, In some embodiments, the amine derivatives include amino, dimethylamino or diethylamino, Because these specific Y groups all have electron-donating properties, they can effectively enhance the fluorescence intensity and alter the emission wavelength of the complex. Specifically, for example, amino groups, as strong electron donors, can increase charge transfer from the ligand to the metal center, often leading to a red-shift in the emission spectrum, i.e., a shift in the luminescence color from blue to red. This can optimize the performance of iridium complexes, improving luminescence efficiency and stability. They can also modulate the emission wavelength by shifting energy levels. The specific combination of these groups depends on the specific objectives and needs.

[0028] In some embodiments, the anion is a hexafluorophosphate ion or a chloride ion. Specifically, for example, the hexafluorophosphate ion is a larger, more symmetrical anion and can effectively surround and stabilize the central metal complex, thereby helping to maintain the stability of the entire complex.

[0029] The present invention provides a method for preparing an iridium complex, comprising the following steps: S10, reacting an iridium compound and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine in a first solvent to obtain an iridium dimer precursor; S20, conducting a coordination substitution reaction between the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent, washing, and purifying to obtain an iridium complex.

[0030] In this preparation step, the iridium complex and the ligand are first reacted in a solvent to pre-form a dimer structure containing an iridium center, providing an active site for the subsequent introduction of the second ligand. This distributed synthesis method helps to control the selectivity of the reaction and the product structure, and can also avoid side reactions caused by the one-step synthesis method.

[0031] In one embodiment, in step S10, the mass ratio of the iridium compound to the 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine is 1:(1-3). This mass ratio range is intended to ensure a moderate excess of the ligand (2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine), thereby increasing the conversion rate in the coordination reaction and ensuring that the iridium compound can fully react.

[0032] In one embodiment, the first solvent includes a mixed solvent of water and 2-methoxyethanol, wherein the mixed solvent of water and 2-methoxyethanol is prepared by using the two solvents in a volume ratio of 1:1, has good dissolving ability, and is conducive to improving reaction efficiency.

[0033] The second solvent includes a mixed solvent of methanol and chloroform, wherein the mixed solvent is prepared from anhydrous methanol and chloroform in a volume ratio of 1:1, which can promote the coordination substitution reaction and help form subsequent products.

[0034] In one embodiment, in step S20, the 2,2'-bipyridine compound is selected from 4,4'-bis(tert-butyl)-2,2'-bipyridine, 4,4'-bis(methoxy)-2,2'-bipyridine, 4,4'-bis(nitrogen, nitrogen-dimethyl)-2,2'-bipyridine or 4,4'-bis(amino)-2,2'-bipyridine. The above 2,2'-bipyridine compounds have different charges or steric properties, which can adjust the electronic structure and spatial configuration of the iridium complex, thereby affecting its fluorescence properties.

[0035] In one embodiment, step S20 includes: S201, performing a coordination substitution reaction on the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent to obtain a reaction mixture; S202, extracting and washing the reaction mixture multiple times to remove impurities, then adding a hexafluorophosphate aqueous solution, and purifying it by filtering and precipitation to obtain an iridium complex.

[0036] In this step, unreacted substances, by-products and other impurities can be effectively removed by extraction and washing multiple times. The purpose of adding an aqueous solution of ammonium hexafluorophosphate is to purify the product by filtration and precipitation, thereby improving the effective separation and purity of the target product. Specifically, the hexafluorophosphate can be potassium hexafluorophosphate or ammonium hexafluorophosphate.

[0037] The present invention provides a fluorescent probe, which includes the iridium complex as described above or the iridium complex prepared by the method for preparing the iridium complex as described above. Since the iridium complex has excellent photophysical properties, it can be widely used in the field of fluorescent probes.

[0038] The present invention provides the application of the fluorescent probe described above in the field of optogenetic imaging. Due to the unique light response characteristics of the iridium complex, it can become a potential optogenetic tool, enabling more precise optical control and signal detection, and can also be used in the field of optogenetic imaging.

[0039] The present invention provides the use of the fluorescent probe described above in cell biology research.

[0040] The specific preparation flow chart is as attached Figure 1 As shown: Step 1: Synthesis of the dimeric complex [(dFCF3ppy)2Ir-Cl]2 Using 2-(4-fluorophenyl)-4-(trifluoromethyl)pyridine (dFCF3ppy) and iridium trichloride (IrCl3) as starting materials, the reaction was heated at 120°C for 12 hours in methoxyethanol (CH3OCH2CH2OH) solvent under argon (Ar) protection to obtain a dimeric iridium (III) complex [(dFCF3ppy)2Ir-Cl]2. The dimeric iridium complex contains two iridium centers, each iridium atom is coordinated with two dFCF3ppy ligands and two chloride ions, and the chloride ions serve as bridging ligands to connect the two iridium atoms to form a double chloride bridge structure.

[0041] Step 2: Synthesis of the monomeric cationic complex [(dFCF3ppy)2Ir(N^N)]⁺ The dimer [(dFCF3ppy)2Ir-Cl]2 synthesized in the first step and a bipyridine (N^N) ligand containing two R groups were heated at 60°C for 12 hours in a mixed solvent of methanol (CH3OH) and chloroform (CHCl3) under argon (Ar) protection to obtain the monomeric iridium (III) cation complex [[(dFCF3ppy)2Ir(N^N)]⁺.

[0042] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0043] Example 1 This embodiment provides a method for preparing a metal iridium complex and a metal iridium complex prepared by the preparation method, and the preparation method comprises the following steps: Step S10, iridium (III) trichloride hydrate (1.6 g, 5.05 mmol) and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine (3 g, 11.58 mmol) were heated under reflux in a mixed solvent of water and 2-methoxyethanol under argon protection to obtain an iridium dimer precursor, i.e., a yellow-green solid iridium (III) μ-chloro-bridged dimer complex [(dFCF3ppy)2-Ir-Cl]2; Step S20: Iridium (III) μ-chlorobridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(tert-butyl)-2,2'-bipyridine (0.40 mmol) were heated at 60°C for 12 hours under argon in a mixture of methanol and chloroform. After cooling to room temperature, the mixture was transferred to a separatory funnel with deionized water and washed three times with n-hexane (30 ml each time). The aqueous layer was heated to 85°C to remove residual n-hexane. To this mixture was added 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g). The precipitate was filtered and dried to obtain the iridium complex as a yellow solid, designated Ir1.

[0044] Example 2 The difference between this embodiment and embodiment 1 is step S20, and the rest are the same. Specifically, the preparation method of step S20 in this embodiment includes: Step S20: Iridium (III) μ-chlorobridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(methoxy)-2,2'-bipyridine (0.40 mmol) were heated at 60°C for 12 hours under argon in a mixed solvent of methanol and chloroform. After cooling to room temperature, the mixture was transferred to a separatory funnel with deionized water and washed three times with n-hexane (30 ml each time). The aqueous layer was heated to 85°C to remove residual n-hexane. To this mixture was added 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g). The precipitate was filtered and dried to obtain the iridium complex as a yellow solid, designated Ir2.

[0045] Example 3 The difference between this embodiment and embodiment 1 is step S20, and the rest are the same. Specifically, the preparation method of step S20 in this embodiment includes: Iridium(III) μ-chlorobridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(nitrogen, nitrogen-dimethyl)-2,2'-bipyridine (0.40 mmol) were heated at 60°C for 12 hours under argon in a methanol and chloroform mixture. After cooling to room temperature, the mixture was transferred to a separatory funnel with deionized water and washed three times with n-hexane (30 ml each time). The aqueous layer was heated to 85°C to remove any residual n-hexane. To this mixture was added 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g). The precipitate was filtered and dried to obtain the iridium complex as a yellow solid, designated Ir3.

[0046] Example 4 The difference between this embodiment and embodiment 1 is step S20, and the rest are the same. Specifically, the preparation method of step S20 in this embodiment includes: Iridium(III) μ-chlorobridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(amino)-2,2'-bipyridine (0.40 mmol) were heated at 60°C for 12 hours under argon in a methanol and chloroform mixture. After cooling to room temperature, the mixture was transferred to a separatory funnel with deionized water and washed three times with n-hexane (30 ml each time). The aqueous layer was heated to 85°C to remove any residual n-hexane. To this mixture was added 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g). The precipitate was filtered and dried to obtain the iridium complex as a yellow solid, designated Ir4.

[0047] Performance Testing 1. For Examples 1 to 4, obtain Figure 2The metal iridium complexes Ir1-Ir4 shown in FIG. 1 show the H NMR spectra of the metal iridium complexes Ir1-Ir4. The solvent used is deuterated DMSO. The test results are as follows: Figures 3 to 6 As shown, Figures 3 to 6 It can be seen that the chemical shift, integral value and peak shape of the metal iridium complex Ir1-Ir4 support and confirm the molecular structure of the iridium complex.

[0048] 2. If Figure 7 Shown are the UV absorption-emission spectra of the metal iridium complexes Ir1-Ir4, 1-2 are the UV-visible absorption spectra of Ir1-Ir4 in DMSO and water, respectively, and the emission spectra of the four iridium complexes 3-6 are Ir1, Ir2, Ir3 and Ir4 in different solvents, respectively.

[0049] UV-visible absorption spectroscopy testing conditions include a working concentration of 5 μM for the iridium complexes Ir1-Ir4, an absorption wavelength of 200-800 nm, and a solvent system consisting of dimethyl sulfoxide and pure water. UV-visible absorption spectra were measured at 298 K using a Shimadzu UV spectrophotometer.

[0050] Phosphorescence emission spectra were measured using a Shimadzu fluorescence spectrophotometer using an iridium complex Ir1-Ir4 at a working concentration of 5 μM, an excitation wavelength of 405 nm, and an emission wavelength of 420-750 nm. The solvent system consisted of toluene, tetrahydrofuran, chloroform, acetonitrile, dimethyl sulfoxide, and pure water.

[0051] Experimental Results: UV-visible absorption spectra of iridium complexes Ir1-Ir4 were measured in dimethyl sulfoxide solutions and pure water at room temperature. Strong absorption peaks in the UV region (250-350 nm) are attributed to transitions in the ligand's neutral region, typically spin-allowed 1π-π* transitions. Lower-energy transitions extending into the visible region (350-450 nm) are generally attributed to spin-allowed 1MLCT transitions and spin-forbidden 3MLCT transitions.

[0052] Experimental Results: The emission spectra of iridium complexes Ir1-Ir4 were tested at room temperature in solvents of varying polarity. A distinct emission peak was observed at 470 nm in these solvents, indicating that light in this wavelength range helps open cellular calcium channels. Furthermore, as the polarity increased, the maximum emission wavelength of the iridium complexes Ir1-Ir4 exhibited a significant red shift.

[0053] like Figure 8As shown, 1 is the superimposed plot of the luminescence lifetime curves of Ir1-Ir4, 2 is the luminescence lifetime curve and fitting curve plot of iridium complex Ir1, 3 is the luminescence lifetime curve and fitting curve plot of iridium complex Ir2, 4 is the luminescence lifetime curve and fitting curve plot of iridium complex Ir3, and 5 is the luminescence lifetime curve and fitting curve plot of iridium complex Ir4.

[0054] Test conditions: the working concentration of iridium complexes Ir1-Ir4 is 5 μM, the excitation wavelength is 365 nm, the solvent is pure water system, the luminescence lifetime is measured by Edinburgh steady-state / transient fluorescence spectrometer, the light source used is hydrogen lamp, the phosphorescence lifetime is measured by pulse laser, and the lifetime value is obtained by iterative calculation fitting of the luminescence lifetime decay curve by professional software provided by Edinburgh company.

[0055] Test results: the luminescence lifetime decay curves of iridium complexes Ir1-Ir4 in pure water at room temperature were tested, and it can be obtained by software fitting that iridium complexes Ir1-Ir4 all have long luminescence lifetime, and from small to large, Ir1 (256.59 ns) < Ir3 (323.57 ns) < Ir2 (453.97 ns) < Ir4 (556.95 ns).

[0056] As shown in Figure 9 , the test conditions: the two-photon absorption spectra of complexes Ir1-Ir4 were measured in a wide spectral range by a typical two-photon laser-induced fluorescence method. Two-photon fluorescence test was carried out in a fluorescence colorimetric quartz cell, and the sample was 100 μM Ir1~Ir4 solution (CH3OH, 298 K). The fluorescence excitation and detection conditions in the experiment were optimized to ensure negligible reabsorption effect, thereby avoiding interference with two-photon absorption measurement. Under the excitation wavelength of 690~800 nm, rhodamine B (Rhodamine B) was used as a reference dye to measure the two-photon absorption cross-section area of iridium complexes.

[0057] Test results: the two-photon absorption cross-section (δ) spectra of four kinds of iridium complexes (Ir1~Ir4) in the wavelength range of 680~800 nm were measured by two-photon excitation fluorescence method, and the results are shown in Figure 9As shown, all complexes exhibit obvious two-photon absorption peaks near 690 nm, in which the delta value of Ir1 is the highest, about 560 GM, and the peak values of Ir2, Ir3 and Ir4 decrease in turn, about 480, 420 and 380 GM, respectively, showing the significant influence of different ligand structures on two-photon absorption ability. With the increase of excitation wavelength, the delta value presents a trend of rapid decline first and then slow recovery, and the valley region is about 740 nm (delta is about 100-150 GM), and then gradually rises in the interval of 760-800 nm, which may be related to the transition of low-energy excited state or specific orbital resonance. The spectrum pattern conforms to the typical two-photon absorption characteristics of metal complexes, indicating that the excitation process is mainly regulated by charge transfer transition (ICT), and the excitation process is stable and has good repeatability. Comprehensive analysis results show that Ir1 has the optimal two-photon absorption performance, which is suitable as the core skeleton for the design of subsequent two-photon fluorescent probes or photosensitizers, and has good potential for structure optimization and application prospect.

[0058] As shown in Figure 10 The test conditions are as follows: human cervical cancer cells HeLa are used as the research object, and the reagent for culturing the cells is DMEM culture solution composed of 10% Gibco fetal bovine serum and 90% DMEM incomplete high-glucose medium (containing 80 μg / mL streptomycin and 80 U / mL penicillin). The temperature in the cell culture box is 37°C, the humidity is 100%, and the atmosphere in the box is air containing 5% CO2. According to different experimental conditions, the cells can be adherent growth in a 96-well plate or incubated in a confocal culture dish, and then the sample incubation is performed after 24 hours. In order to evaluate the cytotoxicity of complexes Ir1-Ir4, the thiazolyl blue (MTT) method is used for related experiments. There is a succinate dehydrogenase in the mitochondria of living cells, which can reduce exogenous MTT to insoluble formazan in DMSO, while dead cells cannot reduce MTT. Based on this principle, living cells and dead cells can be distinguished, so the MTT method is a common method for detecting cell activity. The specific operation of the test is as follows: HeLa cell suspension is inoculated in a 96-well plate, 150 μL is injected into each well, in order to prevent experimental errors caused by evaporation of the solution, the outermost circle is not inoculated with cells, but 150 μL of PBS buffer solution is injected into each well. Then the 96-well plate is transferred to the incubator for 24 hours to allow the cells to adhere to the wall. After the cell density (10 4When the cells were uniformly distributed (per well), the stock solution of complexes Ir1-Ir4 was diluted with DMEM to concentrations of 0 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM, respectively. 150 μL of this solution was then dispensed into different columns of a 96-well plate, with six replicates for each concentration. The cells were incubated for 24 hours. Fresh DMEM was then replaced with 15 μL of MTT solution (5 mg / mL) prepared in PBS (pH 7.4) and incubated for 4 hours. The supernatant was then aspirated, and 150 μL of DMSO was added to each well to dissolve the blue-purple formazan crystals in the viable cells. The cells were allowed to stand for 30 minutes. Finally, the absorbance at 570 nm (OD570) of each well on the 96-well plate was measured using a microplate reader. A higher formazan concentration indicates a higher number of viable cells, and vice versa. Cell survival rate (%) = (OD value of experimental group / OD value of control group) × 100%.

[0059] Test results: After incubation for 24 hours with 0.2μM, 5μM, and 10μM iridium complexes Ir1-Ir4, the cell survival rate of cervical cancer cells HeLa exceeded 80%, indicating that at low concentrations, the iridium complexes Ir1-Ir4 have good biocompatibility. When incubated for 24 hours with high concentrations (greater than 20μM) of iridium complexes Ir2-Ir4 (greater than 20μM), the cell survival rate decreased significantly, indicating that high concentrations of iridium complexes Ir2-Ir4 have high cytotoxicity. However, the iridium complex Ir1 did not cause obvious toxic effects on cervical cancer cells HeLa at concentrations of 0-50μM, and its cell survival rate exceeded 95%, indicating excellent biocompatibility.

[0060] like Figure 11 As shown in the figure, the iridium complexes Ir1-Ir4 were tested for cellular imaging in 293T cells. 293T cells were seeded at a uniform density in the logarithmic growth phase onto a confocal dish. The dish was removed from the incubator when the cells had attached and developed good morphology. The DMEM medium in the dish was then removed with a pipette, and a solution of the iridium complex (5 μM) prepared in DMEM was added to the dish. The dish was incubated in the incubator for 2 hours. The original solution was then discarded from the dish, and the dish was washed three times with PBS to remove excess iridium complex. 1 mL of DMEM was then added to the dish. The dish was then imaged under a single- and two-photon confocal microscope using the single-photon imaging channel (excitation wavelength 405 nm, emission wavelength 450-510 nm) and the two-photon imaging channel (excitation wavelength 700 nm, emission wavelength 450-510 nm).

[0061] Figure 11The test results evaluated the imaging effect of iridium complexes Ir1-Ir4 on 293T cells. After the injection of iridium complexes, strong phosphorescence signals were observed in the cells under a single-photon 405 nm excitation light source, and the phosphorescence intensities of iridium complexes Ir1 and Ir2 were significantly stronger than those of iridium complexes Ir3 and Ir4; under a two-photon 700 nm excitation light source, iridium complex Ir1 showed the strongest phosphorescence intensity, indicating that iridium complex Ir1 has the potential to open calcium ion channels under single / two-photon light source excitation.

[0062] like Figure 12 As shown, the test conditions are as follows: 293T cells were transfected with ChR2-Venus. The blank group did not add the iridium complex Ir-1, while the experimental group used the Ir-1 iridium complex (5 μM). After incubation for 2 hours, the calcium ion indicator Rhod-3 AM (1 μM) was added and incubated for another 40 minutes. The cells were irradiated with a two-photon light source for five minutes. After the calcium ion channels opened and calcium ions flowed in, imaging was performed. The wavelength of the two-photon excitation light source was 700 nm, and the power range was 0-0.8 W / cm 2 , the imaging channel is Rhod-3 AM red channel, the excitation wavelength is 561 nm, and the emission wavelength range is 580-620 nm.

[0063] Test results: The ability of the iridium complex Ir-1 to open calcium ion channels under two-photon light illumination was evaluated. In Figure A (left), no red fluorescence signal was detected in the blank group, while in Figure A (right), a significant red fluorescence signal was detected in the experimental group. This indicates that under two-photon light excitation, the iridium complex Ir-1 emits phosphorescence at around 470 nm, thereby opening calcium ion channels and inducing calcium influx. Figure B shows the effect of different two-photon excitation powers (0-0.8 W / cm 2 ) under the influence of two-photon light power. As the two-photon excitation power increases, the red fluorescence intensity in 293T cells continues to increase. This is because the phosphorescence emission intensity of the iridium complex Ir-1 in 293T cells increases with the increase of two-photon excitation power, thereby triggering more calcium ions to flow in, making the fluorescence intensity of the calcium ion indicator Rhod-3AM stronger. This shows that the concentration of calcium ions flowing into 293T cells is dependent on the two-photon light power, further indicating that the synthesized iridium complex Ir1 has great potential to become a two-photon optogenetic imaging probe.

[0064] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformation made by utilizing the contents of the present invention's description and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. An iridium complex, characterized in that The iridium complex has the following structural formula: , wherein X is an anion and Y is selected from alkane derivatives, ether derivatives or amine derivatives.

2. The iridium complex according to claim 1, wherein The alkane derivatives include tert-butyl, methyl or isopropyl; and / or, The ether derivatives include methoxy, ethoxy or tert-butoxy; and / or, The amine derivatives include amino, dimethylamino or diethylamino; and / or, The anion includes a hexafluorophosphate ion or a chloride ion.

3. A method for preparing the iridium complex according to claim 1 or 2, characterized in that: The following steps are involved: S10, reacting an iridium compound and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine in a first solvent to obtain an iridium dimer precursor; S20, conducting a coordination substitution reaction between the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent, washing, and purifying to obtain an iridium complex.

4. The method for preparing an iridium complex as claimed in claim 3, wherein In the step S10 , the mass ratio of the iridium compound to the 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine is 1:(1-3).

5. The method for preparing the iridium complex according to claim 3, wherein The iridium compound is hydrated iridium trichloride; and / or, The first solvent includes a mixed solvent of water and 2-methoxyethanol; The second solvent includes a mixed solvent of methanol and chloroform.

6. The method for preparing an iridium complex according to claim 3, wherein In step S20, the 2,2'-bipyridine compound is selected from 4,4'-bis(tert-butyl)-2,2'-bipyridine, 4,4'-bis(methoxy)-2,2'-bipyridine, 4,4'-bis(nitrogen, nitrogen-dimethyl)-2,2'-bipyridine or 4,4'-bis(amino)-2,2'-bipyridine.

7. The method for preparing an iridium complex according to claim 3, wherein Step S20 includes: S201, performing a coordination substitution reaction on the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent to obtain a reaction mixture; S202, extracting and washing the reaction mixture multiple times to remove impurities, then adding a hexafluorophosphate aqueous solution, and purifying it by filtering and precipitation to obtain an iridium complex.

8. A fluorescent probe, characterized in that The fluorescent probe includes the iridium complex according to claim 1 or 2 or the iridium complex prepared by the preparation method of the iridium complex according to any one of claims 3 to 7.

9. Use of the fluorescent probe as claimed in claim 8 in the field of optogenetic imaging.

10. Use of the fluorescent probe according to claim 8 in cell biology research.

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