An iridium complex, preparation method and application thereof

Abstract

The application discloses an iridium complex and a preparation method and application thereof, and relates to the technical field of optogenetics. 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. The iridium complex provided by the application has a regular structure, the central metal atom iridium has excellent fluorescence properties, and the fluorine atoms and trifluoromethyl groups introduced in the ligand not only significantly regulate the electronic structure and energy level of the complex, but also enhance the optical physical properties of the complex, and the excellent two-photon absorption capacity is exhibited, which makes the complex have great application potential in the fields of biological imaging and photodynamic therapy.

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:

[0006] , wherein X is an anion, and Y is selected from alkane derivatives, ether derivatives or amine derivatives.

[0007] In an embodiment, the alkane derivatives include tert-butyl, methyl or isopropyl; and / or,

[0008] The ether derivatives include methoxy, ethoxy or tert-butoxy; and / or,

[0009] The amine derivatives include amino, dimethylamino or diethylamino; and / or,

[0010] The anion includes hexafluorophosphate ion or chloride ion.

[0011] The present application provides a preparation method of an iridium complex, comprising the following steps:

[0012] S10, reacting the iridium compound and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine in a first solvent to obtain an iridium dimer precursor;

[0013] S20, performing a coordination substitution reaction on the iridium dimer precursor with a 2,2'-bipyridine compound in a second solvent, washing and purifying to obtain an iridium complex.

[0014] In an embodiment, in the step S10, the mass ratio of the iridium compound to the 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine is 1:(1-3).

[0015] In an embodiment, the iridium compound is hydrated iridium trichloride; and / or,

[0016] The first solvent comprises a mixed solvent of water and 2-methoxyethanol; and / or,

[0017] The second solvent comprises a mixed solvent of methanol and chloroform.

[0018] 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.

[0019] In an embodiment, the step S20 comprises:

[0020] S201, performing a coordination substitution reaction on the iridium dimer precursor with a 2,2'-bipyridine compound in a second solvent to obtain a reaction mixture;

[0021] S202, extracting and washing the reaction mixture multiple times to remove impurities, then adding an aqueous solution of hexafluorophosphate, and purifying by a filtration precipitation method to obtain an iridium complex.

[0022] 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.

[0023] The present application provides an application of the fluorescent probe as described above in the field of optogenetic imaging.

[0024] The present application provides an application of the fluorescent probe as described above in the field of cell biology research.

[0025] The iridium complex provided by the technical scheme of the present application has a regular structure, the central metal atom iridium has excellent luminescent properties, and the introduction of fluorine atoms and trifluoromethyl groups in the ligand not only significantly regulates the electronic structure and energy level of the complex and enhances the photophysical properties thereof, but also, 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 multi-fluorine-substituted bipyridine ligand and the phenylpyridine ligand 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 application show that the iridium (III) complex provided by the present application exhibits excellent two-photon absorption capacity, which makes it have great application potential in the fields of biological imaging and photodynamic therapy. Through 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 treatment activity, and is expected to provide a new strategy for disease diagnosis and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical scheme in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained according to the structures shown in the drawings without creative labor for those skilled in the art.

[0027] Figure 1 The flowchart of the iridium complex prepared in the embodiments of the present application;

[0028] Figure 2 The structural formula diagram of the iridium complex prepared in embodiments 1 to 4 of the present application;

[0029] Figure 3 The nuclear magnetic resonance hydrogen spectrum diagram of the iridium complex Ir1 prepared in embodiment 1 of the present application;

[0030] Figure 4 The nuclear magnetic resonance hydrogen spectrum diagram of the iridium complex Ir2 prepared in embodiment 2;

[0031] Figure 5 The nuclear magnetic resonance hydrogen spectrum diagram of the iridium complex Ir3 prepared in embodiment 3;

[0032] Figure 6 The nuclear magnetic resonance hydrogen spectrum diagram of the iridium complex Ir4 prepared in embodiment 4;

[0033] Figure 7 The ultraviolet absorption-emission spectrum of the iridium complex prepared in embodiments 1 to 4 of the present application, i.e. Ir1-Ir4, in different solvents and the emission spectrum in different solvents;

[0034] Figure 8 The luminescence lifetime curve overlap diagram of the iridium complexes prepared in the embodiments 1-4 of the present application is shown in the following figure:

[0035] Figure 9 The two-photon absorption cross-section area diagram of the iridium complexes prepared in the embodiments 1-4 of the present application is shown in the following figure:

[0036] Figure 10 The cytotoxicity test diagram of the iridium complexes prepared in the embodiments 1-4 of the present application is shown in the following figure:

[0037] Figure 11 The cell imaging effect diagram of the iridium complexes Ir1-Ir4 prepared in the embodiments 1-4 of the present application in 293T cells is shown in the following figure:

[0038] Figure 12 The test diagram of the iridium complex prepared in the embodiment 1 of the present application opening calcium ion channel under the irradiation of two-photon light source is shown in the following figure.

[0039] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present application.

[0041] It should be noted that if the embodiments of the present application 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 condition, etc. between the components in a certain posture, and if the certain posture changes, the directional indications will also change accordingly.

[0042] In addition, if the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can be explicitly or implicitly included at least one of the features. In addition, if "and / or" or "and / or" appears throughout the text, it means that the three parallel schemes include the A scheme, or the B scheme, or the A and B schemes. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the realization of the ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the scope of protection required by the present application.

[0043] At present, metal iridium complex has the advantages of high luminescent efficiency, long phosphorescent lifetime, strong light stability, large stokes shift and adjustable emission wavelength, and is especially suitable for high-resolution deep imaging. However, there is no public document or patent reporting that metal iridium complex is used for two-photon excitation optogenetic imaging probe, so it is urgent to develop a new type of metal iridium probe with two-photon absorption capacity, biocompatibility and targetability, so as to expand the imaging tool box of optogenetics.

[0044] In view of this, the present application provides an iridium complex, which has the following structural formula:

[0045] , wherein X is an anion, Y is selected from alkane derivatives, ether derivatives or amine derivatives.

[0046] In the technical solution of the present application, the iridium complex provided by the present application has a regular structure, the central metal atom iridium has excellent fluorescence properties, and the introduction of fluorine atoms and trifluoromethyl groups in the ligand not only significantly regulates the electronic structure and energy level of the complex, enhances its optical physical properties, more importantly, the introduction of these strong electron-withdrawing groups is beneficial to improve the two-photon absorption cross section of the complex. The multi-fluorine-substituted bipyridine ligand and the phenylpyridine ligand provide a good conjugated skeleton for the complex, which is beneficial to the delocalization of electrons, thereby promoting the two-photon absorption process. In addition, the experimental results of the present application predict that the iridium(III) complex provided by the present application is expected to exhibit excellent two-photon absorption capacity, which makes it have great application potential in the fields of biological imaging and photodynamic therapy. Through 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.

[0047] Further, the iridium complex is constructed by introducing a D-π-Metal-π-A structure to the metal iridium center to build a molecule with a large π conjugated system, which gives it excellent two-photon absorption performance. This design can significantly increase the two-photon absorption cross section of the material because the large π conjugated system helps to enhance the transition probability of electrons between different energy levels, thereby improving the absorption efficiency of light of a specific wavelength. This is crucial for achieving effective two-photon excitation, and using different types of ligands to coordinate with the metal center can produce complexes with different luminescent properties. By precisely controlling the proportion and type of ligands, the color of the emitted light can be continuously adjusted to some extent, enabling the emission of light at different wavelengths such as 470 nm to 632 nm, which is suitable for a variety of photosensitive proteins. The iridium complex probe can work in actual biological environments.

[0048] In some embodiments, the alkane derivative includes a tert-butyl group, a methyl group, or an isopropyl group.

[0049] In some embodiments, the ether derivative includes a methoxy group, an ethoxy group, or a tert-butoxy group; and / or,

[0050] In some embodiments, the amine derivative includes an amino group, a dimethylamino group, or a diethylamino group,

[0051] The above-mentioned specific Y groups can effectively enhance the fluorescence intensity of the complex and change the emission wavelength of the complex because they all have electron-donating effects. Specifically, for example: the amino group as a strong electron donor can increase the charge transfer from the ligand to the metal center, which usually causes a red shift in the emission spectrum, i.e., the color of the emitted light changes from blue to red. This can optimize the performance of the iridium complex, improve the luminescence efficiency and stability, and also change the energy level to adjust the emission wavelength. How to combine and use them depends on the actual target and requirements.

[0052] In some embodiments, the anion is a hexafluorophosphate ion or a chloride ion. Specifically, for example, the hexafluorophosphate ion is a large and highly symmetric anion that can effectively surround and stabilize the central metal complex, thereby helping to maintain the stability of the entire complex.

[0053] The present application provides a preparation method of an iridium complex, comprising the following steps:

[0054] S10, reacting an iridium compound and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine in a first solvent to obtain an iridium dimer precursor;

[0055] S20, carrying out a coordination substitution reaction of the iridium dimer precursor with a 2,2'-bipyridine compound in a second solvent, washing and purifying to obtain an iridium complex.

[0056] In the preparation step, the iridium complex is first reacted with the ligand in a solvent to form a dimer structure containing an iridium center in advance, thereby providing an active site for the subsequent introduction of the second ligand. This distributed synthesis method helps to control the selectivity and structure of the product of the reaction, and can also avoid side reactions caused by one-step synthesis.

[0057] 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). The mass ratio range is to ensure a moderate excess of the ligand (2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine), which can improve the conversion rate in the coordination reaction and ensure that the iridium compound can be fully reacted.

[0058] In an 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 mixing the two solvents in a volume ratio of 1:1, has good solubility, and is beneficial to improve the reaction efficiency.

[0059] The second solvent includes a mixed solvent of methanol and chloroform, wherein the mixed solvent is prepared by mixing anhydrous methanol and chloroform in a volume ratio of 1:1, which can promote the progress of the coordination substitution reaction and is helpful for the formation of the subsequent product.

[0060] 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. The above-mentioned 2,2'-bipyridine compounds have different charge or steric characteristics, which can adjust the electronic structure and spatial configuration of the iridium complex, thereby affecting its fluorescence performance.

[0061] In an embodiment, the step S20 includes:

[0062] S201, performing a coordination substitution reaction on the iridium dimer precursor and the 2,2'-bipyridine compound in a second solvent to obtain a reaction mixture;

[0063] S202, extracting and washing the reaction mixture multiple times to remove impurities, and then adding an aqueous solution of hexafluorophosphate to purify by a filtration precipitation method to obtain the iridium complex.

[0064] In this step, the unreacted raw materials, byproducts and other impurities can be effectively removed by extraction and washing multiple times, and an aqueous solution of ammonium hexafluorophosphate is added to purify by filtration and precipitation, thereby improving the effective separation and purity of the target product.

[0065] The application provides a fluorescent probe comprising the iridium complex or the iridium complex prepared by the preparation method of the iridium complex, and the iridium complex has excellent photophysical properties and can be widely applied in the field of fluorescent probes.

[0066] The application provides application of the fluorescent probe in the field of optogenetic imaging, and the iridium complex has unique light response characteristics, can become a potential optogenetic tool, can realize more fine optical control and signal detection, and can be applied in the field of optogenetic imaging.

[0067] The application provides application of the fluorescent probe in the field of cell biology research.

[0068] The specific preparation flowchart is shown in the accompanying Figure 1

[0069] Step 1: Synthesis of dimeric complex [(dFCF3ppy)2Ir-Cl]2

[0070] 2-(4-fluorophenyl)-4-(trifluoromethyl)pyridine (dFCF3ppy) and iridium trichloride (IrCl3) are used as starting materials, argon (Ar) protection is carried out in a methoxyethanol (CH3OCH2CH2OH) solvent, heating is carried out at 120 DEG C for 12 hours, and a dimeric iridium (III) complex [(dFCF3ppy)2Ir-Cl]2 is obtained, wherein the dimeric iridium complex comprises two iridium centers, each iridium atom is coordinated with two dFCF3ppy ligands and two chloride ions, and the chloride ions are connected with the two iridium atoms as bridging ligands to form a double-chlorine bridge structure.

[0071] Step 2: Synthesis of monomeric cationic complex [(dFCF3ppy)2Ir(N^N)]+

[0072] The dimer [(dFCF3ppy)2Ir-Cl]2 synthesized in the first step and a bipyridine (N^N) ligand containing two R groups are used as starting materials, argon (Ar) protection is carried out in a mixed solvent of methanol (CH3OH) and chloroform (CHCl3), heating is carried out at 60 DEG C for 12 hours, and a monomeric iridium (III) cationic complex [((dFCF3ppy)2Ir(N^N)]+ is obtained.

[0073] ​The technical solutions of the present application will be described in further detail below in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and not used to limit the present application.

[0074] Embodiment 1

[0075] The present embodiment provides a preparation method of a metal iridium complex and a metal iridium complex prepared by the preparation method. The preparation method comprises the following steps:

[0076] In step S10, under argon protection, the hydrated iridium (III) trichloride (1.6 g, 5.05 mmol) and 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine (3 g, 11.58 mmol) are heated to reflux in a mixed solvent of water and 2-methoxyethanol to obtain an iridium dimer precursor, i.e., a yellow-green solid iridium (III) μ-chloro bridged dimer complex [(dFCF3ppy)2-Ir-Cl]2.

[0077] In step S20, under argon protection, the iridium (III) μ-chloro bridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(tert-butyl)-2,2'-bipyridine (0.40 mmol) are heated at 60 degrees Celsius for 12 hours in a mixed solvent of methanol and chloroform. After cooling to room temperature, the mixture is transferred to a separatory funnel with deionized water and washed with n-hexane three times (30 ml each time). The water layer is heated to 85 degrees Celsius to remove the residual n-hexane. 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g) is added to the above mixture, and the precipitate is filtered and dried to obtain a yellow solid iridium complex, which is named as Ir1.

[0078] Embodiment 2

[0079] The difference between the present embodiment and embodiment 1 is in step S20, and the rest is the same. Specifically, the preparation method of step S20 in the present embodiment comprises:

[0080] In step S20, under argon protection, the iridium (III) μ-chloro bridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis(tert-butyl)-2,2'-bipyridine (0.40 mmol) are heated at 60 degrees Celsius for 12 hours in a mixed solvent of methanol and chloroform. After cooling to room temperature, the mixture is transferred to a separatory funnel with deionized water and washed with n-hexane three times (30 ml each time). The water layer is heated to 85 degrees Celsius to remove the residual n-hexane. 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g) is added to the above mixture, and the precipitate is filtered and dried to obtain a yellow solid iridium complex, which is named as Ir1.

[0081] Embodiment 3

[0082] The difference between the present embodiment and embodiment 1 is in step S20, and the rest is the same, specifically, the preparation method of step S20 in the present embodiment includes:

[0083] The iridium (III) μ-chloro bridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis (amino) -2,2'-bipyridine (0.40 mmol) were heated in a mixed solvent of methanol and chloroform under argon protection at 60 degrees Celsius for 12 hours, and after cooling to room temperature, it was transferred to a separatory funnel with deionized water, washed with n-hexane three times (30 ml each time), and the water layer was heated to 85 degrees Celsius to remove the residual n-hexane. 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g) was added to the above mixture, the precipitate was filtered and dried to obtain a yellow solid of the iridium complex, named Ir4.

[0084] Example 4

[0085] The difference between the present embodiment and embodiment 1 is in step S20, and the rest is the same, specifically, the preparation method of step S20 in the present embodiment includes:

[0086] The iridium (III) μ-chloro bridged dimer complex (0.263 g, 0.18 mmol) and 4,4'-bis (amino) -2,2'-bipyridine (0.40 mmol) were heated in a mixed solvent of methanol and chloroform under argon protection at 60 degrees Celsius for 12 hours, and after cooling to room temperature, it was transferred to a separatory funnel with deionized water, washed with n-hexane three times (30 ml each time), and the water layer was heated to 85 degrees Celsius to remove the residual n-hexane. 20 ml of an aqueous solution of potassium hexafluorophosphate (2 g) was added to the above mixture, the precipitate was filtered and dried to obtain a yellow solid of the iridium complex, named Ir4.

[0087] Performance test

[0088] 1. For examples 1-4, the metal iridium complexes Ir1-Ir4 as shown in Figure 2 The nuclear magnetic resonance hydrogen spectrum of the metal iridium complexes Ir1-Ir4, the solvent is deuterated DMSO, and the test results are shown in Figures 3-6 Figures 3-6 It can be seen that the chemical shift, integral value and peak shape of the metal iridium complexes Ir1-Ir4 support and confirm the molecular structure of the iridium complex.

[0089] 2. The ultraviolet absorption-emission spectrum of the metal iridium complexes Ir1-Ir4 is shown in Figure 7 1-2 are the ultraviolet-visible absorption spectra of Ir1-Ir4 in DMSO and water, respectively, and 3-6 are the emission spectra of Ir1, Ir2, Ir3 and Ir4 in different solvents, respectively.

[0090] ​The UV-Vis absorption spectrum test conditions are that the working concentration of iridium complexes Ir1-Ir4 is 5 μM, the absorption wavelength is 200-800 nm, and the solvent system is dimethyl sulfoxide and pure water. The UV-Vis absorption spectrum is measured by a Shimadzu UV-Vis spectrophotometer at 298 K.

[0091] The phosphorescence emission spectrum test conditions are that the working concentration of iridium complexes Ir1-Ir4 is 5 μM, the excitation wavelength is 405 nm, the emission wavelength is 420-750 nm, and the solvent system is toluene, tetrahydrofuran, chloroform, acetonitrile, dimethyl sulfoxide and pure water. All phosphorescence emission spectra are measured by a Shimadzu fluorescence spectrophotometer.

[0092] Experimental results: The UV-Vis absorption spectra of iridium complexes Ir1-Ir4 in dimethyl sulfoxide solution and pure water were tested at room temperature. The strong absorption peaks in the ultraviolet region (250-350 nm) are attributed to the transition of the ligand center state, which is usually a spin-allowed 1π-π* transition. The low-energy transition (350-450 nm) further extended to the visible region is generally attributed to the spin-allowed 1MLCT transition and the spin-forbidden 3MLCT transition.

[0093] Experimental results: The emission spectrum behavior of iridium complexes Ir1-Ir4 in different polar solvents was tested at room temperature. In different polar solvents, a clear emission peak at 470 nm can be observed, which helps to open the calcium ion channel of the cell. And with the increase of polarity, the maximum emission wavelength of iridium complexes Ir1-Ir4 has a significant red shift phenomenon.

[0094] As shown in Figure 8 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.

[0095] 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 an Edinburgh steady-state / transient fluorescence spectrometer, the light source used is a hydrogen lamp, the phosphorescence lifetime is measured by a pulsed laser, and the lifetime value is obtained by iterative calculation fitting of the luminescence lifetime decay curve by professional software provided by Edinburgh company.

[0096] Test results: The luminescence lifetime decay curves of iridium complexes Ir1-Ir4 in pure water were tested at room temperature. By software fitting, it was found that the iridium complexes Ir1-Ir4 all had long luminescence lifetime, and the order from small to large was Ir1 (256.59 ns) < Ir3 (323.57 ns) < Ir2 (453.97 ns) < Ir4 (556.95 ns).

[0097] 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. The two-photon fluorescence test was carried out in a fluorescence colorimetric quartz cell, and the sample was a 100 μM solution of Ir1-Ir4 (CH3OH, 298 K). The fluorescence excitation and detection conditions during 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, the two-photon absorption cross-section of iridium complexes was measured with Rhodamine B as a reference dye.

[0098] Test results: The two-photon absorption cross-section (δ) spectra of four 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 9 . All the complexes showed obvious two-photon absorption peak near 690 nm, among which Ir1 had the highest δ value of about 560 GM, and the peak values of Ir2, Ir3 and Ir4 decreased 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 δ value showed a trend of rapid decline first and then slow recovery, and the valley region was around 740 nm (δ about 100-150 GM), and then gradually increased in the range 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 two-photon absorption characteristics of typical 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 of the results shows that Ir1 has the best two-photon absorption performance, which is suitable as the core skeleton for the design of subsequent two-photon fluorescence probes or photosensitizers, and has good potential for structure optimization and application prospect.

[0099] As shown in Figure 10As shown, the test conditions were as follows: Human cervical cancer cells (HeLa) were used as the research subject. The culture medium consisted of 10% Gibco fetal bovine serum and 90% DMEM incomplete high-glucose medium (containing 80 μg / mL streptomycin and 80 U / mL penicillin). The incubator temperature was 37℃, humidity was 100%, and the atmosphere was air containing 5% CO2. Depending on the experimental conditions, cells could be grown adherently in 96-well plates or incubated in confocal culture dishes, with sample incubation performed after 24 hours. To assess the cytotoxicity of complexes Ir1-Ir4, the MTT assay was used. Living cells have a succinate dehydrogenase in their mitochondria that can reduce exogenous MTT to formazan, which is insoluble in water but soluble in DMSO. Dead cells cannot reduce MTT. Based on this principle, living and dead cells can be distinguished; therefore, the MTT assay is a common method for detecting cell viability. The specific procedure for the test is as follows: HeLa cell suspension was seeded into 96-well plates, 150 μL per well. To prevent experimental errors caused by solution evaporation, no cells were seeded in the outermost ring; instead, 150 μL of PBS buffer was injected into each well. The 96-well plates were then transferred to an incubator and incubated for 24 hours to allow the cells to adhere and grow. The cell density was then increased to [value missing]. 4 When the Ir1-Ir4 complex stock solution was homogeneous, it was diluted with DMEM to prepare solutions with concentrations of 0 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM. Then, 150 μL / well was injected into different columns of a 96-well plate, with six replicates for each concentration, and incubated for 24 hours. Next, fresh DMEM was added to each well, and 15 μL of MTT solution (5 mg / mL) prepared with PBS (pH = 7.4) was added, followed by 4 hours of incubation. Then, the supernatant was aspirated with a pipette, and 150 μL of DMSO was added to each well to dissolve the blue-purple formazan crystals in the live cells, followed by standing for 30 minutes. Finally, the absorbance (OD570) of each well at 570 nm was measured using a microplate reader; a higher formazan concentration resulted in more live cells, and vice versa. Cell viability (%) = (OD value of experimental group / OD value of control group) × 100%.

[0100] Test results: the cell survival rate of cervical cancer cells HeLa incubated with 0.2 μM, 5 μM, 10 μM iridium complexes Ir1-Ir4 for 24 hours is more than 80%, which indicates that the iridium complexes Ir1-Ir4 have good biocompatibility at low concentrations. The cell survival rate significantly decreases when the iridium complexes Ir2-Ir4 are incubated for 24 hours at a high concentration (more than 20 μM), which indicates that the iridium complexes Ir2-Ir4 have high cytotoxicity at high concentrations. The iridium complex Ir1 does not cause obvious toxic effects on cervical cancer cells HeLa at a concentration of 0-50 μM, and the cell survival rate is more than 95%, which indicates that the iridium complex Ir1 has excellent biocompatibility.

[0101] As shown in Figure 11 , the test conditions are as follows: the cell imaging effects of the iridium complexes Ir1-Ir4 are tested in 293T cells. The 293T cells with uniform density and in the logarithmic growth phase are inoculated in a confocal dish. When the cells in the dish grow well, the dish is taken out of the incubator. Then, the DMEM culture solution in the confocal dish is removed with a pipette, and the iridium complex solution (5 μM) prepared with the DMEM culture solution is added to the dish, which is then placed in the incubator for 2 hours. Then, the original solution in the confocal dish is discarded, and the excess iridium complex is removed by washing with PBS solution for three times. Then, 1 mL of DMEM culture solution is added again. The confocal dish is placed under a single / dual-photon confocal microscope for imaging. The single-photon imaging channel is set as an excitation wavelength of 405 nm and an emission wavelength of 450-510 nm, and the dual-photon imaging channel is set as an excitation wavelength of 700 nm and an emission wavelength of 450-510 nm.

[0102] Figure 11 The test results of the iridium complexes Ir1-Ir4 in 293T cell imaging evaluate the cell imaging effects of the iridium complexes Ir1-Ir4. After the iridium complexes are injected, strong phosphorescent signals in the cells can be observed under the single-photon 405 nm excitation light source. The phosphorescent intensity of the iridium complexes Ir1 and Ir2 is obviously stronger than that of the iridium complexes Ir3 and Ir4. Under the dual-photon 700 nm excitation light source, the iridium complex Ir1 exhibits the strongest phosphorescent intensity, which indicates that the iridium complex Ir1 has the potential to open the calcium ion channel under the excitation of the single / dual-photon light source.

[0103] As shown in Figure 12 , the test conditions are as follows: the 293T cells are transfected with ChR2-Venus. The blank group is not added with the iridium complex Ir-1, and the experimental group is selected from the iridium complex Ir-1 (5 μM). After 2 hours of incubation, the calcium ion indicator Rhod-3 AM (1 μM) is added, and the incubation is continued for 40 minutes. The cells are irradiated with a dual-photon light source for five minutes. After the calcium ion channel is opened and the calcium ions flow in, the cells are imaged. The dual-photon excitation light source has a wavelength of 700 nm and a power range of 0-0.8 W / cm 2The imaging channel is a Rhod-3 AM red channel, the excitation wavelength is 561 nm, and the emission wavelength range is 580-620 nm.

[0104] Test results: the ability of iridium complex Ir-1 to open the calcium ion channel under the irradiation of a two-photon light source was evaluated, A figure (left) no red fluorescence signal was detected in the blank group, and A figure (right) a significant red fluorescence signal was detected in the experimental group, indicating that 293T cells emit phosphorescence of about 470 nm under the excitation of a two-photon light source, thereby opening the calcium ion channel and inducing calcium ion influx; B figure shows the fluorescence intensity change of Rhod-3 AM in 293T cells under different two-photon excitation powers (0-0.8 W / cm 2 ) The red fluorescence intensity in 293T cells continuously increased with the increase of two-photon excitation power, which was due to the increase of phosphorescence emission intensity of iridium complex Ir-1 in 293T cells with the increase of two-photon excitation power, thereby causing more calcium ion influx, making the fluorescence intensity of calcium ion indicator Rhod-3 AM stronger, indicating that the concentration of calcium ion influx in 293T cells has two-photon light power dependence, further indicating that the synthesized iridium complex Ir1 has great potential to become a two-photon photo-ge netic imaging probe.

[0105] The above merely describes exemplary embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation made according to the technical concept of the present application, or direct / indirect application in other related technical fields is included in the patent protection scope of the present application.

Claims

1. An iridium complex characterized in that, The iridium complex has the following structural formula: 。 2. A process for the preparation of an iridium complex as claimed in claim 1, characterized in that, The method comprises 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, wherein the iridium compound is hydrated iridium trichloride; S20, performing a coordination substitution reaction on the iridium dimer precursor and a 2,2'-bipyridine compound in a second solvent, and washing and purifying to obtain the iridium complex.

3. The process for the preparation of an iridium complex according to claim 2, characterized in that, In the step S10, the mass ratio of the iridium compound to the 2-(2,4-difluorophenyl)-5-(trifluoromethyl)pyridine is 1:(1-3).

4. The method for preparing the iridium complex according to claim 2, wherein the first solvent comprises a mixed solvent of water and 2-methoxyethanol; and the second solvent comprises a mixed solvent of methanol and chloroform.

4. The method for preparing the iridium complex according to claim 2, wherein the first solvent comprises a mixed solvent of water and 2-methoxyethanol; and the second solvent comprises a mixed solvent of methanol and chloroform. In the step S20, the 2,2'-bipyridine compound is selected from 4,4'-bis(n,n-dimethyl)-2,2'-bipyridine or 4,4'-bis(amino)-2,2'-bipyridine.

5. The process for the preparation of iridium complexes as claimed in claim 2, wherein, The step S20 comprises:

6. The method for preparing the iridium complex as described in claim 2, characterized in that, 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 the iridium complex. The fluorescent probe comprises the iridium complex according to claim 1 or the iridium complex prepared by the method according to any one of claims 2-6.

7. A fluorescent probe, characterized in that, 8. The fluorescent probe according to claim 7 in the preparation of an optogenetic imaging probe.

9. The fluorescent probe according to claim 7 in the preparation of a cell biology research probe. ​

Images