Phosphorescent turn-on bioorthogonal iridium complex probe and preparation and application thereof
By introducing diketone units into phosphorescent iridium complexes, phosphorescent-activated bioorthogonal iridium complex probes were synthesized, solving the problem of severe background signal interference in biolabeling and imaging in existing technologies. This enabled rapid and selective biomolecular labeling and recognition, improving cell imaging performance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING UNIV OF POSTS & TELECOMM
- Filing Date
- 2023-10-26
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies lack efficient phosphorescent-activated orthogonal bioprobes, making it difficult to rapidly and without interference label and identify biomolecules in biological systems, especially in cell imaging where background signal interference is severe.
A phosphorescent-activated bioorthogonal iridium complex probe was designed and synthesized. By conjugating the diketone unit to the C^N ligand of the phosphorescent metal iridium complex, it reacts with the bioorthogonal unit angelicolone, resulting in luminescence from nothing to something, thus achieving efficient labeling and recognition of biomolecules.
This probe significantly reduces background signal interference in biolabeling and imaging, can react rapidly and selectively with biomolecules to enable phosphorescence, and improves the accuracy and efficiency of cell imaging.
Smart Images
Figure CN117447525B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic optoelectronic materials technology. Specifically, it relates to a class of phosphorescently activated bioorthogonal iridium complex probes, their preparation method, and their applications in the fields of biolabeling and imaging. Background Technology
[0002] Bioorthogonal reactions are a class of chemical reactions that can occur in biological systems without interfering with natural biochemical processes. The existence of bioorthogonal reactions has played a crucial role in advancing human life science research, demonstrating broad application prospects in fields such as bioimaging, medical research, and clinical diagnosis. In a bioorthogonal chemical reaction, the two molecules introduced do not affect their respective metabolism in the body, nor do they bind to other molecules. However, it requires that the two molecules introduced into the organism have a sufficiently fast reaction rate so that the molecule to be labeled can be labeled before it is metabolized.
[0003] The azido-alkyne cycloaddition reaction catalyzed by monovalent copper ions (CuAAC) is one of the earliest developed and most widely used bioorthogonal reactions. This reaction exhibits high efficiency and relatively rapid kinetics, and has been extensively applied in the field of bioorthogonal reactions. Furan-2(3H)-ketone derivatives can undergo rapid bioorthogonal reactions with diketones under catalyst-free conditions, exhibiting high reaction rates, with second-order rates reaching as high as 119 M at constant speed. -1 S -1 It can be used in conjunction with ring strain-induced azido-alkynyl cycloaddition reactions and bioorthogonal reactions based on the Diels-Alder (IEDDA) mechanism of reverse electron demand.
[0004] Phosphorescent turn-on probes are probes whose luminescence is quenched before a bioorthogonal reaction occurs, but whose luminescence is turned on after a specific reaction with a bioorthogonal unit. Because of the significant difference in luminescence signal before and after the reaction, using these probes to label and identify biomolecules during cell imaging effectively reduces background signal interference, resulting in significant effects.
[0005] Phosphorescent iridium complexes, as excellent phosphorescent transition metal complexes, possess superior photophysical properties such as high quantum efficiency, tunable emission wavelength, and large Stokes shift, showing promising applications in cancer cell imaging and therapy. They can also serve as a matrix for biological orthogonal probes.
[0006] Based on the above, it is inferred that if diketone units can be successfully introduced into phosphorescent iridium complexes, the bioorthogonal reaction between the diketone units and bioorthogonal units such as furan-2(3H)-ketone derivatives could enable the iridium complexes to emit light, potentially achieving efficient dynamic tracking of target molecules within living cells. The design and synthesis of such complexes could also provide new ideas for developing novel phosphorescently activated bioorthogonal probes, potentially enabling dynamic visualization and tracking of a range of biomolecules and organelles under physiological conditions. Summary of the Invention
[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a class of phosphorescently activated bioorthogonal iridium complex probes, their preparation, and applications. By directly conjugating a diketone unit with electron-withdrawing capability and capable of bioorthogonal reactions to a C^N ligand of a phosphorescent metallic iridium complex rich in excited-state properties, the complex reacts with the bioorthogonal unit—angelicolone—leading to a transition in luminescence from absent to present. This facilitates the efficient identification and labeling of biomolecules in cell imaging.
[0008] The technical solution of this invention is: a class of phosphorescently activated bioorthogonal iridium complex probes, with the following general structural formula:
[0009] ;
[0010] Wherein, R is any one of -H, -CH3, phenyl, or -CH=CH2;
[0011] The N^N ligand can be any one of the following structures:
[0012]
[0013] The preparation route of the above-mentioned phosphorescently activated biological orthogonal iridium complex probe is as follows:
[0014] .
[0015] The synthesis steps for this type of iridium complex are as follows:
[0016] 1) The benzo[h]quinoline derivative and I2O5 were dissolved in acetic acid solution, stirred and refluxed, and deionized water was added to precipitate the product. After post-treatment, benzo[h]quinoline-5,6-dione was obtained.
[0017] 2) Weigh out benzo[h]quinoline-5,6-dione and iridium trichloride trihydrate, add them to an aqueous solution of ethylene glycol diethyl ether under inert gas protection, and stir and reflux under nitrogen protection to obtain an iridium chloride bridged dimer;
[0018] 3) The iridium chloride-bridged dimer was mixed with the N^N ligand and reacted with dichloromethane and methanol as a mixed solvent under heating. Potassium hexafluorophosphate was added at room temperature for ion exchange, and the phosphorescent iridium complex was obtained after purification.
[0019] Further, in step 1), the molar ratio of benzo[h]quinoline derivative to I2O5 is 1:1.2~1.25.
[0020] Furthermore, in step 1), the reflux reaction temperature is 110-120℃ and the reaction time is 3-4 h.
[0021] Furthermore, in step 2), the molar ratio of benzo[h]quinoline-5,6-dione and iridium trichloride trihydrate is 2~2.5:1.
[0022] Furthermore, in step 2), the reflux reaction is carried out at room temperature for 20-24 hours.
[0023] Furthermore, in step 3), the volume ratio of dichloromethane to methanol is 1~3:1.
[0024] Furthermore, in step 3), the heating reaction temperature is 40–45°C and the time is 6–18 h.
[0025] The aforementioned phosphorescently activated bioorthogonal iridium complex probes can be used in biolabeling and imaging. When these iridium complexes react with the bioorthogonal unit angelicin, they exhibit phosphorescent activation.
[0026] The aforementioned phosphorescently activated bioorthogonal iridium complex probes can be used in live-cell imaging to identify and label biomolecules such as proteins, nucleic acids, and polysaccharides in organisms.
[0027] The beneficial effects of this invention are:
[0028] 1. The bioorthogonal iridium complex probe disclosed in this application uses a benzo[h]quinoline-5,6-dione derivative as a C^N ligand. The benzo[h]quinoline-5,6-dione derivative undergoes a coordination reaction with iridium trichloride trihydrate to obtain a cyclometalated iridium chloride-bridged dimer, which is then reacted with an N^N ligand to obtain this type of iridium complex probe. This type of probe can change the excited state energy level of the complex and affect its photophysical properties after reacting with the bioorthogonal unit angelica lactone, thus achieving the effect of phosphorescence activation from nothing to something. Therefore, this type of probe belongs to a true phosphorescence-activating bioorthogonal probe.
[0029] 2. Since the present application discloses a phosphorescently activated bioorthogonal probe, it will show a significant luminescence signal ratio before and after reacting with the bioorthogonal unit - angelica lactone. This feature can minimize background signal interference and facilitate cell imaging and biomolecule recognition.
[0030] 3. The phosphorescent-activated bioorthogonal iridium complex probe disclosed in this application has a simple synthesis approach and high synthesis efficiency. Moreover, this type of bioorthogonal reaction has the advantages of fast reaction rate and high reaction selectivity, which can provide new ideas for the development of novel phosphorescent-activated bioorthogonal materials and has good application prospects in the field of biolabeling and imaging. Attached Figure Description
[0031] Figure 1 Mass spectrometry characterization of the iridium complex prepared in Example 1 before and after the addition of angelicin;
[0032] Figure 2 Mass spectrometry characterization of the iridium complex prepared in Example 2 before and after the addition of angelicin;
[0033] Figure 3 The absorption spectra of the iridium complexes prepared in Examples 1 and 2 in PBS / DMSO mixed solution are shown.
[0034] Figure 4 Cell imaging of the iridium complex prepared in Example 1;
[0035] Figure 5 The emission spectra are shown in PBS / DMSO mixed solution before and after the addition of angelica lactone to the iridium complexes prepared in Examples 1 and 2. Detailed Implementation
[0036] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the essence of the invention are within the scope of the present invention.
[0037] Example 1: Preparation of an iridium complex probe
[0038] 1. Synthesis of cyclometalated iridium chloride bridged dimers
[0039]
[0040] 1) Benzo[h]quinoline (1.5 g, 8.38 mmol) and I₂O₅ (3.49 g, 10.46 mmol) were added to glacial acetic acid (50 mL). The mixture was heated under reflux at 118 °C for 4 h, and deionized water (75 mL) was added to precipitate the product. The solution was left to stand overnight. After filtration, the product was dissolved in chloroform to obtain a dark red solution. The solution was washed with saturated NaHCO₃ (100 mL) and saturated Na₂S₂O₃ (100 mL). The organic layer was dried over anhydrous saturated Na₂SO₄, and the solvent was removed by rotary evaporation. The resulting dark brown solid was benzo[h]quinoline-5,6-dione.
[0041] 2) Benzo[h]quinoline-5,6-dione (1.04 g, 4.99 mmol) and iridium trichloride trihydrate (800 mg, 2.27 mmol) were added to a two-necked flask. A magnetic stir bar and a condenser were added, and the mixture was evacuated and purged with nitrogen. The mixture was circulated three times. Under nitrogen protection, a mixed solvent of ethylene glycol ethyl ether (45 mL) and deionized water (15 mL) was added, and the mixture was refluxed under nitrogen protection for 24 h to obtain a cyclic metallized iridium chloride bridged dimer.
[0042] 2. Synthesis of Iridium Complex Probes
[0043]
[0044] 1,10-phenanthroline (110 mg, 0.61 mmol) and the cyclometalated iridium chloride bridged dimer prepared in step 1 (358 mg, 0.28 mmol) were added to a mixed solvent of dichloromethane (30 mL) and methanol (15 mL) and reacted at 45 °C for 6 hours. After cooling to room temperature, excess potassium hexafluorophosphate (515 mg, 2.8 mmol) was added and stirred at room temperature for 4 hours. After vacuum distillation, excess potassium hexafluorophosphate was extracted to remove the excess potassium hexafluorophosphate and the phosphorescent iridium complex was obtained.
[0045] 1 H NMR (400 MHz, DMSO-d6) δ = 8.94 (d, J = 8.4 Hz, 2H), 8.42 (s, 2H), 8.35 (d, J = 5.2 Hz, 2H), 8.28 (d, J = 7.6 Hz, 2H), 8.08 (dd, J = 5.2, 8.4Hz, 2H), 7.70 (dd, J = 6.0, 14.8 Hz, 4H), 7.25 – 7.14 (m, 4H), 6.84 (d, J =6.8 Hz, 2H).
[0046] Example 2: Preparation of an iridium complex probe
[0047] The synthesis route is as follows:
[0048]
[0049] 2,2-Bipyridine (95 mg, 0.61 mmol) and the cyclometalated iridium chloride bridged dimer prepared in Example 1 (358 mg, 0.28 mmol) were added to a mixed solvent of dichloromethane (30 mL) and methanol (15 mL). The mixture was reacted at 45 °C for 6 hours. After cooling to room temperature, excess potassium hexafluorophosphate (515 mg, 2.8 mmol) was added. The mixture was stirred at room temperature for 4 hours. After vacuum distillation, excess potassium hexafluorophosphate was extracted to remove the excess potassium hexafluorophosphate, and the phosphorescent iridium complex was obtained.
[0050] 1 H NMR (400 MHz, DMSO-d6) δ=8.91 (d, J = 8.0 Hz, 2H), 8.39 – 8.27 (m,4H), 8.02 (d, J = 4.0 Hz, 2H), 7.87 (d, J = 4.0 Hz, 2H), 7.76 – 7.67 (m, 2H),7.64 (d, J = 4.0 Hz, 2H).
[0051] Related performance tests
[0052] Test Example 1: Mass spectrometry characterization of the iridium complex prepared in Example 1 before and after the addition of angelica lactone
[0053] Prepare an iridium complex at a concentration of 10 μM and an angelicin complex at a concentration of 60 μM, and add them together to a mixed solution of PBS / DMSO (99:1, v / v) and react for 30 minutes.
[0054] The results are as follows Figure 1 As shown in the figure, mass spectrometry characterization revealed that the molecular weight of the cycloaddition product formed after the reaction of the iridium complex with angelica lactone was 986.8, which is basically consistent with the expected molecular weight of the product. This indicates that the iridium complex can undergo a highly efficient bioorthogonal reaction with angelica lactone, exhibiting high selectivity and reaction rate.
[0055] Test Example 2: Mass spectrometry characterization of the iridium complex prepared in Example 2 before and after the addition of angelica lactone
[0056] Prepare an iridium complex at a concentration of 10 μM and an angelicin complex at a concentration of 60 μM, and add them together to a mixed solution of PBS / DMSO (99:1, v / v) and react for 30 minutes.
[0057] The results are as follows Figure 2 As shown in the figure, mass spectrometry characterization revealed that the molecular weight of the cycloaddition product formed after the reaction of the iridium complex with angelica lactone was 961.7, which is basically consistent with the expected molecular weight of the product. This indicates that the iridium complex can undergo a highly efficient bioorthogonal reaction with angelica lactone, exhibiting high selectivity and reaction rate.
[0058] Test Example 3: Absorption Spectroscopy Test of Iridium Complexes Prepared in Examples 1 and 2
[0059] Two iridium complex solutions, each with a test concentration of 10 μM, were prepared in cuvettes. The iridium complexes were dissolved in a mixed solution of PBS / DMSO (99:1, v / v).
[0060] The results are as follows Figure 3 As shown, the complex has a strong absorption peak at wavelengths less than 300 nm, while the absorption peak at visible light wavelengths is significantly lower. This phenomenon provides a theoretical basis for the imaging and biomolecular labeling of iridium complexes in cells.
[0061] Test Example 4: Cell Imaging Experiment of Iridium Complex Prepared in Example 1
[0062] 10 μM iridium complex was incubated in HeLa cells for 3 h, and cell imaging was performed under a confocal microscope, such as... Figure 4 As shown, before the reaction, only the cells containing the iridium complex exhibited luminescence quenching. After reacting 10 μM iridium complex with 250 μM angelica lactone in serum-free culture medium for 1 h, the mixture was added to the cells and incubated for 2 h. Cell imaging under a confocal microscope showed that the cells after the reaction exhibited luminescence activation.
[0063] Test Example 5: Emission spectra of iridium complexes prepared in Examples 1 and 2 before and after the addition of angelica lactone
[0064] Two iridium complex solutions, each with a concentration of 10 μM, were prepared in cuvettes. A separate solution of angelica lactone with a concentration of 20 μM was also prepared. Both solutions were dissolved in a PBS / DMSO (99:1, v / v) mixture. The mixtures were then excited with light at a wavelength of 365 nm.
[0065] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that before the addition of angelicin, neither of the two iridium complexes showed any luminescence signal. After undergoing a bioorthogonal reaction with angelicin, both complexes achieved phosphorescence activation, and the luminescence intensity gradually increased with the increase of the test time until the reaction ended, at which point the luminescence intensity reached its strongest point and tended to stabilize.
[0066] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. However, the above description is merely a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments derived by those skilled in the art without departing from the technical solution of the present invention should be covered within the scope of the present invention.
Claims
1. A class of phosphorescently activated bioorthogonal iridium complex probes, characterized in that, The general structural formula is as follows: ； Wherein, R is any one of -H, -CH3, phenyl, or -CH=CH2; The N^N ligand can be any one of the following structures: 。 2. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 1, characterized in that, The preparation route is as follows: 。 3. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 2, characterized in that, The synthesis steps are as follows: 1) The benzo[h]quinoline derivative and I2O5 were dissolved in acetic acid solution, heated and stirred under reflux at 110~120℃, deionized water was added to precipitate the product, and the product was obtained by post-treatment. 2) Weigh out benzo[h]quinoline-5,6-dione derivative and iridium trichloride trihydrate, add them to an aqueous solution of ethylene glycol diethyl ether, and reflux under inert gas protection to obtain iridium chloride bridged dimer; 3) The iridium chloride bridged dimer was mixed with the N^N ligand and reacted with dichloromethane and methanol as a mixed solvent under heating. Potassium hexafluorophosphate was added at room temperature for ion exchange, and the resulting phosphorescent-activated bioorthogonal iridium complex probe was purified.
4. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 1), the molar ratio of benzo[h]quinoline derivative to I2O5 is 1:1.2~1.
25.
5. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 1), the reflux reaction time is 3-4 hours.
6. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 2), the molar ratio of the benzo[h]quinoline-5,6-dione derivative and iridium trichloride trihydrate is 2~2.5:
1.
7. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 2), the reflux reaction time is 20-24 h.
8. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 3), the volume ratio of dichloromethane to methanol is 1~3:
1.
9. The method for preparing the phosphorescently activated bioorthogonal iridium complex probe as described in claim 3, characterized in that, In step 3), the heating reaction temperature is 40~45℃ and the time is 6-18 h.