Biotin orthogonal iridium complex probes containing diketone units, preparation and use thereof
By conjugating diketone units with phosphorescent iridium complexes, the prepared bioorthogonal iridium complex probe exhibits enhanced luminescence intensity after reacting with bioorthogonal units. This solves the problems of imaging accuracy and water washing process in existing technologies, enabling highly sensitive dynamic tracing and imaging within living cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANJING UNIV OF POSTS & TELECOMM
- Filing Date
- 2023-09-28
- Publication Date
- 2026-04-28
AI Technical Summary
In existing biological orthogonal labeling processes, excessive luminescent probes cannot be completely removed, leading to reduced imaging accuracy and cell damage. Furthermore, a rigorous water washing process is required, which affects the temporal resolution and signal-to-noise ratio of the imaging.
A bioorthogonal iridium complex probe containing a diketone unit was designed and conjugated with an N^N ligand of a phosphorescent metal iridium complex. By reacting with the bioorthogonal unit angelicolone, the luminescence intensity of the complex was enhanced, enabling live cell imaging without water washing.
It enables dynamic tracking of target molecules within living cells, improves the signal-to-noise ratio and sensitivity of imaging, reduces background signal interference, simplifies the imaging process, and is applicable to the fields of biomarking and imaging.
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Figure CN117327127B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of organic optoelectronic materials technology. Specifically, it relates to a class of phosphorescence intensity-responsive bioorthogonal iridium complex probes containing diketone units, their preparation method, and their applications in the fields of biolabeling and imaging. Background Technology
[0002] Bioorthogonal reactions are chemical reactions that do not affect biomolecules or interfere with biochemical processes and can be carried out in living systems. They have advantages such as fast reaction rate, high yield, good specificity, and mild reaction conditions, and are widely used in fields such as biolabeling and imaging, drug delivery, and material preparation.
[0003] Furan-2(3H)-one derivatives can undergo rapid bioorthogonal reactions with diketones without a catalyst, exhibiting high reaction rates, with second-order rates reaching as high as 119 M at room temperature. -1 S -1 It can be used in conjunction with ring strain-induced azido-alkynyl cycloaddition (SPAAC) and bioorthogonal reactions based on the Diels-Alder reaction (IEDDA) mechanism.
[0004] Bioorthogonal labeling typically involves introducing functionalized bioorthogonal units into living cells and organisms, followed by recognition using bioorthogonal optical probes with complementary units. To improve labeling efficiency, an excess of luminescent probe is usually added beforehand, followed by rigorous washing to remove unreacted probes and reduce background interference. However, when the probe is inside the cell or organism, it is impossible to guarantee the complete removal of the excess probe, which severely reduces imaging accuracy and leads to problems such as decreased temporal resolution and cell damage.
[0005] Turn-on bioorthogonal probes are quenched before bioorthogonal reactions, and turn on after a specific reaction with a bioorthogonal unit. Therefore, imaging analysis using such probes does not require a strict washing process, which can improve the signal-to-noise ratio of the imaging. They can be used to monitor dynamic changes in real time and are favored by researchers in the fields of labeling and tracing biomolecules such as proteins, lipids, nucleic acids, and glycans, as well as organelle imaging.
[0006] Phosphorescent iridium complexes are widely used in bioimaging due to their advantages such as high quantum yield, good photostability, easily tunable emission wavelength, readily modifiable ligands, and rich excited-state properties. They can serve as the matrix for bioorthogonal probes. If diketone units can be successfully introduced into phosphorescent iridium complexes, and the property of diketone units to undergo bioorthogonal reactions with bioorthogonal units such as furan-2(3H)-ketone derivatives can be used to excite changes in the luminescence intensity of the phosphorescent iridium complexes, it is hoped that waterless imaging can be achieved, thereby enabling dynamic tracking of target molecules within living cells. The design and synthesis of such complexes can also provide new ideas for the development of novel 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 bioorthogonal iridium complex probes containing diketone units, their preparation, and applications. This is achieved by directly conjugating diketone units, which have electron-withdrawing capabilities and can undergo bioorthogonal reactions, to the N-phase of phosphorescent metal iridium complexes rich in excited-state properties. ^ On the N-ligand, the complex reacts with the bioorthogonal unit angelicolone, resulting in a change in the complex with enhanced luminescence intensity. This allows for dynamic tracking of target molecules within living cells without the need for water washing imaging.
[0008] The technical solution of this invention is: a type of bioorthogonal iridium complex probe containing a diketone unit, with the following general structural formula:
[0009]
[0010] The C^N ligand is any one of the following:
[0011]
[0012] The preparation route for the above-mentioned bioorthogonal iridium complex probes containing diketone units is as follows:
[0013]
[0014] The synthesis steps for this type of iridium complex are as follows:
[0015] 1)C ^ The N-ligand was mixed with iridium trichloride trihydrate, and a mixed solvent of ethylene glycol ethyl ether and deionized water was added under inert gas protection. The mixture was stirred and refluxed to obtain a cyclometalated iridium chloride bridged dimer.
[0016] 2) 1,10-phenanthroline-5,6-dione was mixed with cyclometalated iridium chloride-bridged dimer, and stirred under reflux with dichloromethane and methanol as mixed solvents under inert gas protection. After the reaction was completed and cooled, potassium hexafluorophosphate was added for ion exchange to obtain an iridium complex containing a dione unit.
[0017] Furthermore, in step 1), C ^ The molar ratio of N-ligand to iridium trichloride trihydrate is 2.0–2.5:1.
[0018] Further, in step 2), the molar ratio of 1,10-phenanthroline-5,6-dione to the cyclometalated iridium chloride bridged dimer is 1.5 to 2.0:1.
[0019] Furthermore, in step 2), the volume ratio of dichloromethane to methanol is 1 to 3:1.
[0020] Furthermore, in step 2), the stirring reflux temperature is 40-45℃ and the time is 6-18h.
[0021] The above-mentioned bioorthogonal iridium complex probes containing diketone units can be used in biolabeling and imaging. After the iridium complex reacts with the bioorthogonal unit angelicolone, the luminescence intensity of the complex is enhanced. This type of probe is actually a phosphorescence intensity-responsive probe, which can achieve biolabeling and imaging without water washing.
[0022] The above-mentioned bioorthogonal iridium complex probes containing diketone units can be used in live-cell imaging.
[0023] The beneficial effects of this invention are:
[0024] 1. The bioorthogonal iridium complex probe disclosed in this application is constructed by directly connecting a diketone unit in a conjugated manner to an organic ligand of an iridium phosphorus group. After reacting with the bioorthogonal unit-angelicinolone, it can change the excited state energy level of the complex and affect its photophysical properties, thereby achieving a transformation that enhances the luminescence intensity of the complex. It can achieve dynamic tracking of target biomolecules such as proteins, lipids and polysaccharides in living cells without the need for water washing imaging.
[0025] 2. The iridium complex probe containing a diketone unit disclosed in this application is a phosphorescence intensity-responsive probe. When this type of complex is used in the process of biological orthogonal labeling, it can minimize background signal interference, eliminate the need for water washing imaging, and achieve high-sensitivity biological labeling and imaging.
[0026] 3. The bioorthogonal iridium complex probe with diketone units 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, high reaction selectivity and good biocompatibility. It can provide new ideas for the development of novel luminescence intensity-responsive bioorthogonal materials and has good application prospects in the field of biolabeling and imaging. Attached Figure Description
[0027] Figure 1 MALDI-TOF MS images of iridium complex 1 prepared in Example 1 before and after the addition of angelicin;
[0028] Figure 2 MALDI-TOF MS images of iridium complex 2 prepared in Example 2 before and after the addition of angelicin;
[0029] Figure 3 The emission spectra of iridium complex 1 prepared in Example 1 before and after the addition of angelicin are shown.
[0030] Figure 4 The emission spectra of iridium complex 2 prepared in Example 2 before and after the addition of angelica lactone are shown.
[0031] Figure 5 Cell imaging images before and after the addition of angelica lactone to iridium complex 1 prepared in Example 1. Detailed Implementation
[0032] 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.
[0033] Example 1: Preparation of an iridium complex probe containing a diketone unit (iridium complex 1)
[0034] 1. Synthesis of cyclometalated iridium chloride bridged dimers
[0035]
[0036] Weigh 3.12 mmol of 7,8-benzoquinoline and 1.42 mmol of iridium trichloride trihydrate into a two-necked flask, add a magnetic stir bar and a condenser, and seal. Evacuate the flask, purge with nitrogen, and circulate three times. Under nitrogen protection, add 12 mL of ethylene glycol ethyl ether and 4 mL of deionized water, and stir under reflux at 110 °C for 24 h. After the reaction is complete, cool to room temperature, filter, wash three times with deionized water and ethanol, and dry in a vacuum drying oven to obtain the cyclic metallized iridium chloride bridged dimer.
[0037] 2. Synthesis of a bioorthogonal iridium complex probe containing a diketone unit (iridium complex 1)
[0038]
[0039] Weigh 0.27 mmol of cyclometalated iridium chloride-bridged dimer and 0.48 mmol of 1,10-phenanthroline-5,6-dione into a two-necked flask, add a magnetic stir bar and a condenser, and seal. Evacuate the mixture, purge with nitrogen, and circulate three times. Under nitrogen protection, add 40 mL of dichloromethane and 20 mL of methanol, respectively, and stir under reflux at 45 °C for 12 h. After the reaction is complete, cool to room temperature, add potassium hexafluorophosphate (KPF6, 0.81 mmol) to the reaction solution, stir at room temperature for 2 h, evaporate to dryness, extract with dichloromethane, and purify the organic phase by silica gel column chromatography to obtain a light yellow powder complex, i.e., iridium complex 1, with a yield of 25%.
[0040] 1 H NMR (400MHz, DMSO-d6) δ8.65(dd,J=7.6,19.6Hz,4H),8.11(d,J=4.8Hz,2H),8.01(d,J=8.8Hz,4H),7.92(d,J=8.8Hz,2H) ,7.80(t,J=6.8Hz,2H),7.66(dd,J=5.6,8.0Hz,2H),7.59(d,J=7.6Hz,2H),7.21(t,J=7.6Hz,2H),6.25(d,J=7.2Hz,2H).
[0041] Example 2: Preparation of an iridium complex probe containing a diketone unit (iridium complex 2)
[0042] 1. Synthesis of cyclometalated iridium chloride bridged dimers
[0043]
[0044] Weigh 3.12 mmol of 1-phenylpyrazole and 1.42 mmol of iridium trichloride trihydrate into a two-necked flask, add a magnetic stir bar and a condenser, and seal. Evacuate the flask, purge with nitrogen, and circulate three times. Under nitrogen protection, add 12 mL of ethylene glycol ethyl ether and 4 mL of deionized water, and stir under reflux at 110 °C for 24 h. After the reaction is complete, cool to room temperature, filter, wash three times with deionized water and ethanol, and dry in a vacuum drying oven to obtain the cyclic metallized iridium chloride bridged dimer.
[0045] 2. Synthesis of a bioorthogonal iridium complex probe containing a diketone unit (iridium complex 2)
[0046] Weigh 0.27 mmol of cyclometalated iridium chloride-bridged dimer and 0.48 mmol of 1,10-phenanthroline-5,6-dione into a double-necked flask, add a magnetic stir bar and a condenser, and seal. Evacuate the mixture, purge with nitrogen, and circulate three times. Under nitrogen protection, add 40 mL of dichloromethane and 20 mL of methanol, respectively, and stir under reflux at 45 °C for 12 h. After the reaction is complete, cool to room temperature, add potassium hexafluorophosphate (KPF6, 0.81 mmol) to the reaction solution, stir at room temperature for 2 h, evaporate to dryness, extract with dichloromethane, and purify the organic phase by silica gel column chromatography to obtain a yellowish-brown powder complex, i.e., iridium complex 2, with a yield of 48%.
[0047] 1 H NMR (400MHz, DMSO-d6) δ8.92(d,J=2.8Hz,2H),8.69(d,J=8.8Hz,2H),8.19(d,J=6.4Hz,2H),7.89(dd,J=5.6,8.0Hz,2H),7.72(d ,J=7.6Hz,2H),7.20(d,J=2.0Hz,2H),7.09(t,J=6.8Hz,2H),6.89(t,J=7.6Hz,2H),6.75(t,J=2.8Hz,2H),6.23(d,J=5.6Hz,2H).
[0048] Related performance tests
[0049] Test Example 1: MALDI-TOF MS tests of iridium complex 1 and iridium complex 2 before and after the addition of angelica lactone.
[0050] The concentrations of iridium complexes 1 and 2 were both 10 μM, the concentration of angelica lactone was 30 μM, the solvent was PBS / DMSO (99:1, v / v), and the reaction time was 30 min.
[0051] The results are as follows Figure 1 and 2 As shown, the molecular weights of the cycloaddition products formed after the reaction of iridium complex 1 and iridium complex 2 with angelica lactone are 856.9 and 787.6, respectively, which are consistent with the expected molecular weights of the cycloaddition products. This indicates that these complexes can undergo bioorthogonal reactions with angelica lactone and exhibit high selectivity and reaction rates.
[0052] Test Example 2: Emission spectra of iridium complex 1 and iridium complex 2 before and after the addition of angelica lactone.
[0053] The concentrations of iridium complexes 1 and 2 were both 10 μM, the concentration of angelica lactone was 10 μM, the test solvent was PBS / DMSO (99:1, v / v), and the excitation wavelength was 365 nm.
[0054] The results are as follows Figure 3 and 4 As shown, the phosphorescence intensity of the complex increases. Before the addition of angelica lactone, the initial luminescence of the complex molecule is weak due to the electron-withdrawing effect of the bioorthogonal unit. After reacting with the bioorthogonal unit angelica lactone, the photophysical properties of the complex are altered, leading to an increase in luminescence intensity. Therefore, waterless biolabeling and imaging can be achieved by monitoring changes in the luminescence intensity of the complex, providing a possibility for real-time monitoring of biological processes.
[0055] Test Example 3: Cell imaging experiment before and after adding angelica lactone to iridium complex 1.
[0056] HeLa cells were seeded in confocal dishes and incubated for 24 h. Iridium complex 1 (10 μM) was added to the confocal dishes and incubated for 3 h. Then, 100 μM angelica lactone was added directly and incubated for 30 min before laser scanning confocal imaging.
[0057] The results are as follows Figure 5 As shown, when only iridium complex 1 is incubated with cells, weak luminescence is observed in the cell imaging. After adding angelica lactone without water washing, the intensity of luminescence in the cells is observed to increase, indicating that iridium complex 1 and angelica lactone can undergo a bioorthogonal reaction in cells.
[0058] 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 patent scope of the present invention.
Claims
1. A class of bioorthogonal iridium complex probes containing diketone units, characterized in that, The general structural formula of this type of iridium complex is as follows: ； Wherein, the C^N ligand is any one of the following: 。 2. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 1, characterized in that, The synthetic route for iridium complexes is as follows: 。 3. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 2, characterized in that, The synthesis steps of iridium complexes are as follows: 1) The C^N ligand was mixed with iridium trichloride trihydrate, and a mixed solvent of ethylene glycol ethyl ether and deionized water was added under inert gas protection. The mixture was stirred and refluxed to obtain a cyclometalated iridium chloride bridged dimer. 2) 1,10-phenanthroline-5,6-dione was mixed with cyclometalated iridium chloride-bridged dimer, and stirred under reflux with dichloromethane and methanol as mixed solvents under inert gas protection. After the reaction was completed and cooled, potassium hexafluorophosphate was added for ion exchange to obtain an iridium complex containing a dione unit.
4. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 3, characterized in that, In step 1), the molar ratio of C^N ligand to iridium trichloride trihydrate is 2.0~2.5:
1.
5. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 3, characterized in that, In step 2), the molar ratio of 1,10-phenanthroline-5,6-dione to the cyclic metallized iridium chloride bridged dimer is 1.5~2.0:
1.
6. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 3, characterized in that, In step 2), the volume ratio of dichloromethane to methanol is 1~3:
1.
7. The method for preparing a type of bioorthogonal iridium complex probe containing a diketone unit as described in claim 3, characterized in that, In step 2), the stirring reflux temperature is 40~45℃ and the time is 6-18 h.