An iridium metal complex containing imidazole group, its preparation method and application
Patent Information
- Application Number
- CN202611313091.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-27
- Publication Date
- 2026-09-25
AI Technical Summary
[0007]为了克服上述现有技术中存在的无法实现有效的选择性识别，容易产生假阳性信号的缺点和不足，本发明的首要目的在于提供一种含咪唑基团的金属铱(III)配合物；本发明将仿生识别基元——咪唑基团引入铱配合物的辅助配体中，可模拟组氨酸残基在酶活性中心的识别作用，通过咪唑N-H作为强氢键供体、吡啶型氮原子作为氢键受体，与ATP的三磷酸链形成多点、定向的氢键网络，同时辅以静电吸引和π-π堆积作用，为ATP的专一性结合提供可观的结合能和独特的信号转换机制
[0024]本发明针对现有ATP荧光探针背景干扰严重、无法有效区分结构类似物的技术问题，提供了一种含咪唑基团的金属铱(III)配合物作为ATP磷光检测探针。该探针利用其携带的正电荷与ATP的多磷酸链发生静电预富集，再通过咪唑上的N-H基团与磷酸根形成高度定向的多重氢键阵列，并与腺嘌呤基团发生π-π堆积作用，形成稳定的“静电-氢键-堆积”三位一体识别构象；该特异性结合过程使配合物的激发态非辐射去活通道被有效抑制，从而引发显著的磷光“点亮”效应；借助铱配合物微秒级的长磷光寿命，采用时间分辨发光技术，可完全滤除样品和细胞的自发荧光背景，实现对水相、血清及活细胞中ATP的高选择性、高灵敏度磷光定量检测与成像。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioanalytical chemistry and optical sensing technology, specifically relating to a metal iridium(III) complex containing an imidazole group, its preparation method, and its application. Background Technology
[0002] Adenosine triphosphate (ATP), as the most important energy carrier and signaling molecule in living organisms, plays a core regulatory role in many life processes such as energy metabolism, enzymatic reactions, nerve conduction, and apoptosis. Abnormal fluctuations in intracellular ATP concentration are closely related to the occurrence and development of various major diseases such as cardiovascular disease, Parkinson's disease, and malignant tumors. Therefore, developing analytical techniques that can detect ATP in real time, in situ, and with high fidelity in complex biological microenvironments has significant basic research value and clinical diagnostic significance.
[0003] Currently, traditional methods for detecting ATP mainly include high-performance liquid chromatography (HPLC) and bioluminescence methods. HPLC requires expensive, large-scale instruments and complex sample pretreatment, making in-situ real-time detection difficult. While the luciferin-luciferase bioluminescence method offers high sensitivity, it requires expensive reagents, is susceptible to environmental interference, and cannot distinguish between ATP and its hydrolysis products. In recent years, optical detection methods based on small-molecule fluorescent / phosphorescent probes have attracted widespread attention due to their ease of operation and high spatiotemporal resolution.
[0004] However, traditional organic fluorescent probes often face two core challenges: first, their small Stokes shift leads to overlapping excitation and emission spectra, making them prone to self-absorption interference; second, their fluorescence lifetime is in the nanosecond range, highly overlapping with the background autofluorescence lifetime commonly found in biological samples, resulting in a low signal-to-noise ratio and making precise quantification difficult in complex matrices such as serum and cell lysates. Furthermore, because ATP differs only slightly from its structural analogs such as adenosine diphosphate (ADP) and pyrophosphate (PPi), many probes based on purely electrostatic binding cannot achieve effective selective recognition, easily generating false positive signals.
[0005] Iridium(III) complexes possess excellent photophysical properties—including high phosphorescence quantum efficiency, a large Stokes shift exceeding 100 nm, and a long phosphorescence lifetime ranging from hundreds of nanoseconds to microseconds—making them an excellent platform for next-generation optical sensing and bioimaging. Utilizing their long lifetime, time-resolved luminescence techniques can effectively filter out nanosecond-level biological background fluorescence, significantly improving detection sensitivity and signal-to-noise ratio. Simultaneously, their cationic nature naturally leads to electrostatic interactions with negatively charged biomolecules. However, most reported iridium complex probes rely solely on non-specific electrostatic interactions, resulting in insufficient selectivity and specificity, making it difficult to accurately distinguish ATP from other phosphorylated metabolites. The few systems that incorporate metal ion relay recognition strategies suffer from limitations such as cumbersome procedures and potential metal toxicity.
[0006] To overcome the above problems, it is urgent to develop a design strategy that deeply integrates the high specificity of molecular recognition with the long-lasting phosphorescence advantage of iridium complexes. Summary of the Invention
[0007] To overcome the shortcomings and deficiencies of the existing technologies mentioned above, such as the inability to achieve effective selective recognition and the susceptibility to false positive signals, the primary objective of this invention is to provide a metal iridium(III) complex containing an imidazole group. This invention introduces the biomimetic recognition unit—the imidazole group—into the auxiliary ligand of the iridium complex, which can simulate the recognition function of histidine residues in the enzyme active site. Through imidazole NH as a strong hydrogen bond donor and pyridine nitrogen atom as a hydrogen bond acceptor, it forms a multi-point, directional hydrogen bond network with the triphosphate chain of ATP. At the same time, it is supplemented by electrostatic attraction and π-π stacking, providing considerable binding energy and a unique signal conversion mechanism for the specific binding of ATP.
[0008] Another object of the present invention is to provide a method for preparing the above-mentioned metal iridium(III) complex containing an imidazole group.
[0009] Another object of the present invention is to provide an application of the above-mentioned imidazole-containing iridium(III) complex in the preparation of ATP phosphorescence detection probes.
[0010] The objective of this invention is achieved through the following technical solution:
[0011] An imidazole-containing iridium(III) complex, the complex being a compound having the structure shown in formula (I):
[0012]
[0013] (I)
[0014] The preparation method of the above-mentioned imidazole-containing iridium(III) complexes is carried out according to the following steps:
[0015] (1) Methyl 2,2'-bipyridine-4,4'-dicarboxylate and dichlorotetra[2-(2-pyridyl)phenyl]diiridium were added to a mixed solvent of methanol and dichloromethane, and the mixture was heated under reflux in the dark until it became a transparent solution; after removing methanol and dichloromethane by rotary evaporation, the precursor was obtained by silica gel column chromatography.
[0016] (2) Add the precursor obtained in step (1) and 1-(3-aminopropyl)imidazolium to a reaction flask, heat under light and reflux until a transparent solution is obtained; pour in distilled water to terminate the reaction, then add ammonium hexafluorophosphonate, stir at room temperature to generate a precipitate, filter and collect the precipitate to obtain the crude product;
[0017] (3) The crude product obtained in step (2) was extracted with dichloromethane and n-hexane, and then the metal iridium(III) complex containing imidazole group was obtained by silica gel column chromatography.
[0018] In step (1), the molar ratio of methyl 2,2'-bipyridine-4,4'-dicarboxylate to dichlorotetra[2-(2-pyridyl)phenyl]diiridium is 2:1; the reflux temperature is 60°C; and the volume ratio of methanol to dichloromethane in the mixed solvent is 1:1.
[0019] In step (2), the amount of 1-(3-aminopropyl)imidazole used is calculated as 1.0 mL of 1-(3-aminopropyl)imidazole for every 0.1 mmol of dichlorotetra[2-(2-pyridyl)phenyl]diiridium; the amount of distilled water used is calculated as 50 mL of distilled water for every 1.0 mL of 1-(3-aminopropyl)imidazole; and the temperature of the heating reflux is 60°C.
[0020] In step (2), the amount of ammonium hexafluorophosphonate added is 5 times the molar amount of dichlorotetra[2-(2-pyridyl)phenyl]diiridium.
[0021] Step (3) involves using a silica gel column chromatography method with methanol and dichloromethane in a volume ratio of 1:20 as the eluent to elute the target band and obtain a metal iridium(III) complex containing an imidazole group.
[0022] The above-mentioned application of an imidazole-containing iridium(III) complex in the preparation of adenosine triphosphate (ATP) phosphorescent detection probe; the imidazole-containing iridium(III) complex can provide considerable binding energy and a unique signal conversion mechanism for the specific binding of ATP.
[0023] The present invention has the following advantages and beneficial effects compared with the prior art:
[0024] This invention addresses the technical problems of severe background interference and inability to effectively distinguish structural analogs in existing ATP fluorescent probes by providing an iridium(III) complex containing an imidazole group as an ATP phosphorescence detection probe. This probe utilizes its positive charge to electrostatically pre-enrich the polyphosphate chains of ATP, then forms a highly oriented array of multiple hydrogen bonds with phosphate groups via the NH groups on the imidazole, and undergoes π-π stacking interactions with adenine groups, forming a stable "electrostatic-hydrogen bond-stacking" triadic recognition conformation. This specific binding process effectively suppresses the excited-state non-radiative deactivation channel of the complex, thereby triggering a significant phosphorescence "lighting" effect. Leveraging the microsecond-level long phosphorescence lifetime of the iridium complex, time-resolved luminescence technology can completely filter out the autofluorescence background of samples and cells, achieving highly selective and sensitive quantitative detection and imaging of ATP in aqueous phases, serum, and living cells. Attached Figure Description
[0025] Figure 1 This is a high-resolution mass spectrum of the complex Ir-Im of the present invention.
[0026] Figure 2 The complex Ir-Im of this invention in deuterated DMSO (containing 20% D2O) 1 H NMR spectrum.
[0027] Figure 3 The complex Ir-Im of the present invention in deuterated DMSO 13 C10 NMR spectrum.
[0028] Figure 4 The above are aggregation fluorescence characterization diagrams of the complex Ir-Im of the present invention, where a is the relative fluorescence spectrum of the complex Ir-Im in THF / DMSO mixed solvents with different volume percentages of THF; b is the particle size distribution of the complex Ir-Im in a THF / DMSO mixed solvent with a THF volume percentage of 98%.
[0029] Figure 5 The left and right graphs show the fluorescence intensity of the complex Ir-Im at 600 nm as a function of ATP concentration and fluorescence enhancement, respectively.
[0030] Figure 6 This is a graph showing the phosphorescence lifetime measurement of the complex Ir-Im of the present invention before and after binding with ATP.
[0031] Figure 7 This is a scanning electron microscope image of the complex Ir-Im of the present invention bound to ATP.
[0032] Figure 8 The images show laser confocal imaging of the complex Ir-Im of this invention binding to ATP, from left to right: fluorescence image, bright field image, combined image, and combined magnified image. Detailed Implementation
[0033] The following specific embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention.
[0034] Example 1
[0035] A method for preparing a metal iridium(III) complex containing an imidazole group, comprising the following steps:
[0036] (1) 2,2'-bipyridine-4,4'-dicarboxylate (272.2 mg, 1 mmol) and dichlorotetra[2-(2-pyridyl)phenyl]diiridium (53.6 mg, 0.5 mmol) were added to a 1:1 mixture of methanol and dichloromethane (20 mL). The mixture was heated to reflux at 60 °C in the dark until it became a clear solution. After removing methanol and dichloromethane by rotary evaporation, the precursor was obtained by silica gel column chromatography.
[0037] (2) Add the precursor obtained in step (1) and 1-(3-aminopropyl)imidazolium (1 mL) to a reaction flask, heat at 60°C in the dark and reflux until a transparent solution is obtained; pour in 50 mL of distilled water to terminate the reaction, and then add ammonium hexafluorophosphonate in an amount of 5 times the molar amount of dichlorotetra[2-(2-pyridyl)phenyl]diiridium, stir at room temperature to generate a precipitate, filter and collect the precipitate to obtain the crude product;
[0038] (3) The crude product obtained in step (2) was extracted with dichloromethane and n-hexane, and then the target band was eluted by silica gel column chromatography with methanol and dichloromethane in a volume ratio of 1:20 as the eluent to obtain the metal iridium(III) complex containing the imidazole group, referred to as complex Ir-Im.
[0039] The reaction equation for the above preparation method is shown in equation (1).
[0040] (1)
[0042] The molecular structure of the prepared complex Ir-Im was confirmed using high-resolution mass spectrometry (results shown in Figure 1), ¹H NMR (results shown in Figure 2), and ¹³C NMR (results shown in Figure 3). The molecular formula of the complex is C1. 46 H 42 IrN 10 O2PF6; high-resolution mass spectrometry analysis yielded m / z 959.3080, corresponding to [M-PF6].+ The molecular ion peak is consistent with theoretical expectations.
[0043] 1 H NMR (400 MHz, DMSO-D6) δ 9.02 (s, 2H), 8.18 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 5.8 Hz, 2H), 7.93 – 7.82 (m, 6H), 7.67 – 7.59 (m, 4H), 7.17 (s,2H), 7.10 (t, J = 7.3 Hz, 2H), 7.03 (t, J = 7.8 Hz, 2H), 6.92 – 6.84 (m, 4H), 6.15 (d, J = 7.1 Hz, 2H), 4.03 (d, J = 6.9 Hz, 4H), 3.27 (t, J = 6.9 Hz, 4H),1.97 (p, J = 6.7 Hz, 4H).
[0044] 13 C NMR (151 MHz, DMSO) δ 167.30, 164.37, 156.42, 151.22, 150.43,149.78, 144.52, 144.39, 139.75, 138.03, 131.74, 131.15, 128.73, 127.08,125.77, 124.66, 123.51, 120.75, 120.34, 44.62, 37.59, 30.69.
[0045] Based on the above characterization data, the molecular structure of the complex Ir-Im was finally determined as shown in formula (Ⅰ):
[0046]
[0047] (I)
[0048] Test example:
[0049] (A) Aggregation-induced emission properties of the complex Ir-Im
[0050] Weigh 1.0 mg of the Ir-Im complex obtained in Example 1 and dissolve it in 1 mL of DMSO to obtain a 1 mmol / L Ir-Im mother liquor. Dilute the Ir-Im mother liquor with different volume percentages of tetrahydrofuran (THF) in a THF / DMSO mixed solvent to obtain a series of Ir-Im solutions, each with a concentration of 20 μmol / L. The volume percentages of THF in the THF / DMSO mixed solvent were 0%, 70%, 90%, 95%, and 98%, respectively. Measure the fluorescence spectra of the obtained series of Ir-Im solutions under excitation at 385 nm. The results are as follows: Figure 4 As shown, the Ir-Im complex exhibits distinct aggregation-induced emission characteristics. For example... Figure 4 As shown in Figure a, the fluorescence of Ir-Im gradually increases with increasing THF content; when the THF volume percentage is 98%, the luminescence intensity of the Ir-Im complex in the THF / DMSO mixed solvent is 11 times higher than that in DMSO, with the maximum emission peak at 590 nm. Dynamic light scattering (DLS) measurements showed that the aggregated particle size distribution of the Ir-Im complex in the THF / DMSO mixed solvent with a THF volume percentage of 98% was approximately 170 nm. Figure 4 (b).
[0051] (B) Fluorescent titration experiments verified that the fluorescence presentation of Ir-Im is correlated with ATP concentration.
[0052] The 1 mmol / L stock solution of the Ir-Im complex prepared in (A) above was diluted in HEPES buffer (10 mmol / L, pH 7.4) to obtain a 20 μmol / L Ir-Im complex solution. Adenosine triphosphate (ATP) solution was added dropwise to 11 portions of this Ir-Im complex solution to obtain a series of solutions with ATP concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.25, 2.5, 2.75, 3.0, 4.0, and 5.0 mmol / L. The fluorescence spectra of this series of solutions were measured under excitation at 385 nm. The results are as follows. Figure 5As shown, at low ATP concentrations (<2.0 mmol / L), the Ir-Im complex exhibits weak fluorescence. With increasing ATP concentration, the fluorescence of the Ir-Im complex continuously increases, reaching a 60-fold increase at an ATP concentration of 3.0 mmol / L, indicating that the Ir-Im complex has a high sensitivity to ATP concentration. When the ATP concentration exceeds 3.0 mmol / L, the fluorescence of the Ir-Im complex reaches its maximum value. Phosphorescence titration experiments show that the Ir-Im complex exhibits a typical "light-up" phosphorescence response to ATP, and its response characteristics can be summarized as a three-stage pattern: "weak light at low concentration - dramatic enhancement near the threshold - saturation at high concentration." The Ir-Im complex achieves up to 60-fold phosphorescence enhancement near an ATP concentration of 3.0 mmol / L, demonstrating extremely high sensitivity and excellent signal-to-noise ratio potential. This experiment also determined the effective detection concentration window and detection limit of the Ir-Im complex for ATP, laying the analytical foundation for its quantitative detection of ATP in biological samples.
[0053] (C) Measurement of phosphorescence lifetime of the Ir-Im complex with ATP
[0054] The 1 mmol / L stock solution of complex Ir-Im prepared in (A) above was diluted in HEPES buffer (10 mmol / L, pH 7.4) to obtain a 20 μmol / L solution of complex Ir-Im. ATP solution was added to this Ir-Im solution to obtain a 3.0 mmol / L solution of complex Ir-Im containing ATP. The fluorescence lifetime of the Ir-Im solutions before and after the addition of ATP was measured under 405 nm femtosecond laser excitation. The results are as follows. Figure 6 As shown, the phosphorescence decay behavior of the Ir-Im complex before and after binding with ATP exhibits a significant difference. The lifetime of the Ir-Im complex alone in buffer solution is only 7.16 ns, but after the addition of ATP, the phosphorescence lifetime is significantly extended from 7.16 ns to 175.21 ns, an increase of approximately 24.5 times. This result directly demonstrates that in its free state, the Ir-Im complex molecule is highly flexible, undergoes severe non-radiative inactivation, and has an extremely short lifetime; after binding with ATP, the rigidity of the Ir-Im complex molecule is significantly enhanced, non-radiative inactivation is inhibited, and the phosphorescence lifetime is extended to the nanosecond level.
[0055] (D) Scanning electron microscopy (SEM) was used to verify the binding of the iridium complex Ir-Im to ATP.
[0056] Prepare a solution containing the Ir-Im complex at a concentration of 3.0 mmol / L (Ir-Im concentration is 20 μmol / L) as shown in step (C) above. Add 10 μL of this solution to the surface of a silicon wafer, vacuum dry, then perform gold sputtering, and observe the results using a scanning electron microscope. Figure 7 As shown, SEM observation revealed that the complex Ir-Im binds to ATP to form stable, large-sized spherical particles. Energy-dispersive X-ray spectroscopy (EDS) was used to verify the elemental composition of the particles, revealing that Ir is characteristic of the complex and P is characteristic of ATP. This indicates that the particles are formed by the binding of Ir-Im and ATP, providing direct morphological and elemental chemical evidence for the binding of Ir-Im to ATP.
[0057] (E) Laser confocal imaging for imaging analysis of the fluorescence response of probe Ir1 to ATP.
[0058] Prepare a solution containing the Ir-Im complex at a concentration of 3.0 mmol / L (Ir-Im concentration is 20 μmol / L) as shown in step (C) above. Place 2 mL of this solution on a confocal culture dish and observe under a confocal microscope. Excitement wavelength is 405 nm, and fluorescence signals in the 580-620 nm wavelength range are collected. Results are as follows: Figure 8 As shown, the Ir-Im complex itself exhibits weak fluorescence, making it difficult to image clearly under a confocal microscope. However, when ATP solution is added to the Ir-Im solution, uniformly dispersed red fluorescent particles are observed under the confocal microscope, with a particle size of approximately 2 mm after magnification. These results demonstrate that the Ir-Im complex possesses excellent "off-on" phosphorescence imaging characteristics: in the absence of the target analyte ATP, the probe is in a "fluorescence-silent" off state with extremely low background; when ATP is present, the probe is specifically "lit up," generating a high-intensity red phosphorescent particle signal, enabling wash-free, high-contrast ATP imaging analysis.
[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A metal iridium(III) complex containing an imidazole group, characterized in that: The complex is a compound having the structure shown in formula (Ⅰ): （I）。 2. The method for preparing an imidazole-containing iridium(III) complex according to claim 1, characterized in that... Follow these steps: (1) Methyl 2,2'-bipyridine-4,4'-dicarboxylate and dichlorotetra[2-(2-pyridyl)phenyl]diiridium were added to a mixed solvent of methanol and dichloromethane, and the mixture was heated under reflux in the dark until it became a transparent solution; after removing methanol and dichloromethane by rotary evaporation, the precursor was obtained by silica gel column chromatography. (2) Add the precursor obtained in step (1) and 1-(3-aminopropyl)imidazolium to a reaction flask, heat under light and reflux until a transparent solution is obtained; pour in distilled water to terminate the reaction, then add ammonium hexafluorophosphonate, stir at room temperature to generate a precipitate, filter and collect the precipitate to obtain the crude product; (3) The crude product obtained in step (2) was extracted with dichloromethane and n-hexane, and then the metal iridium(III) complex containing imidazole group was obtained by silica gel column chromatography.
3. The method for preparing an imidazole-containing iridium(III) complex according to claim 2, characterized in that: In step (1), the molar ratio of methyl 2,2'-bipyridine-4,4'-dicarboxylate to dichlorotetra[2-(2-pyridyl)phenyl]diiridium is 2:1; the reflux temperature is 60°C; and the volume ratio of methanol to dichloromethane in the mixed solvent is 1:
1.
4. The method for preparing an imidazole-containing iridium(III) complex according to claim 2, characterized in that: In step (2), the amount of 1-(3-aminopropyl)imidazole used is calculated as 1.0 mL of 1-(3-aminopropyl)imidazole for every 0.1 mmol of dichlorotetra[2-(2-pyridyl)phenyl]diiridium; the amount of distilled water used is calculated as 50 mL of distilled water for every 1.0 mL of 1-(3-aminopropyl)imidazole; and the temperature of the heating reflux is 60°C.
5. The method for preparing an imidazole-containing iridium(III) complex according to claim 2, characterized in that: In step (2), the amount of ammonium hexafluorophosphonate added is 5 times the molar amount of dichlorotetra[2-(2-pyridyl)phenyl]diiridium.
6. The method for preparing an imidazole-containing iridium(III) complex according to claim 2, characterized in that: Step (3) involves using a silica gel column chromatography method with methanol and dichloromethane in a volume ratio of 1:20 as the eluent to elute the target band and obtain a metal iridium(III) complex containing an imidazole group.
7. The application of the imidazole-containing iridium(III) complex according to claim 1 in the preparation of adenosine triphosphate phosphorescence detection probe.