Two-channel fluorescent probe for simultaneously detecting A beta and HClO / ClO-, preparation method and application
By developing a dual-channel fluorescence probe for Alzheimer's disease detection, the problem of difficulty in detecting Aβ and HClO/ClO- in the prior art is solved, and independent spectral responses with high selectivity and sensitivity are achieved.
Patent Information
- Application Number
- CN202510318879.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-17
AI Technical Summary
The prior art is difficult to simultaneously and efficiently detect Aβ plaques and HClO/ClO- related to Alzheimer's disease (AD), while single-response fluorescent probes are cumbersome to operate and signals may interfere with each other.
A dual-channel fluorescent probe was developed, with the structural formula of which, by the preparation method, comprises a multi-step reaction, to generate a fluorescent probe capable of independently responding to Aβ and HClO/ClO-.
Independent spectral responses to Aβ and HClO/ClO- are achieved, with high selectivity and sensitivity, and can visualize these two AD pathological factors through two independent fluorescence channels.
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Figure CN120157667A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a dual-channel fluorescent probe for simultaneously detecting Aβ and HClO / ClO - , a preparation method thereof, and an application thereof. Background Art
[0002] Alzheimer's disease (AD) is a neurobiological disease that affects different aspects of cognition. Its main features include early impairment of learning and memory, followed by late impairment of complex attention, cognitive ability, social behavior, and language function, etc. AD is a multifactorial disease, and the exact cause is not yet fully understood. However, research shows that genetic factors, environmental factors, and lifestyle may all be related to its onset. Common pathogenic mechanisms include: accumulation of β-amyloid plaques, neurofibrillary tangles, neuroinflammation, and metal ions and oxidative stress, etc.
[0003] The treatment of Alzheimer's disease mainly focuses on slowing down the progression of symptoms and improving the quality of life of patients. Therefore, early diagnosis is crucial for the development of Alzheimer's disease. Currently, diagnostic methods rely on medical history, clinical evaluation, and a series of examinations (such as cognitive function tests, imaging examinations, cerebrospinal fluid analysis, gene detection). However, the foregoing methods have defects due to their high cost or inaccuracy. Therefore, the development of efficient and sensitive diagnostic methods can promote the progress of AD disease treatment.
[0004] Currently, organic small molecule fluorescent probes have the advantages of high sensitivity, high selectivity, simple operation, low cost, good biocompatibility, etc., and have been increasingly used for the detection of AD-related markers in recent years. Considering the complexity and multifactorial nature of the AD pathogenic mechanism, it is difficult to accurately monitor the disease process by detecting a single biomarker in complex organisms. Therefore, from the perspective of the multifactorial nature of the AD pathogenic mechanism, studying the correlation between Aβ plaques and ROS is of great significance. Although this goal can be achieved by using two single-response fluorescent probes, compared with bifunctional probes, such an operation will be more cumbersome, and at the same time, the two probes may have disadvantages such as mutual interference of fluorescence signals and different distribution sites.
[0005] Currently, only a few bifunctional probes have been developed for detecting Aβ aggregates and ROS (for example, ONOO - or H2O2, ·OH). However, as a member of ROS, there is no effective tool for the bifunctional detection of Aβ aggregates and HClO / ClO - . Summary of the Invention
[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for simultaneously detecting Aβ and HClO / ClO- Dual-channel fluorescent probe, preparation method and application thereof, which solve the problems in the prior art.
[0007] The object of the present invention can be achieved by the following technical solutions:
[0008] Dual-channel fluorescent probe for simultaneously detecting Aβ and HClO / ClO - The structural formula thereof is:
[0009]
[0010] Dual-channel fluorescent probe for simultaneously detecting Aβ and HClO / ClO - Preparation method of the dual-channel fluorescent probe, comprising the following steps:
[0011] S1, under nitrogen protection, dissolve 4-(dimethylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde in ethylene glycol dimethyl ether, add tetrakis(triphenylphosphine)palladium and aqueous sodium carbonate solution, heat and stir the reaction, and then obtain compound 1 after extraction, drying, filtration, concentration under reduced pressure and purification;
[0012] S2, dissolve compound 1 and monoethyl adipate in dichloromethane, and add piperidine; after heating and stirring the reaction, concentrate under reduced pressure to obtain compound 2;
[0013] S3, dissolve methylene blue and sodium carbonate in a mixture of water and dichloromethane, add sodium thiosulfate, reflux and stir, then cool, and then dropwise add a dichloromethane solution of triphosgene for reaction; after quenching, extraction, drying, filtration and concentration under reduced pressure, obtain compound 3;
[0014] S4, dissolve compound 3 and triethylamine in dichloromethane, add 2-(4-aminophenyl)ethylamine and stir the reaction, and then concentrate under reduced pressure to obtain compound 4;
[0015] S5, under nitrogen protection, dissolve compound 2 in dichloromethane, add DIPEA and stir, then add HATU and stir to activate it, and finally add compound 4 for reaction, and then obtain the dual-channel fluorescent probe after concentration under reduced pressure, separation and purification.
[0016] Further, the heating and stirring temperature in S1 is 90 °C; the heating and stirring temperature in S2 is 80 °C.
[0017] Further, in S1, dichloromethane is used for extraction, and silica gel column chromatography is used for purifying compound 1.
[0018] Further, in S3, the reflux and stirring temperature is 40 °C.
[0019] Further, in S3, dichloromethane is used for extraction and ice water is used for quenching.
[0020] Further, in S4, 2-(4-aminophenyl)ethylamine is added at 0 °C.
[0021] Further, the preparation route of the dual-channel fluorescent probe is as follows:
[0022]
[0023] The above-mentioned dual-channel fluorescent probe for simultaneously detecting Aβ and HClO / ClO - is applied in the preparation of an Alzheimer's disease detection kit.
[0024] An Alzheimer's disease detection kit includes the above-mentioned dual-channel fluorescent probe for simultaneously detecting Aβ and HClO / ClO - .
[0025] Advantages of the present invention:
[0026] The dual-channel fluorescent probe of the present invention shows completely independent spectral responses to Aβ plaques and HClO / ClO - and has high selectivity and sensitivity, thus realizing the differential visualization of these two AD pathological factors through two independent fluorescence channels. Description of the Drawings
[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.
[0028] Figure 1 is the fluorescence intensity diagram after the action of the dual-channel fluorescent probe CTAD-MB of the present invention and HClO / ClO - ;
[0029] Figure 2 is the fluorescence intensity (Kd) diagram after the action of the dual-channel fluorescent probe CTAD-MB of the present invention and Aβ42 aggregates;
[0030] Figure 3 is the fluorescence imaging and quantification diagram of the dual-channel fluorescent probe CTAD-MB of the present invention in AD / WT mice. Detailed Embodiments
[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0032] For simultaneous detection of Aβ and HClOClO - A dual-channel fluorescent probe, the structural formula of which is as follows:
[0033]
[0034] The preparation route of this dual-channel fluorescent probe is as follows:
[0035]
[0036] The preparation method of this dual-channel fluorescent probe includes the following steps:
[0037] S1, Under nitrogen protection, dissolve 4-(dimethylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde in ethylene glycol dimethyl ether; add tetrakis(triphenylphosphine)palladium and aqueous sodium carbonate solution to the resulting solution; heat and stir the mixture at 90 °C for 5 h; after the reaction is completed, cool the reaction mixture to room temperature and transfer it to a separatory funnel containing saturated brine; extract the mixture with dichloromethane, dry the combined organic layer over anhydrous sodium sulfate, filter and concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain Compound 1.
[0038] S2, Dissolve Compound 1 and monoethyl adipate in dichloromethane; add piperidine to the resulting solution; heat and stir the mixture at 80 °C; after the reaction is completed, concentrate the mixture under reduced pressure to obtain Compound 2.
[0039] S3, Dissolve methylene blue and sodium carbonate in a mixture of water and dichloromethane; add sodium thiosulfate to the resulting solution; reflux and stir the mixture at 40 °C for 15 - 30 minutes; then cool the mixture to 0 °C, dropwise add a dichloromethane solution of triphosgene, and react at room temperature for 12 h; after the reaction is completed, quench in ice water and extract the mixture with dichloromethane; dry the combined organic layer over anhydrous sodium sulfate, filter and concentrate under reduced pressure; obtain Compound 3.
[0040] S4, Dissolve Compound 3 and triethylamine in dichloromethane; add 2-(4-aminophenyl)ethylamine to the resulting solution at 0 °C; stir the mixture at room temperature; after the reaction is completed, concentrate under reduced pressure to obtain Compound 4.
[0041] S5. Dissolve compound 2 in dichloromethane under nitrogen protection; add DIPEA to the resulting solution. Stir the mixture at room temperature for 10 min, then add HATU and stir for 10 min to activate it. Finally, add compound 4. After the reaction is complete, concentrate under reduced pressure, separate and purify to obtain the target compound (CTAD-MB), namely the dual-channel fluorescent probe.
[0042] The technical solution of the present invention will be described below through the following examples;
[0043] Example 1
[0044] In this example, the preparation of the dual-channel fluorescent probe CTAD-MB is introduced;
[0045] 1) Preparation of compound 1:
[0046] Under nitrogen protection, dissolve 4-(dimethylamino)phenylboronic acid (1.06 g, 6.4 mmol) and 4-bromo-2-hydroxybenzaldehyde (1 g, 5.0 mmol) in ethylene glycol dimethyl ether (35 mL). Add tetrakis(triphenylphosphine)palladium (577.75 mg, 0.5 mmol) and an aqueous solution of sodium carbonate (2 M, 41 mL) to the resulting solution. Heat and stir the mixture at 90 °C for 5 h. After the reaction is complete, cool the reaction mixture to room temperature and transfer it to a separatory funnel containing 100 mL of saturated brine. Extract the mixture with dichloromethane (3 × 150 mL), dry the combined organic layers over anhydrous sodium sulfate, filter and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography to obtain compound 1 as a yellow solid (1.02 g, 84.6%).
[0047] Among them, the 1H NMR data of compound 1 are as follows: 1 H NMR (300 MHz, Chloroform-d) δ 11.18 (s, 1H), 9.88 (s, 1H), 7.64–7.54 (m, 3H), 7.30–7.19 (m, 2H), 6.82 (d, J = 8.4 Hz, 2H), 3.06 (s, 6H).
[0048] 2) Preparation of compound 2:
[0049] Dissolve compound 1 (500 mg, 2.1 mmol) and monoethyl adipate (500 μL, 4.2 mmol) in dichloromethane (15 mL). Add 2 drops of piperidine to the resulting solution. Heat and stir the mixture at 80 °C for 10 h. After the reaction is complete, concentrate the mixture under reduced pressure to obtain compound 2 as a red solid (585.5 mg, 90%).
[0050] Among them, the 1H NMR data of compound 2 are as follows: 11H NMR (300 MHz, Chloroform-d) δ 12.24 (s, 1H), δ 8.93 (s, 1H), 7.81–7.59 (m, 5H), δ 6.93 (s, 2H), 3.10 (s, 6H).
[0051] 3) Preparation of Compound 3:
[0052] Methylene blue (2.1 g, 7.39 mmol) and sodium carbonate (3.13 g, 29.56 mmol) were dissolved in a mixture of water and dichloromethane (2:1). Sodium thiosulfate (5.15 g, 29.56 mmol) was added to the resulting solution. The mixture was refluxed and stirred at 40 °C for 15 - 30 minutes. Then the mixture was cooled to 0 °C, and a dichloromethane solution of triphosgene (1.2 g, 4.43 mmol) was added dropwise, and the reaction was carried out at room temperature for 12 h. After the reaction was completed, it was quenched in ice water, and the mixture was extracted with dichloromethane (3 × 200 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Compound 3 was obtained as a blue solid (2.16 g, 84%).
[0053] 4) Preparation of Compound 4:
[0054] Compound 3 (1.7 g, 4.87 mmol) and triethylamine (3.4 mL, 24.35 mmol) were dissolved in dichloromethane. At 0 °C, 2-(4-aminophenyl)ethylamine (993.5 mg, 7.30 mmol) was added to the resulting solution. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, it was concentrated under reduced pressure. Compound 4 was obtained as a blue solid (1.53 g, 75%).
[0055] Among them, the 1H NMR data of Compound 4 are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 7.20 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 2.8 Hz, 2H), 6.63 (dd, J = 8.9, 2.9 Hz, 2H), 6.49 (d, J = 8.1 Hz, 2H), 5.80 (t, J = 5.7 Hz, 1H), 4.89 (s, 2H), 3.22–3.12 (m, 2H), 2.89 (s, 12H), 2.53 (d, J = 7.4 Hz, 2H).
[0056] 5) Preparation of CTAD-MB:
[0057] Under nitrogen protection, compound 2 (50 mg, 0.16 mmol) was dissolved in dichloromethane. DIPEA (51.7 mg, 0.40 mmol) was added to the resulting solution. The mixture was stirred at room temperature for 10 min, then HATU (73 mg, 0.19 mmol) was added and stirred for 10 min for activation. Finally, compound 4 (76 mg, 0.18 mmol) was added. After the reaction was complete, it was concentrated under reduced pressure, separated and purified to obtain the target compound (14.2 mg, 12%).
[0058] Among them, the 1H NMR data of CTAD-MB are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H), 8.92 (s, 1H), 7.96 (dd, J = 15.1, 8.4 Hz, 1H), 7.75 (dd, J = 8.8, 2.8 Hz, 4H), 7.64 (d, J = 8.1 Hz, 2H), 7.18 (t, J = 9.1 Hz, 4H), 6.84–6.78 (m, 2H), 6.71 (d, J = 2.8 Hz, 2H), 6.64 (dd, J = 8.9, 2.8 Hz, 2H), 5.88 (t, J = 5.6 Hz, 1H), 3.27 (q, J = 6.7 Hz, 2H), 2.98 (d, J = 2.0 Hz, 7H), 2.89 (s, 11H), 2.70 (t, J = 7.3 Hz, 2H).
[0059] Example 2
[0060] In this example, the spectral response of the fluorescent probe CTAD-MB prepared in Example 1 to HClO / ClO - was investigated;
[0061] The emission wavelength of the fluorescent probe CTAD-MB was measured in PBS solution (3% DMSO, 10 mM, pH 7.4). CTAD-MB was dissolved in DMSO to prepare a 1.0 mM stock solution and diluted with PBS. CTAD-MB was incubated with a certain amount of HClO / ClO - solution (containing 10% DMSO, total volume: 1 mL, final concentration of the probe: 10 μM) at 37 °C at the specified pH value, and the fluorescence spectrum was recorded under excitation at 650 nm. To test the time course of the fluorescence reaction of the probe to HClO / ClO - , first ensure that the probe and HClO / ClO– are maintained at the required final concentrations (probe: 10 μM; HClO / ClO - : 10 μM), and the fluorescence intensity at 690 nm was recorded in a timely manner under excitation at 650 nm at 37 °C.
[0062] As Figure 1As shown, with the increase in the concentration of HClO / ClO - (0 - 15 μM), the fluorescence intensity of the CTAD-MB probe at 690 nm increased significantly. When 20 μM HClO / ClO - was added, the fluorescence intensity at 690 nm could be enhanced by about 20 times. Meanwhile, there was a good linear relationship with the fluorescence intensity in the range of 0 - 100 μM, and the detection limit was 19.5 nM. It was proved that the fluorescence probe CTAD-MB showed ideal sensitivity to HClO / ClO - .
[0063] Example 3
[0064] In this example, the measurement of the Kd value of the fluorescence probe CTAD-MB prepared in Example 1 for Aβ42 aggregates was explored;
[0065] 500 nM Aβ42 aggregates were incubated with different concentrations of CTAD-MB, and it was observed that the fluorescence intensity gradually increased from 0 nM to 1000 nM. As Figure 2 shown, CTAD-MB itself had very weak fluorescence at 625 nm. After binding to Aβ42 aggregates, there was a blue shift, and a significant fluorescence enhancement was shown at 540 nm. And the dissociation constant (Kd) of the probe and Aβ42 aggregates is an important factor to evaluate its binding affinity. It was determined by the saturation fluorescence titration method, and the calculated Kd value was Kd = 27 ± 5.0 nM, which was significantly lower than the dissociation constant of ThT (Kd = 890 nM). It was proved that the fluorescence probe CTAD-MB showed ideal sensitivity to Aβ42 aggregates.
[0066] Example 4
[0067] In this example, the fluorescence imaging of the mouse AD model was experimentally explored with CTAD-MB;
[0068] Before background imaging, APP / PS1 transgenic mice (n = 3, C57BL / 6J, APP / PS1, 8 months old, male) and age-matched wild-type mice (n = 3, C57BL / 6J, 8 months old, male) were shaved and intravenously injected with CTAD-MB (5 mg / kg, 10% DMSO, 90% PBS, 200 μL). The fluorescence signals from the brain were recorded at different time points after intravenous injection of CTAD-MB. For measurement, filter sets (λex = 460 nm, λem = 520 nm and λex = 620 nm, λem = 710 nm) were used, and optical images were obtained with a 1 s exposure time. During imaging, the mice were anesthetized with 2.5% isoflurane gas at an oxygen flow rate of (1.5 L / min) to maintain the imaging stage. The imaging data was analyzed by Living Image software.
[0069] As Figure 3 shown in (a) and (b) of - the imaging channel (λ x / λ m = 620 / 670 nm), the relative difference [R(AD / WT)] [R(AD / WT) = F(AD) / F(WT)] between the AD brain and the WT brain reached a maximum of 1.69 times 30 minutes after probe injection. Meanwhile, in APP / PS1 mice, the fluorescence signal intensity increased significantly within 0 - 45 minutes, reached the peak at 30 minutes (Fmax / F0 = 2.35), began to metabolize at 75 minutes, and the fluorescence signal gradually decreased, with metabolism completed within 90 minutes. These results demonstrated the overexpression of HClO / ClO - in AD, which might be due to the promotion of HClO / ClO - expression by Aβ fibers in the AD brain.
[0070] Meanwhile, as shown in (c) and (d) of the figure, in the Aβ imaging channel (λ x / λ m = 460 / 520 nm), after intravenous injection of AH-1 for 20 minutes, the relative difference [F(AD) / F(WT)] reached a maximum of 1.62 times and remained relatively stable within 5 - 45 minutes. It is worth noting that the probe bound to Aβ rapidly, reaching the peak at 5 minutes (Fmax / F0 = 1.75), and after 90 minutes, the fluorescence signal showed relatively no change. Only a slight fluorescence enhancement (Fmax / F0 = 1.18) was observed in the WT group. Therefore, the above results indicate that CTAD-MB can cross the blood-brain barrier smoothly, specifically recognize HClO / ClO - and Aβ fibers in vivo, and the presence of Aβ fibers might promote the upregulation of HClO / ClO - expression.
[0071] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.
[0072] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and all these changes and improvements fall within the scope of the present invention claimed.
Claims
1. For simultaneous detection of Aβ and HClOClO - A dual-channel fluorescent probe, characterized in that Its structural formula is:
2. For simultaneous detection of Aβ and HClO / ClO - A method for preparing a dual-channel fluorescent probe, characterized in that: The following steps are involved: S1, under nitrogen protection, dissolving 4-(dimethylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde in ethylene glycol dimethyl ether, adding tetrakistriphenylphosphine palladium and sodium carbonate aqueous solution, heating and stirring for reaction, extracting, drying, filtering, concentrating under reduced pressure and purifying to obtain compound 1; S2, dissolving compound 1 and monoethyl adipate in dichloromethane, and adding piperidine; After heating and stirring to react, the mixture was concentrated under reduced pressure to obtain compound 2; S3, dissolving methylene blue and sodium carbonate in a mixture of water and dichloromethane, adding sodium thiosulfate, stirring under reflux and cooling, then dropping a dichloromethane solution of triphosgene to react; then quenching, extracting, drying, filtering and concentrating under reduced pressure to obtain compound 3; S4, dissolving compound 3 and triethylamine in dichloromethane, adding 2-(4-aminophenyl)ethylamine to react with stirring, and then concentrating under reduced pressure to obtain compound 4; S5, under nitrogen protection, dissolving compound 2 in dichloromethane, adding DIPEA and stirring, then adding HATU and stirring to activate it, and finally adding compound 4 to react, followed by reduced pressure concentration, separation and purification to obtain the dual-channel fluorescent probe.
3. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, characterized in that: The heating and stirring temperature in S1 was 90°C; the heating and stirring temperature in S2 was 80°C.
4. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, characterized in that: In S1, dichloromethane was used for extraction, and silica gel column chromatography was used for purification of compound 1.
5. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, characterized in that: In S3, the temperature of reflux stirring was 40°C.
6. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, characterized in that: In S3, dichloromethane is used for extraction and ice water is used for quenching.
7. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, characterized in that: In S4, 2-(4-aminophenyl)ethylamine was added at 0°C.
8. The method for simultaneously detecting Aβ and HClO / ClO according to claim 2. - A method for preparing a dual-channel fluorescent probe, wherein the preparation route of the dual-channel fluorescent probe is:
9. The method for simultaneously detecting Aβ and HClO / ClO according to claim 1 - The dual-channel fluorescent probe is used in the preparation of an Alzheimer's disease detection kit.
10. A kit for detecting Alzheimer's disease, characterized in that: The method for simultaneously detecting Aβ and HClO / ClO according to claim 1 - Dual-channel fluorescent probe.