Novel fluorescent probe and application thereof in rapid detection of acetylcholin esterase

Abstract

The invention discloses a novel fluorescent probe and application thereof in rapid detection of acetylcholin esterase, and belongs to the technical field of acetylcholin esterase detection. The novel fluorescent probe can rapidly detect acetylcholin esterase at 520 nm, the response time is short (60 s), the detection limit is as low as 0.014 U / mL, and more importantly, the fluorescent probe can be successfully applied to endogenous and exogenous acetylcholin esterase imaging in cells (such as PC12 cells) and organisms (such as live mice). The invention provides an effective tool for studying acetylcholin esterase in a complex biological environment, and has important significance on deep study and treatment of nervous system diseases and cancers.

Description

Technical Field

[0001] The invention belongs to the technical field of acetylcholinesterase detection, and specifically relates to a novel fluorescent probe capable of rapidly detecting acetylcholinesterase in a biological system, and a preparation method and an application method thereof. Background Art

[0002] Cholinesterases (ChEs) are an important class of enzymes responsible for the hydrolysis of the essential neurotransmitter acetylcholine into choline and acetic acid, and play an important role in the physiological metabolism of the nervous system. There are two types of cholinesterases, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Studies have shown that AChE has a higher catalytic activity towards acetylcholine than BChE. Abnormal expression of AChE can significantly affect the nervous system and may lead to neurological diseases such as depression, schizophrenia, Huntington's disease, Alzheimer's disease, and Parkinson's disease. In addition, abnormal expression of AChE is often observed in patients with liver cancer, suggesting that it may be associated with tumor cell proliferation. Despite the recognition of the importance of AChE in the nervous system and cancer, its exact mechanism of action in these diseases is still unclear. Therefore, there is an urgent need to develop a method for detecting AChE activity in vivo, which is crucial for detailed studies of the mechanism of action of AChE in neurological diseases and related cancers.

[0003] Currently, several methods, including electrochemical sensors, UV-visible spectrophotometry, and chemiluminescence, have been used to detect the activity of cholinesterase. However, these methods usually have problems such as high cost, complex sample processing, lengthy operation procedures, and more importantly, they are not suitable for real-time in situ observation or detection of ChE activity in living tissues. In contrast, fluorescent probes have attracted attention due to their high sensitivity, selectivity, biocompatibility, high spatiotemporal resolution, and real-time in situ observation capabilities. For example, Tang (Detection of acetylcholinesterase andbutyrylcholinesterase in vitro and in vivo using a new fluorescent probe. Chemical Communications 2024, 60 (15), 2082-2085.) developed an OHPD probe that can activate fluorescence to monitor AChE in real time with minimal background interference, and the detection limit is 0.24 U / mL. Lin (Observation of the Elevation of Cholinesterase Activity in Brain Glioma by a Near-Infrared Emission Chemsensor. Analytical Chemistry 2020, 92(19), 13405-13410.) used the near-infrared probe IPAN to reduce the detection limits of AChE and BChE to 0.127 U / mL and 0.0117 U / mL, respectively, and was able to perform fluorescence imaging of AChE in mouse brain slices. Kim (Near-Infrared Fluorescence Probe for Specific Detection of Acetylcholinesterase and Imaging in Live Cells and Zebrafish. ACS Applied Bio Materials 2022, 5 (5), 2232-2239.) used the TCF fluorescent group to develop EW3 for fluorescence imaging of AChE in living zebrafish tissues.

[0004] Although significant progress has been made in the development of small molecule fluorescent probes for detecting ChEs activity, there is still a need to develop fluorescent probes with better performance. Summary of the invention

[0005] In view of this, the present invention aims to develop a fluorescent probe that can observe or detect ChEs activity in cells and living tissues in real time in situ, providing a novel and valuable tool for continuously monitoring the changes of the enzyme in vivo.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions: The first aspect of the present invention provides a novel fluorescent probe which can specifically respond to AChE and has a chemical formula of C 26 H 26 N3O2, the structural formula is as follows: .

[0007] The novel fluorescent probe (hereinafter referred to as probe CNA) provided by the present invention loses the N,N-diformamide group under the action of AChE to generate compound 1, the structural formula of which is shown below: .

[0008] Based on the response mechanism of the probe CNA to AChE, the second aspect provides an application of the probe CNA, which at least includes the following: a) Detecting AChE activity in vivo or in vitro; b) Qualitative or quantitative detection of AChE; b) Fluorescence imaging of AChE in cells or living tissues; c) Real-time monitoring of AChE in vivo.

[0009] The third aspect of the present invention provides a method for preparing a probe CNA, comprising the following steps: S1, compound 1, an inorganic base and dimethylaminoformyl chloride are placed in an organic solvent and stirred for reaction; S2. After the reaction in step S1 is completed, the organic phase is extracted with an organic solvent and concentrated under reduced pressure to obtain the product.

[0010] Preferably, in the above preparation method, the following steps are also included: S3. Purify the product obtained in S2 by column chromatography, and use petroleum ether / ethyl acetate for gradient elution during purification.

[0011] In the above preparation method, the inorganic base is used to provide an alkaline reaction environment, including but not limited to potassium carbonate, cesium carbonate, lithium carbonate, etc. Preferably, the molar ratio of compound 1, potassium carbonate and dimethylcarbamoyl chloride is (0.8-1):(3-4):(4-6).

[0012] In the above preparation method, the organic solvent used includes but is not limited to dichloromethane, tetrahydrofuran, chloroform, etc., among which dichloromethane has the best effect.

[0013] Preferably, in the above preparation method, the reaction temperature of step S1 is room temperature (10-37° C.).

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention synthesizes a fluorescent probe CNA which can be used for rapid detection of acetylcholinesterase, has an emission wavelength of 520 nm, and has the advantages of simple synthesis, rapid reaction, good photostability, high selectivity, good biocompatibility, etc.

[0015] The fluorescent probe provided by the present invention has good cell membrane permeability and is mainly enriched in lysosomes in cells. In view of the key role of lysosomes in waste disposal, foreign body digestion, cell renewal, metabolic regulation and intracellular signal transduction, the probe CNA shows great application potential in lysosomal biological metabolism research.

[0016] The fluorescent probe CNA provided by the present invention has an extremely fast response time to AChE (60s), and the fluorescence intensity is optimal under physiological pH conditions. It can also effectively detect the increase or decrease of AChE in living cells, with a detection limit as low as 0.014 U / mL. Therefore, it can be successfully used for AChE imaging in living cells or living tissues, providing a powerful tool for studying the physiological and pathological effects of AChE in complex biological systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The fluorescent probe CNA of the present invention 1 HNMR spectrum; Figure 2 The fluorescent probe CNA of the present invention 13 CNMR spectrum; Figure 3 The optical reaction detection result of the probe CNA and AChE in Example 2; Figure 4 The response mechanism of the fluorescent probe CNA and AChE of the present invention; Figure 5 The results of the detection of the specific recognition ability of the probe CNA to AChE in Example 3; Figure 6 is a linear relationship diagram between the fluorescence intensity of the probe CNA and the AChE concentration in Example 4; Figure 7 The MTT test results of the probe CNA in Example 5; Figure 8 This is a graph showing the results of the co-localization analysis of probe CNA in PC-12 cells in Example 5; Fig. 9 This is a confocal microscopic image of the probe CNA in Example 5 on exogenous AChE in PC-12 cells; Fig.10 This is a confocal microscopic image of the probe CNA in Example 5 on endogenous AChE in PC-12 cells induced by glutamate; Fig.11 This is a confocal microscopic image of the endogenous AChE of PC-12 cells under the inhibition of neostigmine by the probe CNA in Example 5; Fig.12 This is the fluorescence imaging of the probe CNA in Example 6 on exogenous AChE in living mice. DETAILED DESCRIPTION

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0019] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0020] The preparation of compound 1 used in the following examples is prepared with reference to the document Dyes and Pigments 204 (2022)110443; if no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the field or the product instructions are used; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0021] Example 1 This example provides a method for synthesizing a probe CNA, comprising the following steps: Compound 1 (200 mg, 0.58 mmol), potassium carbonate (243 mg, 1.76 mmol) and dimethylcarbamoyl chloride (252 mg, 2.35 mmol) were dissolved in 20 mL of dichloromethane, stirred at room temperature for 24 h, and then 100 mL of water was added to quench the reaction. The organic phase was extracted three times with dichloromethane (50 mL), and the organic phases were combined and concentrated under reduced pressure. The crude mixture was purified by column chromatography (using a gradient elution of petroleum ether / ethyl acetate from 30:1 to 5:1) to obtain the probe CNA (yield 136.89 mg, yield 56.62%).

[0022] The probe CNA was detected using a Bruker-400 MHz spectrometer (with tetramethylsilane as the internal standard for chemical shift). 1 HNMR spectra and 13 CNMR spectrum, and its 1HNMR spectra and 13 CNMR spectra are Figure 1 and Figure 2 shown. 1 HNMR (400 MHz, CHLOROFORM-d): 9.99 (s, 1H), 8.03 (d, J = 1.7 Hz, 1H), 7.86-7.74 (m, 2H), 7.70 (d, J = 8.7, 1H), 7.51-7.35 (m 2H), 7.17-7.08 (m 2), 6.90 (s, 1H), 2.60 (d, J = 14.8 Hz, 4H), 1.03 (s, 6H). 13 CNMR (101 MHz, chloroform-d): 69.22, 154.77, 153.86, 150.29, 137.07, 134.48,132.79, 131.16, 129.67, 128.79, 123.90, 122.60, 118.59, 113.56, 112.78,78.64, 77.37, 77.06, 76.74, 45.81, 43.03, 39.28, 36.80, 36.56, 32.06, 28.05,8.63. Theoretical calculation: C 26 H 26 N3O2[M+H] + :412.2025, find [M+H] + :412.2012.

[0023] Example 2 For the probe CNA, this example systematically evaluated its optical response to acetylcholinesterase (AChE), including the following: (1) Ultraviolet absorption spectrum.

[0024] CNA solution (10 μM) was prepared in PBS buffer (10 mM, pH 7.4, containing 1% DMSO), AChE (100 U / mL) was added to the CNA solution, and incubated for 10 min. The UV absorption spectra before and after the addition of AChE were measured at room temperature using a Hitachi 3010 spectrophotometer.

[0025] The results are as follows Figure 3 As shown in a: In the absence of AChE, the absorption peak is at 428 nm, but after adding AChE, the absorption peak red-shifts to 435 nm, and the color of the solution changes from colorless to light yellow.

[0026] (2) Fluorescence response of probe CNA to AChE.

[0027] CNA solution (10 μM) was prepared in PBS buffer (10 mM, pH 7.4, containing 1% DMSO) as solvent, AChE (100 U / mL) was added to the CNA solution and incubated for 10 min. The fluorescence response before and after the addition of AChE was detected using a Hitachi F-2500 fluorescence spectrophotometer (λex = 450 nm, slit width dex = dem = 10 nm).

[0028] The results are as follows Figure 3 As shown in b: Under 450 nm excitation, when there is no AChE, the fluorescence emission of the probe CNA is negligible; however, in the presence of AChE, the fluorescence emission at 520 nm increases significantly, and the fluorescence quantum yield (Φ) increases from 0.076 to 0.21.

[0029] (3) Detection of the response time of probe CNA to AChE.

[0030] CNA solution (10 μM) was prepared with PBS buffer (10 mM, pH 7.4, containing 1% DMSO) as solvent. After AChE (100 U / mL) was added to the CNA solution, the changes in fluorescence intensity at different reaction times were detected using a Hitachi F-2500 fluorescence spectrophotometer.

[0031] The results are as follows Figure 3 As shown in Figure c, the fluorescence intensity of the probe CNA reached saturation within 60 seconds and did not increase further. It can be seen that the response time of the probe CNA to AChE is extremely fast, exceeding that of most reported AChE probes, and has the potential for real-time applications.

[0032] (4) Applicable pH range detection of probe CNA.

[0033] CNA solutions (10 μM) were prepared in PBS buffer (10 mM, containing 1% DMSO) with different pH values, and AChE (100 U / mL) was added to each CNA solution and incubated. The fluorescence response before and after the addition of AChE was detected using a Hitachi F-2500 fluorescence spectrophotometer.

[0034] The results are as follows Figure 3 As shown in (d), the fluorescence emission of the probe CNA in the pH range of 2-11 is negligible, but after adding AChE, the fluorescence intensity is significantly enhanced in the pH range of 6.0-8.0. This result shows that the probe is suitable for physiological pH conditions.

[0035] (5) Photostability detection of probe CNA.

[0036] The results of photostability test of probe CNA under 605 nm excitation of 150 W xenon lamp are shown in Figure 3 As shown in e: Under continuous irradiation of a xenon lamp, the probe CNA has excellent optical stability.

[0037] (6) Investigation of the action mechanism of probe CNA and AChE.

[0038] LC-MS (LC-MS, Waters ACQUITY system, acetonitrile and 0.5% formic acid aqueous solution as mobile phase, 45°C) was used to study the reaction of CNA to AChE. The results showed that the retention time of the probe CNA was observed to be 6.08 minutes. When AChE was added to the CNA solution, the retention time shifted to 5.617 min, and the corresponding mass spectrum showed a peak at m / z = 336.17, corresponding to compound 1[M+Na] + This result confirms that compound 1 is formed by the reaction of AChE and probe CNA ( Figure 4 ).

[0039] Computational chemical analysis was also performed to further verify the reaction mechanism. The results showed that the probe CNA and compound 1 had the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). This high energy suppressed the intramolecular charge transfer (ICT) effect, resulting in fluorescence quenching. In addition, the molecular surface electrostatic potential distribution showed that compound 1 had a more uniform electron density distribution throughout the molecule, which was conducive to photon absorption and energy release. In contrast, the electron cloud of the probe CNA was concentrated on the dimethylaminoformyl group, which suppressed the ICT effect and reduced the fluorescence emission. The energy gap between the HOMO and LUMO of the probe CNA was calculated to be 4.91 eV, which is larger than 4.72 eV of compound 1, which is consistent with the experimentally observed absorption red shift and verifies the reliability of the orbital calculation.

[0040] The above results indicate that AChE cleaves the N,N-dicarboxamide group within the probe CNA, releasing a phenolic hydroxyl group; this cleavage restores the intramolecular charge transfer within the entire molecule, leading to fluorescence recovery, demonstrating the effectiveness of the probe CNA in detecting AChE.

[0041] Example 3 In view of the complexity of biological environments, this example uses probe CNA to conduct selective recognition experiments to evaluate its ability to recognize other biologically relevant substances.

[0042] A CNA solution (10 μM) was prepared using PBS buffer (10 mM, pH 7.4, containing 1% DMSO) as a solvent. Common bioactive small molecules were added to the CNA solution, and then fluorescence intensity was detected (the method was the same as in Example 2).

[0043] The results are as follows Figure 5 a, where 1-15 represent glutathione (GSH, 0.5 mM), homocysteine ​​(Hcy, 0.5 mM), cysteine ​​(Cys, 0.5 mM), H2S (0.5 mM), H2O2 (0.5 mM), O 2- (0.5 mM), ONOO - (0.5 mM)、ClO - (0.5 mM), trypsin (100 U / mL), phospholipase (100 U / mL), lysozyme (100 U / mL), phosphatase (100 U / mL), chymosin (100 U / mL), BchE (100 U / mL) and AChE (100 U / mL). From the test results, it can be seen that only AChE caused a significant increase in probe fluorescence, while most other substances did not enhance the probe fluorescence; although BChE also caused a certain degree of fluorescence enhancement, it was only about one-third of the fluorescence enhancement observed with AChE, so it would not significantly impair the selectivity of the probe CNA.

[0044] In order to further evaluate the anti-interference ability of probe CNA to other substances, this example also used the above substances to conduct a competitive reaction experiment. Figure 5 b, where the interfering substances in 1-15 are GSH (1 mM), Hcy (1 mM), Cys (1 mM), H2S (1 mM), H2O2 (0.5 mM), O 2- (0.5 mM), ONOO - (0.5 mM), trypsin (40 U / mL), human serum albumin (40 μg / mL), phospholipase (40 U / mL), lysozyme (40 U / mL), phosphatase (40 U / mL), chymotrypsin (40 U / mL), BchE (40 U / mL) and AChE (40 U / mL). The detection results show that the probe has a high recognition ability in the presence of AChE and other biologically related substances.

[0045] The above results indicate that the probe CNA has the ability to selectively cleave the N,N-diformamide group under complex conditions and has specificity for AChE.

[0046] Example 4 This example verifies the quantitative detection capability of probe CNA for AChE.

[0047] CNA solution (10 μM) was prepared with PBS buffer (10 mM, pH 7.4, containing 1% DMSO) as solvent, AChE was added to the CNA solution and the concentration varied from 0 to 100 U / mL. Finally, a Hitachi F-2500 fluorescence spectrophotometer was used to detect the fluorescence intensity at different AChE concentrations.

[0048] The results are as follows Figure 6 As shown in the figure: With the increase of AChE concentration, the fluorescence intensity of probe CNA at 520 nm gradually increased. After the AChE concentration exceeded 40 U / mL, the fluorescence of the probe tended to stabilize. The fluorescence intensity showed a good linear relationship with the AChE concentration, and the Pearson correlation coefficient was 0.998. The detection limit (LOD) of the probe was determined to be 0.014 U using the formula LOD = 3σ / L, where σ is the standard deviation and L is the signal baseline of the blank sample. This result shows that probe CNA has the ability to quantitatively analyze AChE.

[0049] Example 5 Given that PC-12 cells have abundant AChE activity, this example uses the PC-12 cell detection probe CNA to detect the fluorescence imaging ability of AChE in living cells. The culture medium used for PC-12 cells is RPMI 1640 (Gibco, USA) containing 10% (v / v) fetal bovine serum (Gibco, USA). This example specifically involves the following experiments: (1) Cytotoxicity analysis.

[0050] The MTT assay was used to evaluate the toxicity of probe CNA in PC-12 cells. The results showed that even at the highest concentration of 50 μM, the cell survival rate was over 80% ( Figure 7 ). This indicates that the probe CNA has low toxicity and good biocompatibility.

[0051] (2) Colocalization analysis of intracellular probe CNA.

[0052] PC-12 cells were incubated with 10 μM probe CNA for 30 min, and then replaced with 1 μM lysosomal red fluorescent probe (Lyso-Tracker Red) and incubated for 20 min; after washing 2-3 times with PBS, imaging was performed using a confocal microscope.

[0053] The results are as follows Figure 8 As shown, the colocalization coefficient is 0.87, indicating that the probe CNA has good cell membrane permeability and is mainly enriched in lysosomes.

[0054] (3) Detection of exogenous AChE by probe CNA.

[0055] PC-12 cells were divided into five groups, one of which was added with AChE only, and the other four groups were incubated with 0, 5, 10, and 20 U / mL AChE for 120 min, and then incubated with 10 μM probe for 30 min; washed with PBS 2-3 times, and imaged with a confocal microscope.

[0056] Test results ( Fig. 9 ) showed that only weak fluorescence was observed in PC-12 cells without exogenous AChE, but as the concentration of exogenous AChE increased to 20 U / mL, the fluorescence intensity of living cells gradually increased, eventually reaching 8 times the enhancement of the blank group. It can be seen that the probe CNA can effectively use fluorescence methods to image exogenous AChE.

[0057] (4) Detection of endogenous AChE by probe CNA.

[0058] Western blot confirmed that glutamate (Glu) can induce the expression of AChE in PC-12 cells in a concentration-dependent manner. Therefore, in this case, Glu was used as an inducer to conduct the following experiments: PC-12 cells were divided into three groups and incubated with 0, 5, and 10 μM Glu at 37°C for 48 h, respectively, and then incubated with 10 μM probe at 37°C for 30 min; the cells were washed two to three times with PBS and imaged using a confocal microscope.

[0059] result( Fig.10 ) showed that the fluorescence intensity of PC-12 cells pretreated with Glu increased significantly with the increase of Glu concentration, indicating that the probe CNA can detect the changes in AChE levels caused by Glu.

[0060] (5) Endogenous AChE clearance test.

[0061] Prior art shows that neostigmine is usually used to block AChE expression in PC-12 cells. Therefore, in this example, neostigmine was used as an inhibitor to conduct the following experiments: PC-12 cells were divided into three groups. One group was pretreated with 20 μM inhibitor for 2 h and then incubated with 10 μM probe for 30 min at 37°C. The other two groups were control group (blank group, without probe and inhibitor) and control group (only probe was added for incubation). The cells were washed 2-3 times with PBS and imaged using a confocal microscope.

[0062] The results showed that the probe CNA could not only detect the increase of AChE in living cells, but also effectively identify the decrease ( Fig.11 ).

[0063] In this example of a cell imaging experiment, the probe (green channel) excitation wavelength was set to 430 nm, and the emission light was collected in the 500-700 nm wavelength range.

[0064] Example 6 This example tests the ability of probe CNA to image AChE in living mice. The experimental procedure is as follows: The hair on the back of mice was removed before the experiment. During the experiment, all mice were anesthetized with gas; the probe CNA (00 μL, 100 μL) was injected into the muscles of two groups of mice, and one group was injected with AChE solution (100 μL, 100 U / mL) at the same time.

[0065] Fluorescence imaging (excitation: 500 nm, collection: 560-640 nm) showed that the fluorescence intensity increased significantly by 8 times in mice injected with AChE ( Fig.12 ), indicating the effectiveness of probe CNA in detecting AChE in complex biological systems.

[0066] In summary, the present invention provides a new fluorescent probe CNA that can realize rapid and effective detection of acetylcholinesterase, and the probe has the advantages of high detection sensitivity and low detection limit (0.014 U / mL); more importantly, the present invention successfully uses probe CNA to image AChE in cells and living tissues, so the probe CNA can be used as an effective tool to explore the role of AChE in various nervous system diseases and cancers.

[0067] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A novel fluorescent probe, characterized in that: The fluorescent probe specifically responds to acetylcholinesterase, and the structural formula of the fluorescent probe is as follows: 。 2. Use of the novel fluorescent probe as claimed in claim 1 in detecting acetylcholinesterase activity in vitro or in vivo.

3. Use of the novel fluorescent probe as claimed in claim 1 in fluorescence imaging of acetylcholinesterase in cells or living tissues.

4. Use of the novel fluorescent probe as claimed in claim 1 in real-time monitoring of acetylcholinesterase in vivo.

5. A method for preparing the novel fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: S1. Compound 1, an inorganic base and dimethylaminoformyl chloride are placed in an organic solvent and stirred for reaction; wherein the structural formula of compound 1 is: ； S2: After the reaction of S1 is completed, the organic phase is extracted with an organic solvent and concentrated under reduced pressure to obtain the product.

6. The method according to claim 5, characterized in that The molar ratio of the compound 1, the inorganic base and dimethylaminoformyl chloride is (0.8-1):(3-4):(4-6).

7. The method according to claim 6, characterized in that The inorganic base is selected from potassium carbonate, cesium carbonate and lithium carbonate.

8. The method according to claim 5, characterized in that The following steps are also included: S3. Purify the product obtained in S2 by column chromatography using a petroleum ether / ethyl acetate gradient elution.

9. The method according to claim 5, characterized in that The organic solvent is dichloromethane.

10. The method according to claim 6, characterized in that The reaction temperature in step S1 is 10-37°C.

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