阅读说明：本技术 一种用于检测丁酰胆碱酯酶活性的荧光探针及其合成方法与应用 (Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof ) 是由 丁彩凤 张倩 傅彩霞 滕葆晖 张鹏 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种用于检测丁酰胆碱酯酶活性的荧光探针及其合成方法与应用,其公开了一种在无背景信号检测基础上建立的新型丁酰胆碱酯酶传感策略,在丁酰胆碱酯酶的作用下,实现荧光信号从无到有的转变,进而检测丁酰胆碱酯酶的浓度水平。所述荧光探针通过将丁酰胆碱酯酶的靶向位点引入非发射骨架生成探针P1,避免了检测探针的固有荧光背景,极大地提高了检测灵敏度。在加入丁酰胆碱酯酶后,可以除去环丙基丁酸,进而自发的原位环化,生成荧光产物PF以作为反映BChE活性的指示剂。本发明不仅可以大幅提高检测灵敏度,还具有良好的选择性及生物相容性,在临床诊断领域具有很大的潜力。(The invention discloses a fluorescent probe for detecting butyrylcholinesterase activity and a synthesis method and application thereof, and discloses a novel butyrylcholinesterase sensing strategy established on the basis of background signal-free detection, which realizes the conversion of a fluorescent signal from nothing to nothing under the action of butyrylcholinesterase so as to detect the concentration level of butyrylcholinesterase. The fluorescent probe generates the probe P1 by introducing the target site of butyrylcholinesterase into a non-emission skeleton, so that the inherent fluorescent background of the detection probe is avoided, and the detection sensitivity is greatly improved. After butyrylcholinesterase is added, cyclopropyl butyric acid can be removed, and then spontaneous in-situ cyclization is carried out, so that a fluorescent product PF is generated to serve as an indicator reflecting BChE activity. The invention not only can greatly improve the detection sensitivity, but also has good selectivity and biocompatibility, and has great potential in the field of clinical diagnosis.)

1. A fluorescent probe for detecting butyrylcholine esterase activity is characterized in that the structural formula of the fluorescent probe is as follows:

2. the method for synthesizing a fluorescent probe for detecting butyrylcholinesterase activity according to claim 1, comprising the following steps:

(1) creating an alkaline environment at 0 ℃ with anhydrous dichloromethane as a reaction medium, 4- (diethylamino) -salicylaldehyde and cyclopropanecarbonyl chloride as reactants, and triethylamine, stirring the mixture at room temperature overnight, then performing extraction separation with dichloromethane and deionized water, removing the solvent by distillation under reduced pressure, and purifying the crude product by column chromatography to obtain compound 1;

(2) adding 3-5 drops of acetic acid into absolute ethyl alcohol serving as a reaction medium and 2-aminobenzenethiol and malononitrile serving as reactants to create an acidic environment, stirring and reacting for 4-6 hours at room temperature, extracting and separating, and performing reduced pressure rotary evaporation to obtain a compound 2;

(3) and (2) taking absolute ethyl alcohol as a reaction medium, taking the compound 1 prepared in the step (1) and the compound 2 prepared in the step (2) as reactants, adding piperidine as a catalyst, refluxing for 4-8 h at 60-90 ℃, removing the solvent through reduced pressure rotary evaporation, dissolving the residue in ethyl acetate, washing with dilute hydrochloric acid, saturated sodium bicarbonate solution and saline water, and purifying through silica gel column chromatography to obtain the fluorescent probe P1 for detecting the butyrylcholinesterase activity.

3. The method for synthesizing a fluorescent probe for detecting butyrylcholinesterase activity of claim 2, wherein in step (1), the molar ratio of 4- (diethylamino) -salicylaldehyde to cyclopropane carbonyl chloride to triethylamine is 1: (1.2-1.5): (1.5-2.5).

4. The method for synthesizing a fluorescent probe for detecting butyrylcholinesterase activity according to claim 2, wherein in step (2), the molar ratio of 2-aminobenzenethiol to malononitrile is 1: (1.2-2.5).

5. The method for synthesizing a fluorescent probe for detecting butyrylcholinesterase activity in accordance with claim 2, wherein in said step (3), the molar ratio of compound 1 to compound 2 to piperidine is 1: (1.2-1.5): (1.2-2.5).

6. Use of the fluorescent probe for detecting butyrylcholinesterase activity according to claim 1 or the fluorescent probe synthesized according to any one of claims 2-5 in a solvent system for selectively identifying butyrylcholinesterase.

7. The use of the fluorescent probe for detecting butyrylcholinesterase activity according to claim 6, further comprising the use of the fluorescent probe in the detection of butyrylcholinesterase as a marker and the screening of butyrylcholinesterase inhibitors.

Technical Field

The invention belongs to the technical field of butyrylcholinesterase activity detection, and relates to a method strategy for detecting the activity level of butyrylcholinesterase in cells, tissue slices and organisms. More particularly, it relates to a non-emissive probe and its synthesis method and its application as indicator by spontaneous cyclization to generate fluorophore when detecting butyrylcholinesterase.

Background

Butyrylcholinesterase (BChE) is also called pseudocholinesterase or cholinesterase II. BuChE is released into blood immediately after liver synthesis, so that the BuChE can be used as a sensitive index for evaluating the synthesis function of liver cells, and the measurement of the concentration level of BChE in serum is generally used as a liver function detection test. It is reported that BChE is also closely related to complex neurodegenerative diseases such as Alzheimer's Disease (AD), and BChE level significantly rises with the development of AD, and it is very important to monitor the occurrence of related diseases by tracking BChE activity.

Nowadays, detection methods of BChE activity mainly include enzyme-linked immunosorbent assay (ELISA), raman spectroscopy, pH potentiometry, spectrophotometry, radiochemistry, hydroxylamine colorimetry, 5-dithio-2-nitrobenzoic acid (DTNB) analysis, and the like. With the first three methods, it is difficult to avoid the problems of complicated operation, low sensitivity and poor stability, thus hindering their further application. However, although the DTNB assay uses butyrylcholine as a substrate and has high sensitivity, it may suffer from reduced accuracy due to potential interference with the detection system. And in the hydroxylamine colorimetric method, acetylcholine is used as a reactive substrate, which is disadvantageous for BChE assay, resulting in an inevitable decrease in detection sensitivity.

Therefore, the problem to be solved by those skilled in the art is how to provide a fluorescent probe for detecting butyrylcholinesterase activity with high sensitivity and high selectivity and a synthetic method thereof.

Disclosure of Invention

In view of the above, the present invention provides a non-emissive fluorescent probe for detecting butyrylcholinesterase activity, which is directed to the problems of the prior art.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a fluorescent probe for detecting butyrylcholinesterase activity, wherein the structural formula of the probe is as follows:

the probe introduces a specific reaction site, cyclopropyl butyrate, onto a non-emissive backbone to generate BChE target receptor P1, thereby excluding intrinsic fluorescence in the probe itself. And after BChE is introduced, cyclopropyl butyric acid can be specifically excised, spontaneous cyclization reaction is carried out, and the generated fluorescent product can indicate the activity of BChE. The invention can be visually identified by fluorescence detection equipment, and can also enter living cells, brain tissue slices and organisms to measure endogenous butyrylcholinesterase.

Another purpose of the invention is to provide a synthetic method of a fluorescent probe for detecting butyrylcholinesterase activity.

In order to achieve the purpose, the invention adopts the following technical scheme:

a synthetic method of a fluorescent probe for detecting butyrylcholine esterase activity specifically comprises the following steps:

(1) using anhydrous dichloromethane as a reaction medium, 4- (diethylamino) -salicylaldehyde and cyclopropane carbonyl chloride as reactants, and triethylamine to create an alkaline environment at 0 ℃, stirring the mixture at room temperature overnight, then performing extraction separation by using dichloromethane and deionized water, removing the solvent by reduced pressure distillation, and purifying the crude product by column chromatography (PE: EA ═ 10: 1) to obtain compound 1;

(2) adding 3-5 drops of acetic acid into absolute ethyl alcohol serving as a reaction medium and 2-aminobenzenethiol and malononitrile serving as reactants to create an acidic environment, stirring and reacting for 4-6 hours at room temperature, extracting and separating, and performing reduced pressure rotary evaporation to obtain a compound 2;

(3) using absolute ethyl alcohol as reaction medium, using compound 1 prepared in step (1) and compound 2 prepared in step (2) as reactants, adding piperidine as catalystRefluxing the mixture at 60-90 deg.C for 4-8 h, removing solvent by rotary evaporation under reduced pressure, dissolving the residue in ethyl acetate, washing with dilute hydrochloric acid, saturated sodium bicarbonate solution and brine, purifying by silica gel column chromatography, and purifying with mixture (CH)₂Cl₂: MeOH, 80: 1) and finally obtaining the fluorescent probe P1 for detecting butyrylcholinesterase activity as an elution solvent.

The synthetic route of the fluorescent probe P1 is as follows:

the synthetic method of the fluorescent probe P1 for detecting butyrylcholine esterase activity is simple to operate, convenient and quick to purify and suitable for popularization and application in the market.

The fluorescent probe synthesized by the invention is a non-emission probe, avoids the interference caused by the inherent fluorescence background, can perform spontaneous in-situ substitution reaction after introducing a detection object, generates a green emission fluorophore for indicating the detection object, and can be used for imaging on the level of cells, brain tissue slices and organisms.

In addition, the inventors perform characterization by means of nuclear magnetic resonance hydrogen spectrum, carbon spectrum and the like to indicate that the fluorescent probe P1 is successfully synthesized, and refer to the attached drawings 1 and 2 in the specification.

Preferably, in the step (1), the molar ratio of 4- (diethylamino) -salicylaldehyde to cyclopropane carbonyl chloride to triethylamine is 1: (1.2-1.5): (1.5-2.5).

Preferably, in the step (2), the molar ratio of the 2-aminobenzenethiol to the malononitrile is 1: (1.2-2.5).

Preferably, in the step (3), the molar ratio of the compound 1, the compound 2 and the piperidine is 1: (1.2-1.5): (1.2-2.5).

It should be noted that, aiming at the synthesis reaction of the above-disclosed fluorescent probe for detecting butyrylcholinesterase activity, the inventor obtains various raw material ratios through creative experiments, wherein the ratio of piperidine to triethylamine is particularly important, and the content of piperidine directly affects the degree of reaction progress, which is related to the steps of reaction completion and excessive treatment; and triethylamine influences the acid-base regulation of the reaction, and the reaction is smoothly carried out.

It is still another object of the present invention to provide a specific application of the above fluorescent probe P1 in the detection of butyrylcholinesterase activity.

Particularly comprising the application of the fluorescent probe in selectively recognizing butyrylcholinesterase in a solvent system.

The reaction operation of the fluorescent probe and butyrylcholinesterase is as follows:

adding butyrylcholinesterase into PBS buffer solution containing probe P1, wherein butyrylcholinesterase specifically catalyzes cyclopropyl formate hydrolysis to generate O^-Having nucleophilicity, O^-attack-CN, and then spontaneous in-situ cyclization, to generate the fluorescent product PF. Wherein the fluorescent probe of the invention forms a compound fluorophore PF after reaction with butyrylcholinesterase¹The H NMR spectrum is shown in FIG. 3.

Preferably, the optimal conditions for reacting the probe with butyrylcholinesterase are: the incubation was carried out for 90min at 37 ℃ in PBS buffer at pH 7.4.

The specific reaction equation is as follows:

as shown in fig. 5, the detection limit of the fluorescent probe for butyrylcholinesterase was as low as 0.092 μ g/mL (calculated from LOD 3.3 σ/k), and the detection sensitivity was higher than that of most prior art documents.

In some application scenes, the application of the fluorescent probe in the detection and screening of the butyrylcholinesterase inhibitor by taking butyrylcholinesterase as a marker is also included.

By adopting the technical scheme, the invention has the following beneficial effects:

the synthesized fluorescent probe is used for specific recognition and detection of butyrylcholinesterase, and the structure of the fluorescent probe contains cyclopropyl butyrate of a recognition site of butyrylcholinesterase, the probe is constructed on the basis of a non-emission framework, so that the self fluorescent background interference is avoided, the probe can rapidly cut a cyclopropyl butyrate bond in the presence of butyrylcholinesterase to further perform a cyclization reaction on the probe, and a green fluorescent fluorophore indicator is formed, so that the fluorescent turn-on response of a target object butyrylcholinesterase can be realized.

Preferably, the butyrylcholinesterase is over-expressed in HEK293 cells and is low in expression in some cancer cells, and the synthesized fluorescent probe can enter living cells to detect the butyrylcholinesterase endogenous to the cells.

Preferably, the fluorescent probe can be used for detecting butyrylcholinesterase activity in brain tissue slices and organisms.

According to the technical scheme, compared with the prior art, the invention provides the fluorescent probe for detecting butyrylcholinesterase activity and the synthesis method and application thereof, and the fluorescent probe is a synthesis method of a non-emission probe designed based on zero background and the application thereof in detecting endogenous butyrylcholinesterase in cells, brain tissue slices and organisms, and has the following excellent characteristics:

the invention discloses a synthetic fluorescent probe, which is based on a non-emission skeleton, and forms a target probe by introducing a recognition group of butyrylcholinesterase, wherein the recognition group of the probe is cut in the presence of butyrylcholinesterase to form a fluorophore with green emission property, and the purpose of detecting the level of butyrylcholinesterase is further achieved by the intensity of green fluorescence. The method strategy for detecting the activity of butyrylcholine esterase disclosed by the invention has great market application and popularization values.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 shows a fluorescent probe of the present invention¹H NMR spectrum.

FIG. 2 shows a fluorescent probe of the present invention¹³C NMR spectrum.

FIG. 3 shows the formation of fluorophore by the reaction of the fluorescent probe of the present invention with butyrylcholinesterase¹H NMR spectrum.

FIG. 4 shows an absorption spectrum (a) and a fluorescence emission spectrum (b) of the fluorescent probe of the present invention after reaction with butyrylcholinesterase.

FIG. 5 is a fluorescence spectrum (a) of the fluorescent probe of the present invention reacting with butyrylcholinesterase at different concentrations and a linear response curve (b) of the two reactions.

FIG. 6 shows fluorescence emission spectrum (a) and inhibition efficiency curve (b) of the fluorescent probe of the present invention reacted with butyrylcholinesterase after the addition of an inhibitor.

FIG. 7 is a fluorescent image of the fluorescent probe of the invention used for detecting butyrylcholinesterase activity in three cell strains of HEK293, HeLa and HepG 2.

FIG. 8 is a fluorescence imaging diagram of the fluorescent probe of the invention used for detecting butyrylcholinesterase activity in brain tissue slices.

FIG. 9 is a fluorescence image of the fluorescent probe of the invention used for detecting butyrylcholinesterase activity in nude mice.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a probe for measuring butyrylcholinesterase with high sensitivity and high selectivity as well as a synthetic method and application thereof.

The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention is not to be construed as being limited thereto, and certain insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing disclosure are intended to be included within the scope of the invention.

The invention discloses a probe for measuring butyrylcholine esterase activity, wherein the structural formula of the probe is as follows:

the technical solution of the present invention will be further described with reference to the following specific examples.