CN111592504B - Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof - Google Patents
Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof Download PDFInfo
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Abstract
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.
Description
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 a reaction medium, using 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 brine, purifying through silica gel column chromatography, and using a 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.
Example 1: synthesis of fluorescent probes
1. The synthesis steps are as follows:
(1) 4- (diethylamino) salicylaldehyde (0.97g, 5.0mmol) and triethylamine (1.40mL, 10.0mmol) were dissolved in 20mL of anhydrous dichloromethane at 0 ℃ followed by addition of cyclopropanecarbonyl chloride (0.63g, 6.0mmol) in 10mL of dichloromethane and the mixture was stirred at room temperature overnight; after quenching, the solution was washed with brine 3 times, the organic phase was dried over anhydrous sodium sulfate, then the solvent was removed by distillation under reduced pressure, and the crude product was purified by column chromatography (PE: EA ═ 10: 1) to give compound 1 in 83% yield, and the reaction process was as shown in formula (1):
(2) weighing 1.25g (10.0mmol) of 2-aminobenzenethiol and 0.8g (12.0mmol) of malononitrile, dissolving in 20mL of absolute ethanol, adding 0.5mL of acetic acid, stirring at room temperature for reaction for 5h, extracting and separating, and carrying out reduced pressure rotary evaporation to obtain a compound 2, wherein the reaction process is shown as a formula (2):
(3) compound 1(0.26g, 1.0mmol) and compound 2(0.19g, 1.1mmol) were added to 30mL of ethanol with stirring, followed by a few drops of piperidine as a catalyst; after the mixture was heated at 80 ℃ for 6 hours, the solvent was removed by distillation under the reduced pressure, and the residue was dissolved in ethyl acetate, and then washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and brine, which was purified by silica gel column chromatography using a mixture (CH)2Cl2: MeOH, 80: 1) as an elution solvent, probe P1(0.153g, yield 63%) was obtained, and the reaction sequence was as shown in the formula (3):
2. and (3) testing and analyzing:
FIG. 1 shows probe P11H NMR spectrum, specific peak value of the spectrum is: δ ppm8.89(s,1H),8.13(d, J ═ 7.9Hz,1H),8.00(d, J ═ 8.1Hz,1H),7.73(d, J ═ 8.9Hz,1H),7.53(t, J ═ 7.7Hz,1H),7.41(t, J ═ 7.6Hz,1H),6.80(d, J ═ 8.9Hz,1H),6.47(s,1H),3.50(q, J ═ 6.9Hz,4H),1.92(d, J ═ 4.1Hz,1H),1.15(t, J ═ 7.0Hz,6H),1.08(s,2H),1.02 to 1.00(m,2H), which corresponds to the probe groupThe successful synthesis of the probe can be proved.
FIG. 2 shows probe P113H NMR spectrum, specific peak value of the spectrum is: delta ppm195.18,164.60,162.00,152.82,145.27,144.65,137.48,135.49,135.17,133.31,131.79,131.60,128.96,127.84,127.76,127.51,126.68,123.48,122.64 and 20.76, corresponding to the probe group, further confirming the correct structure of the probe.
In order to further verify the excellent effect of the synthesized fluorescent probe disclosed by the invention in detecting butyrylcholinesterase activity, the inventors also carried out the following experimental operations:
experiment 1: test of probe's ability to react with butyrylcholinesterase in buffer solution
(1) The synthesized probe was adjusted to 2.0 mmol. multidot.L -120. mu.L of the aqueous solution of (4) was added to a buffer solution containing 2mL of PBS (10 mmol. multidot.L)-1pH 7.4) into a centrifuge tube, 4 μ L of butyrylcholinesterase (50 mg/mL) was added-1) Changes in the absorption spectrum and fluorescence emission spectrum were detected after incubation at 37 ℃ for 90 minutes.
FIG. 4 shows an absorption spectrum (a) and a fluorescence emission spectrum (b) before and after the reaction of the probe with butyrylcholinesterase. As can be seen from FIG. 4(a), the maximum UV absorption of the reacted solution is around 490nm, the basic skeleton of the original probe is retained, and from FIG. 4(b), the fluorescence intensity of the original probe is negligible, and when the probe P1 is hydrolyzed by BChE enzyme, a strong fluorescence signal can be detected at 528nm, which provides a sensing method without background detection.
(2) Taking a series of centrifuge tubes, and respectively adding 2ml PBS buffer solution (10 mmol. L)-1pH 7.4) and 20. mu.L of probe were added to the mixture, followed by addition of butyrylcholinesterase (0-300. mu.g/mL) at a concentration of the series-1) Changes in fluorescence emission spectra were detected after incubation at 37 ℃ for 90 minutes, respectively.
FIG. 5 is a titration experiment of probe to butyrylcholinesterase concentration, from which the obvious fluorescent response of P1 to BChE can be easily captured, and even when BChE concentration is 0.5 μ g/mL, an enhanced fluorescent response can be observed, which suggests that the strategy has higher sensing sensitivity. The fluorescence intensity at 528nm gradually increased with increasing BChE concentration, tending to saturate when BChE concentration levels increased to 40 μ g/mL, and the fluorescence intensity at 528nm showed a good linear relationship with BChE concentration within 0.5-7.0 μ g/mL, resulting in a high correlation coefficient and a low limit of detection (LOD) of 0.092 μ g/mL, which is significantly lower than reported in the literature.
This indicates that butyrylcholinesterase can react specifically with the probe to form a fluorophore with green fluorescence to indicate the concentration level of butyrylcholinesterase.
Experiment 2: inhibitor characterization of fluorescent probes
The change in fluorescence spectra was detected after pretreatment with tacrine (butyrylcholinesterase inhibitor) and butyrylcholinesterase (100. mu.g/mL) at various concentrations for 30 minutes, followed by incubation with probe (20. mu.M) for 90 minutes at 37 ℃.
FIG. 6 is a fluorescence spectrum of an inhibitor after butyrylcholinesterase pretreatment and P1 reaction, and when tacrine is introduced, the fluorescence intensity at 528nm is greatly reduced, so that the inhibitor has an obvious inhibition effect. The probe P1 is shown to have high detection sensitivity to BChE, and the IC of the BChE, tacrine corresponding to 100 mug/mL can be calculated from the inhibition efficiency curve50The value was 8.57 nM.
Thus, the probe is indicated to be a fluorescence turn-on response caused by the enzyme-catalyzed reaction of the probe and butyrylcholinesterase.
Experiment 3: determination of butyrylcholinesterase levels in cells by probes
Three cell lines were incubated with P1 (5. mu.M) for 0.5 hours, respectively. Control group: after HEK293 cells were pretreated with tacrine (50. mu.M) for 0.5h and then with 5. mu.M probe P1 for 30 min, the cells were washed with PBS buffer and images taken after 488nm excitation. As can be seen in fig. 7, bright fluorescence was observed in HEK293 cells due to the normal levels of BChE in this cell line, and the weak fluorescence response in HeLa and HepG2 cells further corroborating the conclusion of serum testing that diseases, especially cancer, may lead to decreased BChE activity. As a control, by inhibiting tacrine pretreatment to inhibit BChE activity, similar results to tumor cells were obtained, and weak fluorescent responses confirmed that probe P1 can specifically detect intracellular BChE activity.
Experiment 4: determination of butyrylcholinesterase levels in brain tissue sections by Probe
Incubate with 10 μ M1 for 0.5 hours, and take images of fresh rat brain sections pretreated with or without 50 μ M tacrine for 0.5 hours after 488nm excitation. As can be seen from FIG. 8, after incubation with P1, strong fluorescence was observed in the figure, especially in the region around the vessel cross-section, whereas after pretreatment with inhibitor tacrine, incubation of the control sample with probe P1 produced a sharp contrast, i.e.the sections pretreated with inhibitor showed a large decrease in response intensity. These results confirm that probe P1 can also be used for brain slice testing.
Experiment 5: determination of butyrylcholinesterase levels in organisms by probes
BChE levels in the experimental mice were within the normal range, sufficient to trigger the sensing procedure. One group of mice was treated with probe P1 and after a period of time the brain region of the mice was examined for an emission response, while another group of mice was injected in situ with tacrine to inhibit BChE activity, and then after a period of time the brain region of the mice was examined for an emission response using probe P1.
As can be seen from fig. 9, mice not treated with tacrine can easily capture the emission response of the brain region within 0.5 hours, which demonstrates a rapid detection of endogenous BChE in living organisms. In the next 2-hour study, fluorescence remained generally in the same range, with an accompanying increase in intensity. Whereas, in mice injected with tacrine in situ to inhibit BChE activity, the weak response lasted for more than 2 hours without a significant decrease in intensity.
The result shows that the synthetic fluorescent probe for detecting butyrylcholinesterase activity disclosed by the invention can visualize the expression level of endogenous BChE with high selectivity, and further shows the potential application of the fluorescent probe in pathological research.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
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 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 for selectively recognizing butyrylcholinesterase in a solvent system for non-diagnostic purposes.
7. The use of claim 6, further comprising the use of said fluorescent probe for non-diagnostic purposes in the detection and screening of butyrylcholinesterase inhibitors using butyrylcholinesterase as a marker.
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