CN102964353A - Probe for detecting activity inhibition rate of acetylcholinesterase, application and preparation method - Google Patents
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Abstract
The invention relates to a probe for detecting the activity inhibition rate of acetylcholinesterase, application and a preparation method thereof. The probe has a structure shown as a formula I:
the probe is light yellow and low in fluorescence in a solution, generates strong green fluorescence (the fluorescence emission wavelength is 525 nm) after reacting with Thiocholine (Thiocholine) sulfydryl, has strong selectivity and high sensitivity, and the fluorescence detection method adopting the probe has great application prospect in the field of biomedicine.
Description
Technical field
The present invention relates to a kind of probe for detection of the acetylcholine esterase active inhibiting rate, purposes and preparation method.
Background technology
Alzheimer (AD) is so-called senile dementia, is a kind of lethality nerve degenerative diseases that carries out sexual development, and its cause of disease mainly is because the concentration of the vagusstoff (Ach) in cortex is excessively low; Clinical manifestation constantly worsens for cognitive and memory function, and carrying out property of activity of daily living goes down, and various neuropsychic symptoms and behavior disorder are arranged.Vagusstoff (Ach) is a kind of central neurotransmitter, and acetylcholinesterase (AChE) hydrolysis vagusstoff (Ach) is a significant process of regulating the nervus centralis reactive system.Clinical treatment AD is mainly based on acetylcholinesterase depressant, and therefore needs to detect the technique means of acetylcholine esterase active inhibiting rate.In addition, neural gas, the sterilant that contains organophosphorus and amino formate all are acetylcholinesterase depressant, so the aforementioned techniques means also can be used for detecting neural gas and agricultural chemicals.
The method of traditional detection acetylcholine esterase active inhibiting rate has the Ellmann colorimetric or detects the method for the hydrogen peroxide that the choline oxidation produces, but these detection method sensitivity are low, and interference tends to have powerful connections.
Understand according to the contriver, the detection method of using fluorescent probe has good susceptibility, and angle is started with thus, researches and develops better detection method.
Summary of the invention
Technical problem to be solved by this invention is: overcome the problem that prior art exists, a kind of probe for detection of the acetylcholine esterase active inhibiting rate, purposes are provided, reach the preparation method.
The technology of the present invention design is as follows: known to the contriver, acetyl chloride thiocholine (ATC) can generate thiocholine (Thiocholine) under acetylcholinesterase (AChE) effect, produce the probe of fluorescence as long as preparation can react with the sulfydryl of thiocholine.
The technical scheme that the present invention solves its technical problem is as follows:
A kind of probe for detection of the acetylcholine esterase active inhibiting rate is characterized in that, has suc as formula the structure shown in the I:
Probe of the present invention itself is light yellow and low fluorescence in solution, produce strong green fluorescence (fluorescent emission wavelength 525nm) after reacting with thiocholine (Thiocholine) sulfydryl, and selectivity is strong, and is highly sensitive.
A kind of method for preparing above-mentioned probe is characterized in that, may further comprise the steps:
The first step, will again reaction product be purified suc as formula the fluorescein shown in the A and chloroform, methyl alcohol, crown ether and sodium hydroxide reaction first, obtain the aldehyde of fluorescein list shown in the formula II;
Second step, fluorescein list aldehyde and 2-cyclopentenone that the first step is obtained place the tetrahydrofuran (THF) that contains imidazoles to react, and obtain the probe shown in the formula I.
Further improve and be: the detailed process of the first step is: (1) is put in fluorescein and chloroform, methyl alcohol, 15-hat-5 in the reaction vessel, keep temperature of reaction in 50 ℃-60 ℃ and add sodium hydroxide solution and obtain reaction solution, under this temperature of reaction, continue to stir this reaction solution; (2) when the reaction solution color becomes chocolate, then the cooling stopped reaction adds sulfuric acid acidation and rocks reaction solution, and making reaction solution pH is 2-3, and to make the reaction solution color be tawny and produce precipitation; (3) collecting precipitation adopts silica gel column chromatography to purify after the vacuum-drying, namely get fluorescein list aldehyde; Described silica gel column chromatography adopts the CH of volume ratio 200:3
2Cl
2: CH
3OH is elutriant.
Further improve and be: the detailed process of second step is: (1) adds to fluorescein list aldehyde, 2-cyclopentenone, imidazoles in the tetrahydrofuran (THF), adds deionized water again and mixes, and lasting stirring obtains mixed solution; (2) after mixed solution is concentrated, use the ethyl acetate extraction mixed solution, collected organic layer solution; (3) organic layer solution is obtained probe through the silica gel column chromatography separation; Described silica gel column chromatography adopts the ethyl acetate of volume ratio 25:75: sherwood oil is elutriant.
Preparation method of the present invention can obtain aforementioned target-probe, and step is simple, is easy to realize.
Aforementioned probe is for detection of the purposes of acetylcholine esterase active inhibiting rate.
Compare with existing detection method, adopt the fluorescence detection method of probe of the present invention highly sensitive, at biomedicine field great application prospect is arranged.
Description of drawings
Fig. 1 is the fluorescent reaction time depend on spectra of the embodiment of the invention 2.
Fig. 2 is the fluorescent reaction time depend on spectra of the embodiment of the invention 3.
Fig. 3 is the suppression efficiency figure of the embodiment of the invention 4.
Fig. 4 is the suppression efficiency figure of the embodiment of the invention 5.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.But the invention is not restricted to given example.
The experiment material that relates in the following content and reagent then are commercially available product as not specifying.
Embodiment 1 preparation formula I probe
The first step: (1) is with fluorescein (12mmol) shown in the 4g formula A, 10ml chloroform, 6ml methyl alcohol, 0.06g crown ether (present embodiment adopts 15-hat-5) place in the 100ml flask, keep temperature of reaction to obtain reaction solution in 55 ℃ and the sodium hydroxide solution that adds 20g50%, under this temperature of reaction, continue to stir this reaction solution 5 hours; (2) when the reaction solution color becomes chocolate, inflated with nitrogen protection, then the cooling stopped reaction adds the 10M sulfuric acid acidation, and the limit edged rocks reaction solution, and making reaction solution pH is 2-3, and to make the reaction solution color be tawny and produce precipitation; (3) suction filtration collecting precipitation, dry with vacuum drying oven; (elutriant is the CH of volume ratio 200:3 to adopt the silica gel column chromatography Chromatographic purification
2Cl
2: CH
3OH), namely get formula II fluorescein list aldehyde.
Second step: (1) adds to 360mg fluorescein list aldehyde (1mmol), 164mg2-cyclopentenone (2mmol), 68mg imidazoles (1mmol) in the tetrahydrofuran (THF) (10ml), adding deionized water (10ml) mixes again, continue under the room temperature to stir to obtain mixed solution in 72 hours, with nitrogen protection and lucifuge; (2) with the concentrated mixed solution of underpressure distillation, (3 * 15ml) extraction mixtures, collected organic layer solution is also used Na to use ethyl acetate again
2SO
4Dry; (3) with the concentrated organic layer solution of underpressure distillation, organic layer solution is separated (take the ethyl acetate of volume ratio 25:75: sherwood oil is as elutriant) through silica gel column chromatography, products therefrom is formula I probe, yield is 21%.
Formula I probe
1H NMR (DMSO-d
6, 250MHz) δ (ppm): 10.31 (1H, s), 8.75 (1H, d, J=7.5Hz), 7.88-7.78 (2H, m), 7.69 (1H, s), (7.37 1H, t, J=7.5Hz), 6.79 (1H, d, J=8.25Hz), 6.80-6.76 (2H, m), 6.67-6.65 (2H, m), 5.46 (1H, t, J=8Hz), (2.79-2.65 2H, m), 2.20-2.00 (2H, m).
Formula I probe
13C NMR (DMSO-d6,62.5MHz) δ (ppm): 200.7,168.4,159.6,156.2,151.7,151.4,151.1,149.1,135.7,131.8,131.7,131.4,130.3,129.0,126.2,124.7,124.1,120.0,113.2,112.6,112.5,110.4,109.3,109.0,102.7,82.3,81.9,75.6,36.7,27.4.
The present embodiment operational path is as follows:
The spectral quality of embodiment 2 acetylcholine esterase actives
30 μ l formula I probes (10 μ M), 30 μ L acetyl chloride thiocholines (ATC) (10 μ M) are added in 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffered soln of 10mM, add again the acetylcholinesterase that final concentration is 0.05U/ml (AChE), then test its fluorescent emission time depend on spectra, excite with 485nm when fluorescence emission spectrum is measured, the detection wavelength is 525nm; Exciting with the slit width of launching is 1.5nm.
The result as shown in Figure 1, after adding acetylcholinesterase (AChE), reaction system can be at 525nm place generation green fluorescence, and is swift in response, and namely reacts completely in 5 minutes.
The spectral quality of embodiment 3 different concns acetylcholine esterase actives
Get the series reaction container, add respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffered soln of equivalent 10mM, add respectively again 30 μ l formula I probes (10 μ M), 30 μ L acetyl chloride thiocholines (ATC) (10 μ M); Then add respectively the acetylcholinesterase (AChE) of 0,0.001,0.003,0.006,0.0125,0.025,0.05U/ml, and test respectively the fluorescent emission time depend on spectra, excite with 485nm when fluorescence emission spectrum is measured, the detection wavelength is 525nm; Exciting with the slit width of launching is 1.5nm.
The result as shown in Figure 2, the speed of acetylcholinesterase (AChE) catalyzed reaction is accelerated with enzyme concn.
Get the series reaction container, add respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffered soln of equivalent 10mM, add respectively again 30 μ l formula I probes (10 μ M), 30 μ L acetyl chloride thiocholines (ATC) (10 μ M); Then add respectively the acetylcholinesterase (AChE) of 0.006U/mL and the acetylcholinesterase depressant Neostigmine of 0,0.5,1,5,10nM, the blank tube that does not add enzyme acetylcholine and inhibitor is set simultaneously; Then test respectively the fluorescent emission time depend on spectra, excite with 485nm when fluorescence emission spectrum is measured, the detection wavelength is 525nm; Exciting with the slit width of launching is 1.5nm.
Calculate the suppression efficiency of acetylcholinesterase depressant Neostigmine in each reaction vessel according to following formula:
F
(inhibitor)For adding the fluorescence intensity of enzyme acetylcholine and inhibitor; F
(noinhbitor)For only adding the not fluorescence intensity of inhibiting of enzyme acetylcholine; F
0For not adding the fluorescence intensity of enzyme acetylcholine and inhibitor.
Calculate according to a conventional method and can draw, acetylcholinesterase depressant Neostigmine is to the inhibiting rate IC of acetylcholine esterase active
50=4.1nM.
The result as shown in Figure 3, the inhibiting rate of acetylcholinesterase depressant Neostigmine increases with inhibitor concentration.
Embodiment 5 acetylcholinesterase depressant Tacrine are to the detection of acetylcholine esterase active inhibiting rate
Get the series reaction container, add respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffered soln of equivalent 10mM, add respectively again 30 μ l formula I probes (10 μ M), 30 μ L acetyl chloride thiocholines (ATC) (10 μ M); Then add respectively the acetylcholinesterase (AChE) of 0.006U/mL and the acetylcholinesterase depressant Tacrine of 0,10,50,100,150nM, the blank tube that does not add enzyme acetylcholine and inhibitor is set simultaneously; Then test respectively the fluorescent emission time depend on spectra, excite with 485nm when fluorescence emission spectrum is measured, the detection wavelength is 525nm; Exciting with the slit width of launching is 1.5nm.
Calculate the suppression efficiency of acetylcholinesterase depressant Tacrine in each reaction vessel according to following formula:
F
(inhibitor)For adding the fluorescence intensity of enzyme acetylcholine and inhibitor; F
(noinhbitor)For only adding the not fluorescence intensity of inhibiting of enzyme acetylcholine; F
0For not adding the fluorescence intensity of enzyme acetylcholine and inhibitor.
Calculate according to a conventional method and can draw, acetylcholinesterase depressant Tacrine is to the inhibiting rate IC of acetylcholine esterase active
50=43.5nM.
The result as shown in Figure 4, the inhibiting rate of acetylcholinesterase depressant Tacrine increases with inhibitor concentration.
Claims (5)
1. the probe for detection of the acetylcholine esterase active inhibiting rate is characterized in that, has suc as formula the structure shown in the I:
2. a method for preparing the described probe of claim 1 is characterized in that, may further comprise the steps:
The first step, will again reaction product be purified suc as formula the fluorescein shown in the A and chloroform, methyl alcohol, crown ether and sodium hydroxide reaction first, obtain the aldehyde of fluorescein list shown in the formula II;
Second step, fluorescein list aldehyde and 2-cyclopentenone that the first step is obtained place the tetrahydrofuran (THF) that contains imidazoles to react, and obtain probe shown in the formula I.
3. described method according to claim 2, it is characterized in that, the detailed process of the first step is: (1) is put in fluorescein and chloroform, methyl alcohol, 15-hat-5 in the reaction vessel, keep temperature of reaction in 50 ℃-60 ℃ and add sodium hydroxide solution and obtain reaction solution, under this temperature of reaction, continue to stir this reaction solution; (2) when the reaction solution color becomes chocolate, then the cooling stopped reaction adds sulfuric acid acidation and rocks reaction solution, and making reaction solution pH is 2-3, and to make the reaction solution color be tawny and produce precipitation; (3) collecting precipitation adopts silica gel column chromatography to purify after the vacuum-drying, namely get fluorescein list aldehyde; Described silica gel column chromatography adopts the CH of volume ratio 200:3
2Cl
2: CH
3OH is elutriant.
4. described method according to claim 2, it is characterized in that the detailed process of second step is: (1) adds to fluorescein list aldehyde, 2-cyclopentenone, imidazoles in the tetrahydrofuran (THF), adds deionized water again and mixes, and lasting stirring obtains mixed solution; (2) after mixed solution is concentrated, use the ethyl acetate extraction mixed solution, collected organic layer solution; (3) organic layer solution is obtained probe through the silica gel column chromatography separation; Described silica gel column chromatography adopts the ethyl acetate of volume ratio 25:75: sherwood oil is elutriant.
5. the described probe of claim 1 is for detection of the purposes of acetylcholine esterase active inhibiting rate.
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| CN110408069A (en) * | 2019-08-02 | 2019-11-05 | 西北大学 | A kind of molecularly imprinted polymer cladding carbon dots fluorescence probe and its preparation method and application |
| CN113004220A (en) * | 2021-03-12 | 2021-06-22 | 南京工业大学 | Esterase detection fluorescent probe, preparation method and application |
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| CN114853779A (en) * | 2022-06-09 | 2022-08-05 | 南京工业大学 | Fluorescent probe for detecting biological thiol in cells and organisms and quickly labeling thiol protein specifically, preparation method and application |
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