CN114539078B - Active small molecular probe for specifically sorting monoamine oxidase, preparation method and application thereof - Google Patents

Active small molecular probe for specifically sorting monoamine oxidase, preparation method and application thereof Download PDF

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CN114539078B
CN114539078B CN202210161073.3A CN202210161073A CN114539078B CN 114539078 B CN114539078 B CN 114539078B CN 202210161073 A CN202210161073 A CN 202210161073A CN 114539078 B CN114539078 B CN 114539078B
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吴琼
吴宛霞
李林
姬文辉
杨雅
李�杰
杜威
黄维
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Nanjing Tech University
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Abstract

The invention discloses an active small molecular probe for specifically sorting monoamine oxidase, a preparation method and application thereof. The structural formula of the in vitro specific probe is shown as follows
Figure DDA0003514715270000011
When R is 1 Is composed of
Figure DDA0003514715270000012
When the in vitro specific probe has a structural formula
Figure DDA0003514715270000013
The in vitro specific probe is specifically combined with MAO-A and is marked as C-TCO; r 1 Is composed of
Figure DDA0003514715270000014
When the in vitro specific probe has a structural formula
Figure DDA0003514715270000015
The in vitro specific probe binds specifically to MAO-B, denoted PA-TCO. The trans-cyclooctene structure of the specific probe generates a bioorthogonal reaction with a dye with a 1,2,4, 5-tetrazine derivative structure, a product can generate strong fluorescence, and the activity of monoamine oxidase A/B in different biological systems is determined by fluorescence change. The probe is prepared by a full-chemical synthesis method, the raw materials are cheap and easy to obtain, the preparation process is simple and easy to synthesize, the protein can be specifically identified, the detection speed is high, and the probe is used for in-vitro semi-quantitative typing detection of monoamine oxidase.

Description

Active small molecule probe for specifically sorting monoamine oxidase, preparation method and application thereof
Technical Field
The invention belongs to the field of organic/biochemical technology, and particularly relates to an active small molecular probe for specifically sorting monoamine oxidase, and a preparation method and application thereof.
Background
Proteins are essential substances for all organisms and can be used as important biomarkers for health conditions and disease states, and rapid detection of proteins is critical for diagnosis and treatment of diseases. The method commonly used today for detecting protein level expression is western blotting, which is a protein detection technique for detecting a specific antigen with a specific antibody, and is now widely used in many fields such as gene expression research at protein level, antibody activity detection, and early diagnosis of diseases. The detection method has many disadvantages, such as long incubation time of the antibody, high antibody cost and complicated operation process. The existing other methods for detecting protein comprise immunofluorescence, liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay and the like, and the methods still cannot get rid of antibodies, require expensive instruments and professional technicians, and are difficult to realize quantitative detection and the like. Therefore, there is a need to develop a low-cost, high-throughput, high-specificity, low-usage, or even antibody-free protein detection method for analyzing proteins in complex samples.
Monoamine oxidase (MAOs) is A protein expressed on the outer mitochondrial membrane, and MAOs are classified into two subtypes, MAO-A and MAO-B, according to their binding specificity to natural substrates or probes, and abnormally expressed MAO-A or MAO-B causes neurodegenerative diseases and disorders, such as the overexpression of MAO-A in humans, which is strongly linked to schizophreniA and depression, and MAO-B is abundantly expressed in the brains of patients with parkinson's disease and alzheimer's disease. However, MAO-A and MAO-B are highly homologous, with A degree of gene similarity of up to 90% and A degree of active amino acid sequence similarity of up to 70%, which are difficult to distinguish. Therefore, there is a need to develop a specific differentiation method to realize typing and early prognosis of neurodegenerative diseases at the genetic level.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an active small molecule probe for specifically detecting monoamine oxidase, and a preparation method and application thereof. The probe subversively replaces the incubation and application of the antibody in the traditional detection by a preparation method of full chemical synthesis, the raw materials are easy to obtain, the preparation process is simple, the subsequent detection process does not need fussy operation, and the monoamine oxidase can be specifically identified.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
the structural formula of the small molecular probe for specifically sorting the activity of monoamine oxidase is shown as follows
Figure BDA0003514715250000011
Wherein R is 1 Is composed of
Figure BDA0003514715250000021
As an improvement, when R1 is
Figure BDA0003514715250000022
When the in vitro specific probe is used, the structural formula is shown as follows,
Figure BDA0003514715250000023
the in vitro specific probe is specifically combined with MAO-A and is marked as C-TCO; r1 is
Figure BDA0003514715250000024
When the in vitro specific probe has the structural formula of->
Figure BDA0003514715250000025
The in vitro specific probe binds specifically to MAO-B, denoted PA-TCO.
The preparation method of the in vitro specific probe is that when R1 is
Figure BDA0003514715250000026
When the in vitro specific probe has the structural formula of->
Figure BDA0003514715250000027
The preparation method is specifically marked as CL-TCO and comprises the following steps:
the first step is as follows: the reaction time required in the synthesis process of the 2-chloro-4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol is 4-10h; the second step is that: heating reflux time of the synthetic process of the tert-butyl (2-chloro-4-methylphenoxy) dimethylsilane is 2-8h; the third step: the reaction time of the synthesis process of the (4- (bromomethyl) -2-chlorophenoxy) (tert-butyl) dimethylsilane is 5-10h; the fourth step: (E) -the reaction time of the synthesis process of tert-butyl (2-chloro-4- ((cyclooct-4-en-1-yloxy) methyl) phenoxy) dimethylsilane is 2-10h; the fifth step: (E) -synthesis of N- (3- (2-chloro-4- ((cyclooct-4-en-1-oxy) methyl) phenoxy) propyl) -N-methylpropan-2-yn-1-amine (E) -2-chloro-4- ((cyclooct-4-en-1-propoxy) methyl) phenol, 1, 3-dibromopropane are dissolved in acetonitrile, potassium carbonate is added, reflux is carried out for 2-4h under nitrogen protection, N-methylpropan-2-amine is added after cooling to room temperature, and the potassium carbonate is stirred for 22-48h at 40 ± 10 ℃;
when R2 is
Figure BDA0003514715250000031
When the in vitro specific probe has a structural formula
Figure BDA0003514715250000032
The specific preparation method is marked as PA-TCO and comprises the following steps:
the first step is as follows: (E) The reaction process of the-cycloocta-4-ene-1-yl (4-nitrophenyl) carbonate is carried out for 10-20h at room temperature; the second step is that: (E) The reaction process of the-cycloocta-4-en-1-yl (2-bromoethyl) carbamate is carried out for 14 to 22 hours at room temperature; the third step: stirring 4- ((methyl (prop-2-yne-1-yl) amino) methyl) phenol at room temperature for 30-90min during the reaction process, and adding sodium acetate to react for 4-10h; the fourth step: (E) During the reaction of-cyclooct-4-en-1-yl (2- (4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenoxy) ethyl) carbamate, 4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol, (E) -cyclooct-4-en-1-yl (2-bromoethyl) carbamate and potassium carbonate are dissolved in acetonitrile solution, and heated under nitrogen for reflux for 10-20h.
200 μ L of a monoamine oxidase reaction system including PBS buffer with pH =6-8, monoamine oxidase A/B (20-80 μ g/mL), CL-TCO/PA-TCO (20-50 μ M) was prepared in advance, and pre-incubated at 37 ℃ for 0.5-2h with shaking. The procedure for separating the probe-protein conjugate with the separation column was as follows: remove the bottom seal of the column and release the lid (without removing the lid). The column was placed in a 1.5-2.0mL collection tube and centrifuged for 1-30min to remove the stock solution.
Sample loading: the column was placed in A new 0.5-5.0m centrifuge tube, and 30-130. Mu. of MAO-A/MAO-B solution and C-TCO/PA-TCO solution were slowly added dropwise to the center of the column packed resin bed, centrifuged for 2-30min, and MAO-A/B sample recognized by the specific probe was collected.
The application of the in vitro specific probe in vitro typing detection of monoamine oxidase A/B is characterized in that a trans-cyclooctene structure of the specific probe can perform bio-orthogonal reaction with a dye with a 1,2,4, 5-tetrazine structure, the specific probe does not have fluorescence, the fluorescence of a dye molecule with the 1,2,4, 5-tetrazine structure is weak, the product can generate stronger fluorescence, and the activity of monoamine oxidase A/B in different biological systems can be determined through fluorescence change. And (2) reacting the specifically bound protein-probe solution with a dye solution with a 1,2,4, 5-tetrazine structure in equal volume, wherein the concentration of the protein solution modified by the specific probe is 5-50 times of that of the dye solution with the 1,2,4, 5-tetrazine structure, the reaction time is 5-30min, the reaction temperature is 20-37 ℃, after the reaction, a fluorescence scanning detection (Ex =430 nm) is carried out, and the fluorescence value is increased, so that the probe is proved to successfully and specifically recognize the monoamine oxidase.
Has the advantages that:
compared with the prior art, the in vitro specific probe and the preparation method and the application thereof have the following advantages:
1. high specificity: the synthesized specific probe can be covalently combined with a target protein to specifically recognize the protein;
2. the method is cheap and fast: the specific probe C-TCO/PA-TCO of the MAO-A/MAO-B can be obtained by chemical synthesis, the synthesis raw materials are cheap and easy to obtain, and the synthesis process is simple and easy to implement;
3. the detection step does not need complex antibody incubation and cleaning processes, and the detection process is rapid.
Drawings
FIG. 1 shows the molecular structure of the specific probe C-TCO for MAO-A;
FIG. 2 shows the molecular structure of specific probe PA-TCO for MAO-B;
FIG. 3 (A) is A scheme for the synthesis of the specific probe C-TCO for MAO-A;
FIG. 3 (B) is nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum of compound B2;
FIG. 3 (c) is a nuclear magnetic hydrogen spectrum, a carbon spectrum and a high-resolution mass spectrum of the compound B3;
FIG. 3 (d) is a nuclear magnetic hydrogen spectrum, a carbon spectrum and a high-resolution mass spectrum of the compound B5;
FIG. 3 (e) is a nuclear magnetic hydrogen spectrum, a carbon spectrum and a high-resolution mass spectrum of the compound B6;
FIG. 3 (f) is nuclear magnetic hydrogen, carbon and high resolution mass spectra of compound CL-TCO;
FIG. 4 (a) is a synthesis route of specific probe PA-TCO for MAO-B;
FIG. 4 (b) is nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum of compound C3;
FIG. 4 (C) is a nuclear magnetic hydrogen spectrum, a carbon spectrum and a high resolution mass spectrum of compound C4;
FIG. 4 (d) is a nuclear magnetic hydrogen spectrum, a carbon spectrum and a high resolution mass spectrum of compound C6;
FIG. 4 (e) is nuclear magnetic hydrogen, carbon and high resolution mass spectra of compound PA-TCO;
FIG. 5 is a schematic diagram of a mechanism for simulating specific probe recognition by molecular docking;
FIG. 6 is A fluorescence spectrum of the bioorthogonal reaction of MAO-A-C-TCO with 1,2,4, 5-tetrazine structural dye;
FIG. 7 is a fluorescence spectrum of the bio-orthogonal reaction of MAO-B-PA-TCO with 1,2,4, 5-tetrazine structural dye.
Detailed Description
Example 1 chemical Synthesis of the specific Probe C-TCO MAO-A
The CL-TCO structure is shown in figure 1, and the synthesis process is shown in figure 3 (a).
The first step is as follows: dissolving 2-chloro-4- ((methyl (prop-2-yne-1-yl) amino) methyl) phenol (1 eq.) and tert-butyldimethylchlorosilane (1.5 eq.) in dichloromethane, adding dropwise triethanolamine under the protection of ice bath and nitrogen, reacting for 6h, adding water after the reaction is finished, and extracting with dichloromethane. The organic phase is washed with 1M dilute hydrochloric acid. Adding anhydrous sodium sulfate and drying. Purification by flash column chromatography followed by spin drying gave tert-butyl (2-chloro-4-methylphenoxy) dimethylsilane. The spectrum analysis is shown in FIG. 3 (b), wherein i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high resolution mass spectrum.
The second step is that: dissolving tert-butyl (2-chloro-4-methylphenoxy) dimethylsilane (1 eq.) in carbon tetrachloride, adding N-bromosuccinimide (1 eq.) under the condition of ice bath, and heating and refluxing for 3h. Adding water for extraction. Drying over anhydrous sodium sulfate gave (4- (bromomethyl) -2-chlorophenoxy) (tert-butyl) dimethylsilane. The spectrum analysis is shown in FIG. 3 (c), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high resolution mass spectrum.
The third step: trans-cyclooctenol (1.5 eq.) was dissolved in N, N-dimethylformamide, sodium hydride (2 eq.) was slowly added under ice bath conditions, stirred at room temperature for 30min, then 4-hydroxy trans-cyclooctene (1 eq.) was added, and reacted for 5h. After the reaction, water was added to quench the reaction, followed by extraction with dichloromethane. Dried over anhydrous sodium sulfate. Purification by flash column chromatography and spin-drying gave (E) -tert-butyl (2-chloro-4- ((cyclooct-4-en-1-yloxy) methyl) phenoxy) dimethylsilane. The spectrum analysis is shown in FIG. 3 (d), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high resolution mass spectrum.
The fourth step: (E) -tert-butyl (2-chloro-4- ((cyclooct-4-en-1-yloxy) methyl) phenoxy) dimethylsilane (1 eq.) was dissolved in tetrahydrofuran and tetrabutylammonium fluoride (1.2 eq.) was added dropwise under ice-bath conditions. Stir at rt for 2h. After the reaction is finished, water is added for quenching. Extraction was carried out with dichloromethane. Dried over anhydrous sodium sulfate. Purification by flash column chromatography and spin-drying gave (E) -2-chloro-4- ((cyclooct-4-en-1-propoxy) methyl) phenol. The spectrum analysis is shown in FIG. 3 (e), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high-resolution mass spectrum.
The fifth step: (E) -2-chloro-4- ((cyclooct-4-en-1-propoxy) methyl) phenol (1 eq.) and 1, 3-dibromopropane (2 eq.) were dissolved in acetonitrile, and potassium carbonate (1 eq.) was added and refluxed under nitrogen for 3h. After cooling to room temperature, N-methylpropan-2-amine (3 eq.) and potassium carbonate (3 eq.) were added and stirred at 40 ℃ for 24h. The solvent was spin dried and extracted with water and ethyl acetate. Dried over anhydrous sodium sulfate. Purification by flash column chromatography, spin-drying to give the final compound CL-TCO. The spectrum analysis is shown in FIG. 3 (f), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high resolution mass spectrum.
Example 2 chemical Synthesis of PA-TCO as specific Probe MAO-B
The PA-TCO structure is shown in FIG. 2, and the synthesis process is shown in FIG. 4 (a).
The first step is as follows: 4-Hydroxytrans-cyclooctene and pyridine (1.2 eq.) were dissolved in dichloromethane in an ice bath and 4-nitrophenylcarbonyl chloride (1.2 eq.) was slowly added dropwise and stirred at room temperature for 12h. After the reaction was completed, water and methylene chloride were added for extraction, and the organic phase was washed with 1M aqueous sodium carbonate solution, dried over anhydrous sodium sulfate, purified by flash column chromatography, and spin-dried to obtain (E) -cyclooct-4-en-1-yl (4-nitrophenyl) carbonate. The spectrum analysis is shown in FIG. 4 (b), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high-resolution mass spectrum.
The second step is that: 2-bromoethyl-1-amine (6 eq.) was dissolved in dichloromethane and (E) -cyclooct-4-en-1-yl (4-nitrophenyl) carbonate (1 eq.) was slowly added dropwise under nitrogen protection and stirred at room temperature for 16h. After the reaction is completed, extraction is added. The organic phase was washed with aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, purified by flash column chromatography, and spin-dried to give (E) -cyclooct-4-en-1-yl (2-bromoethyl) carbamate. The spectrum analysis is shown in FIG. 4 (c), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high-resolution mass spectrum.
The third step: 4-hydroxybenzaldehyde (1 eq.) is dissolved in tetrahydrofuran, 4-hydroxybenzaldehyde-1 (1.2 eq.) is added to the reaction mixture under nitrogen protection, the mixture is stirred at room temperature for 30min, and then sodium acetate (1.4 eq.) is added to the mixture for reaction for 5h. The reaction mixture was quenched with saturated sodium bicarbonate. Extract with ethyl acetate (4 × 20 mL). The organic layers were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Purification by flash column chromatography, spin-dried to give 4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol. The spectrum analysis is shown in FIG. 4 (d), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high-resolution mass spectrum.
The fourth step: 4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol (1 eq.), (E) -cyclooct-4-en-1-yl (2-bromoethyl) carbamate (1.2 eq.), potassium carbonate (2 eq.) were dissolved in acetonitrile solution and heated under reflux for 12h under nitrogen. After the reaction was complete, the solvent was spin dried. 1M dilute hydrochloric acid was added to adjust the pH to 7.0. Dichloromethane was added for extraction. Drying with anhydrous sodium sulfate, purifying by flash column chromatography, and spin-drying to obtain final product PA-TCO. The spectrum analysis is shown in FIG. 4 (e), (i) is NMR hydrogen spectrum, (ii) is NMR carbon spectrum, and (iii) is high resolution mass spectrum.
Example 3 simulation of the recognition of specific probes by molecular docking (the mechanism of recognition of specific probes for MAO-A/MAO-B is the same)
As shown in FIG. 5, 200. Mu.L of a monoamine oxidase reaction system including PBS buffer with pH =7.4, monoamine oxidase A/B (50. Mu.g/mL), CL-TCO/PA-TCO (20. Mu.M) was prepared in advance, and preincubated at 37 ℃ with shaking for 1h.
Example 4 purification of specifically bound protein-probe conjugates by separation column
Placing a separation column: the bottom seal of the column was removed (the lid was released) and placed in a 2.0mL collection tube and centrifuged at 1500 Xg for 1min to remove the stock solution. The column is marked on the upwardly inclined side of the compacted resin and the mark is kept outward during all subsequent centrifugation steps.
Sample loading: a0.5 mL fresh centrifuge tube was placed on the column, the lid removed, and the 200. Mu.L reaction was slowly placed in the center of the compacted resin bed and centrifuged at 1500 Xg for 2min to collect the specifically bound protein-probe conjugate.
Example 5 bioorthogonal reaction fluorescence assay
(1) A dye solution of 1 μ M1,2,4, 5-tetrazine structure (the 1,2,4, 5-tetrazine structure is as follows) was prepared with PBS buffer solution of pH =7.4
Figure BDA0003514715250000061
Tz-TER), 100 μ of 10 μ M specifically bound MAO-A-CL-TCO conjugate solution was mixed with 100 μ of 1 μ M1,2,4, 5-tetrazine structured dye solution, and shaken at 37 ℃ for 20min. After 20min of reaction, fluorescence scan detection (Ex =505 nm) was performed, and the results are shown in fig. 6.
(2) mu.M dye solution of 1,2,4, 5-tetrazine structure was prepared with PBS buffer pH =7.4, 100. Mu.L of 10. Mu.M specifically bound MAO-B-PA-TCO conjugate solution was mixed with 100. Mu.L of 1. Mu.M dye solution of 1,2,4, 5-tetrazine structure, and shaken at 37 ℃ for 20min. After 20min of reaction, fluorescence scan detection (Ex =505 nm) was performed, and the results are shown in fig. 7.
If the probe does not specifically bind to the protein, the protein sample separated from the separation column will be a protein sample without the probe, and will not undergo bioorthogonal reaction with the dye of 1,2,4, 5-tetrazine structure, and will not produce fluorescence enhancement effect. If the probe binds specifically to the protein, a sample of the protein-probe conjugate that will be specifically bound that is separated from the separation column will undergo a bio-orthogonal reaction with the dye of the 1,2,4, 5-tetrazine structure, resulting in an increase in fluorescence. As can be seen from FIGS. 6 and 7, the fluorescence intensity after the reaction was increased, confirming that all the proteins in the sample separated from the rotary spin column had been specifically bound by the probe. The active small molecule probe provided by the invention can specifically detect monoamine oxidase.
In conclusion, the specific probe (E) -N- (3- (2-chloro-4- ((cyclooctane-4-ene-1-oxy) methyl) phenoxy) propyl) -N-methylpropane-2-alkyne-1-amine of MAO-A is designed, and is represented by C-TCO (the C-TCO molecular structure is shown in figure 1), the characteristic structure of the probe, namely propargylamine substituted chlorophenol can be used as A targeting part, and the MAO-A is specifically, quantitatively and covalently bound; a specific probe (E) -cyclooctane-4-ene-1-yl (2- (4- ((methyl (prop-2-yne-1-yl) amino) methyl) phenoxy) ethyl) carbamate of MAO-B is also designed, PA-TCO is used for representing below (the molecular structure of the PA-TCO is shown in figure 2), the characteristic structure of the probe, namely propargylamine substituted phenol can be used as a targeting moiety, and the mechanism of the specific probe for identifying the protein by covalent binding with MAO-B is specifically quantified and shown in figure 5. Meanwhile, the structures of the two probe molecules also comprise a trans-cyclooctene structure, so that the trans-cyclooctene structure can perform bio-orthogonal reaction with the dye molecules with 1,2,4, 5-tetrazine structures, the fluorescence of the product after the reaction is enhanced, and the activity of monoamine oxidase A/B in different biological systems can be measured through fluorescence change.
In view of the fast reaction rate and high fluorescence characteristic of the 1,2,4, 5-tetrazine structure in the chemical reaction, after the specific quantitative covalent binding of MAO-A/MAO-B and the corresponding probe, the dye molecule with the 1,2,4, 5-tetrazine structure and the trans-cyclooctene structure of the specific probe C-TCO/PA-TCO modified on MAO-A/MAO-B can generate the bio-orthogonal chemical reaction, and the fluorescence of the product is enhanced. The monoamine oxidase A/B activity in different biological systems can be determined by fluorescence change.

Claims (3)

1. The small molecular probe for specifically detecting the activity of monoamine oxidase is characterized in that the structural formula of the in vitro specific probe is shown as follows
Figure QLYQS_1
Wherein R is 1 Is->
Figure QLYQS_2
When the in vitro specific probe is used, the structural formula of the in vitro specific probe is shown as
Figure QLYQS_3
When the probe is used, the probe is marked as C-TCO, and the in vitro specific probe is specifically combined with MAO-A; or R 1 Is composed of
Figure QLYQS_4
In which the in vitro specific probe has the formula->
Figure QLYQS_5
The in vitro specific probe binds specifically to MAO-B, denoted PA-TCO.
2. The method for preparing the small molecule probe for specifically detecting monoamine oxidase according to claim 1, wherein R1 is
Figure QLYQS_6
In which the in vitro specific probe has the formula->
Figure QLYQS_7
The specific preparation method is as follows: the first step is as follows: dissolving 1eq of 2-chloro-4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol and 1.5eq of tert-butyldimethylchlorosilane in dichloromethane, adding dropwise triethanolamine under ice bath and nitrogen protection, reacting for 4-10h, adding water after the reaction is finished, extracting with dichloromethane, washing an organic phase with 1M dilute hydrochloric acid, adding anhydrous sodium sulfate, drying, purifying by flash column chromatography, and spin-drying to obtain tert-butyl (2-chloro-4-methylphenoxy) dimethylsilane; the second step: dissolving tert-butyl (2-chloro-4-methylphenoxy) dimethylsilane 1eq in carbon tetrachloride, adding N-bromosuccinimide 1eq under the ice bath condition, heating and refluxing for 3h, adding water for extraction, and drying anhydrous sodium sulfate to obtain (4- (bromomethyl) -2-chlorophenoxy) (tert-butyl) dimethylsilane; the third step: dissolving trans-cyclooctenol 1.5eq in N, N-dimethylformamide, slowly adding sodium hydride 2eq under the ice bath condition, stirring at room temperature for 30min, adding 4-hydroxy trans-cyclooctene 1eq, and reacting for 5-10h; after the reaction is finished, adding water to quench the reaction, extracting the reaction product by dichloromethane, drying the reaction product by anhydrous sodium sulfate, purifying the reaction product by flash column chromatography, and spin-drying the purified product to obtain (E) -tert-butyl (2-chloro-4- ((cycloocta-4-ene-1-oxyl) methyl) phenoxy) dimethylSilane; the fourth step: dissolving 1eq of (E) -tert-butyl (2-chloro-4- ((cyclooct-4-ene-1-oxyl) methyl) phenoxy) dimethyl silane in tetrahydrofuran, dropwise adding 1.2eq of tetrabutylammonium fluoride under the ice bath condition, stirring for 2-10h at room temperature, adding water after the reaction is finished, and quenching, and extracting by using dichloromethane; drying over anhydrous sodium sulfate, purifying by flash column chromatography, and spin-drying to obtain (E) -2-chloro-4- ((cyclooct-4-en-1-propoxy) methyl) phenol; the fifth step: dissolving 1eq of (E) -2-chloro-4- ((cyclooct-4-ene-1-propoxy) methyl) phenol and 2eq of 1, 3-dibromopropane in acetonitrile, adding 1eq of potassium carbonate, refluxing for 3h under the protection of nitrogen, cooling to room temperature, adding 3eq of N-methylpropane-2-amine and 3eq of potassium carbonate, and stirring for 22-48h at the temperature of 3eq.40 ℃; spin-drying the solvent, extracting with water and ethyl acetate; drying with anhydrous sodium sulfate; purifying by flash column chromatography, and spin-drying to obtain final compound CL-TCO; when R1 is->
Figure QLYQS_8
When the in vitro specific probe has the structural formula of->
Figure QLYQS_9
The specific preparation method is marked as PA-TCO and comprises the following steps: under the ice bath condition, dissolving 4-hydroxy trans-cyclooctene and pyridine at 1.2eq in dichloromethane, slowly dropwise adding 4-nitrophenylcarbonyl chloride at 1.2eq, stirring at room temperature for 10-20h, adding water and dichloromethane for extraction after the reaction is completed, washing an organic phase with 1M sodium carbonate aqueous solution, drying with anhydrous sodium sulfate, purifying by flash column chromatography, and spin-drying to obtain (E) -cyclooct-4-en-1-yl (4-nitrophenyl) carbonate; the second step is that: dissolving 2-bromoethyl-1-amine 6eq in dichloromethane, slowly dropwise adding (E) -cyclooct-4-en-1-yl (4-nitrophenyl) carbonate 1eq under the protection of nitrogen, stirring at room temperature for 14-22h, and adding for extraction after the reaction is completed; washing the organic phase with aqueous sodium bicarbonate solution, drying over anhydrous sodium sulfate, purifying by flash column chromatography, and spin-drying to obtain (E) -cycloocta-4-en-1-yl (2-bromoethyl) carbamate; the third step: dissolving 4-hydroxybenzaldehyde 1eq in tetrahydrofuran, adding 4-hydroxybenzaldehyde 1.2eq under nitrogen protection, stirring the reaction mixture at room temperature for 30min, and adding sodium acetate 1.4eqReaction for 4-10h, quenching the reaction mixture with saturated sodium bicarbonate, extracting with ethyl acetate at 4 × 20mL, combining the organic layers, washing with saturated brine, drying over anhydrous sodium sulfate, purifying by flash column chromatography, and spin-drying to give 4- ((methyl (prop-2-yn-1-yl) amino) methyl) phenol; the fourth step: dissolving 1eq of 4- ((methyl (prop-2-yne-1-yl) amino) methyl) phenol, 1.2eq of (E) -cyclooct-4-ene-1-yl (2-bromoethyl) carbamate and 2eq of potassium carbonate in acetonitrile solution, heating and refluxing for 10-20h under the protection of nitrogen, after the reaction is completed, drying the solvent in a rotary manner, adding 1M diluted hydrochloric acid to adjust the pH to 7.0, adding dichloromethane for extraction, drying anhydrous sodium sulfate, purifying by flash column chromatography, and drying in a rotary manner to obtain the final product PA-TCO.
3. The use of the active small molecule probe according to claim 1 or 2 for detecting monoamine oxidase a/B, wherein the trans-cyclooctene structure of the specific probe produces strong fluorescence by bioorthogonal reaction with the dye having 1,2,4, 5-tetrazine structure, and the activity of monoamine oxidase a/B in various biological systems is measured by fluorescence change, and the specifically bound protein-probe solution is reacted with the dye solution having 1,2,4, 5-tetrazine structure in equal volumes, wherein the concentration of the protein solution modified by the specific probe is 5-50 times that of the dye solution having 1,2,4, 5-tetrazine structure, the reaction time is 5-30min, the reaction temperature is 20-37 ℃, and the fluorescence value of the reaction per unit time is measured as an evaluation index of monoamine oxidase a/B activity.
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