Solid-phase probe for marking terminal alkyne compound as well as synthesis method and application of solid-phase probe

Abstract

The invention discloses a solid-phase probe for marking a terminal alkyne compound as well as a synthesis method and application of the solid-phase probe, and belongs to the technical field of analysis and detection. The solid-phase probe is prepared by taking a cyclodextrin polymer material as a carrier and a molecular probe as a carrier and loading the carrier onto the carrier through a subject-object recognition effect, and is marked as alpha-CDP-d0 / d5-Azo-N3. The solid-phase probe is characterized in that a benzene ring in the structure of the solid-phase probe is used as an isotope group, an azobenzene unit is used as a light-operated switch, imidazolium salt is used as a mass spectrum signal enhancement label, and an azido group is used as a reaction group. After chemical labeling, a labeled product can be dissociated from a solid-phase carrier under the irradiation of ultraviolet light, and rapid detection and screening of the natural terminal alkyne compound in a complex system can be realized by combining a matrix-assisted laser desorption ionization mass spectrometry technology, so that directional separation of the natural terminal alkyne compound is guided. The method has the characteristics of good selectivity to terminal alkyne compounds, strong mass spectrum signals of labeled products, simplicity in operation, mild reaction conditions and the like.

Description

Technical Field

[0001] The invention relates to a solid-phase probe for labeling a terminal alkyne compound and a synthesis method and application thereof, belonging to the technical field of analysis. Background Art

[0002] With the continuous development of scientific research and technology, the ways to obtain new drug molecules have also become more diversified, but natural products are still an important source for the discovery of new chemical entities. According to statistics, nearly 50% of the new drugs launched globally from 1981 to 2019 were derived from active compounds in plants or developed based on their pharmacophores (J. Nat. Prod., 2020, 83, 770-803). There are a large number of natural compounds with diverse structures in natural resources. Screening active natural compounds from them is an important way to discover lead compounds that are urgently needed in drug research and development. Compounds with acetylene groups at the end, also known as acetylene or terminal acetylenic compounds, are widely distributed in plants, fungi, animals, and marine organisms. They are an important structural source for the discovery of drug lead compounds. Most of them show excellent biological activity, and studies have shown that acetylene is the main pharmacophore in such drug and pesticide molecules (J.Med. Chem., 2020, 63, 5625-5663, Nature, 2019, 567, 420-424). Such as the antihypertensive drug paroxysmal hydrochloride, synthetic steroid hormone drugs norethindrone and ethinyl estradiol, anticancer drugs erlotinib and icotinib, herbicide fluazifop, etc. However, the content of terminal acetylene natural compounds in natural resources is very low, and they are extremely unstable, easily oxidized and photolyzed, and cannot exist stably under acidic and alkaline conditions, making their analysis and separation in complex matrices such as plant extracts very challenging (J. Nat. Prod. 2022, 85, 105–114; J. Agric. Food Chem., 2023, 71,14814-14824), which is one of the reasons why the number of natural terminal acetylene compounds discovered in recent years is very limited.

[0003] In order to solve the above problems, a solid-phase probe system based on covalent bonding has been developed in recent years. This technology modifies molecular probes to the surface of a solid-phase carrier through covalent bonds to prepare solid-phase probes, uses the solid-phase probe system to specifically identify and label compounds containing specific functional groups, and breaks the covalent bonds under certain conditions to release the labeled products for subsequent detection. This technology has improved the recognition, extraction performance and detection sensitivity of trace compounds in complex systems to a certain extent. However, the solid-phase carrier only plays a supporting role and cannot be reused after the covalent bond is broken. In addition, the multi-step chemical reaction, drastic connection and breaking conditions, and complex extraction process result in low extraction efficiency, poor repeatability and recovery rate of the existing solid-phase probe system and the corresponding sample pretreatment technology, which cannot meet the enrichment and detection needs of trace target compounds in complex systems. In addition, the preparation method of the existing solid-phase probe is complicated and difficult to synthesize, which greatly limits its application. Summary of the invention

[0004] The purpose of the present invention is to provide a solid-phase probe for labeling terminal alkyne compounds and a synthesis method thereof. The synthesis method has the characteristics of simple solid-phase probe preparation, strong selectivity, fast analysis speed, high accuracy, strong labeled product signal, etc.

[0005] 1. Solid-phase probes for labeling terminal alkyne compounds and their synthesis methods The solid-phase probe consists of two parts. The first part is a cyclodextrin polymer material, which uses α-cyclodextrin as a monomer and tetrafluoroterephthalonitrile as a crosslinker to construct a block polymer material with a dense ultra-microporous structure through polymerization reaction, abbreviated as α-CDP.

[0006] The second part is the molecular probe, the structure of which is as follows: , Among them: When X=H, it is called d 0-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 0-Azo-N3; when X=D, it is called d 5-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 5-Azo-N3.

[0007] Using α-CDP and molecular probes d 0 / d The non-covalent host-guest recognition between 5-Azo-N3 enables the preparation of solid-phase probes, which are denoted as α-CDP- d 0-Azo-N3 or α-CDP- d 5-Azo-N3; The method for synthesizing the solid phase probe of the present invention comprises the following steps: First, the preparation of the solid phase carrier α-CDP includes the following steps: (1) Take α-cyclodextrin as the monomer, mix it with potassium carbonate and cross-linking agent (tetrafluoroterephthalonitrile) and place them in a dry three-necked flask, the molar ratio of α-cyclodextrin to potassium carbonate is 1:5~1:20 (preferably 1:8~1:20), and the molar ratio of α-cyclodextrin to cross-linking agent tetrafluoroterephthalonitrile is 1:1~1:5 (preferably 1:2~1:5). Add a mixed solution of anhydrous tetrahydrofuran and anhydrous N,N-dimethylformamide in a volume ratio of 9:1, protect with inert gas, and heat at 85°C for 12~48 h (preferably 24~48 h); (2) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 7500 rpm for 10 min to collect the yellow precipitate. After being soaked and washed multiple times with methanol, water, tetrahydrofuran, and dichloromethane, the precipitate is dried in a vacuum oven to obtain a yellow solid powder, namely, cyclodextrin polymer material (α-CDP).

[0008] Secondly, molecular probes d 0 / d The synthesis of 5-Azo-N3 comprises the following steps: (1) The first step of reduction reaction: 4-nitrobenzyl alcohol and ammonium chloride are dissolved in a mixed solvent of 2-ethylene glycol monomethyl ether / water (10:1, v / v) under argon protection, and zinc powder is added at room temperature. The molar ratio of 4-nitroaniline to ammonium chloride is 1:1~1:2, and the molar ratio of 4-nitroaniline to zinc powder is 1:1~1:4. After stirring for 1~5 hours, the suspension is filtered to obtain a yellow-green clear solution. The filtrate is placed in an ice-water bath, and an FeCl3•6H2O aqueous solution is added. The molar ratio of 4-nitroaniline to FeCl3•6H2O is 1:1~1:3. The aqueous solution is extracted three times with dichloromethane, and the extracted solution is spin-dried and vacuum-dried at 45°C to obtain intermediate I, which is recorded as 4-nitrosobenzyl alcohol; (2) The second step Mills reaction: intermediate I, d 0 / d 5-Aniline and acetic acid were added to the ethanol solution and stirred at room temperature for 6 to 10 hours. d 0 / d The molar ratio of 5-aniline is 1:1~1:2.5, and the molar ratio of intermediate I to acetic acid is 1:5~1:20. After the reaction is completed, water is added to the solution, and the precipitate obtained is filtered and dried in vacuum at 60°C. The intermediate II is obtained by purification by silica gel column chromatography (dichloromethane:acetone = 40:1, v / v), which is recorded as d 0 / d 5-4-Hydroxymethylazobenzene; (3) The third step: Appel reaction: weigh intermediate II and carbon tetrabromide in a single-necked flask, add dry tetrahydrofuran, stir to dissolve, then add triphenylphosphine, react at room temperature for 4-8 hours, the molar ratio of intermediate II to carbon tetrabromide is 1:1-1:3, and the molar ratio of intermediate II to triphenylphosphine is 1:1-1:4. After the reaction is completed, filter, collect the filtrate, remove the solvent under reduced pressure, and purify it by silica gel column chromatography (petroleum ether: ethyl acetate = 40:1) to obtain intermediate III, which is recorded as d 0 / d 5-4-Bromomethylazobenzene; (4) The fourth step is substitution reaction: add imidazole, sodium hydroxide and ultrapure water to a round-bottom flask. Dissolve intermediate III in tetrahydrofuran, the molar ratio of intermediate III to imidazole is 1:1~1:3, and the molar ratio of intermediate III to sodium hydroxide is 1:1~1:2. Add it dropwise to the above mixture under argon protection, heat the reaction at 60°C for 10~15h, remove the solvent under reduced pressure, and extract the crude product three times with dichloromethane / water (4:1, v / v). The organic layer is concentrated and dried under vacuum. Purify by silica gel column chromatography (dichloromethane: methanol = 25:1, v / v) to obtain intermediate IV. d 0 / d 5-1-(4-(phenyldiazenyl)benzyl)imidazole; (5) The fifth step of salt formation reaction: intermediate IV and 1,3-dibromopropane are dissolved in acetonitrile, the molar ratio of intermediate IV to 1,3-dibromopropane is 1:5-1:20, heated to reflux for 20-25 h, the solvent is removed under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol = 7:1, v / v) to obtain intermediate V, which is recorded as d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide; (6) Step 6: Substitution reaction: Dissolve intermediate V and NaN3 in DMF, with the molar ratio of intermediate V to NaN3 being 1:1~1:6. Heat at 50°C for 10~15h, pour in a large amount of distilled water, extract with n-butanol, and wash with water several times. Remove the solvent under reduced pressure to obtain the target compound, which is recorded as d 0 / d 5-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 0 / d 5-Azo-N3.

[0009] Finally, α-CDP was dispersed into the molecular probe solution ( d 0-Azo-N3 or d5-Azo-N3), oscillation adsorption, using the host-guest recognition between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure, the molecular probe is loaded on α-CDP, and after washing and freeze-drying, a solid-phase probe is obtained, which is recorded as α-CDP- d 0-Azo-N3 or α-CDP- d 5-Azo-N3.α-CDP and Molecular Probes d 0 / d The mass ratio of 5-Azo-N3 is 1:0.01~1:0.8 (preferably 1:0.2~1:0.6), the concentration of the molecular probe solution is 0.1~2.0 mg / mL (preferably 0.5~1.5 mg / mL), the pH of the molecular probe solution is 3~10 (preferably 5~8), the temperature of the oscillating adsorption is 20~60°C (preferably 25~35°C), and the time of the oscillating adsorption is 5~60 min (preferably 20~45 min).

[0010] The synthesis mechanism of molecular probes is as follows: In the first step, under the acidic conditions provided by ammonium chloride, the electrons of the zinc powder are continuously transferred to the nitro group of 4-nitrobenzyl alcohol through a single electron transfer mechanism. The nitro group loses a water molecule and is reduced to a nitroso group to obtain intermediate I (4-nitrosobenzyl alcohol). In the second step, intermediate I and d 0 / d 5-Aniline generates aromatic azo compounds under the catalysis of acetic acid. d 0 / d The lone pair of electrons on the nitrogen atom of 5-aniline attacks the nitroso group, and after losing a water molecule, intermediate II is generated ( d 0 / d 5-4-hydroxymethylazobenzene); in the third step, triphenylphosphine reacts with carbon tetrabromide to be activated, and then the oxygen atom of the hydroxyl group on the intermediate II attacks the bromotriphenylphosphine to obtain the phosphinium oxide salt, and the bromide ion generates S N 2 reaction, triphenylphosphine leaves as a leaving group to obtain intermediate III ( d 0 / d 5-4-bromomethylazobenzene); the fourth step, using d 0 / d The nucleophilic substitution reaction between 5-4-bromomethylazobenzene and imidazole prepared intermediate IV. Under the action of inorganic base, the nitrogen atom on imidazole attacked d 0 / d The carbon atom in 5-4-bromomethylazobenzene carries a positive charge, and the bromide ion leaves as a leaving group to give intermediate IV ( d 0 / d5-1-(4-(phenyldiazenyl)benzyl)imidazole); Step 5, intermediate IV reacts with 1,3-dibromopropane under heating conditions to generate a quaternary ammonium salt ion to obtain intermediate V ( d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide); Step 6, the target product is prepared by the nucleophilic substitution reaction between sodium azide and intermediate V. The N atom of sodium azide attacks the Br atom of intermediate V to obtain the target product d 0 / d 5-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide ( d 0 / d 5-Azo-N3).

[0011] The preparation mechanism of solid phase probe is as follows: Cyclodextrin polymer is used as a solid phase carrier. It is obtained by cross-linking α-cyclodextrin as a monomer molecule with a rigid aromatic cross-linking agent tetrafluoroterephthalonitrile. This overcomes the problem of polymer molecular chains being entangled with each other and forms a cyclodextrin polymer (α-CDP) with an ultra-microporous structure and selective adsorption of molecular probes. d 0 / d The strong host-guest recognition effect of the azobenzene unit in the 5-Azo-N3 structure can achieve efficient loading of probe molecules and realize the preparation of solid-phase probes.

[0012] 2. Application of solid-phase probes labeled with terminal alkyne compounds in the detection and screening of natural terminal alkyne compounds Another object of the present invention is to provide an application of the above-mentioned solid-phase probe in the detection and screening of natural terminal alkyne compounds. The solid-phase probe combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) technology can realize rapid detection and screening of natural terminal alkyne compounds.

[0013] The method for detecting and screening natural terminal alkyne compounds comprises the following steps: (1) Take 1 mg of plant extract and add it to two separate 1.5 mL centrifuge tubes. Add the solid phase probe α-CDP- d 0-Azo-N3 and α-CDP- d5-Azo-N3, the amount of solid phase probe is 1~10 mg / mL (preferably 1~5 mg / mL), then sodium ascorbate and copper sulfate pentahydrate aqueous solutions are added respectively, the concentration of sodium ascorbate is 0.05~2 mg / mL (preferably 0.2~1.0 mg / mL), the concentration of copper sulfate pentahydrate is 5~400 μg / mL (preferably 50~200 mg / mL), diluted to 1 mL with ultrapure water, ultrasonically dispersed, heated and shaken for chemical labeling. The ultrasonic dispersion time is 0.5~5 min (preferably 0.5~2 min), the heating reaction temperature is 25~70°C (preferably 25~45°C), the shaking speed is 600~1500 rpm (preferably 1200~1500rpm), and the labeling time is 10~120 min (preferably 30~120 min); (2) Centrifuge and wash to remove impurities, add ultrapure water to disperse, spot 1 μL of sample solution on the target plate, irradiate with 365 nm ultraviolet light for 1 to 60 min (preferably 5 to 30 min), spot 1 μL of matrix solution, and wait for matrix-assisted laser desorption ionization mass spectrometry detection; the matrix refers to any one of 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), α-cyano-4-hydroxycinnamic acid (CHCA), 9-aminoacridine (9-AA), 3-hydroxy-2-pyridinecarboxylic acid (3-HPA) and 2,4,6-trihydroxyacetophenone (THAP), preferably α-cyano-4-hydroxycinnamic acid (CHCA), and the concentration of the matrix solution is 2 to 20 mg / mL (preferably 5 to 12 mg / mL); (3) Matrix-assisted laser desorption ionization mass spectrometry was performed to observe a characteristic isotope mass spectrometry peak with a mass-to-charge ratio (m / z) difference of approximately 5.0314. This isotope characteristic mass spectrometry peak can be used to accurately screen terminal alkyne compounds.

[0014] The detection and assisted screening mechanisms are as follows: In situ reduction of Cu by sodium ascorbate 2+ Producing catalyst Cu + , using molecular probes loaded on solid-phase probes d 0 / dThe 1,3-dipolar cycloaddition reaction between the azide group in 5-Azo-N3 and the acetylene group of the terminal alkyne compound forms a 1,4-disubstituted 1,2,3-triazole labeled product. After the CuAAC labeling reaction, the solid phase probe after the reaction is centrifuged and washed several times to eliminate the influence of the molecular probe on the detection in the solution, and effectively reduce the matrix interference caused by the complex system. The washed solid phase probe is spotted on the target plate of MADLI-MS. Under the irradiation of ultraviolet light, the azobenzene unit in the labeled product can be used as a photo-controlled switch to achieve the transformation from trans to cis configuration and dissociate from the solid phase support, which is conducive to the subsequent MALDI-MS detection. Through the copper-catalyzed azide-alkynyl cycloaddition (CuAAC) reaction, isotope groups and mass spectrometry labels can be introduced into the labeled product, generating a characteristic isotope mass spectrum peak with a mass-to-charge ratio (m / z) difference of 5.0314. This isotope characteristic mass spectrum peak can be used to accurately detect terminal alkyne compounds. Through calculation, the accurate molecular weight of unlabeled front-end alkyne compounds can be obtained. Combined with the multi-stage mass spectrometry fragmentation rules, it is expected to achieve structural analysis of terminal alkyne compounds. Labeling and detection of natural resource extracts can assist in screening natural resources containing terminal alkyne compounds through isotope characteristic mass spectrometry peaks, thereby improving the efficiency of discovering terminal alkyne compounds. In addition, positively charged imidazole salts as mass spectrometry labels can greatly improve the sensitivity of labeled products in mass spectrometry positive ion mode detection.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The solid-phase probe for labeling terminal alkyne compounds described in the present invention and its synthesis method and application are to use cyclodextrin polymer as a solid-phase carrier, and load the molecular probe onto the carrier through the host-guest recognition effect, thereby constructing a solid-phase probe. Compared with the traditional solid-phase probe based on covalent bond binding, the present method adopts the host-guest recognition effect to load the molecular probe, does not require multi-step, complicated synthesis steps, simplifies the preparation process of the solid-phase probe, and greatly improves the loading amount of the molecular probe. Thanks to the organic combination of the solid-phase carrier and the molecular probe, the solid-phase probe has the advantages of easy separation of the solid-phase carrier, promoting efficient reaction, simplifying the separation and purification steps of the labeled product, etc. At the same time, the loaded molecular probe can realize the highly selective labeling of terminal alkyne compounds in complex systems through the CuAAC reaction, and introduces the isotope group and the mass spectrum label into the labeled product through the labeling reaction, giving it an isotope characteristic signal, and improving the sensitivity of the labeled product in mass spectrometry detection. Under the irradiation of ultraviolet light, it is easy to realize the release of the labeled product, and carry out subsequent MALDI-MS detection. In addition, combined with the multi-stage mass spectrometry fragmentation law, it is expected to realize the structural identification of terminal alkyne compounds. Compared with the traditional labeled product release process, this method does not require further post-processing and does not involve drastic breaking conditions (strong acid, strong base, organic solvent, etc.). It has the advantages of simple operation, strong labeled product signal, and obvious isotope characteristic signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the FT-IR spectrum of the cyclodextrin polymer (α-CDP) prepared in the embodiment of the present invention.

[0017] Figure 2 This is a SEM image of the α-CDP prepared in an embodiment of the present invention.

[0018] Figure 3 Molecular probe prepared for the embodiment of the present invention d NMR of 0-Azo-N3 1 H NMR spectrum.

[0019] Figure 4 Molecular probe prepared for the embodiment of the present invention d High-resolution mass spectrum of 0-Azo-N3.

[0020] Figure 5 Molecular probe prepared for the embodiment of the present invention d NMR of 5-Azo-N3 heavy labeling reagent 1 H NMR spectrum.

[0021] Figure 6 Molecular probe prepared for the embodiment of the present invention d High-resolution mass spectrum of 5-Azo-N3.

[0022] Figure 7 The solid phase probe α-CDP- d 0-Azo-N3 and α-CDP- d MALDI-MS mass spectrum of 5-Azo-N3.

[0023] Figure 8 The present invention is a MALDI-MS mass spectrum of terminal alkyne compounds in plant extracts labeled by two solid-phase probes of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further explained below with reference to specific embodiments. Example

[0025] First, the synthesis of solid phase support α-CDP (1) Mix 1.2 g of α-cyclodextrin, 4.0 g of potassium carbonate, and 1.0 g of tetrafluoroterephthalonitrile in a dry three-necked flask, add 90 mL of anhydrous tetrahydrofuran and 10 mL of anhydrous N,N-dimethylformamide solution, and heat at 85 °C for 24 h under inert gas protection; (2) After the reaction is completed, the mixture is cooled to room temperature and centrifuged at 7500 rpm for 10 min to collect the yellow precipitate. After being soaked and washed multiple times with methanol, water, tetrahydrofuran, and dichloromethane, the precipitate is dried in a vacuum oven to obtain a yellow solid powder, namely, cyclodextrin polymer material (α-CDP).

[0026] Secondly, molecular probes d 0 / d Synthesis of 5-Azo-N3 d Synthesis of 0-Azo-N3: (1) Synthesis of Intermediate Ⅰ (4-nitrosobenzyl alcohol) 7.4 g of 4-nitrobenzyl alcohol and 3.3 g of ammonium chloride were dissolved in 220 mL of a mixed solvent of 2-ethylene glycol monomethyl ether / water (10:1) under argon protection, and then 7.8 g of zinc powder was added at room temperature. After stirring for 3 h, the suspension was filtered to obtain a yellow-green clear solution. The filtrate was placed in an ice-water bath, and 50 mL of 13.3 g of FeCl3•6H2O aqueous solution was added and stirred overnight. The obtained aqueous solution was extracted three times with dichloromethane, and the extracted solution was spin-dried and 45 o C and dried in vacuo to obtain 5.4 g of intermediate Ⅰ (4-nitrosobenzyl alcohol).

[0027] (2) Intermediate Ⅱ ( d Synthesis of 0-4-hydroxymethylazobenzene 5.4 g of intermediate I, 2.6 mL of aniline, and 36 mL of acetic acid were added to 150 mL of ethanol solution and stirred at room temperature for 8 h. After the reaction was completed, water was added to the solution, and the precipitate was filtered and then heated to 60 °C. o C and dried in vacuum. After silica gel column chromatography (dichloromethane:acetone = 40:1, v / v), 3.3 g of yellow-brown solid was obtained, namely intermediate Ⅱ ( d 0-4-hydroxymethylazobenzene), yield 84%.

[0028] (3) Intermediate ⅠII ( d Synthesis of 0-4-bromomethylazobenzene Weigh 1.0 g of intermediate ⅠI and 2.4 g of carbon tetrabromide in a single-necked flask, add 8 mL of dry THF, stir to dissolve, then add 2.0 g of triphenylphosphine, and react at room temperature for 6 h. After the reaction is completed, filter, collect the filtrate, and remove the solvent under reduced pressure. After silica gel column chromatography (petroleum ether: ethyl acetate = 40:1), 0.66 g of orange-yellow solid, namely intermediate ⅠII ( d 0-4-bromomethylazobenzene), yield 51% (4) Intermediate IV ( dSynthesis of 1-(4-(phenyldiazenyl)benzyl)imidazole Add 0.3 g imidazole, 0.2 g sodium hydroxide and 5 mL ultrapure water into a round-bottom flask. Dissolve 0.5 g intermediate III in tetrahydrofuran and add dropwise to the above mixture under argon protection. o After heating at 40 °C for 12 h, the solvent was removed under reduced pressure. The crude product was extracted three times with CH2Cl2 / H2O (4:1). The organic layer was concentrated and dried under vacuum. 0.4 g of orange-yellow solid was obtained by silica gel column chromatography (CH2Cl2 / MeOH = 25:1), namely intermediate Ⅳ ( d 0-1-(4-(phenyldiazenyl)benzyl)imidazole), yield 86%.

[0029] (5) Intermediate V ( d Synthesis of 3-(3-bromopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide) 0.3 g of intermediate ⅠV and 2.3 g of 1,3-dibromopropane were dissolved in 25 mL of CH3CN, refluxed for 24 h, the solvent was dried, and purified by silica gel column chromatography (CH2Cl2 / MeOH = 7:1) to obtain 0.5 g of orange solid, namely intermediate V ( d 0-3-(3-bromopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide), yield 87%.

[0030] (6) Target product d Synthesis of 0-Azo-N3 0.2 g of intermediate V and 0.2 g of NaN3 were dissolved in 30 mL of DMF. o After heating at C for 12 h, a large amount of distilled water was poured in, extracted with n-butanol, and washed with water several times. The solvent was removed under reduced pressure to obtain 0.08 g of orange solid, which was the target product. d 0-Azo-N3, yield 44%.

[0031] d Synthesis of 5-Azo-N3: With the above d The synthesis of 0-Azo-N3 was compared with that of Molecular Probes d The synthesis of 5-Azo-N3 is carried out in the second step with d 5-Aniline is the reaction raw material, and the other synthetic steps are the same.

[0032] Finally, the solid-phase probe α-CDP- d 0-Azo-N3 and α-CDP- d Preparation of 5-Azo-N3 Take 50 mg of α-CDP and add it to 30 mL of molecular probe solution ( d 0-Azo-N3 and d 5-Azo-N3, pH = 7, concentration of 1 mg / mL), oscillated and adsorbed at 40 ° C for 45 min, and the molecular probe was loaded on α-CDP by utilizing the host-guest recognition between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure. After washing and freeze-drying, a solid-phase probe was obtained, which was recorded as α-CDP- d 0-Azo-N3 and α-CDP- d 5-Azo-N3.

[0033] Product structure characterization Characterization of solid support α-CDP: Fourier transform-infrared (FT-IR) spectrum ( Figure 1 ):2244 cm -1 The CN group introduced after cross-linking of cyclodextrin and tetrafluoroterephthalonitrile, 1636 cm -1 The absorption peak at corresponds to the stretching vibration of CC of the benzene ring, indicating that tetrafluoroterephthalonitrile has been successfully cross-linked to α-CD, proving the successful synthesis of the solid-phase supported α-CDP.

[0034] Scanning Electron Microscope (SEM) Characterization: Figure 2 The SEM morphology of α-CDP shows that the morphology of α-CDP is an irregular block structure with a rough surface and the characteristics of an ultra-microporous structure. This structure is conducive to the adsorption of molecular probes, thereby realizing the rapid preparation of solid-phase probes.

[0035] Molecular probes d 0-Azo-N3 Product Characterization: H NMR 1 H NMR ( Figure 3 ): 1 H NMR (400 MHz, DMSO- d 6) δ 9.61 (s, 1H), 8.04– 7.81 (m, 6H), 7.67 (d, J = 8.1 Hz, 2H), 7.63 – 7.58(m, 3H), 5.60 (s, 2H), 4.29 (t, J = 7.0 Hz, 2H), 3.45 (t, J = 6.5 Hz, 2H), 2.09 (p, J = 6.8 Hz, 2H). High resolution mass spectrometry ( Figure 4):calcd. for C 19 H 20 N7 + , 346.1775 [M – Br] + ; found,346.1777. From the above characterization, it can be seen that this example successfully synthesized the molecular probe d 0-Azo-N3.

[0036] Molecular probes d 5-Azo-N3 Product Characterization: H NMR 1 H NMR ( Figure 5 ): 1 H NMR (400 MHz, DMSO- d 6) δ 9.56 (s, 1H), 7.96– 7.88 (m, 4H), 7.66 (d, J = 8.1 Hz, 2H), 5.59 (s, 2H), 4.29 (t, J = 7.0 Hz, 2H),3.45 (t, J = 6.5 Hz, 2H), 2.09 (p, J = 6.8 Hz, 2H). High resolution mass spectrometry ( Figure 6 ):calcd. for C 19 H 15 D5N7 + , 351.2089 [M – Br] + ; found:351.2083. From the above characterization, it can be seen that this example successfully synthesized the molecular probe d 5-Azo-N3.

[0037] Solid phase probe α-CDP- d Characterization of 0-Azo-N3: Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) characterization ( Figure 7 a): Solid phase probe α-CDP- d 0-Azo-N3 is prepared by using d The non-covalent host-guest interaction between 0-Azo-N3 and α-CDP. The solid phase probe was analyzed by MALDI-MS and a mass peak with m / z of 346.182 was found, corresponding to d Mass spectrum peak of 0-Azo-N3.

[0038] From the above characterization, it can be seen that the solid phase probe α-CDP- d 0-Azo-N3.

[0039] Solid phase probe α-CDP- d Characterization of 5-Azo-N3: Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) characterization ( Figure 7 b): The solid phase probe was analyzed by MALDI-MS, and a mass spectrum peak with m / z of 351.211 was found, corresponding to d Mass spectrum peak of 5-Azo-N3.

[0040] From the above characterization, it can be seen that the solid phase probe α-CDP- d 5-Azo-N3.

[0041] Application in the detection and auxiliary screening of natural terminal alkyne compounds The two solid-phase probes α-CDP- d 0-Azo-N3 and α-CDP- d 5-Azo-N3 is used to label and screen terminal alkyne compounds. The specific steps are as follows: (1) Take 1 mg of the herbal medicine Golden Button ( Spilanthes callimorpha ) were added to two separate 1.5 mL centrifuge tubes, and the solid phase probe α-CDP- d 0-Azo-N3 and α-CDP- d 5-Azo-N3, the dosage of solid phase probe is 1 mg / mL, then sodium ascorbate and copper sulfate pentahydrate aqueous solutions are added respectively, the concentration of sodium ascorbate is 0.5 mg / mL, the concentration of copper sulfate pentahydrate is 100 μg / mL, diluted to 1 mL with ultrapure water, ultrasonically dispersed for 1 min, heated and shaken at 40 °C (1200 rpm) for chemical labeling, and the labeling time is 90 min; (2) Centrifuge and wash to remove impurities, add 100 μL ultrapure water to disperse, spot 1 μL of sample solution on the target plate, irradiate with 365 nm ultraviolet light for 30 min, then spot 1 μL of CHCA matrix solution (CHCA concentration is 10 mg / mL), and wait for matrix-assisted laser desorption ionization mass spectrometry detection; (3) Matrix-assisted laser desorption ionization mass spectrometry was performed to observe a characteristic isotope mass spectrometry peak with a mass-to-charge ratio (m / z) difference of approximately 5.0314. This isotope characteristic mass spectrometry peak can be used to accurately screen terminal alkyne compounds.

[0042] The two solid-phase probes were used to label the terminal alkyne compounds in the plant extracts through CuAAC reaction. After the labeling reaction, the labeled products were dissociated from the solid-phase carrier by ultraviolet irradiation for the next MALDI-MS detection. Figure 8 As shown in the figure, a group of mass spectrometry peaks with isotopic characteristics (the mass-to-charge ratio m / z difference is 5.0307, ​​which is close to the theoretical value of 5.0314) were found, proving that the crude extract contains at least one terminal alkyne compound, proving that natural terminal alkyne compounds exist in the plant. Through retrosynthetic analysis, the chemical structure of the original terminal alkyne compound can be obtained ( Figure 8 ).

Claims

1. A solid-phase probe for labeling a terminal alkyne compound, characterized in that: The solid-phase probe is prepared by using cyclodextrin polymer material as a carrier and molecular probe as a carrier, and loading the carrier onto the carrier through host-guest recognition, and is denoted as α-CDP- d 0-Azo-N3 or α-CDP- d 5-Azo-N3; Among them, the cyclodextrin polymer material is a block polymer material with a dense ultra-microporous structure prepared by polymerization reaction using α-cyclodextrin as a monomer and tetrafluoroterephthalonitrile as a crosslinker, which is abbreviated as α-CDP; The structure of the molecular probe is as follows: ， When X=H, it is called d 0-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 0-Azo-N3; when X=D, it is called d 5-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 5-Azo-N3.

2. A method for preparing a solid-phase probe for labeling a terminal alkyne compound as claimed in claim 1, characterized in that: The method is to disperse α-CDP into a molecular probe solution, perform oscillation adsorption, utilize the host-guest recognition effect between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure to load the molecular probe on α-CDP, and obtain a solid-phase probe after washing and freeze-drying.

3. The method for preparing a solid-phase probe for labeling a terminal alkyne compound according to claim 2, characterized in that: The mass ratio of the α-CDP to the molecular probe is 1:0.01-1:0.8, the concentration of the molecular probe solution is 0.1-2.0 mg / mL, the pH value is 3-10, the oscillation adsorption temperature is 20-60° C., and the oscillation adsorption time is 5-60 min.

4. The method for preparing a solid-phase probe for labeling a terminal alkyne compound according to claim 2, characterized in that: The preparation of the cyclodextrin polymer material comprises the following steps: (1) α-cyclodextrin is used as a monomer, mixed with potassium carbonate and tetrafluoroterephthalonitrile and placed in a dry three-necked flask, and a mixed solution of anhydrous tetrahydrofuran and anhydrous N,N-dimethylformamide in a volume ratio of 9:1 is added, and the mixture is heated at 85°C for 12 to 48 hours under inert gas protection; (2) After the reaction is completed, cool to room temperature, centrifuge at 7500 rpm for 10 min to collect the yellow precipitate, wash it with methanol, water, tetrahydrofuran, and dichloromethane multiple times, and dry it in a vacuum oven to obtain a yellow solid powder, namely the cyclodextrin polymer material, recorded as: α-CDP.

5. The method for preparing a solid-phase probe for labeling a terminal alkyne compound according to claim 4, characterized in that: The molar ratio of α-cyclodextrin to potassium carbonate is 1:5-1:20, and the molar ratio of α-cyclodextrin to tetrafluoroterephthalonitrile is 1:1-1:

5.

6. The method for preparing a solid-phase probe for labeling a terminal alkyne compound according to claim 2, characterized in that: The synthesis of the molecular probe comprises the following steps: (1) The first step of reduction reaction: 4-nitrobenzyl alcohol and ammonium chloride are dissolved in a mixed solvent of diethylene glycol monomethyl ether / water under argon protection, zinc powder is added at room temperature, and the reaction is stirred for 1-5 hours. The suspension is filtered to obtain a yellow-green clear solution. The filtrate is placed in an ice-water bath, and an aqueous FeCl3•6H2O solution is added. The solution is extracted three times with dichloromethane. The extracted solution is spin-dried and vacuum-dried at 45°C to obtain intermediate I, which is recorded as 4-nitrosobenzyl alcohol. (2) The second step Mills reaction: intermediate I, d 0 / d 5-Aniline and acetic acid were added to the ethanol solution and stirred at room temperature for 6-10 hours. After the reaction was completed, water was added to the solution. The precipitate was filtered and vacuum dried at 60°C. The intermediate II was purified by silica gel column chromatography and recorded as d 0 / d 5-4-Hydroxymethylazobenzene; (3) The third step Appel reaction: weigh intermediate II and carbon tetrabromide in a single-necked flask, add dry tetrahydrofuran, stir to dissolve, then add triphenylphosphine, react at room temperature for 4-8 hours. After the reaction is completed, filter, collect the filtrate, remove the solvent under reduced pressure, and purify it by silica gel column chromatography to obtain intermediate III, which is recorded as d 0 / d 5-4-Bromomethylazobenzene; (4) Step 4: Substitution reaction: imidazole, sodium hydroxide and ultrapure water were added to a round-bottom flask, and the intermediate III was dissolved in tetrahydrofuran. The mixture was added dropwise to the mixture under argon protection. The mixture was heated at 60°C for 10-15 hours, and the solvent was removed under reduced pressure. The crude product was extracted three times with dichloromethane / water. The organic layer was concentrated and dried under vacuum, and purified by silica gel column chromatography to obtain the intermediate IV, which was recorded as d 0 / d 5-1-(4-(phenyldiazenyl)benzyl)imidazole; (5) The fifth step of salt formation reaction: intermediate IV and 1,3-dibromopropane were dissolved in acetonitrile, heated under reflux for 20-25 h, the solvent was removed under reduced pressure, and purified by silica gel column chromatography to obtain intermediate V, which was recorded as d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide; (6) Step 6: Substitution reaction: Dissolve the intermediate V and NaN3 in DMF, heat at 50°C for 10-15h, pour in a large amount of distilled water, extract with n-butanol, wash with water several times, and remove the solvent under reduced pressure to obtain the target compound, which is recorded as d 0 / d 5-3-(3-azidopropyl)-1-(4-(phenyldiazenyl)benzyl)imidazol-3-ium bromide, abbreviated as d 0 / d 5-Azo-N3.

7. The method for preparing a solid-phase probe for labeling a terminal alkyne compound according to claim 6, characterized in that: In the reduction reaction of step (1), the molar ratio of 4-nitroaniline to ammonium chloride is 1:1-1:2, the molar ratio of 4-nitroaniline to zinc powder is 1:1-1:4, and the molar ratio of 4-nitroaniline to FeCl3•6H2O is 1:1-1:3; In the Mills reaction described in step (2), intermediate I and d 0 / d The molar ratio of 5-aniline is 1:1 to 1:2.5, and the molar ratio of intermediate I to acetic acid is 1:5 to 1:20; In the Appel reaction in step (3), the molar ratio of intermediate II to carbon tetrabromide is 1:1 to 1:3, and the molar ratio of intermediate II to triphenylphosphine is 1:1 to 1:4; In the substitution reaction in step (4), the molar ratio of intermediate III to imidazole is 1:1-1:3, and the molar ratio of intermediate III to sodium hydroxide is 1:1-1:2; In the salt-forming reaction in step (5), the molar ratio of intermediate IV to 1,3-dibromopropane is 1:5 to 1:20; In the substitution reaction described in step (6), the molar ratio of intermediate V to NaN3 is 1:1 to 1:

6.

8. Use of the solid phase probe labeled with a terminal alkyne compound as claimed in claim 1 in the detection of natural terminal alkyne compounds, characterized in that: The following steps are involved: (1) Take 1 mg of plant extract and add it to two separate 1.5 mL centrifuge tubes. Add the solid phase probe α-CDP- d 0-Azo-N3 and α-CDP- d 5-Azo-N3, then add an aqueous solution of sodium ascorbate and copper sulfate pentahydrate, dilute to 1 mL with ultrapure water, disperse by ultrasound, and heat and oscillate for chemical labeling; (2) Centrifuge and wash to remove impurities, add ultrapure water to disperse, spot 1 μL of sample solution on the target plate, irradiate with 365 nm ultraviolet light, spot 1 μL of matrix solution, and wait for matrix-assisted laser desorption ionization mass spectrometry detection; (3) Matrix-assisted laser desorption ionization mass spectrometry was performed to observe a characteristic isotope mass spectrometry peak with a mass-to-charge ratio difference of approximately 5.0314. This isotope characteristic mass spectrometry peak can be used to accurately detect terminal alkyne compounds and assist in the screening of natural resources containing terminal alkyne compounds.

9. Use of the solid phase probe labeled with a terminal alkyne compound as claimed in claim 8 in the detection of natural terminal alkyne compounds, characterized in that: In step (1), the amount of the solid phase probe is 1-10 mg / mL, the concentration of the sodium ascorbate solution is 0.05-2 mg / mL, the concentration of the copper sulfate pentahydrate solution is 5-400 μg / mL, the ultrasonic dispersion time is 0.5-5 min, the heating reaction temperature is 25-70 ° C, the oscillation speed is 600-1500 rpm, and the labeling time is 10-120 min.

10. Use of the solid phase probe labeled with a terminal alkyne compound as claimed in claim 8 in the detection of natural terminal alkyne compounds, characterized in that: In step (2), the 365 nm ultraviolet light irradiation time is 1 to 60 min, the matrix is ​​any one of 2,5-dihydroxybenzoic acid, 1,5-diaminonaphthalene, α-cyano-4-hydroxycinnamic acid, 9-aminoacridine, 3-hydroxy-2-pyridinecarboxylic acid and 2,4,6-trihydroxyacetophenone, and the concentration of the matrix solution is 2 to 20 mg / mL.

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