A solid-phase probe for labeling terminal alkynes, its synthesis method and application

Abstract

This invention discloses a solid-phase probe for labeling terminal alkynes, its synthesis method, and its application, belonging to the field of analytical detection technology. The solid-phase probe is prepared by loading a molecular probe onto a cyclodextrin polymer material as a carrier through host-guest recognition, and is denoted as α-CDP- d 0 / d 5-Azo-N3. This solid-phase probe is characterized by its structure using a benzene ring as an isotopic group, an azobenzene unit as a light-controlled switch, an imidazolium salt as a mass spectrometry signal enhancement tag, and an azide group as a reactive group. After chemical labeling, the labeled product can dissociate from the solid-phase support under ultraviolet light irradiation. Combined with matrix-assisted laser desorption / ionization mass spectrometry (MALS), it enables rapid detection and screening of natural terminal alkyne compounds in complex systems, thereby guiding their targeted separation. This invention features good selectivity for terminal alkyne compounds, strong mass spectrometric signal of the labeled product, simple operation, and mild reaction conditions.

Description

Technical Field

[0001] This invention relates to a solid-phase probe for labeling terminal alkyne compounds, its synthesis method, and its application, belonging to the field of analytical technology. Background Technology

[0002] With the continuous development of scientific research techniques, the pathways to obtaining novel drug molecules have become increasingly diversified, but natural products remain an important source for the discovery of new chemical entities. Statistics show that nearly 50% of new drugs launched globally between 1981 and 2019 originated from active compounds in plants or were developed based on their pharmacophores (J. Nat. Prod., 2020, 83, 770-803). Natural resources contain a large number of structurally diverse natural compounds, and screening for active natural compounds is a crucial pathway for lead compound discovery in drug development. Compounds with terminal alkyne groups, also known as ethynyl or terminal alkyne compounds, are widely distributed in plants, fungi, animals, and marine organisms, and are an important structural source for drug lead compound discovery. Most exhibit excellent biological activity, and research indicates that the ethynyl group is the main pharmacophore in these drugs and pesticide molecules (J. Med. Chem., 2020, 63, 5625-5663, Nature, 2019, 567, 420-424). Examples include the antihypertensive drug phenoxyphene hydrochloride, the synthetic steroid hormones norethindrone and ethinylestradiol, the anticancer drugs erlotinib and icotinib, and the herbicide propyzamide. However, the content of natural terminal alkynes in natural resources is very low, and they are extremely unstable, easily oxidized and photodegraded, and cannot exist stably under acidic or alkaline conditions. This makes 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). This is also one of the reasons why the number of naturally occurring terminal alkynes discovered in recent years has been very limited.

[0003] To address the aforementioned issues, solid-phase probe systems based on covalent bonds have been developed in recent years. This technology prepares solid-phase probes by covalently modifying molecular probes onto the surface of a solid support. The solid-phase probe system specifically identifies and labels compounds containing specific functional groups, and under certain conditions, the covalent bonds are broken to release the labeled products for subsequent detection. This technology has improved the identification, extraction performance, and detection sensitivity of trace compounds in complex systems to some extent. However, the solid support only serves a supporting function and cannot be reused after the covalent bonds are broken. Furthermore, the multi-step chemical reactions, the intense bonding and breaking conditions, and the complex extraction process result in low extraction efficiency, poor repeatability, and low recovery rates for existing solid-phase probe systems and corresponding sample pretreatment techniques, failing to meet the needs for enrichment and detection of trace target compounds in complex systems. In addition, the preparation methods of existing solid-phase probes are complex and difficult to synthesize, significantly limiting their application. Summary of the Invention

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

[0005] I. Solid-phase probes for labeling terminal alkynes and their synthesis methods

[0006] The solid-phase probe consists of two parts. The first part is a cyclodextrin polymer material, which is a blocky polymer material with a dense ultraporous structure constructed by polymerization reaction using α-cyclodextrin as monomer and tetrafluoroterephthalonitrile as crosslinking agent. It is abbreviated as α-CDP.

[0007] The second part is the molecular probe, the structure of which is as follows:

[0008] ,

[0009] Where: when X=H, it is called d 0-3-(3-azidopropyl)-1-(4-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 0-Azo-N3; when X=D, it is called d 5-3-(3-azidopropyl)-1-(4-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 5-Azo-N3.

[0010] Using α-CDP with molecular probes d 0 / d The non-covalent host-guest recognition between 5-Azo-N3 molecules enables the fabrication of a solid-phase probe, denoted as α-CDP-. d 0-Azo-N3 or α-CDP-d 5-Azo-N3;

[0011] The method for synthesizing the solid-phase probe of the present invention includes the following steps:

[0012] First, the preparation of the solid support α-CDP includes the following steps:

[0013] (1) Using α-cyclodextrin as a monomer, mix it with potassium carbonate and a crosslinking agent (tetrafluoroterephthalonitrile) 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 the crosslinking 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, and heat at 85°C for 12~48 h (preferably 24~48 h) under inert gas protection.

[0014] (2) After the reaction is complete, cool to room temperature, centrifuge at 7500 rpm for 10 min to collect the yellow precipitate, wash it repeatedly with methanol, water, tetrahydrofuran and dichloromethane, and dry it in a vacuum oven to obtain a yellow solid powder, namely cyclodextrin polymer material (α-CDP).

[0015] Secondly, molecular probes d 0 / d The synthesis of 5-Azo-N3 includes the following steps:

[0016] (1) First step reduction reaction: 4-Nitrobenzyl alcohol and ammonium chloride were dissolved in a mixed solvent of 2-ethylene glycol monomethyl ether / water (10:1, v / v) under argon protection. Zinc powder was added at room temperature. The molar ratio of 4-nitroaniline to ammonium chloride was 1:1~1:2, and the molar ratio of 4-nitroaniline to zinc powder was 1:1~1:4. After stirring for 1~5h, the suspension was filtered to obtain a clear yellow-green solution. The filtrate was placed in an ice-water bath, and FeCl3•6H2O aqueous solution was added. The molar ratio of 4-nitroaniline to FeCl3•6H2O was 1:1~1:3. The aqueous solution was extracted three times with dichloromethane. The extracted solution was evaporated to dryness and dried under vacuum at 45℃ to obtain intermediate I, denoted as 4-nitrosobenzyl alcohol.

[0017] (2) Second step Mills reaction: intermediate I, d 0 / d 5-Aniline and acetic acid were added to an ethanol solution and stirred at room temperature for 6–10 hours. Intermediate I and... d 0 / dThe molar ratio of 5-aniline was 1:1 to 1:2.5, and the molar ratio of intermediate I to acetic acid was 1:5 to 1:20. After the reaction was complete, water was added to the solution, and the precipitate was dried under vacuum at 60°C after filtration. Intermediate II was obtained by silica gel column chromatography (dichloromethane:acetone = 40:1, v / v), denoted as […]. d 0 / d 5,4-Hydroxymethylazobenzene;

[0018] (3) Third step Appel reaction: Weigh intermediate II and carbon tetrabromide into a single-necked flask, add dry tetrahydrofuran, stir to dissolve, then add triphenylphosphine, and react at room temperature for 4-8 h. 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 complete, filter, collect the filtrate, remove the solvent under reduced pressure, and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 40:1) to obtain intermediate III, denoted as... d 0 / d 5-4-Bromomethylazobenzene;

[0019] (4) Fourth step of substitution reaction: Imidazole, sodium hydroxide, and ultrapure water were added to a round-bottom flask. Intermediate III was dissolved in tetrahydrofuran, with a molar ratio of intermediate III to imidazole of 1:1 to 1:3 and a molar ratio of intermediate III to sodium hydroxide of 1:1 to 1:2. This solution was added dropwise to the above mixture under argon protection. After heating at 60°C for 10 to 15 hours, the solvent was removed under reduced pressure. The crude product was extracted three times with dichloromethane / water (4:1, v / v). The organic layer was concentrated and dried under vacuum. Intermediate IV was obtained by silica gel column chromatography (dichloromethane:methanol = 25:1, v / v). d 0 / d 5-1-(4-(phenyldiazeninyl)benzyl)imidazolium;

[0020] (5) Fifth step, salt formation reaction: Intermediate IV and 1,3-dibromopropane were dissolved in acetonitrile, with a molar ratio of 1:5 to 1:20. The mixture was heated under reflux for 20 to 25 hours, and the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography (dichloromethane:methanol = 7:1, v / v) to obtain intermediate V, denoted as... d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazetenyl)benzyl)imidazol-3-onium bromide;

[0021] (6) Sixth step of substitution reaction: Intermediate V and NaN3 are dissolved in DMF, with a molar ratio of intermediate V to NaN3 of 1:1 to 1:6. After heating at 50°C for 10 to 15 hours, a large amount of distilled water is added, and the mixture is extracted with n-butanol and washed several times with water. The solvent is removed under reduced pressure to obtain the target compound, denoted as […]. d0 / d 5-3-(3-azidopropyl)-1-(4-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 0 / d 5-Azo-N3.

[0022] Finally, α-CDP was dispersed into the molecular probe solution ( d 0-Azo-N3 or d In 5-Azo-N3), oscillatory adsorption occurs, utilizing the host-guest recognition between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure to achieve molecular probe loading on α-CDP. After washing and freeze-drying, a solid-phase probe is obtained, denoted as α-CDP- d 0-Azo-N3 or α-CDP- d 5-Azo-N3. α-CDP and molecular probe d 0 / d The mass ratio of 5-Azo-N3 is 1:0.01 to 1:0.8 (preferably 1:0.2 to 1:0.6), the concentration of the molecular probe solution is 0.1 to 2.0 mg / mL (preferably 0.5 to 1.5 mg / mL), the pH of the molecular probe solution is 3 to 10 (preferably 5 to 8), the temperature for oscillation adsorption is 20 to 60℃ (preferably 25 to 35℃), and the time for oscillation adsorption is 5 to 60 min (preferably 20 to 45 min).

[0023] The synthesis mechanism of molecular probes is as follows:

[0024]

[0025] The first step involves zinc powder continuously transferring electrons to the nitro group of 4-nitrobenzyl alcohol via a single-electron transfer mechanism under acidic conditions provided by ammonium chloride. The nitro group loses a water molecule and is reduced to a nitroso group, yielding intermediate I (4-nitrosobenzyl alcohol). The second step involves intermediate I and... d 0 / d 5-Aniline reacts with acetic acid to form aromatic azo compounds. d 0 / d The lone pair of electrons on the nitrogen atom of 5-aniline attacks the nitroso group, losing a water molecule to form intermediate II. d 0 / d (5,4-hydroxymethylazobenzene); In the third step, the reaction of triphenylphosphine and carbon tetrabromide is activated, and then the oxygen atom of the hydroxyl group on intermediate II attacks the bromotriphenylphosphine to give the oxophosphonium salt, and the bromide ion undergoes S... N In reaction 2, triphenylphosphine departs as a leaving group, yielding intermediate III ( d 0 / d5,4-bromomethylazobenzene); Step 4, utilizing d 0 / d The nucleophilic substitution reaction between 5-4-bromomethylazobenzene and imidazole prepares intermediate IV. Under the action of an inorganic base, the nitrogen atom on the imidazole attacks... d 0 / d In 5-4-bromomethylazobenzene, the positively charged carbon atom undergoes a bromide ion departure as a leaving group, yielding intermediate IV. d 0 / d 5-1-(4-(phenyldiazeninyl)benzyl)imidazolium); In the fifth step, intermediate IV undergoes a salt-forming reaction with 1,3-dibromopropane under heating conditions to generate quaternary ammonium salt ions, yielding intermediate V ( d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazetenyl)benzyl)imidazol-3-onium bromide); Step 6: The target product is prepared by a 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-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide ( d 0 / d 5-Azo-N3).

[0026] The preparation mechanism of solid-phase probes is as follows:

[0027] Cyclodextrin polymers, used as solid-phase supports, are obtained by crosslinking α-cyclodextrin monomers with tetrafluoroterephthalonitrile, a rigid aromatic crosslinking agent. This overcomes the problem of polymer chain entanglement, forming α-CDPs with ultraporous structures and selective adsorption of molecular probes. d 0 / d The strong host-guest recognition of the azobenzene unit in the 5-Azo-N3 structure enables efficient loading of probe molecules, facilitating the preparation of solid-phase probes.

[0028] II. Application of solid-phase probes labeled with terminal alkynes in the detection and screening of natural terminal alkynes.

[0029] Another objective of this invention is to provide the 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 achieve rapid detection and screening of natural terminal alkyne compounds.

[0030] The method for detecting and screening natural terminal alkyne compounds includes the following steps:

[0031] (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- to each centrifuge tube. d 0-Azo-N3 and α-CDP- d The solid-phase probe, 5-Azo-N3, is used at a concentration of 1–10 mg / mL (preferably 1–5 mg / mL). Then, aqueous solutions of sodium ascorbate and copper sulfate pentahydrate are added, with the sodium ascorbate concentration being 0.05–2 mg / mL (preferably 0.2–1.0 mg / mL) and the copper sulfate pentahydrate concentration being 5–400 μg / mL (preferably 50–200 mg / mL). The solution is diluted to 1 mL with ultrapure water, ultrasonically dispersed, and chemically labeled by heating and shaking. 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 1200–1500 rpm), and the labeling time is 10–120 min (preferably 30–120 min).

[0032] (2) Centrifuge and wash to remove impurities, add ultrapure water to disperse, spot 1 μL of sample solution onto the target plate, irradiate with 365 nm ultraviolet light for 1~60 min (preferably 5~30 min), spot 1 μL of matrix solution, and wait for matrix-assisted laser desorption / ionization mass spectrometry for 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 2~20 mg / mL (preferably 5~12 mg / mL).

[0033] (3) Matrix-assisted laser desorption / ionization mass spectrometry was performed to observe the characteristic isotope mass spectrum peak with a mass-to-charge ratio (m / z) difference of approximately 5.0314. This isotope characteristic mass spectrum peak can be used to accurately screen terminal alkyne compounds.

[0034] The detection and assisted screening mechanisms are as follows:

[0035] Cu was reduced in situ using sodium ascorbate. 2+ Catalyst Cu + 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 ethynyl group of the terminal alkyne compound forms a 1,4-disubstituted 1,2,3-triazole-labeled product. After CuAAC labeling, the solid-phase probe is separated by centrifugation and washed multiple times to eliminate the influence of the molecular probe in solution on detection, while effectively reducing matrix interference from complex systems. The washed solid-phase probe is spotted onto the target plate of MADLI-MS. Under ultraviolet light irradiation, the azobenzene unit in the labeled product acts as a photo-switching mechanism, enabling the trans-to-cis configuration transition and dissociation from the solid-phase support, which is beneficial for subsequent MALDI-MS detection. The monovalent copper-catalyzed azide-alkynyl cycloaddition (CuAAC) reaction can introduce isotopic groups and mass spectrometry tags into the labeled product, generating a characteristic isotopic mass spectrometry peak with a mass-to-charge ratio (m / z) difference of 5.0314. This characteristic isotopic mass spectrometry peak can be used for accurate detection of terminal alkyne compounds. Calculations can yield the accurate molecular weight of unlabeled terminal alkyne compounds. Combined with the fragmentation patterns of multi-stage mass spectrometry, structural analysis of terminal alkyne compounds is expected. Labeling and detecting natural resource extracts, using isotopic characteristic mass spectrometry peaks, can aid in screening natural resources containing terminal alkyne compounds, improving the discovery efficiency. Furthermore, using positively charged imidazole salts as mass spectrometry tags can significantly enhance the sensitivity of labeled products in positive ion mass spectrometry detection.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention discloses a solid-phase probe for labeling terminal alkynes, its synthesis method, and its application. It uses a cyclodextrin polymer as a solid-phase support, loading a molecular probe onto the support through host-guest recognition, thereby constructing a solid-phase probe. Compared to traditional solid-phase probes based on covalent bonds, this method uses host-guest recognition to load the molecular probe, eliminating the need for multi-step, complex synthesis steps, simplifying the solid-phase probe preparation process, and significantly increasing the molecular probe loading capacity. Thanks to the organic combination of the solid-phase support and the molecular probe, this solid-phase probe possesses advantages such as easy separation of the solid-phase support, efficient reaction promotion, and simplified separation and purification steps of the labeled product. Simultaneously, the loaded molecular probe can achieve highly selective labeling of terminal alkynes in complex systems through the CuAAC reaction, and introduces isotopic groups and mass spectrometry tags into the labeled product through the labeling reaction, endowing it with isotopic characteristic signals and improving the sensitivity of the labeled product in mass spectrometry detection. Under ultraviolet light irradiation, the labeled product is easily released and subsequently detected by MALDI-MS. Furthermore, combined with the multi-stage mass spectrometry fragmentation rules, it is expected to achieve structural identification of terminal alkynes. Compared with traditional labeled product release processes, this method does not require further post-processing and does not involve severe fracture conditions (strong acids, strong bases, organic solvents, etc.). It has the advantages of simple operation, strong labeled product signal, and obvious isotopic characteristic signal. Attached Figure Description

[0038] Figure 1 The FT-IR spectrum of the cyclodextrin polymer (α-CDP) prepared in the embodiments of the present invention.

[0039] Figure 2 SEM image of α-CDP prepared in an embodiment of the present invention.

[0040] Figure 3 Molecular probes prepared for embodiments of the present invention d NMR of 0-Azo-N3 1 H NMR spectrum.

[0041] Figure 4 Molecular probes prepared for embodiments of the present invention d High-resolution mass spectrum of 0-Azo-N3.

[0042] Figure 5 Molecular probes prepared for embodiments of the present invention d NMR of 5-Azo-N3 heavy labeling reagent 1 H NMR spectrum.

[0043] Figure 6 Molecular probes prepared for embodiments of the present invention d High-resolution mass spectrum of 5-Azo-N3.

[0044] Figure 7 The solid-phase probe α-CDP- prepared for the embodiments of the present invention d 0-Azo-N3 and α-CDP- d MALDI-MS mass spectrum of 5-Azo-N3.

[0045] Figure 8 MALDI-MS mass spectra of terminal alkyne compounds in plant extracts labeled using the two solid-phase probes of this invention. Detailed Implementation

[0046] The present invention will be further explained and described below with reference to specific embodiments. Example

[0047] First, the synthesis of solid-phase α-CDP.

[0048] (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.

[0049] (2) After the reaction is complete, cool to room temperature, centrifuge at 7500 rpm for 10 min to collect the yellow precipitate, wash it repeatedly with methanol, water, tetrahydrofuran and dichloromethane, and dry it in a vacuum oven to obtain a yellow solid powder, namely cyclodextrin polymer material (α-CDP).

[0050] Secondly, molecular probes d 0 / d Synthesis of 5-Azo-N3

[0051] d Synthesis of 0-Azo-N3:

[0052] (1) Synthesis of intermediate I (4-nitrosobenzyl alcohol)

[0053] 7.4 g of 4-nitrobenzyl alcohol and 3.3 g of ammonium chloride were dissolved in 220 mL of a 10:1 mixture of 2-ethylene glycol monomethyl ether and water under argon protection. Then, 7.8 g of zinc powder was added at room temperature, and the mixture was stirred for 3 h. The suspension was filtered to obtain a clear, yellow-green solution. The filtrate was placed in an ice-water bath, and 50 mL of 13.3 g of FeCl3•6H2O aqueous solution was added. The mixture was stirred overnight. The resulting aqueous solution was extracted three times with dichloromethane. The extracted solutions were then evaporated to dryness. o Vacuum drying at C yielded 5.4 g of intermediate I (4-nitrosobenzyl alcohol).

[0054] (2) Intermediate ⅠI ( d Synthesis of 0-4-hydroxymethylazobenzene

[0055] 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 complete, water was added to the solution, and the mixture was filtered. The resulting precipitate was then heated at 60 °C. o Vacuum dried at C. After silica gel column chromatography (dichloromethane:acetone = 40:1, v / v), 3.3 g of a yellowish-brown solid was obtained, namely intermediate II. d 0-4-hydroxymethylazobenzene), yield 84%.

[0056] (3) Intermediate I II ( d Synthesis of 0-4-bromomethylazobenzene

[0057] Weigh 1.0 g of intermediate II and 2.4 g of carbon tetrabromide into 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 complete, filter, collect the filtrate, remove the solvent under reduced pressure, and perform silica gel column chromatography (petroleum ether:ethyl acetate = 40:1) to obtain 0.66 g of orange-yellow solid, i.e., intermediate II. d 0-4-bromomethylazobenzene), yield 51%.

[0058] (4) Intermediate IV ( d Synthesis of 0-1-(4-(phenyldiazetenyl)benzyl)imidazolium

[0059] Add 0.3 g imidazole, 0.2 g sodium hydroxide, and 5 mL of ultrapure water to a round-bottom flask. Dissolve 0.5 g of intermediate I / II in tetrahydrofuran and add it dropwise to the above mixture under argon protection. 60 o After heating at 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. Silica gel column chromatography (CH2Cl2 / MeOH = 25:1) gave 0.4 g of an orange-yellow solid, namely intermediate IV. d 0-1-(4-(phenyldiazeninyl)benzyl)imidazolium), yield 86%.

[0060] (5) Intermediate V ( d Synthesis of 0-3-(3-bromopropyl)-1-(4-(phenyldiazetenyl)benzyl)imidazol-3-onium bromide

[0061] 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 evaporated, and the solution was purified by silica gel column chromatography (CH2Cl2 / MeOH = 7:1) to give 0.5 g of orange solid, i.e., intermediate V. d 0-3-(3-bromopropyl)-1-(4-(phenyldiazetenyl)benzyl)imidazol-3-onium bromide), yield 87%.

[0062] (6) Target product d Synthesis of 0-Azo-N3

[0063] Dissolve 0.2 g of intermediate V and 0.2 g of NaN3 in 30 mL of DMF, 50 o After heating at C for 12 h, a large amount of distilled water was added, followed by extraction with n-butanol and washing with water several times. The solvent was removed under reduced pressure to obtain 0.08 g of orange solid, which is the target product. d 0-Azo-N3, yield 44%.

[0064] d Synthesis of 5-Azo-N3:

[0065] With the above d Compared to the synthesis of 0-Azo-N3, molecular probes d The synthesis of 5-Azo-N3 was carried out in the second step. d 5-Aniline is used as the starting material, and the rest of the synthesis steps are the same.

[0066] Finally, the solid-phase probe α-CDP- d 0-Azo-N3 and α-CDP- d Preparation of 5-Azo-N3

[0067] Take 50 mg of α-CDP and add it to 30 mL of molecular probe solution. d 0-Azo-N3 and d In 5-Azo-N3 (pH=7, concentration 1 mg / mL), the molecular probe was adsorbed by shaking at 40 °C for 45 min. The host-guest recognition between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure was utilized to load the molecular probe onto α-CDP. After washing and freeze-drying, a solid-phase probe was obtained, denoted as α-CDP- d 0-Azo-N3 and α-CDP- d 5-Azo-N3.

[0068] Product structure characterization

[0069] Characterization of solid-phase support α-CDP:

[0070] Fourier transform-infrared (FT-IR) spectrum Figure 1 ): 2244 cm -1 The CN group is introduced after crosslinking cyclodextrin and tetrafluoroterephthalonitrile, 1636 cm -1 The absorption peak at that point corresponds to the stretching vibration of the benzene ring C-C, indicating that tetrafluoroterephthalonitrile has been successfully crosslinked onto α-CD, proving the successful synthesis of solid-phase α-CDP.

[0071] Scanning electron microscopy (SEM) characterization: Figure 2 The SEM morphology of α-CDP shows that it has an irregular blocky structure with a rough surface and features an ultraporous structure. This structure is conducive to the adsorption of molecular probes, thereby enabling the rapid preparation of solid-phase probes.

[0072] Molecular probe d Characterization of 0-Azo-N3 products:

[0073] 1H NMR spectrum 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).

[0074] High-resolution mass spectrometry (HS-MS) Figure 4 ): calcd. for C 19 H 20 N7 + , 346.1775 [M – Br] + ; found, 346.1777.

[0075] The above characterization results demonstrate that the molecular probe was successfully synthesized in this embodiment. d 0-Azo-N3.

[0076] Molecular probe d Characterization of 5-Azo-N3 products:

[0077] 1H NMR spectrum 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).

[0078] High-resolution mass spectrometry (HS-MS) Figure 6 ): calcd. for C 19 H 15 D5N7 + , 351.2089 [M – Br] + ; found: 351.2083.

[0079] The above characterization results demonstrate that the molecular probe was successfully synthesized in this embodiment. d 5-Azo-N3.

[0080] Solid-phase probe α-CDP- d Characterization of 0-Azo-N3:

[0081] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) characterization Figure 7 a): Solid-phase probe α-CDP- d The preparation of 0-Azo-N3 utilizes d Non-covalent host-guest interaction between 0-Azo-N3 and α-CDP. Analysis of this solid-phase probe using MALDI-MS revealed a mass spectrum peak at m / z 346.182, corresponding to... d The mass spectrum peak of 0-Azo-N3.

[0082] The above characterization results show that the solid-phase probe α-CDP- was successfully synthesized in this embodiment. d 0-Azo-N3.

[0083] Solid-phase probe α-CDP- d Characterization of 5-Azo-N3:

[0084] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) characterization Figure 7b): Analysis of the solid-phase probe using MALDI-MS revealed a mass spectrum peak at m / z 351.211, corresponding to... d The mass spectrum peak of 5-Azo-N3.

[0085] The above characterization results show that the solid-phase probe α-CDP- was successfully synthesized in this embodiment. d 5-Azo-N3.

[0086] Applications of natural terminal alkyne compounds in detection and assisted screening

[0087] The two solid-phase probes α-CDP- prepared in the above embodiments were used. d 0-Azo-N3 and α-CDP- d The labeling and screening of terminal alkyne compounds using 5-Azo-N3 were carried out using the following steps:

[0088] (1) Take 1 mg of the herbal beauty button ( ) Spilanthes callimorpha The extract was added to two separate 1.5 mL centrifuge tubes, and the solid-phase probe α-CDP- was added to each tube. d 0-Azo-N3 and α-CDP- d 5-Azo-N3 was used as a solid-phase probe at a concentration of 1 mg / mL. Then, aqueous solutions of sodium ascorbate and copper sulfate pentahydrate were added, with the concentrations of sodium ascorbate (0.5 mg / mL) and copper sulfate pentahydrate (100 μg / mL) respectively. The solutions were diluted to 1 mL with ultrapure water, ultrasonically dispersed for 1 min, and chemically labeled by heating and shaking at 40 °C (1200 rpm) for 90 min.

[0089] (2) Centrifuge and wash to remove impurities, add 100 μL of ultrapure water to disperse, spot 1 μL of sample solution onto the target plate, irradiate with 365 nm ultraviolet light for 30 min, then spot 1 μL of CHCA matrix solution. The concentration of CHCA is 10 mg / mL. Wait for matrix-assisted laser desorption / ionization mass spectrometry to detect it.

[0090] (3) Matrix-assisted laser desorption / ionization mass spectrometry was performed to observe the characteristic isotope mass spectrum peak with a mass-to-charge ratio (m / z) difference of approximately 5.0314. This isotope characteristic mass spectrum peak can be used to accurately screen terminal alkyne compounds.

[0091] Two solid-phase probes were used to label terminal alkyne compounds in plant extracts via a CuAAC reaction. After the labeling reaction, the labeled products were dissociated from the solid support by ultraviolet light irradiation for further MALDI-MS detection. Figure 8As shown, a set of mass spectral peaks with isotopic characteristics (mass-to-charge ratio m / z difference of 5.0307, ​​close to the theoretical value of 5.0314) was found, proving that the crude extract contains at least one terminal alkyne compound, and confirming the presence of a naturally occurring terminal alkyne compound 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 terminal alkyne compounds, characterized in that, This solid-phase probe uses a cyclodextrin polymer material as a carrier and a molecular probe as the substrate. The substrate is loaded onto the carrier through host-guest recognition between the cyclodextrin units in the polymer material and the azobenzene units in the molecular probe structure. It is denoted as α-CDP- d 0-Azo-N3 or α-CDP- d 5-Azo-N3; Among them, cyclodextrin polymer material is a blocky polymer material with a dense ultra-microporous structure prepared by polymerization reaction using α-cyclodextrin as monomer and tetrafluoroterephthalonitrile as crosslinking agent, 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-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 0-Azo-N3; when X=D, it is called d 5-3-(3-azidopropyl)-1-(4-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 5-Azo-N3.

2. A method for preparing a solid-phase probe of a labeled terminal alkyne compound as described in claim 1, characterized in that, The process involves dispersing α-CDP into a molecular probe solution, oscillating and adsorbing it, and utilizing the host-guest recognition between the cyclodextrin unit in α-CDP and the azobenzene unit in the molecular probe structure to load the molecular probe onto α-CDP. After washing and freeze-drying, a solid-phase probe is obtained.

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

4. The method for preparing a solid-phase probe of a labeled terminal alkyne compound as described in claim 2, characterized in that, The preparation of the cyclodextrin polymer material includes the following steps: (1) Using α-cyclodextrin as a monomer, mix it with potassium carbonate and tetrafluoroterephthalonitrile and place it in a dry three-necked flask. Add a mixed solution of anhydrous tetrahydrofuran and anhydrous N,N-dimethylformamide in a volume ratio of 9:

1. Under inert gas protection, heat the mixture at 85°C for 12~48h. (2) After the reaction is complete, cool to room temperature, centrifuge at 7500 rpm for 10 min to collect the yellow precipitate, wash it repeatedly with methanol, water, tetrahydrofuran and dichloromethane, and dry it in a vacuum oven to obtain a yellow solid powder, which is the cyclodextrin polymer material, denoted as: α-CDP.

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

5.

6. The method for preparing a solid-phase probe of a labeled terminal alkyne compound as described in claim 2, characterized in that, The synthesis of the molecular probe includes the following steps: (1) First step reduction reaction: 4-nitrobenzyl alcohol and ammonium chloride were dissolved in a mixed solvent of diethylene glycol monomethyl ether / water under argon protection. Zinc powder was added at room temperature and the mixture was stirred for 1-5 h. The suspension was filtered to obtain a clear yellow-green solution. The filtrate was placed in an ice-water bath and FeCl3•6H2O aqueous solution was added. The solution was extracted three times with dichloromethane. The extracted solution was evaporated to dryness and dried under vacuum at 45 °C to obtain intermediate I, denoted as 4-nitrosobenzyl alcohol. (2) Second step Mills reaction: intermediate I, d 0 / d 5-Aniline and acetic acid were added to an ethanol solution and stirred at room temperature for 6–10 h. After the reaction was complete, water was added to the solution, and the mixture was filtered. The resulting precipitate was dried under vacuum at 60 °C and purified by silica gel column chromatography to obtain intermediate II, denoted as […]. d 0 / d 5,4-Hydroxymethylazobenzene; (3) Third step Appel reaction: Weigh intermediate II and carbon tetrabromide into 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 complete, filter, collect the filtrate, remove the solvent under reduced pressure, and purify by silica gel column chromatography to obtain intermediate III, denoted as... d 0 / d 5,4-Bromomethylazobenzene; (4) Fourth step of substitution reaction: Imidazole, sodium hydroxide and ultrapure water were added to a round-bottom flask. Intermediate III was dissolved in tetrahydrofuran and added dropwise to the above mixture under argon protection. The reaction was heated at 60°C for 10-15 h. 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 intermediate IV, denoted as... d 0 / d 5-1-(4-(phenyldiazeninyl)benzyl)imidazolium; (5) Fifth step: 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, denoted as... d 0 / d 5-3-(3-bromopropyl)-1-(4-(phenyldiazetenyl)benzyl)imidazol-3-onium bromide; (6) Sixth step substitution reaction: Dissolve intermediate V and NaN3 in DMF, heat at 50°C for 10-15 h, pour in a large amount of distilled water, extract with n-butanol, wash with water several times, remove solvent under reduced pressure to obtain the target compound, denoted as […]. d 0 / d 5-3-(3-azidopropyl)-1-(4-(phenyldiazeninyl)benzyl)imidazol-3-onium bromide, abbreviated as d 0 / d 5-Azo-N3.

7. The method for preparing a solid-phase probe of a labeled terminal alkyne compound as described in claim 6, characterized in that, In the reduction reaction described in step (1), the molar ratio of 4-nitroaniline to ammonium chloride is 1:1 to 1:2, the molar ratio of 4-nitroaniline to zinc powder is 1:1 to 1:4, and the molar ratio of 4-nitroaniline to FeCl3•6H2O is 1:1 to 1:

3. In the Mills reaction described in step (2), intermediate I reacts with... 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 described 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 described in step (4), the molar ratio of intermediate III to imidazole is 1:1 to 1:3, and the molar ratio of intermediate III to sodium hydroxide is 1:1 to 1:

2. In the salt formation reaction described 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. The application of a solid-phase probe for labeling terminal alkynes as described in claim 1 in the detection of natural terminal alkynes, characterized in that, Includes the following steps: (1) Take 1 mg of the plant extract and add it to two separate 1.5 mL centrifuge tubes. Add the solid-phase probe α-CDP- to each centrifuge tube. d 0-Azo-N3 and α-CDP- d 5-Azo-N3 was then added to an aqueous solution of sodium ascorbate and copper sulfate pentahydrate, diluted to 1 mL with ultrapure water, ultrasonically dispersed, and chemically labeled by heating and shaking. (2) Centrifuge and wash to remove impurities, add ultrapure water to disperse, spot 1 μL of sample solution onto 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 to detect it. (3) Matrix-assisted laser desorption / ionization mass spectrometry was performed to observe the characteristic isotope mass spectrum peak with a mass-to-charge ratio difference of 5.0314. This isotope characteristic mass spectrum peak can be used to accurately detect terminal alkyne compounds and assist in screening natural resources with terminal alkyne compounds.

9. The application of the solid-phase probe for labeled terminal alkyne compounds as described in claim 8 in the detection of natural terminal alkyne compounds, characterized in that, In step (1), the amount of solid probe used is 1~10 mg / mL, the concentration of sodium ascorbate solution is 0.05~2 mg / mL, the concentration of 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℃, the oscillation speed is 600~1500 rpm, and the labeling time is 10~120 min.

10. The application of the solid-phase probe for labeled terminal alkyne compounds as described in claim 8 in the detection of natural terminal alkyne compounds, characterized in that, In step (2), the irradiation time with 365nm ultraviolet light is 1~60min. 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. The concentration of the matrix solution is 2~20mg / mL.

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