Cyclodextrin bonded covalent organic framework chiral stationary phase for chiral amino acid separation and preparation method of cyclodextrin bonded covalent organic framework chiral stationary phase

Abstract

The invention discloses a cyclodextrin bonded covalent organic framework chiral stationary phase for chiral amino acid separation and a preparation method of the cyclodextrin bonded covalent organic framework chiral stationary phase. The method comprises the following steps: modifying silica gel microspheres by using a silane coupling agent to obtain silanized silica gel microspheres, reacting the silanized silica gel microspheres with a covalent organic framework by using a one-pot method to prepare covalent organic framework modified silica gel microspheres, and carrying out a thiol-alkyne click strategy by using vulcanized cyclodextrin as a functional molecule to obtain the covalent organic framework modified silica gel microspheres. The cyclodextrin bonded covalent organic framework chiral stationary phase is prepared; the cyclodextrin bonded covalent organic framework has strong chiral recognition capability, a packed column of the cyclodextrin bonded covalent organic framework can realize baseline separation of 12 dansyl amino acids within 30 minutes in liquid chromatography, the separation degrees of valine, isoleucine and leucine respectively reach 5.017, 4.351 and 3.782, the preparation process is simple to operate, the reaction condition is mild, the cost is low, and the cyclodextrin bonded covalent organic framework is suitable for industrial production. The practical application prospect is realized.

Description

Technical Field

[0001] The invention relates to the field of chiral covalent organic frameworks, and in particular to a novel cyclodextrin-bonded covalent organic framework chiral stationary phase for separation of chiral amino acids and a preparation method thereof. Background Art

[0002] Covalent organic frameworks (COFs) are two-dimensional or three-dimensional porous materials with all-organic composition, which form highly ordered networks through condensation reactions of elements such as boron, carbon, nitrogen, oxygen and silicon. Due to their high specific surface area, low density, inherent porosity, adjustable pore size and easily customizable structure, these materials show unique properties in many fields, such as catalysis, drug delivery, gas storage and adsorption, sensor design and chromatography technology. In particular, COFs show great research and application potential in asymmetric catalysis and enantioselective recognition. By selecting appropriate chiral selectors and introducing them into COFs, chiral covalent organic frameworks (CCOFs) with chiral recognition and separation functions can be developed. In recent years, the scientific community has successfully synthesized a variety of CCOFs by adopting bottom-up and post-modification strategies and applied them to stationary phases for chromatographic separation. In particular, cyclodextrin-bonded covalent organic frameworks have attracted widespread attention due to their excellent performance in chiral recognition.

[0003] Chirality is a fundamental property of nature and is widely present in key molecules that make up organisms, such as amino acids, enzymes, and nucleotides. This stereochemical diversity not only gives molecules unique properties, but also plays a vital role in specific interactions in biological processes, thereby affecting the structure and function inside organisms. It is well known that although a pair of chiral enantiomers are almost identical in physical and chemical properties, their biological activities may be completely different, and sometimes even show opposite effects or toxicity. For example, amino acids in the human body are a new type of small molecule chiral biomarker, and their enantiomeric composition is a key indicator in disease prediction and diagnosis research. Therefore, the establishment of chiral amino acid analysis methods is imminent.

[0004] In summary, it is of great significance to develop a new type of cyclodextrin-bonded covalent organic framework chiral stationary phase for the separation of chiral amino acids. Summary of the invention

[0005] The present invention aims to provide a cyclodextrin bonded covalent organic framework chiral stationary phase for separation of chiral amino acids and a preparation method thereof.

[0006] The present invention modifies silica microspheres by a silane coupling agent, then bonds a covalent organic framework to the surface of the silica microspheres by a one-pot method, and then bonds mono(6-mercapto-6-deoxy)beta-cyclodextrin (β-CD-SH) to the covalent organic framework by a "click" reaction strategy to obtain a cyclodextrin-bonded covalent organic framework chiral stationary phase. The chiral stationary phase of the present invention is filled into a stainless steel column and can be used for high performance liquid chromatography enantiomer separation.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing a cyclodextrin-bonded covalent organic framework chiral stationary phase comprises the following steps:

[0009] (1) placing silica gel microspheres in a hydrochloric acid solution, stirring at room temperature for activation for 6 to 48 hours, washing with purified water until neutral, and freeze-drying to obtain activated silica gel microspheres (SiO2);

[0010] The particle size of silica microspheres is 2 to 10 μm;

[0011] The hydrochloric acid solution is prepared by mixing concentrated hydrochloric acid and water in a volume ratio of 4:1 to 1:1;

[0012] (2) dispersing the active silica microspheres obtained in step (1) in anhydrous toluene, adding γ-glycidyloxypropyltrimethoxysilane (GPTS), reacting at 110-140° C. for 12-36 hours under a nitrogen atmosphere, then cooling to room temperature, washing (using ethanol and water in sequence), and freeze-drying to obtain silanized silica microspheres (SiO2-GPTS);

[0013] Preferably, the mass volume ratio of activated silica microspheres to γ-glycidyloxypropyltrimethoxysilane is 2 to 5:10, g / mL;

[0014] (3) mixing the silanized silica microspheres obtained in step (2), 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde (BPTA), 1,3,5-tri(4-aminophenyl)benzene (TPB), and acetonitrile, uniformly dispersing by ultrasonication, adding AcOH under stirring, reacting at 25 to 50° C. for 1 to 3 days, washing after the reaction (using acetonitrile and water to wash thoroughly), and freeze-drying to obtain covalent organic framework-modified silica microspheres (SiO2-COF);

[0015] Preferably, the mass ratio of silanized silica microspheres, 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde, and 1,3,5-tris(4-aminophenyl)benzene is 2-5:0.2-0.5:0.2-0.5;

[0016] (4) adding the covalent organic framework-modified silica microspheres obtained in step (3) to 1,4-dioxane for ultrasonic dispersion, then adding a solution of β-CD-SH dissolved in DMSO, reacting at 100-130° C. for 8-24 hours under a nitrogen atmosphere, washing after the reaction (using acetonitrile and water in sequence), and freeze-drying to obtain the cyclodextrin-bonded covalent organic framework chiral stationary phase (SiO2-COF-S-CD);

[0017] Preferably, the mass ratio of the covalent organic framework-modified silica microspheres to β-CD-SH is 2 to 5:1.

[0018] The invention relates to a cyclodextrin bonded covalent organic framework chiral stationary phase prepared by the preparation method.

[0019] The cyclodextrin bonded covalent organic framework chiral stationary phase of the present invention is filled into a chromatographic column, which can be used for high performance liquid chromatography to separate chiral amino acids; the chiral amino acids include but are not limited to dansyl amino acids, and the dansyl amino acids are specifically exemplified by dansyl valine, dansyl isoleucine, and dansyl leucine; Figure 1 As shown, the selected 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde has an alkyne side chain, which can react with two molecules of β-SH-CD to prepare a chiral stationary phase similar to cyclodextrin; through the synergistic effect of the two molecules of cyclodextrin, it helps to improve the separation effect of dansyl amino acids.

[0020] The specific column filling method is as follows:

[0021] The cyclodextrin bonded covalent organic framework chiral stationary phase is ultrasonically dispersed in isopropanol, methanol is used as the replacement liquid, the column packing machine pressure is increased to 40MPa and maintained for 2 hours, and the chiral stationary phase is loaded into the chromatographic column. After the filling is completed, the chromatographic column needs to be balanced with the mobile phase for two hours before the chromatographic experiment can be carried out;

[0022] The specifications of the chromatographic column are inner diameter: 4.6 mm, column length: 150 mm.

[0023] The beneficial effects of the present invention are:

[0024] The invention discloses a novel stationary phase of cyclodextrin-bonded covalent organic framework-modified silica microspheres as a filler for high performance liquid chromatography. The covalent organic framework-modified silica microspheres are first prepared by a one-pot Schiff base reaction method, and then β-CD-SH is bonded to the framework by a thiol-alkyne "click" reaction, thereby obtaining a novel chiral high performance liquid chromatography filler.

[0025] The filler prepared by the present invention has the advantages of high mechanical strength, excellent physical and chemical properties and strong chiral separation ability, and can efficiently separate chiral dansyl amino acid compounds in HILIC mode, with fast separation speed and high selectivity. The preparation process of the present invention has mild conditions, simple operation, high reactant yield, good material stability, significant separation effect for dansyl amino acids, and high economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of the synthesis of cyclodextrin-bonded covalent organic framework chiral stationary phase.

[0027] Figure 2 : Infrared spectra (IR) of SiO2, SiO2-GPTS, SiO2-COF and SiO2-COF-S-CD.

[0028] Figure 3 : Scanning electron microscope (SEM) images of SiO2-GPTS and SiO2-COF.

[0029] Figure 4 : Thermogravimetric (TGA) curves of SiO2-COF and SiO2-COF-S-CD.

[0030] Figure 5 : High performance liquid chromatography (HPLC) chart of dansyl valine.

[0031] Figure 6 : High performance liquid chromatography (HPLC) chart of dansyl isoleucine.

[0032] Figure 7 : High performance liquid chromatography (HPLC) chart of dansylleucine. DETAILED DESCRIPTION

[0033] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0034] The high performance liquid chromatograph used in the embodiment of the present invention is a Shimadzu analytical LC-20A high performance liquid chromatograph (Shimadzu, Japan), including a SPD-20AUV ultraviolet detector, a dual LC-20ATvp liquid phase pump, a CTO-10ASvp column oven, a LabSolutions chromatography workstation, a 20 μL injection loop, etc.

[0035] Silica gel microspheres were purchased from Qingdao Haisu New Material Technology Co., Ltd., 2-10 μm, 100A.

[0036] β-CD-SH can be prepared according to a known method, see Chen, S.; Zhu, J.; Ma, C.; Zhu, B.; Qiu, H.; Tong, S., Mercapto-β-cyclodextrin covalent organic frameworks forenantioselective liquid-liquid extraction. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2024, 687.

[0037] Embodiment 1:

[0038] A novel stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD) of silica spheres modified by cyclodextrin bonded covalent organic framework material is prepared, which specifically comprises the following steps:

[0039] (1) Weigh 3.0 g of silica gel microspheres, add them into 60 mL of a mixed solution of hydrochloric acid / water (1:1), stir and activate for two days, wash with pure water until neutral, and freeze-dry for 24 h to obtain activated silica gel microspheres (SiO2).

[0040] (2) Weigh 2.80 g of the activated silica microspheres (SiO2) prepared in step (1), add them to 60 mL of anhydrous toluene for ultrasonic suspension, add 10 mL of γ-glycidyloxypropyltrimethoxysilane (GPTS), and react at 120° C. for 24 h under nitrogen protection. After the reaction, cool to room temperature, wash with ethanol and water in turn, and freeze-dry to remove excess water to obtain silica microspheres modified with a silanization agent (SiO2-GPTS).

[0041] (3) Take 2.80g of the silica microspheres modified with silanization reagent (SiO2-GPTS) prepared in step (2), add 276.8mg of 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde (BPTA), 294mg of 1,3,5-tris(4-aminophenyl)benzene (TPB), and 140mL of acetonitrile, dissolve by ultrasonication, then add 12mL of AcOH (12M) under stirring, and react at 25°C for 3 days. After the reaction, wash with acetonitrile and water in turn, freeze-dry to remove excess water, and obtain silica microspheres modified with covalent organic framework (SiO2-COF).

[0042] (4) Take 2.50g of the covalent organic framework modified silica microspheres (SiO2-COF) prepared in step (3), add them to 30mL of 1,4-dioxane, and disperse them by ultrasonication; dissolve 1g of β-CD-SH in 4mL of DMSO, and then pour the solution into the reaction system, and react at 130°C for 8h under a nitrogen atmosphere. After the reaction, wash with acetonitrile and water in turn, and freeze-dry to remove excess water, to obtain a new type of cyclodextrin-bonded covalent organic framework material modified silica sphere stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD).

[0043] The activated silica microspheres (SiO2), silica microspheres modified with silanization reagents (SiO2-GPTS), silica microspheres modified with covalent organic frameworks (SiO2-COF), and silica microspheres modified with cyclodextrin-bonded covalent organic frameworks (SiO2-COF-S-CD) prepared in Example 1 were characterized by infrared spectroscopy. The results are shown in Figure 1. Figure 2 As shown, at 3420cm -1 、1640cm -1 and 1090cm -1 The change of the characteristic absorption peak at indicates the successful progress of each step of the reaction, proving that the method of the present invention can indeed bond the novel cyclodextrin-bonded covalent organic framework to the surface of silica microspheres.

[0044] Further, each step in the preparation process of the novel cyclodextrin bonded covalent organic framework modified silica sphere stationary phase prepared in Example 1 was characterized by scanning electron microscopy, elemental analysis and thermogravimetric analysis. Figure 3 ,The SEM images show that the surface of the covalent organic framework modified silica microspheres (SiO2-COF) obviously has a layer of COF particles, while the surface of the silanization reagent modified silica microspheres (SiO2-GPTS) is relatively smooth; Figure 4 The thermogravimetric curves show that the thermogravimetric loss of cyclodextrin-bonded covalent organic framework-modified silica spheres is significantly higher than that of silica microspheres (SiO2-COF) modified by cyclodextrin, which is attributed to the thermal decomposition of cyclodextrin; finally, the change in the sulfur content in the elemental analysis of Table 1 further proves the successful preparation of the new cyclodextrin-bonded covalent organic framework-modified silica sphere stationary phase.

[0045] Table 1 Elemental analysis results of SiO2-COF and SiO2-COF-S-CD

[0046]

[0047] Chromatographic Separation of Dansyl Amino Acids Using Cyclodextrin-Bonded Covalent Organic Framework-Modified Silica Spherical Stationary Phase Packing

[0048] Take 2.0g of the liquid chromatography filler SiO2-COF-S-CD prepared in Example 1, ultrasonically disperse it in 20mL of isopropanol, use methanol as the replacement liquid, increase the pressure of the column packing machine to 40MPa and maintain it for 2h, and load the filler into the chromatographic column. The specifications of the chromatographic column are inner diameter: 4.6mm, column length: 150mm. After the filling is completed, the chromatographic column needs to be equilibrated with the mobile phase for two hours before the chromatographic experiment can be carried out.

[0049] When investigating the chromatographic retention behavior of chiral enantiomers of dansyl amino acids on the stationary phase, acetonitrile / formic acid water (0.1%) was selected as the liquid chromatography mobile phase, with a flow rate of 0.8 mL / min, a column temperature of 25°C, and a detection wavelength of 212 nm. Sixteen dansyl amino acids were successfully separated, and baseline separation of twelve dansyl amino acids was achieved within 30 minutes. Figure 5-Figure 7 The typical chromatogram of the separation of dansyl valine, dansyl isoleucine and dansyl leucine proves that the prepared new liquid chromatography filler can effectively separate dansyl amino acid drugs.

[0050] Embodiment 2:

[0051] A novel stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD) of silica spheres modified by cyclodextrin bonded covalent organic framework material is prepared, which specifically comprises the following steps:

[0052] (1) Weigh 3.0 g of silica gel microspheres, add them into 60 mL of a mixed solution of hydrochloric acid / water (2:1), stir and activate for 12 h, wash with pure water until neutral, and freeze-dry for 24 h to obtain activated silica gel microspheres (SiO2).

[0053] (2) Weigh 2.80 g of the activated silica microspheres (SiO2) prepared in step (1), add them to 60 mL of anhydrous toluene for ultrasonic suspension, add 10 mL of γ-glycidyloxypropyltrimethoxysilane (GPTS), and react at 140° C. for 12 h under nitrogen protection. After the reaction, cool to room temperature, wash with ethanol and water in turn, and freeze-dry to remove excess water to obtain silica microspheres modified with a silanization agent (SiO2-GPTS).

[0054] (3) Take 2.80g of the silica microspheres modified with silanization reagent (SiO2-GPTS) prepared in step (2), add 276.8mg of 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde (BPTA), 294mg of 1,3,5-tris(4-aminophenyl)benzene (TPB), and 140mL of acetonitrile, dissolve by ultrasonication, then add 12mL of AcOH (12M) under stirring, and react at 50°C for 1 day. After the reaction, wash with acetonitrile and water in turn, freeze-dry to remove excess water, and obtain silica microspheres modified with covalent organic framework (SiO2-COF).

[0055] (4) Take 2.50g of the covalent organic framework modified silica microspheres (SiO2-COF) prepared in step (3), add them to 30mL of 1,4-dioxane, and disperse them by ultrasonication; dissolve 1g of β-CD-SH in 4mL of DMSO, and then pour the solution into the reaction system, and react at 120°C for 12h under a nitrogen atmosphere. After the reaction, wash with acetonitrile and water in turn, and freeze-dry to remove excess water, to obtain a new type of cyclodextrin-bonded covalent organic framework material modified silica sphere stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD).

[0056] Embodiment 3:

[0057] A novel stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD) of silica spheres modified by cyclodextrin bonded covalent organic framework material is prepared, which specifically comprises the following steps:

[0058] (1) Weigh 3.0 g of silica gel microspheres, add them into 60 mL of a mixed solution of hydrochloric acid / water (4:1), stir and activate for 6 h, wash with pure water until neutral, and freeze-dry for 24 h to obtain activated silica gel microspheres (SiO2).

[0059] (2) Weigh 2.80 g of the activated silica microspheres (SiO2) prepared in step (1), add them to 60 mL of anhydrous toluene for ultrasonic suspension, add 10 mL of γ-glycidyloxypropyltrimethoxysilane (GPTS), and react at 110° C. for 36 h under nitrogen protection. After the reaction, cool to room temperature, wash with ethanol and water in turn, and freeze-dry to remove excess water to obtain silica microspheres modified with a silanization agent (SiO2-GPTS).

[0060] (3) Take 2.80g of the silica microspheres modified with silanization reagent (SiO2-GPTS) prepared in step (2), add 276.8mg of 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde (BPTA), 294mg of 1,3,5-tris(4-aminophenyl)benzene (TPB), and 140mL of acetonitrile, dissolve by ultrasonication, then add 12mL of AcOH (12M) under stirring, and react at 40°C for 2 days. After the reaction, wash with acetonitrile and water in turn, freeze-dry to remove excess water, and obtain silica microspheres modified with covalent organic framework (SiO2-COF).

[0061] (4) Take 2.50g of the covalent organic framework modified silica microspheres (SiO2-COF) prepared in step (3), add them to 30mL of 1,4-dioxane, and disperse them by ultrasonication; dissolve 1g of β-CD-SH in 4mL of DMSO, and then pour the solution into the reaction system, and react at 100°C for 24h under a nitrogen atmosphere. After the reaction, wash with acetonitrile and water in turn, and freeze-dry to remove excess water, to obtain a new type of cyclodextrin-bonded covalent organic framework material modified silica sphere stationary phase high performance liquid chromatography filler (SiO2-COF-S-CD).

[0062] Comparative Example:

[0063] Chinese patent CN202310017073.0, Tang Yimin et al. reported a method for preparing a cyclodextrin-modified covalent organic framework for capillary electrochromatography chiral stationary phase; Chinese patent CN202211279932.5, Wang Lujun et al. reported a method for preparing a cyclodextrin-bonded covalent organic framework chiral stationary phase using thiophene "click" chemistry.

[0064] There has never been a report on the preparation of a cyclodextrin covalent organic framework stationary phase by a thiol-alkyne "click" reaction, and its use in the chiral separation of dansyl amino acids. This method prepares a new type of cyclodextrin-bonded covalent organic framework chiral stationary phase, and its filling column achieves extensive separation of dansyl amino acids in chiral separation, among which the separation degrees of dansyl valine, dansyl isoleucine and dansyl leucine reach 5.017, 4.351 and 3.782, respectively. The cyclodextrin-bonded covalent organic framework material has a strong chiral recognition ability and can be used as a chiral stationary phase for liquid chromatography to separate dansyl amino acids, and the preparation process is simple to operate, the reaction is mild, and the cost is low.

Claims

1. A method for preparing a cyclodextrin bonded covalent organic framework chiral stationary phase, characterized in that: The following steps are involved: (1) placing silica gel microspheres in a hydrochloric acid solution, stirring and activating at room temperature for 6 to 48 hours, washing with pure water until neutral, and freeze-drying to obtain activated silica gel microspheres; (2) dispersing the active silica microspheres obtained in step (1) in anhydrous toluene, adding γ-glycidyloxypropyltrimethoxysilane, reacting at 110-140° C. for 12-36 hours under a nitrogen atmosphere, then cooling to room temperature, washing, and freeze-drying to obtain silanized silica microspheres; (3) mixing the silanized silica microspheres obtained in step (2), 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde, 1,3,5-tri(4-aminophenyl)benzene, and acetonitrile, uniformly dispersing by ultrasonication, adding AcOH under stirring, reacting at 25 to 50° C. for 1 to 3 days, washing after the reaction, and freeze-drying to obtain covalent organic framework-modified silica microspheres; (4) adding the covalent organic framework-modified silica microspheres obtained in step (3) into 1,4-dioxane for ultrasonic dispersion, then adding a solution of β-CD-SH dissolved in DMSO, reacting at 100-130° C. for 8-24 h under a nitrogen atmosphere, washing after the reaction, and freeze-drying to obtain the cyclodextrin-bonded covalent organic framework chiral stationary phase.

2. The method for preparing the cyclodextrin bonded covalent organic framework chiral stationary phase according to claim 1, characterized in that: In step (1), the particle size of the silica microspheres is 2 to 10 μm.

3. The method for preparing the cyclodextrin bonded covalent organic framework chiral stationary phase according to claim 1, characterized in that: In step (1), the hydrochloric acid solution is prepared by mixing concentrated hydrochloric acid and water in a volume ratio of 4:1 to 1:

1.

4. The method for preparing the cyclodextrin bonded covalent organic framework chiral stationary phase according to claim 1, characterized in that: In step (2), the mass volume ratio of the activated silica microspheres to γ-glycidyloxypropyltrimethoxysilane is 2 to 5:10, g / mL.

5. The method for preparing the cyclodextrin bonded covalent organic framework chiral stationary phase according to claim 1, characterized in that: In step (3), the mass ratio of silanized silica microspheres, 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde, and 1,3,5-tris(4-aminophenyl)benzene is 2-5:0.2-0.5:0.2-0.

5.

6. The method for preparing the cyclodextrin bonded covalent organic framework chiral stationary phase according to claim 1, characterized in that: In step (4), the mass ratio of the covalent organic framework-modified silica microspheres to β-CD-SH is 2 to 5:

1.

7. A cyclodextrin bonded covalent organic framework chiral stationary phase prepared according to the preparation method of any one of claims 1 to 6.

8. Use of the cyclodextrin bonded covalent organic framework chiral stationary phase as claimed in claim 7 in the separation of chiral amino acids by high performance liquid chromatography.

9. The use according to claim 8, characterized in that The cyclodextrin bonded covalent organic framework chiral stationary phase is filled into a chromatographic column for separation of chiral amino acids by high performance liquid chromatography; the chiral amino acid is one of the following: dansyl valine, dansyl isoleucine, and dansyl leucine.

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