Chiral stationary phase

Abstract

A chiral stationary phase comprises a porous framework material and biomolecules. The porous framework material includes one of the metal-organic framework (MOF) material, the covalent organic framework (COF) material and the hydrogen-bonded organic framework (HOF) material. The biomolecules are biological chiral resolving agents. A pore size of the porous framework material is 0.2-15 nm. The porous framework material serves as a solid carrier. The biomolecules are loaded into the porous framework material. The porous framework material is modified with one or more of carboxyl, hydroxyl, amino, aldehyde, double bonds and mercapto groups.

Claims

1. A chiral stationary phase, comprising porous framework materials and biomolecules, wherein: the porous framework materials include at least one of metal-organic framework materials (MOFs), covalent organic framework materials (COFs) and hydrogen-bonded organic framework materials (HOFs); the biomolecules are biological chiral resolving agents; a pore size of the porous framework materials is 0.2-15 nm; the porous framework materials serve as solid carrier; the biomolecules are loaded into the porous framework materials; and the porous framework materials are modified with one or more of carboxyl, hydroxyl, amino, aldehyde, double bonds and mercapto groups.

2. The chiral stationary phase in claim 1, wherein the biomolecules have one or more of carboxyl, hydroxyl, amino and mercapto groups.

3. The chiral stationary phase in claim 2, wherein the porous framework materials are modified with one or more of carboxyl, hydroxyl, amino, aldehyde, double bonds and mercapto groups.

4. The chiral stationary phase in claim 1, wherein the biological chiral resolving agents are one of chiral proteins and chiral macrocyclic antibiotics.

5. The chiral stationary phase in claim 4, wherein the porous framework materials are modified with one or more of carboxyl, hydroxyl, amino, aldehyde, double bonds and mercapto groups.

6. The chiral stationary phase in claim 4, wherein: the chiral proteins are one or more of lysozyme, bovine serum albumin (BSA), cytochrome C, trypsin, papain, pepsin and avidin; and the chiral macrocyclic antibiotics are one or more of vancomycin, norvancomycin, avoparcin, ristocetin A, rifamycin B, teicoplanin, kanamycin, fradiomycin and streptomycin.

7. The chiral stationary phase in claim 6, wherein the porous framework materials are modified with one or more of carboxyl, hydroxyl, amino, aldehyde, double bonds and mercapto groups.

8. The chiral stationary phase in claim 1, wherein: the metal-organic frameworks (MOFs) are one of PCN-777, PCN-600, PCN-333, PCN-222, MIL-101, MIL-100, ZIF-8, ZIF-90, ZPF-1, ZPF-2 and Tb-MOF; the covalent organic frameworks (COFs) are one of PICOF-1, PICOF-2, PICOF-3, COF-1, COF.sub.TTA-DHTA, COF—NH.sub.2, PTDB-NH.sub.2, PTPA-NH.sub.2, COF—OH and [HOOC]X—COFs; and the hydrogen bonded organic frameworks (HOFs) are one of HOF-1, HOF-2, HOF-3, HOF-4, HOF-5, HOF-6, HOF-7 and MPM-1-Br.

9. The chiral stationary phase in claim 2, wherein: the metal-organic frameworks (MOFs) are one of PCN-777, PCN-600, PCN-333, PCN-222, MIL-101, MIL-100, ZIF-8, ZIF-90, ZPF-1, ZPF-2 and Tb-MOF; the covalent organic frameworks (COFs) are one of PICOF-1, PICOF-2, PICOF-3, COF-1, COF.sub.TTA-DHTA, COF—NH.sub.2, PTDB-NH.sub.2, PTPA-NH.sub.2, COF—OH and [HOOC]X—COFs; and the hydrogen bonded organic frameworks (HOFs) are one of HOF-1, HOF-2, HOF-3, HOF-4, HOF-5, HOF-6, HOF-7 and MPM-1-Br.

10. The chiral stationary phase in claim 6, wherein: the metal-organic frameworks (MOFs) are one of PCN-777, PCN-600, PCN-333, PCN-222, MIL-101, MIL-100, ZIF-8, ZIF-90, ZPF-1, ZPF-2 and Tb-MOF; the covalent organic frameworks (COFs) are one of PICOF-1, PICOF-2, PICOF-3, COF-1, COF.sub.TTA-DHTA, COF—NH.sub.2, PTDB-NH.sub.2, PTPA-NH.sub.2, COF—OH and [HOOC]X—COFs; and the hydrogen bonded organic frameworks (HOFs) are one of HOF-1, HOF-2, HOF-3, HOF-4, HOF-5, HOF-6, HOF-7 and MPM-1-Br.

11. The chiral stationary phase in claim 1, wherein: the metal-organic frameworks (MOFs) are one of PCN-777, PCN-600, PCN-333, PCN-222, MIL-101, MIL-100, ZIF-8, ZIF-90, ZPF-1, ZPF-2 and Tb-MOF; the covalent organic frameworks (COFs) are one of PICOF-1, PICOF-2, PICOF-3, COF-1, COFTTA-DHTA, COF—NH.sub.2, PTDB-NH.sub.2, PTPA-NH.sub.2, COF—OH and [HOOC]X—COFs; the hydrogen bonded organic frameworks (HOFs) are one of HOF-1, HOF-2, HOF-3, HOF-4, HOF-5, HOF-6, HOF-7 and MPM-1-Br; the chiral proteins are one or more of lysozyme, bovine serum albumin (BSA), cytochrome C, trypsin, papain, pepsin and avidin; and the chiral macrocyclic antibiotics are one or more of vancomycin, norvancomycin, avoparcin, ristocetin A, rifamycin B, teicoplanin, kanamycin, fradiomycin and streptomycin.

Description

BRIEF DESCRIPTION OF FIGURES

(1) The following detailed descriptions, given by way of example, and not intended to limit the present invention solely thereto, will be best be understood in conjunction with the accompanying figures:

(2) FIG. 1 is schematic diagram of chiral stationary phase for chiral separation with porous framework material as support for chiral separation agent;

(3) FIG. 2 is the covalent and adsorption curve of enzyme chiral resolution agents immobilized into COF-1, and the adsorption curve of traditional material MCM-41 for enzyme chiral resolution agents (protein-COF-1 means the protein is covalently fixed to COF-1; protein @ COF-1 means protein is adsorbed fixed to COF-1; protein @ MCM-41 means protein is adsorbed fixed to MCM-41);

(4) FIG. 3 is the data diagram of the final loading amount of enzyme chiral resolution agents in various materials after 24 hours (protein-COF-1 means the protein is covalently fixed to COF-1; protein @ COF-1 means protein is adsorbed fixed to COF-1; protein @ MCM-41 means protein is adsorbed fixed to MCM-41);

(5) FIG. 4 is the covalent immobilization curve of vancomycin into covalent organic framework COF-1;

(6) FIG. 5 is the cross linking curve of norvancomycin into covalent organic framework COF.sub.TTA-DHTA;

(7) FIG. 6 is the adsorption curve of enzyme chiral resolution agents into metal-organic frameworks;

(8) FIG. 7 is the thermal stability, solvent stability and mechanical stability of the enzyme after immobilization (the activity of free enzyme is 100%). In the figure, free protein refers to free enzyme and protein-COF-1 refers to the protein is covalently immobilized in COF-1;

(9) FIG. 8 is the data of 5 times of reuse of the enzyme after immobilization;

(10) FIG. 9 is the resolution and repeated injection diagram of DL-threonioic acid on the chiral column that prepared with the enzyme loaded into COF material as the chiral stationary phase. Specification of chromatographic column: 100×4.6 mm (i.d.), mobile phase: pH 6.5 phosphoric acid buffer solution 98%/isopropanol 2%, flow rate: 0.5 mL/min, detection wavelength: 214 nm, temperature: 25° C.;

(11) FIG. 10 is the resolution of chlorpheniramine on a chiral column that prepared with the enzyme loaded COF material as the chiral stationary phase. Specification of chromatographic column: 100×4.6 mm (i.d.), mobile phase: pH 6.5 phosphoric acid buffer solution 98%/isopropanol 2%, flow rate: 0.5 mL/min, detection wavelength: 214 nm, temperature: 25° C.; and

(12) FIG. 11 is resolution of neutral benzoin drugs on a chiral column that prepared with enzyme loaded COF material as chiral stationary phase. Specification of chromatographic column: 100×4.6 mm (i.d.), mobile phase: methanol 80%/acetonitrile 20%, flow rate: 0.5 mL/min, detection wavelength: 214 nm, temperature: 25° C.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(13) Referring to FIG. 1-FIG. 11, the synthesis of porous framework materials, characterization test of various properties and loading method of chiral resolution agent are as follows.

(14) Embodiments 1-8 are the loading of biomolecules into porous framework materials.

Embodiment 1: The Synthesis of Covalent Organic Framework COF-1 that Used for Covalent Bonded Chiral Resolution Agents, the Specific Steps are as Follows

(15) The ligands 1,3,5-tris-(4-aminobenzene) triazine (TAPT 0.10 mmol) and pyromellitic acid tincture (PMDA 0.15 mmol) were added to the thick wall glass tube (O.D.×I.D=10×8 mm.sup.2), then 0.5 mL mesitylene, 0.5 mL N-methylpyrrolidone and 0.05 mL isoquinoline were added, frozen rapidly in liquid nitrogen, vacuumized, and then sealed with hydrogen oxygen machine flame. The sealed glass tube was put into an oven at 200° C. for 5 days to react, and then yellow solid product COF-1 was obtained.

(16) Solvent Activation of Porous Framework Material COF-1:

(17) The obtained solid was washed several times with anhydrous tetrahydrofuran, until the supernatant was colorless. Then 100 mL anhydrous tetrahydrofuran was used as solvent of Soxhlet extraction for 24 hours, and the yellow solid was activated at 80° C. under vacuum for 8 hours, and then the product was stored in the glove box for the next experimental operation.

Embodiment 2: Covalent Organic Framework COF-1 as Carrier for Covalent Immobilization of Enzyme

(18) Activation of Carboxylic Acids in Covalent Organic Framework COF-1.

(19) Weigh 10 mg of COF-1 to 20 mL of glass reaction bottle, add 5 mL of MES buffer solution containing equal amount of EDC and NHS, shake in the shaker at room temperature for 2 hours, then filter out COF-1, and wash 3-5 times with 5 ml of MES buffer solution of 0.1M pH 6.0, and then dry at room temperature.

(20) Covalent Immobilization of Enzyme on Activated COF-1.

(21) Weigh 10 mg activated COF-1, add into 20 mL glass reaction flask, then add 2 mL of MES buffer solution containing 3 mg/mL of enzyme to the vial, shake in the shaker at 37° C. As shown in FIG. 1, the ultraviolet absorption of supernatant is tested every other period of time to calculate the amount of enzyme loaded on the material, and the covalent immobilization curve of enzyme into COF-1 is drawn. FIG. 2 is the final loading amount of enzyme on the material after 24 hours.

(22) In FIG. 1 and FIG. 2, the adsorption process of enzyme into porous framework material COF-1 and traditional material MCM-41 is as follows:

(23) Weigh 10 mg of COF-1 and MCM-41 respectively, directly add 2 mL of MES buffer solution of 0.1 M, pH 7.0 containing 3 mg/mL of enzyme, shake it in a shaker at 37° C., as shown in FIG. 1, like COF-1 covalent immobilized enzyme, test the ultraviolet absorption of the supernatant at intervals to calculate the amount of enzyme adsorbed by each material.

Embodiment 3: Covalent Immobilization of Vancomycin with Covalent Organic Framework COF-1 as Carrier

(24) Covalent Immobilization of Vancomycin on Activated COF-1.

(25) Weigh 5 mg of activated COF-1, add into 10 mL of centrifuged tube, then add 2 mL aqueous solution containing 5 mg/mL vancomycin to the centrifuged tube, shake it in a shaker at 37° C. As shown in FIG. 3, the ultraviolet absorption of supernatant is tested every time interval to calculate the vancomycin content loaded on the material, and the covalent immobilization curve of vancomycin by COF-1 is drawn.

Embodiment 4: The Synthesis of the Covalent Organic Framework COF.SUB.TTA-DHTA

(26) The ligands TTA (0.05 mmol) and DHTA (0.075 mmol) were weighed respectively and added to the thick wall heat-resistant glass tube (O.D.×I.D=10×8 mm.sup.2), then 0.5 mL mesitylene, 1 mL 1,4-dioxane and 0.1 mL 6 M acetic acid were added, frozen rapidly in liquid nitrogen, vacuumized, and then sealed with a hydrogen oxygen machine flame. The sealed glass tube was put into an oven at 120° C. for 3 days to react, and then red solid product COF.sub.TTA-DHTA was obtained.

Embodiment 5: The Crosslinking of Norvancomycin on Covalent Organic Framework COF.SUB.TTA-DHTA

(27) The Activation of Covalent Organic Framework COF.sub.TTA-DHTA.

(28) Weigh 40 mg of COF.sub.TTA-DHTA, and disperse it in 2 mL of tetrahydrofuran solution. Then add 40 mg of cyanuric chloride, and react at room temperature for 3 hours. After the reaction, the activated material is washed with tetrahydrofuran, ethanol and water for several times and dried.

(29) The crosslinking of norvancomycin.

(30) Weigh 5 mg of activated COF.sub.TTA-DHTA, add into 10 mL centrifuge tube, then add 2 mL of aqueous solution containing 5 mg/mL norvancomycin to the centrifuge tube, shake it in the shaking table at 37° C. As shown in FIG. 4, the ultraviolet absorption of supernatant is tested every time interval to calculate the amount of norvancomycin loaded on the material, and the cross-linking fixed curve of norvancomycin into COF.sub.TTA-DHTA is drawn.

Embodiment 6: Adsorption Loading of Enzyme by Metal-Organic Framework

(31) The Synthesis of Metal-Organic Framework.

(32) Dissolve 50 mg of 4,4′,4′-s-triazine-2,4,6-triphenic acid (H.sub.3TATB) and 200 mg of AlCl.sub.3.Math.6H.sub.2O into 10 mL N, N-dimethylformamide, and then add 1 mL of trifluoromethanesulfonic acid. Put the above mixture into the oven at 135° C. for 2 days to obtain white solid. The white precipitate was collected by centrifugation and washed several times with fresh N, N-dimethylformamide. Until the supernatant was colorless and the white product, PCN-333, was collected. Yield: 80%.

(33) Adsorption Immobilization of Enzyme on Metal-Organic Framework.

(34) Weigh 10 mg PCN-333, then add 2 mL of MES buffer solution containing 3 mg/mL of enzyme directly, shake in a shaker at 37° C., as shown in FIG. 5, test the ultraviolet absorption of the supernatant every other period of time to calculate the amount of enzyme adsorbed on the solid.

Embodiment 7: Embedding of Enzyme by Metal-Organic Framework

(35) Weigh 5 mg of BSA, 1.552 g of 2-methylimidazole in 5.4 ml of deionized water, 80 mg of zinc nitrate hexahydrate in 0.6 ml of deionized water, mix the two solutions, react at 30° C. for 10 min to obtain BSA @ ZIF-8, and the entrapment rate of BSA is more than 90%.

Embodiment 8: Adsorption Immobilization of Vancomycin by Hydrogen Bonded Organic Framework

(36) The Synthesis of Hydrogen Bonded Organic Framework.

(37) Weigh 11 mg of adenine to be dissolved in 12 ml of methanol, 8.8 mg of copper bromide to be dissolved in 12 ml of isopropanol, and add the methanol solution of adenine to the test tube, slowly drop the isopropanol solution of copper bromide to cover the methanol solution, and then allow the reaction to stand for one week at room temperature. The obtained material was washed several times with methanol to obtain MPM-1-Br.

(38) Adsorption Immobilization of Vancomycin by Hydrogen Bonded Organic Framework

(39) Weigh 5 mg of MPM-1-Br, add 2 mL water solution containing 5 mg/mL vancomycin directly, shake in the shaker at 37° C., as shown in FIG. 6, test the ultraviolet absorption of the supernatant every other period of time to calculate the amount of vancomycin adsorbed.

(40) Embodiments 9-11 are stability tests of novel chiral stationary phases.

Embodiment 9: Stability Test Experiment of Novel Chiral Stationary Phase

(41) Thermal Stability Test of Enzyme after Covalent Immobilization.

(42) The covalent bonded enzyme and the same amount of free enzyme were simultaneously placed in an oven at 80° C. for 1 hour. After cooling, 150 μg/mL chitosan substrates were added respectively. After incubation at 50° C. for 30 minutes, the supernatant was filtered. The supernatant was reacted with the developer potassium iron dense for 15 minutes at 100° C., and then the reaction solution was tested at 420 nm. Compared with the activity of the immobilized enzyme and the free enzyme, the thermostability of the immobilized enzyme is improved significantly, which is still about 80% activity, while the free enzyme has no activity after heat treatment.

(43) Solvent Stability Test of Enzyme after Covalent Immobilization.

(44) The covalent bonded enzyme and the same amount of free enzyme were treated in methanol solution for one hour at the same time, and then the enzyme activity was tested according to the method of enzyme activity test in the heat stability test. The results showed that the immobilized enzyme' activity was still above 85% and the activity of free enzyme was below 40% after methanol treatment.

(45) Mechanical stability test of enzyme after covalent immobilization.

(46) The covalent bonded enzyme and the same amount of free enzyme were treated at the same time under the condition of ultrasonic 40 kHz for 30 min, and then the enzyme activity test was carried out according to the method of enzyme activity test under the condition of thermal stability test. The results showed that the immobilized enzyme activity was still above 90% and the activity of free enzyme was reduced to below 30% after treated with ultrasonic.

(47) Reusability Test of Enzyme after Covalent Immobilization.

(48) The results showed that the enzyme activity did not decrease significantly after five cycles.

(49) All the above stability tests showed that the enzyme stability was significantly improved after covalent immobilization, so the chiral stationary phase prepared by this method had good stability and reusability.

Embodiment 10: The Preparation of the HPLC Chiral Stationary Phase with Porous Framework Material COF-1 as Carrier to Covalent Immobilize Enzyme

(50) According to the method of covalent immobilization of enzyme in embodiment 2, 1 g COF-1 was loaded with enzyme, and then freeze-dried. The prepared materials were added into 50 mL ethanol, dispersed by ultrasound for 10 min, and the uniform chiral stationary phase packing was loaded into the homogenate tube. Ethanol was used as the substitution liquid, and then pressed into the empty tube column of ferrule chromatography by pneumatic pump. The column specification is 100×4.6 mm (i.d.). The filling pressure is 5000 psi.

(51) Preparation of Chiral Monolithic Capillary Column by In Situ Method:

(52) 5 m×0.32 mm capillary column was repeatedly washed and activated by 1 M sodium hydroxide, water, 0.1 M hydrochloric acid, water and methanol, then 50% 3-aminopropyltriethoxysilane was injected into the column and reacted at 40° C. for 10 hours to obtain amino activated capillary column. Dissolve 1,3,5-tris-(4-aminobenzene) triazine (0.1 mmol) and p-benzenedicaldehyde (0.15 mmol) in 0.4 mL mesitylene and 1.6 mL, 4-dioxane, add 5 mg BSA, pour the mixed solution into the capillary tube, and then add 0.4 mL 3.75 mg/mL trifluoromethylsulfonic acid solution, mix with ultrasound, and allow the reaction to stand for 30 minutes, At the end of the reaction, the Chiral Monolithic Capillary Column with BSA was obtained by repeatedly washing with methanol solution.

Embodiment 11: Preparation of Mobile Phase and Samples

(53) Acid, alkaline and neutral samples were selected for the test. The sample was first prepared into 2 mg/mL by water or ethanol. Before the test, the sample was diluted to 20 μg/mL using mobile phase, and the injection volume was 10 μL.

(54) Mobile phase: pH 6.5, 20 mM phosphoric acid buffer solution is prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate, and then 2% isopropanol is added as the organic regulator, and filtrate with filter membrane (0.22 μm) before use; organic mobile phase: methanol, acetonitrile, ultrasonic for 30 minutes before use. The flow rate is 0.5 mL/min.

(55) TABLE-US-00001 TABLE 1 separation results of chiral samples under reversed phase conditions. Retention time retention time Sample (t1/min) (t1/min) Rs α DL-Leucine 4.32 5.61 1.42 1.15 DL-Tryptophan 4.35 5.65 1.41 1.29 DL-Threonine 4.30 5.63 1.43 1.31 Chlorpheniramine 3.97 4.69 1.64 1.18

(56) Conclusion: from the results of separation in the above table, it can be seen that the chiral column has better separation efficiency of these acidic and alkaline samples in Table 1, and can be compared with the existing commercial chiral columns.

(57) TABLE-US-00002 TABLE 2 separation results of chiral samples in polar organic phase. Retention time retention time Sample (t1/min) (t1/min) Rs α Benzoin 1 4.00 6.51 2.41 1.34 Benzoin 2 4.70 10.86 3.71 1.57 Benzoin 1: acetonitrile 100%, flow rate 0.5 mL/min; Benzoin 2: methanol/acetonitrile, 80/20 (V/V), flow rate 0.5 mL/min.

(58) Conclusion: it can be seen from the separation results in the above table that the chiral stationary phase has good separation efficiency on benzoin under the condition of polar organic phase.

(59) TABLE-US-00003 TABLE 3 separation results of chiral samples in normal phase Retention time retention time Sample (t1/min) (t1/min) Rs α 1-phenyl-2-isopropanol 24.5 32.1 1.78 1.29 1-phenyl-1-pentanol 23.2 29.6 1.58 1.21
Specification of chromatographic column: 150×4.6 mm (i.d.), mobile phase: n-hexane 99%/isopropanol 1%, flow rate: 0.2 mL/min, detection wavelength: 214 nm, temperature: 25° C.

(60) Conclusion: it can be seen from the separation results in the above table that the chiral stationary phase has a good separation effect on organic small molecules under the condition of normal phase.

(61) TABLE-US-00004 TABLE 4 repeatability of the chiral column Reinjection (8) Day-to-Day Column-to-Column Relative standard 0.068 0.14 0.12 deviation (RSD %)

(62) Conclusion: from the data in Table 4, it can be concluded that the stability and reusability of the chiral column are very good, and the difference between different batches is small.

Embodiment 12: Commercial Cost of the Chiral Column with Enzyme and Macrocyclic Antibiotic as Chiral Resolution Agent and Covalent Organic Framework as Carrier

(63) The production of a 4.6×100 mm enzyme immobilized chiral column requires the material (COF-1) with chiral resolution agent 0.8 g, and the cost is:

(64) TABLE-US-00005 Material cost: 187 CNY Benzenetetracarboxylic Empty Column Aminobenzonitrile anhydride TfOH Lysozyme 180 CNY 1000 CNY/Kg 260 CNY/Kg 900 CNY/Kg 450 CNY/100 g 180 CNY 0.8 CNY/800 mg 0.169 CNY/650 mg 3.6 CNY/4 g 2.25 CNY/0.5 g Salary Cost: Material synthesis personnel 1 person × 3 days = 600 CNY(Calculated according to the average wage of graduate staff of per present) Environmental costs: 27 CNY Triethylbenzene isoquinoline N-methylpyrrolidone Tetrahydrofuran Ethanol 880 CNY/2.5 L 330 CNY/Kg 277 CNY/2.5 L 340 CNY/10 L 10 CNY/L 3.52 CNY/10 mL 0.33 CNY/1 mL 1.1 CNY/10 mL 17 CNY/500 mL 5 CNY/500 mL Total: 814 CNY

(65) At present, the price of imported Daicel enzyme immobilized chiral column with the same specification on the market is 14000-16000 CNY; the price of domestic chiral column with the same specification on the market is 12000 CNY. The cost of preparing an enzyme based chiral column by the method of this application is only 814 CNY.

(66) Having described at least one of the embodiments of the claimed invention with reference to the accompanying drawings, it will be apparent to those skills that the invention is not limited to those precise embodiments, and that various modifications and variations can be made in the presently disclosed system without departing from the scope or spirit of the invention. Thus, it is intended that the present disclosure cover modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents. Specifically, one or more limitations recited throughout the specification can be combined in any level of details to the extent they are described to improve the invention.