CN-118834562-B - Poria cocos acidic polysaccharide chiral stationary phase and application thereof

Abstract

The invention relates to the technical field of chiral stationary phases of liquid chromatographic columns, in particular to a poria cocos acidic polysaccharide chiral stationary phase and application thereof. The poria acidic polysaccharide and the derivatives thereof have the advantages of spectrum and high-efficiency chiral resolution capability, can realize the effective resolution of chiral compounds in normal phase chromatographic mode, realize the common application of single-configuration medicaments for further obtaining single-configuration medicaments, enrich the range of chiral selection agents based on the novel glycosyl stationary phase provided by cellulose, amylose, beta-cyclodextrin and other traditional glycosyl stationary phases, and have the characteristics of high separation speed, good resolution effect, good stability, reusability and the like.

Inventors

• ZHANG MEI
• Zhang guoqiong
• YU JIE
• YANG XINGXIN
• LI LINZHE
• YANG ZERONG
• QI YAN
• RUAN DEQING
• WANG JIAO

Assignees

• 云南中医药大学

Dates

Publication Date

20260508

Application Date

20240722

Claims (4)

1. 1. The poria cocos acidic polysaccharide chiral stationary phase is characterized by comprising a chromatographic matrix and a chiral selector coated on the chromatographic matrix; The chromatographic matrix refers to 3-aminopropyl triethoxy silanized silica gel; The chiral selector is selected from a chiral selector based on pachyman, a chiral selector based on 3, 5-dimethylphenyl isocyanate-derived pachyman and a chiral selector based on 1, 6-hexamethylene diisocyanate-derived pachyman; The chiral selector based on pachyman acid polysaccharide is prepared by extracting the medicinal residues with water and ethanol, adding 70 times of 0.5mol/LNaOH, stirring and extracting for 1h, filtering with a cloth funnel, neutralizing filtrate with 20% hydrochloric acid, standing overnight, centrifuging the suspension at 4000r/min, discarding supernatant, repeatedly washing gel-like precipitate with distilled water, desalting, lyophilizing to obtain pachyman acid polysaccharide, weighing 0.5g pachyman acid polysaccharide in a 50mL round bottom flask, adding 20mLN, dissolving N-dimethylformamide at 90deg.C for 24h, and adding 0.1g anhydrous lithium chloride, and continuously stirring for 2.5 h; 1.0g of freeze-dried pachyman is weighed and dried for 2 hours at 90 ℃, 40mL of anhydrous pyridine is added for swelling for 2.5 hours, 3.8mL of 3, 5-dimethylphenyl isocyanate is added into a nitrogen protection system for refluxing for 24 hours at 90 ℃, 1L of methanol is used for precipitation after cooling to room temperature, filtration and washing with methanol and n-hexane until no pyridine smell exists, vacuum drying is carried out for 5 hours at 60 ℃, 0.5g of product is weighed and put into a 50mL round bottom flask, and 20mL of N-dimethylformamide is added for dissolution at 60 ℃ to obtain the chiral selector based on 3, 5-dimethylphenyl isocyanate-derived pachyman; 1.0g of freeze-dried pachyman is weighed, dried in vacuum for 2 hours at 90 ℃, added into 40mL of dimethyl sulfoxide to swell for 2.5 hours, added with 3.8mL of 1, 6-hexamethylene diisocyanate for refluxing for 24 hours at 90 ℃ in a nitrogen protection system, cooled to room temperature, precipitated with 1L of methanol, filtered, washed with methanol and n-hexane until no pyridine smell exists, dried in vacuum for 5 hours at 60 ℃, weighed 0.5g of product is put into a 50mL round bottom flask, and 20mL of LN and N-dimethylformamide are added to dissolve at 60 ℃ to obtain the chiral selector based on 1, 6-hexamethylene diisocyanate-derived pachyman; The preparation method of the chiral stationary phase comprises the steps of weighing 10g of silica gel with the particle size of 5 mu m, adding 100mL of 10% diluted hydrochloric acid into a 250mL round bottom flask, heating at 90 ℃ for 8h, filtering by a sand core funnel, washing to neutrality by distilled water, drying filter residues under reduced pressure at 180 ℃ for 5h, transferring 8g of spherical silica gel into a 250mL two-necked flask, slowly adding 80mL of anhydrous benzene, 1.2mL of anhydrous pyridine, 1.6mL of 3-aminopropyl triethoxysilane, refluxing for 24h under the condition of 80 ℃ under the protection of nitrogen, filtering after cooling, washing by proper amount of methanol, acetone and normal hexane, vacuum drying at 60 ℃ for 5h to obtain 3-aminopropyl triethoxysilane silica gel, dropwise coating the 3-aminopropyl triethoxysilane silica gel with a small amount of 3, 5-dimethylphenyl isocyanate derivatization acidic polysaccharide-based chiral selector and 1, 6-hexamethylene diisocyanate derivatization acidic polysaccharide chiral selector respectively, repeatedly coating the 3-aminopropyl triethoxysilane with 2.5g of 3-aminopropyl acidic polysaccharide for 3-6 ℃ for 30min, and finally, dropwise coating the silica gel under the condition of vacuum drying at 60 ℃ for 30 min; according to the method for preparing the chromatographic column by using the chiral stationary phase, 1.2g of each of three stationary phases is weighed, a proper amount of n-hexane and isopropanol with the volume ratio of 9:1 are added and pass through a 250-mesh sieve, after the particle size is uniform, 200mL of n-hexane and isopropanol with the volume ratio of 9:1 are used as displacement liquid, after the column is filled under the pressure of 40Mpa for 2min, the pressure is slowly reduced to 15-20 MPa and stabilized for 30min, the coated chiral chromatographic column is obtained after the stabilization under the normal pressure for 30min, the n-hexane and isopropanol with the volume ratio of 9:1 are used as mobile phases on a high-performance liquid chromatograph, the elution is carried out at the flow rate of 0.1mL/min, and after the baseline is stable, the poria cocos acidic polysaccharide chiral chromatographic column and the polysaccharide chiral chromatographic column derived from 3, 5-dimethylphenyl isocyanate and 1, 6-hexamethylene diisocyanate are obtained.
2. 2. The use of the pachyman acidic polysaccharide chiral stationary phase of claim 1 as a liquid chromatographic column in chiral resolution.
3. 3. The application of the chiral chromatographic column of pachyman acid polysaccharide in benzoin, 1- (1-naphthyl) -ethanol, biffurol and tertagine compounds, the application of the chiral chromatographic column of polysaccharide derived from 3, 5-dimethylphenyl isocyanate in chiral resolution of ketoprofen, flurbiprofen, ofloxacin and praziquantel compounds, and the application of the chiral chromatographic column of polysaccharide derived from 1, 6-hexamethylene diisocyanate in chiral resolution of warfarin sodium, propranolol hydrochloride, sulpiride and amlodipine compounds.
4. 4. A process for separating a mixture of enantiomers, comprising the steps of: s1, passing a composition comprising an enantiomer mixture through a chromatographic column comprising the pachymic acid polysaccharide chiral stationary phase of claim 1; S2, contacting a composition comprising an enantiomer mixture with the chiral selector of claim 1, wherein each component of the composition is equilibrated on a chromatographic column; s3, separating the enantiomer mixture by utilizing the difference of retention time of each component on the chromatographic column.

Description

Poria cocos acidic polysaccharide chiral stationary phase and application thereof Technical Field The invention relates to the technical field of chiral stationary phases of liquid chromatographic columns, in particular to a poria cocos acidic polysaccharide chiral stationary phase and application thereof. Background Chirality is the most common phenomenon in nature, and is the fundamental property of chiral substances, particularly in that substances and enantiomers thereof show mirror image differences between the left hand and the right hand, and stereospecific recognition and enantiomer identification of chiral molecules are fundamental phenomena of life science, namely that chirality of constituent substances (nucleic acids, amino acids, proteins and the like) of a life system has different degrees of influence on bioactive compounds involved in interaction of life activities, and sensitivity of the life system to stereochemistry is represented on activity differences of a pair of enantiomers, and the finding also suggests that single optical isomer drugs are greatly demanded in clinical treatment. The existing chiral resolution research is mainly in the analysis field, only a small part of pure optical isomer medicaments can be prepared by a chemical synthesis method, chiral resolution of natural compounds becomes a reliable source of pure optical medicaments, and kurarinone, kurarinol, taxol, silymarin, luteolin and the like show better pharmacological activity in cell experiments and animal experiments, but the pharmacological effects of different optical isomers of the natural compounds are not studied in detail, and the bottleneck is that the preparation of the pure optical compounds has great working difficulty in milestone significance. In chiral resolution research, polysaccharide derivatives chiral stationary phase has the most wide application, the resolution capability is the best in the existing commercial columns, amylose derivatives and cellulose derivatives are widely used for filling chromatographic columns, and cyclodextrin can be used as chiral mobile phase additive and CSPs in chiral HPLC separation to initiate great research and discussion. Most chiral columns sold in the market are prepared by coating or chemically fixing derivatives of polysaccharides such as cellulose, amylose and the like on silica gel, wherein cellulose tri (3-chloro-4-methylphenyl carbamate) can effectively split 5 beta-adrenergic blockers such as bisoprolol, carvedilol and the like, cellulose 6-benzoic acid-2, 3-phenyl carbamate and cellulose tri (3, 5-dimethylphenylcarbamate) can split compounds such as flavanone, atenolol and the like, but cellulose 6-benzoic acid-2, 3-phenyl carbamate can better separate flavanone and benzoin, amylose tri (3-chloro-5-methylphenyl carbamate) has a good splitting effect on each flavanone, giovanni and the like can fix the amylose tri (3-chloro-5-methylphenyl carbamate) on aminopropyl alkylated (silica) in a covalent fixing manner to prepare chirality, and the chiral analysis of the racemic phase of barbituric acid, the racemate and the like under a capillary electrochromatography system shows the chiral analysis of the target property and the neutral property of CSP. Beta-cyclodextrin and its derivative are used as chiral stationary phase of high performance liquid chromatography to split 7 kinds of flavone and 9 kinds of triazole enantiomer successfully. The wide application of glycosyl chiral stationary phases such as beta-cyclodextrin, cellulose, amylose and the like reveals the huge chiral resolution potential of the saccharide stationary phase. The pachyman is a linear glucan formed by connecting beta-1, 3-glycosidic bonds, the content of the pachyman in the pachyman is up to 80%, the polysaccharide has extremely poor water solubility, the research is mainly focused on pharmacological activity and structural modification, more activity of the compound is expected to be found through the structural modification, and the pachyman, cellulose and amylose are the same as the glucan, so that chiral resolution research of the pachyman has not been reported. Disclosure of Invention Aiming at overcoming the defects of the prior art, the invention provides the pachyman acidic polysaccharide chiral stationary phase and the application thereof, wherein pachyman acidic polysaccharide and pachyman acidic polysaccharide derivatives are used as chiral selective agents to be coated on chromatographic matrixes to be used as chromatographic column chiral stationary phases, and the chiral stationary phases show excellent chiral resolution performance. In order to achieve the above object, in a first aspect, the present invention provides a poria acid polysaccharide chiral stationary phase comprising a chromatographic matrix and a chiral selector coated on the chromatographic matrix; The chiral selector is selected from pachyman acid polysaccharide and pachyman acid polysaccharide derivativ