CN103301653B - A kind of separation method of sulfonic acid combination - Google Patents
A kind of separation method of sulfonic acid combination Download PDFInfo
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- CN103301653B CN103301653B CN201210064562.3A CN201210064562A CN103301653B CN 103301653 B CN103301653 B CN 103301653B CN 201210064562 A CN201210064562 A CN 201210064562A CN 103301653 B CN103301653 B CN 103301653B
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Abstract
A kind of separation method of sulfonic acid combination, with the mixture of hydroxy compounds (phenolic hydroxy group and alcoholic extract hydroxyl group) substrate after chemically derived or biological conversion for raw material, by the chromatographic column that composite is filled, utilize the eluant, eluent of different pH value to realize the quick separating of hydroxy compounds and sulfonic acid combination acidity dependence thereof simultaneously.The present invention has applicability widely when preparing sulfonic acid combination, is suitable for purifying and the preparation of all sulfonic acid combination containing phenol (alcohol) hydroxyl but not containing acidic-group.
Description
Technical field
The invention belongs to medical art, specifically provide a kind of preparation method of sulfonic acid combination.
Background technology
Drug metabolism is the main mode of body defenses foreign substance invasion, is considered to the important biochemical barrier that body resists external nuisance.Sulfonic acid is that II that in body, a class is important combines metabolic response in conjunction with metabolism, and play an important role (Naunyn Schmiedebergs Arch Pharmacol 2004 in the biology of endogenous Small molecular (as steroid, catecholamines, peanut acids etc.) and exogenous material (as medicine) transforms; 369:55-68; Toxicol Sci 2006; 90:5-22.).Most drug is after sulfonic acid association reaction, and the sulfonated bond of generation is compared with substrate has higher solubility, is therefore easier to excrete, thus plays the effect reducing drug effect and toxicity.But some compounds can form reactive intermediate after carrying out sulfonic acid combination, thus be combined and the generation of induced cancer (Chem Biol Interact 1998 by covalent bond with the large molecule such as DNA; 109:221-235).Therefore, the sulfonic acid combination preparing medicine has very important significance to safe and effective medication clinically.
The synthetic method of sulfonic acid combination is mainly divided into two classes: chemical derivatization and biotransformation method.The maximum advantage of the sulfonated derivatization method of chemistry be reaction rate fast, be applicable to large-scale production, but its severe reaction conditions (usually needing strong acid and strong base or high temperature), for the substrate of partial chemical poor stability, chemically derivedly often produce multiple accessory substance.Compared with chemical method, bioanalysis has good applicability, can be widely used in the preparation of different types of structure substrate, but often higher than chemical method on preparation cost.Therefore, for the preparation of the sulfonated product of specific substrates, chemical method or bioanalysis can be selected to reach the object of preparation target sulfonic acid combination according to substrate structure and chemical attribute.But it is not single to be that chemical method or bioanalysis all exist reaction end-body set member, the problems such as target product purity is lower.How to realize preparing to the fast purifying of target sulfonic acid combination monomer the committed step having become sulfonic acid combination and prepared.But, still lack the effective ways of quick separating sulfonic acid combination monomer at present, be therefore necessary to develop a kind of practicality, fast and the high technology selecting separate targets sulfonic acid combination.
Summary of the invention
The object of this invention is to provide a kind of separation method of sulfonic acid combination, the method is not only simple to operate, and can obtain a large amount of sulfonic acid metabolites fast.
The invention provides a kind of separation method of sulfonic acid combination, the method is that substrate is through chemically derived or biotransformation, form the mixture of substrate and its sulfonic acid combination, with this mixture for raw material, by the chromatographic column that composite is filled, utilize the eluant, eluent of different pH value to realize the quick separating of mixture material simultaneously.
The separation method of sulfonic acid combination provided by the invention, the requirement of described method successively:
First chemical synthesis or bio-conversion processes is carried out: chemically derived or containing sulfonated transferase the biological transformation system such as sulfonated is converted into sulfonic acid combination by all or part of for substrate and obtain sample to be separated.Enzyme used is specially: recombinant expressed sulfonic acid transferase (that is: SULT), or for the microsome containing SULT enzymatic activity that derives from animal tissue or containing MC cell liquid (S9), animal tissue is preferably from the tissue of the strong metabolic capability such as liver, kidney, intestines; Described substrate is containing phenol (alcohol) hydroxyl but not containing the compound of acidic-group;
Next carries out separation and purification treatment: the response sample obtained by above-mentioned biotransformation is by the enrichment of flash chromatography post and obtain target sulfonic acid combination after purifying; Described flash chromatography post filler used is the mixed fillers containing reverse phase silica gel and anion exchange resin, and the volume ratio of two kinds of fillers is between 0.2-5; Used silica gel is bonded with phenyl, a kind of carbochain in C8 and C18, and silica gel particle diameter, between 10-200 μm, requires containing a kind of basic group in primary amine groups, secondary amine, tertiary amine groups, season amino in anionic ion-exchange resins used.
The separation method of sulfonic acid combination provided by the invention, described biotransformation needs the lithium salts etc. of 3 '-AMP-5 '-phosphosulfate ester or its correspondence providing sulfonic acid group for biological catalytic process, and its mole dosage added is 1-100 times of substrate mole dosage; Reaction is carried out in the buffer salt solution that pH value is 6-8, and keeps the conversion temperature of 30-50 DEG C (preferred 35-40 DEG C).
The separation method of sulfonic acid combination provided by the invention, in the filler of described flash chromatography post, silica gel bonded have different length carbochain, preferred C8 and C 18 of carbon chain lengths, the preferred 40-60 μm of granularity.Anion exchange resin is alkalescent or strong basic ion exchange resin, and preferably contain the ion exchange resin of primary amine groups or secondary amine group, resin particle diameter is 0.2-1mm.The gross mass of two kinds of fillers is 20 times that are not less than substrate and metabolite gross mass.
The separation method of sulfonic acid combination provided by the invention, described separation and purification process is that mixture material is dissolved the column cap being placed on chromatographic column, then uses water, organic solvent, the drip washing of acidifying organic solvent respectively.Described organic solvent is preferably alcohol.Described organic solvent is one in methyl alcohol, ethanol, isopropyl alcohol, acetonitrile, acetone or mixed solvent, is preferably alcohol.The acid used in described acidifying organic solvent is lower boiling organic acid, is preferably formic acid or acetic acid.
The separation method of sulfonic acid combination provided by the invention, after described chemical synthesis or biological conversion sample are placed in flash chromatography post column cap, successively through the process of water wash, alcohol drip washing and the drip washing of acidifying alcohol, water wash is intended to water-soluble substances in elution of reactive liquid, alcohol drip washing is intended to other liposoluble substance in wash-out substrate and reactant liquor, and the drip washing of acidifying alcohol is intended to wash-out sulfonic acid combination.Alcohol used is the fatty alcohols such as methyl alcohol, ethanol or isopropyl alcohol; Acid used is the higher boil organic acid such as formic acid or acetic acid, and in alcohol, the molar concentration of acid is 0.01-0.1mol/L.In water-washing process, the consumption of water is not less than 2 times of column volumes; Alcohol is specially methyl alcohol or ethanol, and the node of alcohol drip washing is no longer containing substrate in eluent; The consumption of acidifying alcohol is 5 times of column volume.
The separation method of sulfonic acid combination provided by the invention, described chemical synthesis or biology transform sample upper prop and successively by after washing, alcohol wash, acidifying alcohol wash, through high performance liquid chromatography and liquid phase-Mass Spectrometer Method, metabolite is enriched in acidified methanol; Then through low pressure evaporation or inert gas (N
2) dry up, obtain the metabolite of purity > 95%.
Sulfonic acid is the reaction mediated by sulfonic acid transferase (SULT) in conjunction with metabolism, based on the defect of traditional biological legal system for sulfonic acid combination, the present invention with hydroxy compounds through chemical derivatization or biological transform after mixture for raw material, by the chromatographic column that ion exchange resin and reverse phase filler composite are filled, utilize the eluant, eluent p-sulfonic acid bond of different pH value to carry out the quick separating of acidity dependent form simultaneously.Not only simple to operate, and a large amount of sulfonic acid metabolites can be obtained fast.
Separation method with existing sulfonic acid combination is compared, and tool of the present invention has the following advantages:
1) efficiently easy and simple to handle, from enrichment in animal body, also need not need more purification;
2) there is applicability widely, be suitable for all separation containing the SULT substrate corresponding sulfonic acid combination of phenolic hydroxyl group simultaneously not containing acidic-group.
Accompanying drawing explanation
Fig. 1 is for utilizing the biological flow chart transforming (chemically derived) and prepare sulfonated pregnenolone;
Fig. 2 is the high-efficient liquid phase chromatogram of resibufogenin sulfonic acid combination;
Fig. 3 is the mass spectrogram of resibufogenin sulfonic acid combination;
Fig. 4 is structure and the mass spectrogram of 8-sulfonic group dephnetin;
Fig. 5 is structure and the mass spectrogram of 7-sulfonic group dephnetin.
Detailed description of the invention
Embodiment 1, the preparation of pregnenolone sulfonic acid combination, as follows:
1) through biotransformation method preparative separation sample: pregnenolone is dissolved in the storage liquid being made into 5mg/ml in the middle of methyl alcohol, take out the phosphatic buffer salt that 20ml adds pH7.4, rats'liver endochylema (RLC) is added successively after stirring, add sulfonic donor PAPS, in system, resibufogenin final concentration is 0.10mg/ml, final concentration of protein is 0.5mg/ml, PAPS final concentration 0.2mM, and 37 DEG C of waters bath with thermostatic control are stirred and hatched 2h.Reactant liquor is transferred to-80 DEG C of refrigerator and cooled but after reaching 50% by HPLC detection substrate conversion ratio.Extract reaction solution 100ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have phenyl, and particle diameter is 40 μm, and consumption is 2.5g, and ion exchange resin contains secondary amino group, and materials are 2.5g.Successively use 10ml water wash, the drip washing of 10ml methyl alcohol, the carboxylic acid methyl alcohol drip washing of 30ml.2) through chemical derivatization preparative separation sample: sulfur trioxide-Trimethylamine compound (0.137g, 0.96mmol) be dissolved in pyridine (5ml) and pregnenolone (0.126g, 0.039mmol) at room temperature mix, mixture was in 55 DEG C of reflux heatings 12 hours.Question response mixture is cooled to room temperature, shifts and enrichment of reducing pressure.Reactant mixture redissolves and be splined on flash chromatography post after methyl alcohol (100ml).The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have phenyl, and particle diameter is 40 μm, and consumption is 25g, and ion exchange resin contains secondary amino group, and materials are 25g.Successively use 100ml water wash, the drip washing of 100ml methyl alcohol, the carboxylic acid methyl alcohol drip washing (Fig. 1) of 300ml.
Acidified methanol leacheate N under room temperature
2dry up, biotransformation method obtains sulfonic acid combination 7.9mg, and chemical derivatization obtains sulfonic acid combination 110mg.
Embodiment 2, the preparation of resibufogenin sulfonic acid combination, as follows:
Resibufogenin is dissolved in the storage liquid being made into 5mg/ml in the middle of dimethyl sulfoxide (DMSO) (DMSO), take out the phosphatic buffer salt that 20ml adds pH7.4, add people after stirring successively to recombinate single enzyme rhSULT2A1, add sulfonic donor PAPS, in system, resibufogenin final concentration is 0.10mg/ml, final concentration of protein is 0.25mg/ml, PAPS final concentration 0.2mM, and 37 DEG C of waters bath with thermostatic control are stirred and hatched 2h.Reactant liquor is transferred to-80 DEG C of refrigerator and cooled but after reaching 50% by HPLC detection substrate conversion ratio.Extract reaction solution 100ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have phenyl, and particle diameter is 40 μm, and consumption is 2.5g, and ion exchange resin contains secondary amino group, and materials are 2.5g.Successively use 10ml water wash, the drip washing of 10ml methyl alcohol, the carboxylic acid methyl alcohol drip washing of 30ml.Acidified methanol leacheate N under room temperature
2dry up to obtain sulfonic acid combination 7mg, HPLC purity > 95% (Fig. 2 and Fig. 3).
Embodiment 3, the sulfonated metabolite preparation of dephnetin
Dephnetin is dissolved in methyl alcohol the storage liquid being made into 5mg/ml, take out the phosphatic buffer salt that 30ml adds pH7.4, rat intestine S9 (RIS9) is added successively after stirring, add sulfonic donor PAPS, in system, dephnetin final concentration is 0.10mg/ml, final concentration of protein is 0.5mg/ml, PAPS final concentration 0.2mM, and 37 DEG C of waters bath with thermostatic control are stirred and hatched 1h.Reactant liquor is transferred to-80 DEG C of refrigerator and cooled but after reaching 50% by HPLC detection substrate conversion ratio.Extract reaction solution 100ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have phenyl, and particle diameter is 40 μm, and consumption is 4g, and ion exchange resin contains secondary amino group, and materials are 4g.Successively use 20ml water wash, the drip washing of 20ml methyl alcohol, the carboxylic acid methyl alcohol drip washing of 40ml, realizes the enrichment to sulfonated products.Acidified methanol leacheate N under room temperature
2dry up concentrated, utilize and prepare high-efficient liquid relative to two further separation and purification of metabolite, obtained dephnetin-7-sulfuric ester 5.1mg (Fig. 5) and dephnetin-8-sulfuric ester 3.9mg (Fig. 4).
Claims (9)
1. prepare a method for sulfonic acid combination, it is characterized in that:
(1) chemically derived or biotransformation: by substrate through chemically derived or biotransformation, forms the mixture of substrate and its sulfonic acid combination;
(2) separation and purification process: using mixture obtained above as raw material, by the chromatographic column that composite is filled, utilizes the eluant, eluent of different pH value to realize the quick separating of mixture material simultaneously;
Filler in described chromatographic column is the mixed fillers of bonding reverse phase silica gel and anion exchange resin simultaneously, and the volume ratio of above-mentioned two kinds of fillers is between 0.2-5; The particle diameter of two kinds of filler particles is between 10-200 μm;
Described substrate for containing phenolic hydroxyl group or/and alcoholic extract hydroxyl group, and does not contain the compound of acidic-group;
Described anion exchange resin is containing anion exchange resin a kind of in primary amine groups, secondary amine, tertiary amine groups, quaternary ammonium group.
2. according to the method preparing sulfonic acid combination described in claim 1, it is characterized in that: described biology transforms and refers to that substrate generates sulfonic acid combination through the catalysis of the living things system containing sulfonated transferase; Transferase source used comprises following one of several or combination: recombinant expressed sulfonic group transferase SULT, the animal tissue's homogenate containing SULT enzyme, the cell liquid containing SULT enzyme.
3. according to the method preparing sulfonic acid combination described in claim 1 or 2, it is characterized in that: the lithium salts requiring to add 3'-AMP-5'-phosphosulfate ester or its correspondence in described biotransformation, its mole added is 1-100 times of substrate; Reaction is carried out in the buffer salt solution that pH value is 6-8, and keeps the conversion temperature of 30-50 DEG C.
4. according to the method preparing sulfonic acid combination described in claim 1, it is characterized in that: the consumption of the filler in described chromatographic column is 20-100 times of material quality.
5. according to the method preparing sulfonic acid combination described in claim 1, it is characterized in that: the separation and purification process of described step (2) is that mixture material is dissolved the column cap being placed on chromatographic column, then uses water, organic solvent, the drip washing of acidifying organic solvent respectively.
6. according to the method preparing sulfonic acid combination described in claim 5, it is characterized in that: described organic solvent is one in following solvents or mixed solvent: methyl alcohol, ethanol, isopropyl alcohol, acetonitrile, acetone.
7. according to the method preparing sulfonic acid combination described in claim 5, it is characterized in that: the acid used in described acidifying organic solvent is lower boiling organic acid.
8. according to the method preparing sulfonic acid combination described in claim 7, it is characterized in that: the acid used in described acidifying organic solvent is formic acid or acetic acid.
9. according to the method preparing sulfonic acid combination described in claim 5, it is characterized in that: described water wash process institute water consumption is not less than 2 times of column volumes; Organic solvent lessivation organic solvent amount used is not less than 2 times of column volumes; The amount of acidifying organic solvent lessivation acidifying organic solvent used is not less than 5 times of column volumes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0967272A2 (en) * | 1998-06-23 | 1999-12-29 | Seikagaku Corporation | Novel sulfotransferase |
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| JP2005507640A (en) * | 2001-03-28 | 2005-03-24 | マサチューセッツ・インスティチュート・オブ・テクノロジー | Method for 6-O sulfation of glucosaminyl and 6-O sulfated polysaccharide preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0967272A2 (en) * | 1998-06-23 | 1999-12-29 | Seikagaku Corporation | Novel sulfotransferase |
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| Sherry S. Lamb."Biosynthesis of Sulfated Glycopeptide Antibiotics by Using the Sulfotransferase StaL".《Chemistry Biology》.2006,第13卷(第2期),第171-181页. * |
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Effective date of registration: 20180301 Address after: 215600 A 207 room A building center of Zhangjiagang Free Trade Zone, Suzhou Free Trade Zone, Jiangsu Patentee after: Zhangjiagang Institute of industrial technology, Dalian Institute of Chemical Physics, China Academy of Sciences Address before: 116023 Zhongshan Road, Liaoning, No. 457, Patentee before: Dalian Institute of Chemical Physics, Chinese Academy of Sciences |
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