CN103301653A - Separation method of sulfonic acid combination - Google Patents
Separation method of sulfonic acid combination Download PDFInfo
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- CN103301653A CN103301653A CN2012100645623A CN201210064562A CN103301653A CN 103301653 A CN103301653 A CN 103301653A CN 2012100645623 A CN2012100645623 A CN 2012100645623A CN 201210064562 A CN201210064562 A CN 201210064562A CN 103301653 A CN103301653 A CN 103301653A
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Abstract
The invention discloses a separation method of a sulfonic acid combination. The separation method of the sulfonic acid combination comprises the following step of: separating the acidity dependence of a hydroxy compound as well as a sulfonic acid combination thereof rapidly by taking a mixture of a substrate of the hydroxy compound (containing phenolic hydroxyl group and alcoholic hydroxyl group) after chemical derivation or biological conversion as raw materials in virtue of chromatographic columns filled by composite materials, and meanwhile through utilizing eluents with different pH values. The separation method of the sulfonic acid combination provided by the invention has wide application in preparing the sulfonic acid combination, and is suitable for the purification and preparation of all sulfonic acid combinations which contain phenolic (alcohol) hydroxyl group, but not contain acidity groups.
Description
Technical field
The invention belongs to medical technical field, a kind of preparation method of sulfonic acid bond is provided especially.
Background technology
Drug metabolism is the main mode of body defence foreign substance invasion, is considered to the important biochemical barrier that body is resisted external nuisance.Sulfonic acid is the important II of the class metabolic response that combines in the body in conjunction with metabolism, and (Naunyn Schmiedebergs Arch Pharmacol 2004 plays an important role in the bio-transformation of the little molecule of endogenous (as steroidal class, catecholamines, peanut acids etc.) and exogenous material (as medicine); 369:55-68; Toxicol Sci 2006; 90:5-22.).Most drug is behind the sulfonic acid association reaction, and the sulfonated bond of generation is compared with substrate has higher solubility, therefore is easier to excrete, thereby plays the effect that reduces drug effect and toxicity.But some compounds carry out sulfonic acid in conjunction with after can form reactive intermediate, thereby induce generation (the Chem Biol Interact 1998 of cancer by covalent bonds with big molecule such as DNA; 109:221-235).Therefore, the sulfonic acid bond of preparation medicine has very important significance to safe and effective medication clinically.
The synthetic method of sulfonic acid bond mainly is divided into two classes: chemical derivatization and biotransformation method.The advantage of the sulfonated derivatization method maximum of chemistry be reaction rate fast, be fit to large-scale production, but its severe reaction conditions (usually needing strong acid and strong base or high temperature) for the substrate of part poor chemical stability, chemically derivedly often produces multiple accessory substance.Compare with chemical method, bioanalysis has favorable applicability, can be widely used in the preparation of different types of structure substrate, but often is higher than chemical method on preparation cost.Therefore, for the preparation of the sulfonated product of specific substrates, can select chemical method or bioanalysis to reach the purpose of preparation target sulfonic acid bond according to substrate structure and chemical attribute.But, be problems such as chemical method or bioanalysis all exist reaction end-body set member not single, and target product purity is lower.How to realize the fast purifying preparation of target sulfonic acid bond monomer has been become the committed step of sulfonic acid bond preparation.Yet, still lack the effective ways of quick separation sulfonic acid bond monomer at present, so be necessary to develop a kind of practicality, the quick and high technology of selecting separate targets sulfonic acid bond.
Summary of the invention
The separation method that the purpose of this invention is to provide a kind of sulfonic acid bond, this 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 bond, this method is that substrate is through chemically derived or biotransformation, form the mixture of substrate and its sulfonic acid bond, be raw material with this mixture, by the chromatographic column that composite is filled, utilize the quick separation of the eluant, eluent realization mixture material of different pH values simultaneously.
The separation method of sulfonic acid bond provided by the invention, the requirement of described method is successively:
At first carry out chemical synthesis or bio-conversion processes: the chemically derived or bio-transformation system that contains sulfonated transferase such as sulfonated is converted into the sulfonic acid bond and obtains sample to be separated substrate is all or part of.Used enzyme is specially: recombinant expressed sulfonic acid transferase (that is: SULT), or for to derive from the microsome that contains the SULT enzymatic activity of animal tissue or to contain MC cell liquid (S9), animal tissue is preferably from the tissue of strong metabolic capability such as liver, kidney, intestines; Described substrate is to contain phenol (alcohol) hydroxyl but the compound that do not contain acidic-group;
Next carries out separation and purification treatment: the response sample that above-mentioned biotransformation is obtained passes through to obtain target sulfonic acid bond behind the enrichment of flash chromatography post and the purifying; The used filler of described flash chromatography post is the mixed fillers that contains reverse phase silica gel and anion exchange resin, and the volume ratio of two kinds of fillers is between 0.2-5; The used silica gel bonding has phenyl, a kind of carbochain among C8 and the C18, and the silica gel particle diameter is between 10-200 μ m, requires to contain a kind of basic group in primary amine groups, secondary amine, tertiary amine groups, the season amino in the used anionic ion-exchange resins.
The separation method of sulfonic acid bond provided by the invention, described biotransformation needs 3 '-AMP-5 '-phosphoric acid sulfuric ester or its corresponding lithium salts etc. providing sulfonic acid group for biological catalytic process, the mole dosage of its adding be the substrate mole dosage 1-100 doubly; Be reflected at the pH value and carry out in the buffer salt solution of 6-8, and keep 30-50 ℃ conversion temperature (preferred 35-40 ℃).
The separation method of sulfonic acid bond provided by the invention, in the filler of described flash chromatography post, silica gel bonded have different length carbochain, the preferred C8 of carbon chain lengths and C 18, the preferred 40-60 μ of a granularity m.Anion exchange resin is alkalescent or strong basic ion exchange resin, preferably contains the ion exchange resin of primary amine groups or secondary amine group, and the resin particle diameter is 0.2-1mm.The gross mass of two kinds of fillers is to be not less than substrate and metabolite gross mass 20 times.
The separation method of sulfonic acid bond provided by the invention, described separation and purification process is for being placed on the mixture material dissolving column cap of chromatographic column, water, organic solvent, the drip washing of acidifying organic solvent respectively then.Described organic solvent is preferably alcohol.Described organic solvent is a kind of or mixed solvent in methyl alcohol, ethanol, isopropyl alcohol, acetonitrile, the acetone, is preferably alcohol.Employed acid is lower boiling organic acid in the described acidifying organic solvent, is preferably formic acid or acetic acid.
The separation method of sulfonic acid bond provided by the invention, after described chemical synthesis or bio-transformation sample place flash chromatography post column cap, priority is through the process of water wash, pure drip washing and the drip washing of acidifying alcohol, water wash is intended to water-soluble substances in the elution of reactive liquid, alcohol drip washing is intended to other liposoluble substance in wash-out substrate and the reactant liquor, and the drip washing of acidifying alcohol is intended to wash-out sulfonic acid bond.Used alcohol is fatty alcohols such as methyl alcohol, ethanol or isopropyl alcohol; Used acid is higher boil organic acids such as formic acid or acetic acid, and the molar concentration of acid is 0.01-0.1mol/L in the alcohol.The consumption of water is not less than 2 times of column volumes in the water-washing process; Alcohol is specially methyl alcohol or ethanol, and the node of pure drip washing is no longer to contain substrate in the eluent; The consumption of acidifying alcohol is 5 times of column volume.
The separation method of sulfonic acid bond provided by the invention, described chemical synthesis or bio-transformation sample upper prop and successively by washing, after alcohol washes, acidifying alcohol washes, through high performance liquid chromatography and liquid phase-Mass Spectrometer Method, metabolite is enriched in the acidifying methyl alcohol; Then through low pressure evaporation or inert gas (N
2) dry up, namely get the metabolite of purity＞95%.
Sulfonic acid is the reaction that is mediated by sulfonic acid transferase (SULT) in conjunction with metabolism, the defective that is equipped with the sulfonic acid bond based on the traditional biological legal system, the present invention is raw material with the mixture of hydroxy compounds after chemical derivatization or bio-transformation, by the chromatographic column of ion exchange resin and the filling of reverse phase filler composite, utilize the eluant, eluent p-sulfonic acid bond of different pH values to carry out the quick separation of acidity dependent form simultaneously.Not only simple to operate, and can obtain a large amount of sulfonic acid metabolites fast.
Separation method with existing sulfonic acid bond is compared, and the present invention has following advantage:
1) easy and simple to handle efficient, do not need enrichment in the animal body, do not need the multistep purifying yet;
2) have applicability widely, be suitable for all and contain the separation that phenolic hydroxyl group does not contain the corresponding sulfonic acid bond of SULT substrate of acidic-group simultaneously.
Description of drawings
Fig. 1 is for utilizing the flow chart of the sulfonated pregnenolone of bio-transformation (chemically derived) preparation;
Fig. 2 is the high-efficient liquid phase chromatogram of resibufogenin sulfonic acid bond;
Fig. 3 is the mass spectrogram of resibufogenin sulfonic acid bond;
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.
The specific embodiment
Embodiment 1, and the preparation of pregnenolone sulfonic acid bond is as follows:
1) prepare sample separation through biotransformation method: pregnenolone is dissolved in the storage liquid that is made into 5mg/ml in the middle of the methyl alcohol, take out the phosphatic buffer salt that 20ml adds pH7.4, add rats'liver endochylema (RLC) after the stirring successively, add sulfonic donor PAPS, the resibufogenin final concentration is 0.10mg/ml in the system, final concentration of protein is 0.5mg/ml, PAPS final concentration 0.2mM, and 37 ℃ of waters bath with thermostatic control are stirred and are hatched 2h.HPLC detects and reactant liquor is transferred to-80 ℃ of refrigerator and cooled but after substrate conversion efficiency reaches 50%.Extract reaction solution 100ml and directly go up the flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have a phenyl, and particle diameter is 40 μ m, and consumption is 2.5g, and ion exchange resin contains secondary amino group, and materials are 2.5g.Successively use the 10ml water wash, the drip washing of 10ml methyl alcohol, the drip washing of 30ml formic acid methyl alcohol.2) prepare sample separation through chemical derivatization: (0.137g, (0.126g 0.039mmol) at room temperature mixes sulfur trioxide-Trimethylamine compound, and mixture is in 55 ℃ of heating 12 hours that reflux with pregnenolone 0.96mmol) to be dissolved in pyridine (5ml).The question response mixture is cooled to room temperature, shifts and the decompression enrichment.Reactant mixture redissolves and be splined on the flash chromatography post behind methyl alcohol (100ml).The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have a phenyl, and particle diameter is 40 μ m, and consumption is 25g, and ion exchange resin contains secondary amino group, and materials are 25g.Successively use the 100ml water wash, the drip washing of 100ml methyl alcohol, 300ml formic acid methyl alcohol drip washing (Fig. 1).
Acidifying methyl alcohol leacheate N under room temperature
2Dry up, biotransformation method makes sulfonic acid bond 7.9mg, and chemical derivatization makes sulfonic acid bond 110mg.
Resibufogenin is dissolved in the storage liquid that is made into 5mg/ml in the middle of the dimethyl sulfoxide (DMSO) (DMSO), take out the phosphatic buffer salt that 20ml adds pH7.4, add people single enzyme rhSULT2A1 that recombinates after the stirring successively, add sulfonic donor PAPS, the resibufogenin final concentration is 0.10mg/ml in the system, final concentration of protein is 0.25mg/ml, PAPS final concentration 0.2mM, and 37 ℃ of waters bath with thermostatic control are stirred and are hatched 2h.HPLC detects and reactant liquor is transferred to-80 ℃ of refrigerator and cooled but after substrate conversion efficiency reaches 50%.Extract reaction solution 100ml and directly go up the flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have a phenyl, and particle diameter is 40 μ m, and consumption is 2.5g, and ion exchange resin contains secondary amino group, and materials are 2.5g.Successively use the 10ml water wash, the drip washing of 10ml methyl alcohol, the drip washing of 30ml formic acid methyl alcohol.Acidifying methyl alcohol leacheate N under room temperature
2Dry up sulfonic acid bond 7mg, HPLC purity＞95% (Fig. 2 and Fig. 3).
Embodiment 3, the sulfonated metabolite preparation of dephnetin
Dephnetin is dissolved in the storage liquid that is made into 5mg/ml in the methyl alcohol, take out the phosphatic buffer salt that 30ml adds pH7.4, add rat intestine S9 (RIS9) after the stirring successively, add sulfonic donor PAPS, the dephnetin final concentration is 0.10mg/ml in the system, final concentration of protein is 0.5mg/ml, PAPS final concentration 0.2mM, and 37 ℃ of waters bath with thermostatic control are stirred and are hatched 1h.HPLC detects and reactant liquor is transferred to-80 ℃ of refrigerator and cooled but after substrate conversion efficiency reaches 50%.Extract reaction solution 100ml and directly go up the flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have a phenyl, and particle diameter is 40 μ m, and consumption is 4g, and ion exchange resin contains secondary amino group, and materials are 4g.Successively use the 20ml water wash, the drip washing of 20ml methyl alcohol, the drip washing of 40ml formic acid methyl alcohol realizes the enrichment to sulfonated products.Acidifying methyl alcohol leacheate N under room temperature
2Dry up concentratedly, utilize the efficient liquid phase of preparation to two further separation and purification of metabolite, make dephnetin-7-sulfuric ester 5.1mg (Fig. 5) and dephnetin-8-sulfuric ester 3.9mg (Fig. 4).
Claims (11)
1. the separation method of a sulfonic acid bond is characterized in that:
(1) chemically derived or biotransformation: substrate through chemically derived or biotransformation, is formed the mixture of substrate and its sulfonic acid bond;
(2) separation and purification process: the above-mentioned mixture that obtains as raw material, by the chromatographic column that composite is filled, is utilized the quick separation of the eluant, eluent realization mixture material of different pH values simultaneously.
2. according to the separation method of the described sulfonic acid bond of claim 1, it is characterized in that: described substrate or/and alcoholic extract hydroxyl group, and does not contain the compound of acidic-group for containing phenolic hydroxyl group.
3. according to the separation method of the described sulfonic acid bond of claim 1, it is characterized in that: described bio-transformation refers to that the substrate process contains the catalysis generation sulfonic acid bond of the living things system of sulfonated transferase; Used transferase source comprises following one of several or combination: recombinant expressed sulfonic group transferase SULT, contain the SULT enzyme animal tissue's homogenate, contain the cell liquid of SULT enzyme.
4. according to the separation method of claim 1 and 3 described sulfonic acid bonds, it is characterized in that: requirement adding 3 in the described biotransformation '-AMP-5 '-phosphoric acid sulfuric ester or its corresponding lithium salts, the mole of its adding is 1-100 times of substrate; Be reflected at the pH value and carry out in the buffer salt solution of 6-8, and keep 30-50 ℃ conversion temperature.
5. according to the separation method of the described sulfonic acid bond of claim 1, it is characterized in that: the filler in the described chromatographic column is the mixed fillers of while bonding reverse phase silica gel and anion exchange resin, 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 μ m.
6. according to the separation method of claim 1 and 5 described sulfonic acid bonds, it is characterized in that: the consumption of the filler in the described chromatographic column is 20-100 times of raw material quality.
7. according to the separation method of the described sulfonic acid bond of claim 1, it is characterized in that: the separation and purification process of described step (2) is for being placed on the mixture material dissolving column cap of chromatographic column, water, organic solvent, the drip washing of acidifying organic solvent respectively then.
8. according to the separation method of the described sulfonic acid bond of claim 7, it is characterized in that: described organic solvent is a kind of or mixed solvent in the following solvents: methyl alcohol, ethanol, isopropyl alcohol, acetonitrile, acetone.
9. according to the separation method of the described sulfonic acid bond of claim 1, it is characterized in that: employed acid is lower boiling organic acid in the described acidifying organic solvent.
10. according to the separation method of the described sulfonic acid bond of claim 9, it is characterized in that: employed acid is formic acid or acetic acid in the described acidifying organic solvent.
11. the separation method according to the described sulfonic acid bond of claim 7 is characterized in that: described water wash process institute water consumption is not less than 2 times of column volumes; The used organic solvent amount of organic solvent lessivation is not less than 2 times of column volumes; The amount of the used acidifying organic solvent of acidifying organic solvent lessivation is not less than 5 times of column volumes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0967272A2 (en) * | 1998-06-23 | 1999-12-29 | Seikagaku Corporation | Novel sulfotransferase |
| US20040191870A1 (en) * | 2001-03-28 | 2004-09-30 | Robert Rosenberg | Methods of 6-0 sulfating polysaccharides and 6-0 sulfated polysaccharide preparations |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0967272A2 (en) * | 1998-06-23 | 1999-12-29 | Seikagaku Corporation | Novel sulfotransferase |
| US20040191870A1 (en) * | 2001-03-28 | 2004-09-30 | Robert Rosenberg | Methods of 6-0 sulfating polysaccharides and 6-0 sulfated polysaccharide preparations |
Non-Patent Citations (1)
| Title |
|---|
| SHERRY S. LAMB: ""Biosynthesis of Sulfated Glycopeptide Antibiotics by Using the Sulfotransferase StaL"", 《CHEMISTRY & BIOLOGY》 * |
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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 |