CN102628075A - Method for producing chiral amino acid by penicillin acylase resolution and product thereof - Google Patents
Method for producing chiral amino acid by penicillin acylase resolution and product thereof Download PDFInfo
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Abstract
The invention discloses a method for producing chiral amino acid by penicillin acylase resolution, comprising the following steps of: performing acetylation reaction between DL-amino acid and phenacetyl chloride in water and under the action of NaOH, adjusting pH of the solution to 0.5-2.0, filtering and recovering an N-DL-phenylacetyl-amino acid solid, dissolving in water, adding a cation exchange resin for adsorption reaction, adjusting pH of the solution to 7.5-8.0, adding penicillin acylase for enzymatic resolution to obtain a L-amino acid or D-amino acid solid, adding the amino acid product into an alcohol solvent of 60-70 DEG C until it is oversaturated, cooling for crystallization, filtering, and recovering the solid product. Through ion exchange resin adsorption of an intermediate p-phenylacetyl amino acid in present technologies and high-temperature recrystallization of the final product amino acid alcoholic solvent and with the combination of two operations. The chiral content of the final product amino acid is significantly raised and reaches more than 99.9%.
Description
Technical field
The present invention is specifically related to a kind of Semacylase that utilizes and splits method of producing chiral amino acid and products thereof.
Background technology
Alpha-non-natural amino acid acts on increasing in pharmaceutical developments.At present new drug development to chiral purity require increasingly highly, much require chirality content will reach 99.9%.The production technique of alpha-non-natural amino acid has chemistry to split with enzyme process to split two kinds of technologies, and wherein enzyme process splits because mild condition, and high specificity pollutes for a short time, and chemical fractionation has remarkable advantages.No matter chemistry splits and the enzyme process fractionation, and chirality content all is the major consideration of weighing the quality of resolution process.
Semacylase splits and produces alpha-non-natural amino acid is recent research focus, can be used for splitting ALFA-amino acid and BETA-amino acid, is the main technology that enzyme process splits alpha-non-natural amino acid, in particular for the amino acid whose fractionation of BETA-.The Semacylase that uses has immobilized Semacylase and two kinds of forms of free Semacylase.Technological process mainly was two steps: the first step amino acid reacts with phenyllacetyl chloride under solvent and alkaline environment, and amino acid becomes the acid of N-DL-phenylacetylamino through acidylate, isolates-N-DL-phenylacetylization-amino acid after acid cooling with sour regulation system then; Second step was prepared into solution for resolution reaction with the above-mentioned N-DL-acetylize-amino acid that obtains, and added Semacylase then, obtained L-amino acid or D-amino acid behind the resolution reaction.
Discover; General Semacylase splits in the technology of preparation chiral amino acid; Amino acid chiral content after the fractionation receives two controlling factors; One is the phenylacetylization-DL-content of amino acids that splits usefulness, and general phenylacetylization-DL-amino acid all contains a certain amount of unreacting material DL-amino acid; In addition, in the acid adjustment crystallisation process, acid also can make phenylacetylization-DL-amino acid hydrolysis the becoming DL-amino acid of part; After containing the amino acid whose phenylacetylization of DL--DL-amino acid fractionation like this, inevitably there are a certain amount of D type or L-type amino acid simultaneously in the product; So all influence and split the amino acid whose chirality content in back.
The another one factor is the specificity of Semacylase and splits the back because heating and pH value change the racemization that causes.After the fractionation, high temperature decolouring, the high temperature that pass through the long period concentrate and crystallisation process, and these factors all can cause the reduction of chirality content, and generally speaking, this process can cause chirality content to reduce by 0.1～3% percentage point.If without refining, the amino acid chiral content after general enzyme splits is difficult to reach 99.9% 90%～99.5%.
Simultaneously, generally all hope to obtain two chiral amino acids of D-type and L-type simultaneously through enzyme method technique; Generally speaking, after N-phenylacetyl-DL-amino acid split through Semacylase, product was L-amino acid and N-phenylacetyl-D-amino acid; If react not thorough; Although the amino acid whose chirality content of L-can be higher, the amino acid whose chirality content of N-phenylacetyl-D-then can be very not high, so the amino acid whose chirality content of the D-that obtains after the hydrolysis can be lower.As yellow hat worn by a Taoist priest China " synthetic chemistry " (Vol 15,2007, the research in P69-72), the D-phenylalanine(Phe) chirality content that uses immobilized penicillin acylated enzyme to split out is 91.4%.And for example Li Tian child (Vol 15,11,2008, split to produce the D-L-Ala with immobilized penicillin acylated enzyme in P1066-1069), and the optical purity of D-L-Ala is 95.3% in " fine chemistry industry ".The production technique of other document records, chirality content is the highest also only to reach 99%, can't reach more than 99.9%.If therefore require a technology to obtain two products that chirality content is high simultaneously, then must study the mode that improves amino acid chiral content.
Summary of the invention
The technical problem that the present invention will solve is to split production technique to existing Semacylase; The comparatively low defective of chiral amino acid chirality content of preparation; Provide a kind of new Semacylase to split the method for producing chiral amino acid; This method comprises that middle product N-DL-phenylacetylization-amino acid is carried out ion exchange resin to be made with extra care, and the amino acid whose high temperature re-crystallization step of the finished product.The finished product chirality content for preparing through this method is very high, can reach 99.9%.The present invention provides the product that utilizes this method prepared simultaneously.
The inventor is in order to improve the chirality content that utilizes Semacylase to split the alpha-non-natural amino acid of preparation technology's production, and the recrystallizing technology to acylation process, resolution process and chiral amino acid has carried out big quantity research and improvement respectively.The contriver finds through conventional Semacylase fractionation preparation amino acid technology acetylize intermediate product being carried out ion exchange resin refining pleasantly surprisedly; And the amino acid whose alcoholic solvent of the finished product carried out the high temperature recrystallization; The amino acid whose chirality content of the finished product is significantly improved; Reach 99.9%, thereby accomplished the present invention.
Therefore, the technical scheme that solves the problems of the technologies described above of the present invention is following.
One of technical scheme that the present invention takes is: a kind of Semacylase splits the method for producing chiral amino acid, comprising following steps:
(1) in water, under the NaOH effect, DL-amino acid and phenyllacetyl chloride carry out acetylization reaction, and the pH value of gained reaction soln is adjusted to 0.5～2.0, and filtered and recycled N-DL-phenylacetylization-amino acid solid is dissolved in water;
(2) in step (1) gained solution, add Zeo-karb and carry out absorption reaction, filtering then and collecting filtrating is N-DL-phenylacetylization-amino acid solution;
(3) step (2) gained pH value of filtrate is adjusted to 7.5～8.0; Add Semacylase and carry out the enzymatic resolution reaction,, add the alcoholic solvent decrease temperature crystalline concentrating behind the gained reaction soln removal Semacylase; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; The filtered and recycled solid promptly gets L-amino acid, collects filtrating;
(4) filtrating of step (3) gained is concentrated the back and regulate pH value to 0.5～2.0, the filtered and recycled solid is N-phenylacetyl-D-amino acid, adds the acid reaction that is hydrolyzed in this solid, and hydrolysate is concentrated, crystallization, and the filtered and recycled solid promptly gets D-amino acid;
(5) step (3) or (4) gained L-amino acid or D-amino acid being joined temperature is in 60～70 ℃ the alcoholic solvent; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; Be added to hypersaturated state; With this solution decrease temperature crystalline, the filtered and recycled solid product gets final product then.
The method of the invention step (1) is in water, and under the NaOH effect, DL-amino acid and phenyllacetyl chloride carry out acetylization reaction, and the pH value of gained reaction soln is adjusted to 0.5～2.0, and filtered and recycled N-DL-phenylacetylization-amino acid solid is dissolved in water.Wherein said DL-amino acid is DL-Amino Acid, and promptly L type and D type amino acid respectively account for half, and specific rotation is zero amino acid.DL-amino acid according to the invention preferably is selected from DL-methionine, DL-L-Ala, DL-L-glutamic acid, DL-aspartic acid, DL-phenylalanine(Phe), DL-phenylglycine, DL-valine, DL-leucine, DL-Methionin, DL-ornithine, DL-Terleu, DL-in fluorophenyl glycine and the DL-o-chlorobenzene glycine one or more.Wherein said NaOH can be selected from caustic soda, caustic soda, ion film caustic soda, sodium hydroxide solution and the liquid caustic soda one or more, more preferably adopts sheet alkali and sodium hydroxide solution.The mol ratio of wherein said DL-amino acid and NaOH is preferably 1: 1～and 1: 4, more preferably mol ratio is 1: 1.The water yield that adds in the N-DL-phenylacetylamino acid crude be so that the DL-amino acid of phenylacetylization can fully dissolve is as the criterion, and suitably excessive, more preferably dissolves for adding 2～10 times of water of mass ratio.
The method of the invention step (2) is in step (1) gained solution, to add Zeo-karb to carry out absorption reaction, and filtering then and collecting filtrating is N-DL-phenylacetylization-amino acid solution.Wherein the Zeo-karb consumption generally calculates according to the mole exchange capacity; According to the corresponding acidic cation-exchange resin that adds of the free aminoacids content that detects; Preferably the Zeo-karb add-on is more than 1 times of free amino acid theoretical amount; More preferably, Zeo-karb exchange capacity molal quantity of the present invention is 1.5～5 times of total free aminoacids molal quantity.The cationic exchange resin adsorption reaction times according to the invention, promptly gained filtrating was detected through thin-layer chromatography (TLC) preferably for stopping after the absorption reaction fully, in the time of can not detecting total free aminoacids in the solution till, the absorption reaction time more preferably is 1～30 minute.Zeo-karb according to the invention preferably is selected from one or more in strongly acidic cationic exchange resin, weakly acidic cation-exchange resin and the large hole cation exchanger resin; More preferably adopt the highly acidic cation macroporous resin.
The method of the invention step (3) is that step (2) gained pH value of filtrate is adjusted to 7.5～8.0; Add Semacylase and carry out the enzymatic resolution reaction,, add the alcoholic solvent decrease temperature crystalline concentrating behind the gained reaction soln removal Semacylase; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; The filtered and recycled solid promptly gets L-amino acid, collects filtrating.Semacylase of the present invention is meant: be used for the side chain of catalysis penicillium mould and the hydrolysis reaction of the amido linkage between the parent nucleus; To obtain the class of enzymes of penicillium mould parent nucleus 6-APA (6-amino-penicillanic acid); This enzyme can be applicable to synthesis of chiral peptide, chiral separation etc.Said Semacylase comprises penicillin G acylase, V-cillin acylase and penbritin acylase according to the Substratspezifitaet of enzyme.Preferably, technical scheme according to the invention adopts immobilized penicillin acylated enzyme.The consumption of said immobilized penicillin acylated enzyme confirms the ratio vigor of different aminoacids according to Semacylase, and general consumption is to transform 2～5 times of the theoretical amount that finishes in 24 hours.Consider the thoroughness of fractionation, what the time of above-mentioned fractionation was preferable is 12～48 hours, more preferably is 24 hours.Said immobilized penicillin acylated enzyme resolution reaction temperature preferably is 25～40 ℃, and better temperature of reaction is 30 ℃.Wherein the immobilized penicillin acylated enzyme resolution reaction preferably the pH value be 7.0～8.0, better pH value is 7.5, can keep through in reaction solution, adding sheet alkali.Described alcohol reagent preferably is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols, and better alcoholic solvent is an ethanol.
The method of the invention step (4) is the filtrating of step (3) gained to be concentrated the back regulate pH value to 0.5～2.0, and the filtered and recycled solid is N-phenylacetyl-D-amino acid, adds the acid reaction that is hydrolyzed in this solid; Hydrolysate is concentrated; Crystallization, the filtered and recycled solid promptly gets D-amino acid.Wherein said enrichment step preferably method is: the filtrating of gained is concentrated into liquid concentrator occurs stopping to concentrate behind the solid, according to one times of moisturizing of residue liquid concentrator volume, and then be concentrated into liquid concentrator and occur stopping to concentrate behind the solid.Wherein said acid is hydrochloric acid preferably, more preferably is the 2N hydrochloric acid of 10 times of volumes.
The method of the invention step (5) is that step (3) or (4) gained L-amino acid or D-amino acid are joined temperature is in 60～70 ℃ the alcoholic solvent; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; Be added to hypersaturated state; With this solution decrease temperature crystalline, the filtered and recycled solid product gets final product then.Wherein said alcoholic solvent preferably is selected from methyl alcohol, ethanol, and one or more in the terepthaloyl moietie, more preferably alcoholic solvent is a methyl alcohol.The preferable temperature of said alcoholic solvent is 60～70 ℃, and the better ground temperature of said methanol solvate is 66 ℃; Said Tc preferably is 45～60 ℃, and Tc more preferably is 55～60 ℃.
Two of the technical scheme that the present invention takes is: through the chiral amino acid product of method for preparing.The chirality content of wherein said amino acid the finished product is preferably 99.9%.
Three of the technical scheme that the present invention takes is: the application of above-mentioned chiral amino acid product in the preparation chiral drug.
On the basis of this area general knowledge, above-mentioned each optimum condition, but arbitrary combination promptly get each preferred embodiments of the present invention.
Raw material that the present invention is used or reagent except that specifying, all commercially available getting.
Than prior art; Beneficial effect of the present invention is following: the present invention has carried out ion exchange resin absorption and the finished product amino acid alcoholic solvent high temperature recrystallization through existing Semacylase is split preparation technology's intermediate product to phenylacetyl amino acid; And through the two procedures coupling; The finished product amino acid chiral content is significantly improved, reach more than 99.9%.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." normal temperature " described in the embodiment is meant the temperature of the operation room that makes an experiment, and is generally 25 ℃.
The title specification of raw materials used source and equipment is:
Immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive
-1): Hunan Fu Laige company;
Zeo-karb is available from traditional Chinese medicines reagent company.
Embodiment 1
In the 130ml deionized water, add DL-2-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) and the 6.5mol/L NaOH aqueous solution (30mL); After dripping fully, stir 2h stirring at normal temperature 3h, drip 2.0mol/L hydrochloric acid; To pH value of solution=3, filtration drying gets white powdery solid N-phenylacetyl-DL-2-phenylalanine(Phe) 26.0 grams; Analyze through HPLC, content is 98.6%.
In Erlenmeyer flask, add deionized water 1000mL, add N-phenylacetyl-DL-2-phenylalanine(Phe) 28.3g,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 7.5; Filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters; Obtain L-phenylalanine(Phe) 8 grams, analyze through chirality HPLC, chirality EE is 98.4%.Filtrating is continued to be concentrated into liquid concentrator and is occurred stopping to concentrate behind the solid, occurs stopping to concentrate behind the solid according to one times of moisturizing reconcentration to the liquid concentrator that remains the liquid concentrator volume, and the cooling back transfers pH2.0 to filter; Drying obtains N-phenylacetyl-D-2-phenylalanine(Phe) 12 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-2-phenylalanine(Phe) 7.1 grams, through analyzing, chirality content is 91.5%.
Embodiment 2
In the 130ml deionized water, add DL-3-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) and the 6.5mol/L NaOH aqueous solution (30mL); After dripping fully, stir 2h, stirring at normal temperature 3h; Drip 2.0mol/L hydrochloric acid, to pH value of solution=3.0, filtration drying; Get white powdery solid N-phenylacetyl-DL-3-phenylalanine(Phe) 25.5 grams, analyze through HPLC, content is 98.5%.
In Erlenmeyer flask, add deionized water 1000mL, add N-phenylacetyl-DL-3-phenylalanine(Phe) 28.3g,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 7.5; Filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling and filters; Obtain L-3-phenylalanine(Phe) 7.9 grams, through analyzing, chirality EE is 98.4%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-3-phenylalanine(Phe) 12.1 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-3-phenylalanine(Phe) 7.2 grams, through analyzing, chirality content is 92.5%.
Embodiment 3
In the 130ml deionized water, add DL-2-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=3, filtration drying gets white powdery solid N-phenylacetyl-DL-2-phenylalanine(Phe) 26.0 grams; Add 2000ml water, add 2 gram strongly acidic cationic exchange resins then, stirred 2 minutes; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is filtering IPA2750 after 7.5,24 hours, is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters to obtain L-phenylalanine(Phe) 7.5 grams, through analyzing, chirality EE is 99.4%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-2-phenylalanine(Phe) 12.1 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-2-phenylalanine(Phe) 7.1 grams, through analyzing, chirality content is 92.5%.
Embodiment 4
In the 130ml deionized water, add DL-3-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) and the 6.5mol/L NaOH aqueous solution (30mL); After dripping fully, stir 2h, stirring at normal temperature 3h; Drip 2.0mol/L hydrochloric acid, to pH value of solution=3, filtration drying; Get white powdery solid N-phenylacetyl-DL-3-phenylalanine(Phe) 25.5 grams, add 2000ml water, add 2 gram weakly acidic cation-exchange resins (available from reagent company of traditional Chinese medicines group) then; Stirred 2 minutes; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 7.5; Filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters; Obtain L-3-phenylalanine(Phe) 7.8 grams, through analyzing, chirality EE is 99.3%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-3-phenylalanine(Phe) 12.0 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-3-phenylalanine(Phe) 7.2 grams, through analyzing, chirality content is 94.5%.
Embodiment 5
In the 130ml deionized water, add DL-2-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=3, filtration drying gets white powdery solid N-phenylacetyl-DL-2-phenylalanine(Phe) 26.0 grams; Add 2000ml water, add 2 gram strongly acidic cationic exchange resins then, stirred 2 minutes; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, keep pH7.5 with sheet alkali simultaneously, filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters, and obtains L-phenylalanine(Phe) 7.5 grams, and through analyzing, chirality EE is 99.4%; Above L-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 58 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.4 gram L-phenylalanine(Phe)s, optical purity 99.9%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-2-phenylalanine(Phe) 12.1 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-2-phenylalanine(Phe) 7.1 grams, through analyzing, chirality content is 92.5%.Above D-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 58 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 6.7 grams, optical purity 99.9%.
Embodiment 6
In the 130ml deionized water, add DL-3-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=2.0, filtration drying gets white powdery solid N-phenylacetyl-DL-3-phenylalanine(Phe) 25.5 grams; Add 2000ml water, add 2 gram weakly acidic cation-exchange resins then, stirred 2 minutes; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 7.5
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 7.5; Filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters; Obtain L-3-phenylalanine(Phe) 7.8 grams, through analyzing, chirality EE is 99.3%; Above L-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 55 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.7 grams, optical purity 99.9%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-3-phenylalanine(Phe) 12.0 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-3-phenylalanine(Phe) 7.2 grams, through analyzing, chirality content is 94.5%.Above D-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 70 ℃, keeps 10 minutes, cools to 58 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.1 grams, optical purity 99.9%.
Embodiment 7
With the DL-Terleu is the process implementing of raw material use-case 1, and after the phenylacetylization, measuring content is 98.5%; After the chiral separation, L-Terleu optical purity is 98.6%, and D-Terleu optical purity is 98.2%.
Embodiment 8
With the DL-Terleu is the process implementing of raw material use-case 3, and after the phenylacetylization, strongly acidic macroporous cation exchange resin is refining, and after the fractionation, L-Terleu optical purity is 99.1%, and D-Terleu optical purity is 98.8%.
Embodiment 9
With the DL-Terleu is the process implementing of raw material use-case 5, and after the phenylacetylization, the slightly acidic large hole cation exchanger resin is refining, and behind the ethanol high temperature recrystallization, L-Terleu optical purity is 99.9%, and D-Terleu optical purity is 99.9%.
Embodiment 10
In the 130ml deionized water, add DL-3-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 16.0 grams (0.4mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=0.5, filtration drying gets white powdery solid N-phenylacetyl-DL-3-phenylalanine(Phe) 25.5 grams; Add 50ml water, add 2 gram weakly acidic cation-exchange resins then, stirred 1 minute; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 8.0
-1, Hunan Fu Laige company) and 1.0g, in 30 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 8.0; Filtering IPA2750 after 24 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters; Obtain L-3-phenylalanine(Phe) 7.8 grams, through analyzing, chirality EE is 99.3%; Above L-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 60 ℃, keeps 10 minutes, cools to 45 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.7 grams, optical purity 99.9%.Filtrating continues to be concentrated into small volume, the moisturizing reconcentration once, it is 0.5 that pH is transferred in the cooling back, filters; Drying obtains N-phenylacetyl-D-3-phenylalanine(Phe) 12.0 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-3-phenylalanine(Phe) 7.2 grams, through analyzing, chirality content is 94.5%.Above D-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 60 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.1 grams, optical purity 99.9%.
Embodiment 11
In the 130ml deionized water, add DL-2-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 4.0 grams (0.1mol), 0 ℃ drip simultaneously phenyllacetyl chloride (5mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=1.5, filtration drying gets white powdery solid N-phenylacetyl-DL-2-phenylalanine(Phe) 26.0 grams; Add 2000ml water, add 2 gram strongly acidic cationic exchange resins then, stirred 30 minutes; Cross and filter to remove resin,, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then then with sheet alkali adjustment pH to 7.0
-1, Hunan Fu Laige company) and 1.0g, in 25 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is filtering IPA2750 after 7.5,12 hours, is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters, and obtains L-phenylalanine(Phe) 7.5 grams, through analyzing, chirality EE is 99.4%; Above L-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 58 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.4 grams, optical purity 99.9%.Filtrating continues to be concentrated into small volume, and the moisturizing reconcentration is once transferred pH2.0 after the cooling, filter; Drying obtains N-phenylacetyl-D-2-phenylalanine(Phe) 12.1 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-2-phenylalanine(Phe) 7.1 grams, through analyzing, chirality content is 92.5%.Above D-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 58 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 6.7 grams, optical purity 99.9%.
Embodiment 12
In the 130ml deionized water, add DL-3-phenylalanine(Phe) 16.5 grams (0.1mol) and sheet alkali 8.0 grams (0.2mol), 0 ℃ drip simultaneously phenyllacetyl chloride (15mL, 0.11mol) with the 6.5mol/L NaOH aqueous solution (30mL), drip complete after; Stir 2h, stirring at normal temperature 3h drips 2.0mol/L hydrochloric acid; To pH value of solution=2.0, filtration drying gets white powdery solid N-phenylacetyl-DL-3-phenylalanine(Phe) 25.5 grams; Add 50ml water, add 2 gram weakly acidic cation-exchange resins then, stirred 25 minutes; Cross and filter to remove resin, adjust to pH7.0 with sheet alkali then, add immobilized penicillin acylated enzyme IPA2-750 (enzyme 100～130Ug alive then
-1, Hunan Fu Laige company) and 1.0g, in 40 ℃ of resolution reactions, using sheet alkali to keep pH simultaneously is 8.0; Filtering IPA2750 after 48 hours is concentrated into 300ML then, adds the ethanol crystallisation by cooling, filters; Obtain L-3-phenylalanine(Phe) 7.8 grams, through analyzing, chirality EE is 99.3%; Above L-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 60 ℃, keeps 10 minutes, cools to 45 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.7 grams, optical purity 99.9%.Filtrating continues to be concentrated into small volume, the moisturizing reconcentration once, it is 0.5 that pH is transferred in the cooling back, filters; Drying obtains N-phenylacetyl-D-3-phenylalanine(Phe) 12.0 grams, adds 10 times of volume 2N hydrochloric acid hydrolysis, concentrates; Drying is filtered in crystallization; Obtain D-3-phenylalanine(Phe) 7.2 grams, through analyzing, chirality content is 94.5%.Above D-3-phenylalanine(Phe) adds 20 times of methyl alcohol, is heated to reflux 66 ℃, keeps 10 minutes, cools to 60 ℃ then, keeps 5 minutes, keeps this temperature to filter, and obtains 7.1 grams, optical purity 99.9%.
Should be understood that after having read foregoing of the present invention those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a Semacylase splits the method for producing chiral amino acid, it is characterized in that, may further comprise the steps:
(1) in water, under the NaOH effect, DL-amino acid and phenyllacetyl chloride carry out acetylization reaction, and the pH value of gained reaction soln is adjusted to 0.5～2.0, and filtered and recycled N-DL-phenylacetylization-amino acid solid is dissolved in water;
(2) in step (1) gained solution, add Zeo-karb and carry out absorption reaction, filtering then and collecting filtrating is N-DL-phenylacetylization-amino acid solution;
(3) step (2) gained pH value of filtrate is adjusted to 7.5～8.0; Add Semacylase and carry out the enzymatic resolution reaction,, add the alcoholic solvent decrease temperature crystalline concentrating behind the gained reaction soln removal Semacylase; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; The filtered and recycled solid promptly gets L-amino acid, collects filtrating;
(4) filtrating of step (3) gained is concentrated the back and regulate pH value to 0.5～2.0, the filtered and recycled solid is N-phenylacetyl-D-amino acid, adds the acid reaction that is hydrolyzed in this solid, and hydrolysate is concentrated, crystallization, and the filtered and recycled solid promptly gets D-amino acid;
(5) step (3) or (4) gained L-amino acid or D-amino acid being joined temperature is in 60～70 ℃ the alcoholic solvent; Said alcoholic solvent is selected from one or more in methyl alcohol, ethanol, terepthaloyl moietie, n-propyl alcohol, Virahol and the butanols; Be added to hypersaturated state; With this solution decrease temperature crystalline, the filtered and recycled solid product gets final product then.
2. method according to claim 1; It is characterized in that the said DL-amino acid of step (1) is selected from DL-methionine, DL-L-Ala, DL-L-glutamic acid, DL-aspartic acid, DL-phenylalanine(Phe), DL-phenylglycine, DL-valine, DL-leucine, DL-Methionin, DL-ornithine, DL-Terleu, DL-in fluorophenyl glycine and the DL-o-chlorobenzene glycine one or more.
3. the method for claim 1 is characterized in that, the mol ratio of said DL-amino acid of step (1) and NaOH is 1: 1～1: 4, and said dissolving step is that N-DL-phenylacetylization-amino acid solid is dissolved in mass ratio is in 2～10 times the water.
4. the method for claim 1; It is characterized in that; The said Zeo-karb of step (2) is selected from one or more in strongly acidic cationic exchange resin, weakly acidic cation-exchange resin and the large hole cation exchanger resin, and described Zeo-karb consumption is 1.5～5 times (mol ratios) of total free aminoacids amount in the aqueous solution.
5. the described method of claim 1 is characterized in that, the time of the said resin cation(R.C.) absorption reaction of step (2) is 1～30 minute.
6. the method for claim 1; It is characterized in that the said Semacylase of step (3) is an immobilized penicillin acylated enzyme, the time of said enzymatic resolution reaction is 12～48 hours; The temperature of resolution reaction is 25～40 ℃, and the pH value of resolution reaction is 7.0～8.0.
7. the method for claim 1; It is characterized in that; The said enrichment step of step (4) is a secondary concentration: filtrating being concentrated into of gained occurred stopping to concentrate behind the solid; According to one times of moisturizing of residue liquid concentrator volume, and then be concentrated into liquid concentrator and occur stopping to concentrate behind the solid, the wherein said adding acid reaction that is hydrolyzed is to add the hydrochloric acid reaction that is hydrolyzed.
8. the method for claim 1 is characterized in that, the said Tc of step (5) is 45～60 ℃.
9. the chiral amino acid product for preparing like each said method of claim 1～8.
10. like the application of the said chiral amino acid product of claim 9 in the preparation chiral drug.
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Denomination of invention: A method and product for separating and producing chiral amino acids using penicillin acylase Effective date of registration: 20230703 Granted publication date: 20140813 Pledgee: The Bank of Shanghai branch Caohejing Limited by Share Ltd. Pledgor: SHANGHAI HANHONG TECHNOLOGY CO.,LTD. Registration number: Y2023310000341 |