WO2020173453A1 - 使用电渗析技术拆分光学异构体的方法 - Google Patents

使用电渗析技术拆分光学异构体的方法 Download PDF

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WO2020173453A1
WO2020173453A1 PCT/CN2020/076711 CN2020076711W WO2020173453A1 WO 2020173453 A1 WO2020173453 A1 WO 2020173453A1 CN 2020076711 W CN2020076711 W CN 2020076711W WO 2020173453 A1 WO2020173453 A1 WO 2020173453A1
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optical isomer
electrodialysis
ionizable
racemate
butyrolactone
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PCT/CN2020/076711
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English (en)
French (fr)
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邱贵森
苏金环
曾聪明
蒋泰隆
陈彦
刘文杰
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上海艾美晶生物科技有限公司
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Priority to US17/434,418 priority Critical patent/US20220154240A1/en
Priority to EP20763387.6A priority patent/EP3933043A4/en
Publication of WO2020173453A1 publication Critical patent/WO2020173453A1/zh

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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/001Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by metabolizing one of the enantiomers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/42Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
    • B01D61/44Ion-selective electrodialysis
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/58Multistep processes
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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02Oxygen as only ring hetero atoms
    • C12P17/04Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42Hydroxy-carboxylic acids
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/04Specific process operations in the feed stream; Feed pretreatment
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/18Details relating to membrane separation process operations and control pH control
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/26Further operations combined with membrane separation processes
    • B01D2311/2643Crystallisation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2311/00Details relating to membrane separation process operations and control
    • B01D2311/26Further operations combined with membrane separation processes
    • B01D2311/2676Centrifugal separation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14Ultrafiltration; Microfiltration
    • B01D61/145Ultrafiltration
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D61/00Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14Ultrafiltration; Microfiltration
    • B01D61/147Microfiltration

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to the use of biocatalysis technology and electrodialysis technology to separate optical isomers from racemates.
  • BACKGROUND Chirality is an essential attribute of nature, and many biological macromolecules and biologically active substances have chirality characteristics. Although the chemical composition of two or more different configurations of chiral substances is exactly the same, there are often differences in physiological activities. Usually only one configuration has the required activity, and the other configurations have little or no effect, and may even have toxic side effects. .
  • pantothenic acid also known as pantothenic acid, is one of the B vitamins and a component of coenzyme A. It participates in the metabolism of protein, fat, and sugar, and plays an important role in material metabolism.
  • D-configuration D-pantothenic acid vitamin B5
  • pantothenic acid is unstable, its commercial form is mainly D-calcium pantothenate.
  • Resolution is one of the main ways to obtain optically pure chiral compounds. Compared with traditional chemical resolution, enzymatic resolution does not require expensive resolution reagents, has mild reaction conditions, good optical selectivity, is environmentally friendly, and can perform some reactions that cannot be performed by chemical methods. Enzymatic resolution is more and more respected by scientific researchers from various countries due to its significant advantages, and there have been many successful industrialization cases.
  • D-Pantolactone is an important chiral intermediate in the production of pantothenic acid series products such as calcium D-pantothenate, D-panthenol, and D-pantetheine.
  • the industrial synthesis of D-pantoic acid lactone mostly adopts a technical route that combines a chemical method with a hydrolytic enzyme resolution method. That is, the chemical method produces racemic DL-pantoate lactone, and then uses D-pantoate lactone hydrolase for hydrolytic resolution.
  • the clear liquid of the resolution reaction is first extracted with an organic solvent to extract L-pantoate lactone
  • the aqueous phase (containing D-pantoic acid) is acid lactonized and then extracted with an organic solvent, then desalted, decolorized, and refined by recrystallization.
  • free or immobilized cells are used to resolve DL-pantoic acid lactone, and then extracted with dichloromethane, the aqueous phase is acidified with hydrochloric acid and then extracted with dichloromethane, and the solvent is recovered to obtain D-pantoic acid.
  • the crude ester was recrystallized from acetone/isopropyl ether to obtain qualified D-pantoic acid lactone.
  • This process needs to be improved
  • a large amount of organic solvents are used in the extraction and purification process of D-pantoic acid obtained by the enzyme reaction, which brings environmental and cost problems, and the crude D-pantoic acid lactone needs to be recrystallized and refined.
  • Electrodialysis technology is an electrochemical separation process that uses ion exchange membranes and DC electric fields to separate electrolyte components from aqueous solutions.
  • This method can make up for the shortcomings of the existing chiral resolution process, utilize the different ionization degrees of the optical isomers in the chiral resolution product, and replace the traditional organic solvent extraction process with electroosmosis technology to improve the yield of the product. Rate and product quality. Reduce production costs.
  • the present invention provides a method for separating optical isomers from racemates by electrodialysis, which comprises: a) reacting the racemates in the presence of a catalyst to form ionizable optical isomers including the first optical isomer And a mixture of the non-ionized form of the second optical isomer; b) subjecting the mixture to electrodialysis to allow the ionizable form of the first optical isomer and the second optical isomer to be The non-ionized form is separated; and
  • racemate refers to a mixture of two or more optical isomers with different optical properties.
  • a compound with one chiral center may have two optical isomers, one with the chiral center in the R configuration and the other with the chiral center in the S configuration.
  • the racemate includes both optical isomers of R configuration and optical isomers of S configuration.
  • different optical isomers may exist in equal molar amounts (that is, the optical activity is offset), or may exist in unequal molar amounts.
  • the racemate has a hydrolyzable functional group.
  • the hydrolyzable functional groups include, but are not limited to, ester bonds, amide bonds, and the like.
  • the functional group can generate an ionizable group after being hydrolyzed.
  • the ionizable group refers to a group that can ionize in an aqueous solution, for example, a carboxyl group, an amino group, and the like. Ionizable groups will produce charged groups after ionization, such as negatively charged carboxylate, positively charged Ammonia ions, etc.
  • the chiral center in the racemate may be located in the hydrolyzable functional group, or may be located near the hydrolyzable functional group, for example, on the atom adjacent to the hydrolyzable functional group, or 1, 2, or 3 atoms away from it.
  • the catalyst can specifically react with the specific optical isomer in the racemate (for example, hydrolyze the hydrolyzable functional group therein) to make it into an ionizable form.
  • "Ionizable form" in this application means that it ionizes to form charged groups in an aqueous solution.
  • the ionizable form may include ionizable groups, such as carboxyl, amino, and the like.
  • the catalyst may not catalyze the second optical isomer in the racemate, so that it maintains its non-ionized form.
  • "Non-ionized form” in this application means that it does not ionize in an aqueous solution and does not have a charged group.
  • the non-ionized form contains non-ionized groups, such as esters (such as lactones in racemates), amides, ethers, and the like.
  • the racemate has a ring structure, and the hydrolyzable functional group may be in the ring structure. Exemplary ring structures are, for example, lactone, lactam. These ring functional groups can react to open the ring.
  • the ring structure in the non-ionized form of the second optical isomer is closed.
  • the ring structure in the ionizable form of the first optical isomer is ring-opened.
  • the ring structure is ring-opened in the non-ionized form of the second optical isomer, and/or the ring structure is ring-opened in the ionizable form of the first optical isomer
  • the ring structure is closed.
  • the chiral center may or may not be on a ring atom.
  • the racemate is an ester.
  • Exemplary racemic esters include, 3-cyclohexene
  • the racemate is a lactone.
  • Lactone refers to the internal ester bond (-C(O)O) produced by dehydration of carboxyl and hydroxyl in the molecular structure. Usually the ester bond inside the molecule is in the ring structure. Examples of lactones such as DL racemic pantoic lactone, P-butyrolactone, ⁇ -butyrolactone, a-hydroxy-butyrolactone, P-hydroxyt-butyrolactone, a-acetyl- Y -butyrolactone Lactone, n-butylphthalide, etc.
  • the catalyst comprises an enzyme composition.
  • the enzyme composition contains an enzyme capable of specifically reacting with a certain optical isomer. For example, it specifically reacts with the D-configuration optical isomer, or specifically reacts with the L-configuration optical isomer.
  • the enzyme composition contains an ester hydrolase. In some embodiments, the ester hydrolase specifically catalyzes ⁇ D-configuration lactone.
  • ester hydrolases include, for example, D-pantoate lactone hydrolase, Novozyme 435 lipase, P-butyrolactone hydrolase, Y -butyrolactone hydrolase, ⁇ -hydroxy- Y -butyrolactone Hydrolase, p-hydroxyf-butyrolactone hydrolase, a-acetyl-Y-butyrolactone hydrolase, n-butylphthalide hydrolase, etc.
  • D-pantoate lactone hydrolase it can specifically hydrolyze the D-configuration pantoate lactone in the racemate, so that the lactone structure is hydrolyzed to form an independent intramolecular carboxyl group and Hydroxyl groups, in which carboxyl groups can be ionized in an aqueous solution, can be charged and are in an ionizable form.
  • D-pantoate lactone hydrolase cannot hydrolyze the L-configuration pantoate lactone in the racemate, so the L-configuration pantoate lactone still maintains the lactone structure after the catalyzed reaction. Form of ionization.
  • Novozyme 435 lipase can specifically hydrolyze the R-configuration of 3-cyclohexene-1-carboxylic acid methyl ester in the racemate to form 3-cyclohexene-1-carboxylic acid. Can be ionized. Since methyl 3-cyclohexene-1-carboxylate in the S-configuration cannot be hydrolyzed, it still remains in a non-ionized form.
  • the enzyme composition contains lactamase.
  • the lactamase specifically catalyzes lactams in D-configuration.
  • Exemplary lactamases are, for example, P-lactamase, lactamase.
  • P-lactamase As an example, it can specifically hydrolyze the D-configuration P-lactam in the racemate, so that the lactam structure is hydrolyzed to form independent carboxyl and amino groups in the molecule. It can be ionized in an aqueous solution, so it can be charged and is an ionizable form.
  • the P-lactamase cannot hydrolyze the L-configuration p-lactam in the racemate, so the L-configuration p-lactam still maintains the lactam structure after the catalytic reaction, which is a non-ionized form.
  • the racemate of the present invention is DL-pantoate lactone
  • the first optical isomer is D-pantoate lactone
  • the second optical isomer Is L-pantoate lactone
  • the ionizable form of the first optical isomer is D-pantoic acid
  • the non-ionized form of the second optical isomer is L-pantoate lactone.
  • the racemate is methyl 3-cyclohexene-1-carboxylate
  • the first optical isomer is methyl (R)-3-cyclohexene-1-carboxylate
  • the second optical isomer is (S) ..
  • the ionizable form of the first optical isomer is (R) -3 -cyclohexene- 1-Formic acid
  • the non-ionized form of the second optical isomer is (S:)-3-cyclohexene-1-methyl carboxylate.
  • the racemate is a-hydroxy-Y-butyrolactone
  • the first optical isomer is (R)-a-hydroxy-Y-butyrolactone
  • the The two optical isomers are (S)-a-hydroxy-Y-butyrolactone
  • the The ionizable form of the first optical isomer is (R)- ⁇ -hydroxy-Y-butyric acid
  • the non-ionizable form of the second optical isomer is (S)- ⁇ -hydroxybutyrolactone.
  • the racemate is P-hydroxy-Y-butyrolactone
  • the first optical isomer is (R)-P-light-based butyrolactone
  • the second optical The isomer is (S)-P-hydroxy t-butyrolactone
  • the ionizable form of the first optical isomer is (R) -P-hydroxy-Y-butyric acid
  • the second optical isomer The non-ionized form of the body is (S) -P-hydroxybutyrolactone.
  • the racemate is a-acetyl-Y-butyrolactone
  • the first optical isomer is (R)-a-acetyl-Y-butyrolactone
  • the second optical isomer is (S)-a-acetyl-Y-butyrolactone
  • the ionizable form of the first optical isomer is (R)-a-acetyl_Y_butyric acid
  • the non-ionized form of the second optical isomer is (S)-a-acetyl based on butyrolactone.
  • Any form of enzyme with selective catalytic function for optical isomers can be used.
  • the enzyme composition may contain a purified enzyme, a cell expressing the enzyme, or a cell lysate expressing the enzyme.
  • the cell expressing the enzyme may be any suitable host cell, and may be a prokaryotic cell such as bacteria, or a eukaryotic cell such as yeast, animal cells, and the like.
  • the cell lysate can be any lysate component containing enzymes, such as a cell lysate.
  • the enzyme composition is immobilized on a substrate.
  • the applicable substrate may include materials for immobilizing enzymes, such as magnetic particles, macroporous resins, etc.; it may also include materials for immobilizing cells, such as calcium alginate, gels, and the like.
  • the step a) maintains the pH value in the range of 7.0 to 7.5 during the reaction, for example, the pH value is maintained at 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or any two of the above values Any number in the range. In some embodiments, 15N NH 3 H 2 0 titration is used to maintain the pH. In some embodiments, the temperature in step a) during the reaction is maintained between 20 ° C and 40 ° C, for example, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C.
  • step a) the reaction time of step a) is 1-10 hours, for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or any value between any two numerical ranges above.
  • step b) it further includes: removing the residue of the catalyst in the mixture.
  • the residues include cell debris, proteins and other macromolecules
  • Those skilled in the art can use conventional separation means to remove the residue of the catalyst in the mixture according to their actual needs, for example, one or more of various means such as filtration, centrifugation, microfiltration, ultrafiltration and the like.
  • the filtration is achieved by using filter paper or filter cloth.
  • the filter paper or filter cloth described in the present invention may be commercially available filter paper or filter cloth, for example, filter paper or filter cloth produced by GE Healthcare Life Sciences, Spy Pure, Asahi Kasei, etc.
  • the pore size of the filter paper or filter cloth is 10 ⁇ 150(j_m, for example 10[j_m, 20(j_m, 30(j_m, 40(j_m, 50(j_m, 60(j_m, 70(j_m, 80 (j_m, 90(j_m, 100(j_m, 110(j_m, 120(j_m, 130(j_m, 140(j_m, 150(j_m, or any value between any two numerical ranges above.
  • a suitable filter paper or filter cloth pore size is selected to remove the catalyst residue.
  • the centrifugation is achieved by using a centrifugal separator.
  • the centrifugal separator can be a commercially available centrifugal separator, such as Guangzhou Fuyi Liquid Separation Technology Co., Ltd., Yantai Chengbo Machinery Technology Co., Ltd., Dongjing Yaotian Electric Technology Co., Ltd., TEMA System, Kyte, Heinkel, GEA, etc.
  • the centrifugal separator produced by the company.
  • the centrifugal speed is 1000rpm ⁇ 2000rpm, such as 1000rpm, 1100rpm, 1200rpm, 1300rpm, 1400rpm, 1500rpm,
  • the centrifugation time is 2-15 minutes, for example, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes or any value between any two numerical ranges above.
  • the microfiltration is achieved by passing the mixture through a microfiltration membrane.
  • the microfiltration membrane described in the present invention may be a commercially available microfiltration membrane, for example, a series of microfiltration hollow fiber membranes produced by GE Healthcare Life Sciences, Spy Pure, Asahi Kasei and other companies.
  • the pore size of the microfiltration membrane is ⁇ 0.6 (j _m, such as 0.1pm, 0.15 (j _m, 0.2 (j _m, 0.22 (j _m, 0.25 (j _m,
  • the ultrafiltration is achieved by passing the mixture through an ultrafiltration membrane.
  • the ultrafiltration membrane described in the present invention may be a commercially available ultrafiltration membrane, for example, the ultrafiltration hollow fiber membrane series produced by GE Healthcare Life Sciences, Spectra, and Asahi Kasei.
  • the ultrafiltration membrane is a hollow fiber ultrafiltration membrane with a pore size of 10kD ⁇ 500kD, for example, a pore size of 10kD, 20kD, 30kD, 40kD, 50kD, 60kD, 70kD, 80kD, 90kD, 100kD, 150kD, 200kD, 250kD,
  • the method of the present invention further includes purifying and/or concentrating the ionizable form of the separated first optical isomer, and/or purifying and/or concentrating the separated second optical isomer The non-ionized form of the isomer.
  • the isolated ionizable form of the first optical isomer and/or the non-ionized form of the second optical isomer may be further purified.
  • the ionizable form of the first optical isomer and/or the non-ionized form of the second optical isomer can be extracted by using a suitable solvent.
  • a suitable solvent for example, an organic solvent (for example, ethyl acetate) can be added to the collected (R) -3-cyclohexene-1-carboxylic acid, and the organic phase can be collected to obtain purified (R) . 3 -cyclohexene- 1-Formic acid.
  • an organic solvent for example, ethyl acetate
  • the organic phase can be collected to obtain purified (S) -3- Methyl cyclohexene-1-carboxylate.
  • the isolated and/or purified ionizable form of the first optical isomer and/or the non-ionized form of the second optical isomer may be further concentrated.
  • the concentration is achieved by reduced pressure, for example, the ionizable form of the isolated and/or purified first optical isomer and/or the isolated and/or purified
  • the non-ionized form of the second optical isomer is pumped into the concentration equipment for concentration under reduced pressure.
  • the present invention further includes converting the non-ionized form of the second optical isomer into the racemate.
  • the non-ionized form of the second optical isomer may have been separated by the method provided in this application, or further purified, or further concentrated. For example, when the racemate is an ester, the separated non-ionized form (ie, ester) of the second optical isomer can be racemized to obtain racemates with different chiral isomers.
  • the present invention further comprises converting the ionizable form of the isolated first optical isomer into a non-ionized form.
  • the isolated (and/or purified or concentrated) ionizable form of the first optical isomer can be further reacted to restore the ionizable groups therein to hydrolyzable functional groups.
  • the ionizable form of the separated first optical isomer is D-pantoic acid, which can be lactonized to obtain D-pantoic acid lactone, so that the The ionizable group (ie, carboxyl) is restored to a hydrolyzable functional group (ie, lactone).
  • the ionizable group ie, carboxyl
  • a hydrolyzable functional group ie, lactone
  • the electrodialysis treatment is performed in an electrodialysis cell, wherein the electrodialysis cell has a desalination chamber and a concentration chamber separated by ion exchange membranes.
  • the ion exchange membrane is a homogeneous membrane or a heterogeneous membrane.
  • anions and cations are directed to the anode and The cathode moves.
  • the ion exchange membrane is an anion exchange membrane, such as a Q membrane. In some embodiments, the ion exchange membrane is a cation exchange membrane, such as an S membrane. In some embodiments, the ion exchange membrane is a cation exchange membrane and an anion exchange membrane. In certain embodiments, the cation exchange membrane allows the passage of cations and repels and blocks the passage of anions. In certain embodiments, the anion exchange membrane allows the passage of anions and repels the passage of cations.
  • the compartment formed between the cation exchange membrane and the anode, and between the anion exchange membrane and the cathode is a concentration compartment
  • the compartment formed between the cation membrane and the anion membrane is a desalination compartment.
  • cation exchange membranes and anion exchange membranes are commercially available, for example, from Novasep Company, Eurodia Company, Shandong Tianwei Membrane Technology Co., Ltd., Zhejiang Qianqiu Environmental Water Treatment Co., Ltd., and the like.
  • the electrodialysis treatment includes placing the mixture in the desalination chamber, placing a solvent in the concentration chamber, and energizing the electrodialysis cell to make the desalination chamber The ionizable form of the first optical isomer migrates into the solvent in the concentration chamber.
  • the flow rate is adjusted to adjust the pressure of the concentration chamber and the desalination chamber, so that the pressure of the concentration chamber is 1, 2, 3, 4, 5 times the pressure of the desalination chamber. Times or any value between any two numerical ranges above.
  • the electrodialysis treatment is performed under constant voltage conditions until the conductivity of the desalination chamber is less than 30
  • the constant voltage is 10V, 15V, 20V, 25V, 30V, 35V, 40V, 45V, 50V # o
  • the solvent includes pure water.
  • the electrodialysis treatment is performed in an electrodialysis cell.
  • step b) of the method of the present invention can be repeated in the electrodialysis cell, thereby improving the separation efficiency.
  • the concentration chamber clear liquid can be pumped into the desalination chamber of the electrodialysis cell in the electrodialysis device to repeat the electrodialysis step in the electrodialysis cell.
  • the electrodialysis treatment is performed in more than one electrodialysis cell connected in series.
  • the pressures of the concentration chamber and the desalination chamber between different electrodialysis cells are the same. In some embodiments, the pressures of the concentration chamber and the desalination chamber between different electrodialysis cells are different. In some embodiments, the voltage between different electrodialysis cells is the same. In some embodiments, the voltages between different electrodialysis cells are different.
  • the purity of the optical isomer obtained by resolution using the method of the present invention is greater than 90%, for example, greater than
  • the purity of the optical isomers obtained by resolution using the method of the present invention is expressed by the ee value.
  • Regular technical means for example, HPLC method
  • measure or calculate the ee value. For example, if a racemate contains two optical isomers A and B, then the ee value 8%-B%.
  • One of the advantages of the present invention lies in the use of biocatalysis (for example, enzyme catalysis) technology combined with electrodialysis technology, the use of enzyme catalysis to produce products with different ionization degrees, the use of electrodialysis technology to resolve the racemate optical Isomer, mild reaction conditions, reducing operation steps;
  • biocatalysis for example, enzyme catalysis
  • Electrodialysis separation Use homogeneous membrane module B (size: 10*30cm; number of membrane pairs: 5 pairs), pump the ultrafiltrate clear liquid into the electrodialysis desalination chamber, and put 2L of pure water in the concentration chamber and adjust The flow makes the pressure of the three chambers equal, and runs at a constant pressure of 10V until the conductivity of the desalination chamber is less than 10 (Vs/cm; Pump the clear liquid of the concentration chamber into the desalination chamber of the secondary electrodialysis device, put 2L of pure water in the concentration chamber, adjust the flow rate to make the pressure of the three chambers equal, and operate at a constant pressure of 10V until the conductivity of the desalination chamber is ⁇ 10 (Vs/cm;
  • Electrodialysis separation Use heterogeneous membrane stack Z (size: 10*20cm; number of membrane pairs: 10 pairs), pump the ultrafiltrate clear liquid into the electrodialysis desalination chamber, put 3L pure water in the concentration chamber, adjust The flow rate makes the concentration chamber pressure 3 times that of the desalination chamber.
  • the pretreatment liquid enzymatic conversion first filtered with filter paper, then with a 0.4 M m of microfiltration membrane filters;
  • Electrodialysis separation Use homogeneous membrane stack S (size: 10*20cm; number of membrane pairs: 10 pairs), pump the ultrafiltrate clear liquid into the electrodialysis desalination chamber, and put 1 L of pure water in the concentration chamber, Adjust the flow rate to make the pressure of the three chambers equal, and run at a constant pressure of 14V until the conductivity of the desalination chamber drops to ⁇ 100
  • Concentration chamber clear liquid treatment The electrodialysis concentration chamber clear liquid is injected into the concentration equipment to reduce pressure and concentrate to approximately
  • Desalination chamber clear liquid treatment Combine the three-stage desalination chamber clear liquid, and pump into the concentration equipment to reduce pressure and concentrate to approximately 400ml, add an equal volume of ethyl acetate for extraction, Muji organic phase vacuum distillation to obtain (S)! cyclohexene-1-carboxylic acid methyl ester, the ee value is 74.1%, the yield is about 41.2%.

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Abstract

一种通过电渗析从外消旋体中拆分光学异构体的方法。具体地,将电渗析技术应用于酶拆分工艺中,主要应用于酶拆分后产物的分离。以D-泛解酸内酯制备工艺为例,关键点在于通过电渗析方法从酶拆分液中分离D-泛解酸和L-泛解酸内酯,取代了现有的有机溶剂提取方法,工艺方法简单易行,D-泛解酸收率高,纯度好,大大减少了有机溶剂的使用量,降低了生产成本,环境友好,可以很大程度的改善工人的工作环境,提高运行安全指数。

Description

使用电渗析技术拆分光学异构体的方法
技术领域
本发明属于生物技术领域,具体涉及使用生物催化技术和电渗析技术从外消 旋体中拆分光学异构体。 背景技术 手性是自然界的本质属性,许多生物大分子和生物活性物质都具有手性特征。 手性物质的两个或多个不同构型虽化学成分完全一样,但生理活性往往存在差别, 通常只有一个构型具有所需活性, 其他构型作用很小, 或者无作用, 甚至可能有 毒副作用。 例如泛酸 (pantothenic acid), 又名本多生酸, 为 B族维生素之一, 是 辅酶 A 的组成部分, 参与蛋白质、 脂肪、 糖的代谢, 在物质代谢中起着重要的 作用。 其活性成分为 D-构型的右旋泛酸 (维生素 B5 ), 但因泛酸不稳定, 其商 品形式主要为 D-泛酸钙。 拆分是光学纯手性化合物的主要获得途径之一。与传统化学法拆分相比, 酶 法拆分不需使用价格昂贵的拆分试剂,反应条件温和,光学选择性好,环境友好, 且还能进行一些化学法无法进行的反应。酶法拆分凭借其显著的优势越来越受到 各国科研人员的推崇, 已经有很多工业化的成功案例。 例如, D-泛解酸内酯 (D-Pantolactone) 是生产 D-泛酸 f丐、 D-泛醇、 D-泛硫 乙胺等泛酸系列产品的重要手性中间体。 目前工业化合成 D-泛解酸内酯多采用 的就是化学法与水解酶拆分法相结合的技术路线。即化学法生产消旋的 DL-泛解 酸内酯, 然后用 D-泛解酸内酯水解酶进行水解拆分, 拆分反应的清液先用有机 溶剂萃取出 L-泛解酸内酯和未反应的 D-泛解酸内酯, 水相 (含有 D-泛解酸)加 酸内酯化后再用有机溶剂萃取, 然后脱盐、 脱色, 用重结晶方法精制。 例如 CN1313402A中, 利用游离或固定化细胞拆分 DL-泛解酸内酯, 然后用二氯甲烷 萃取, 水相盐酸酸化后再用二氯甲烷萃取, 回收溶剂后得到的 D-泛解酸内酯粗 品在丙酮 /异丙醚中重结晶, 得到合格的 D-泛解酸内酯。 这个工艺存在有待提高 之处, 例如, 酶反应得到的 D-泛解酸提取精制过程中要用到大量的有机溶剂进 行提取, 带来环境和成本问题, 且 D-泛解酸内酯粗品要重结晶精制, 收率低, 成 本鳥。 电渗析技术是利用离子交换膜和直流电场的作用,从水溶液分离出电解质组 分的一种电化学分离过程。 发明内容 本发明提供了一种全新的拆分光学异构体的方法。该方法可以弥补现有手性 拆分工艺的不足, 利用手性拆分产物中光学异构体的可离子化程度不同, 用电渗 拆技术取代传统的有机溶剂提取工艺 丛而提高产物的收率和产品质量. 降低生 产成本。 本发明提供了一种通过电渗析从外消旋体中拆分光学异构体的方法, 包括: a) 将外消旋体在催化剂的存在下反应形成包括第一光学异构体的可电 离形式和第二光学异构体的非电离形式的混合物; b) 将所述混合物进行电渗析处理, 以允许所述第一光学异构体的可电 离形式和所述第二光学异构体的非电离形式得以分离; 和
C) 收集分离的所述第一光学异构体的可电离形式, 和 /或收集分离的所 述第二光学异构体的非电离形式。 在本申请中, “外消旋体”是指, 具有两种或两种以上的具有不同旋光性质 的光学异构体的混合物。例如, 具有一个手性中心的化合物可以具有两种光学异 构体, 一种具有 R构型的手性中心, 另一种具有 S构型的手性中心。 对于该化 合物而言,其外消旋体既包括 R构型的光学异构体也包括 S构型的光学异构体。 在本申请所述的外消旋体中, 不同的光学异构体可以以相等的摩尔量存在(即旋 光性抵消), 也可以以不等的摩尔量存在。 在某些实施方式中, 所述外消旋体具有可水解的官能团。可水解的官能团例 如, 但不限于, 酯键、 酰胺键等。 在某些实施方式中, 所述官能团被水解后可以 生成可电离的基团。 可电离的基团是指在水溶液中会电离的基团, 例如, 羧基、 氨基等。 可电离基团在电离后会产生带电基团, 例如带负电的羧酸根、 带正电的 氨离子等。在某些实施方式中, 所述外消旋体中的手性中心可以位于可水解的官 能团中, 或者也可以位于可水解的官能团附近, 例如在可水解的官能团相邻的原 子上, 或者在与其间隔 1个、 2个或 3个原子的位置。 在本发明的方法中,所述催化剂可以特异性地与所述外消旋体中的特定的光 学异构体反应(例如水解其中的可水解的官能团), 使之成为可电离形式。“可电 离形式”在本申请中是指在水溶液中会电离形成带电基团。 在某些实施方式中, 所述可电离形式可以包含可电离基团, 例如羧基、 氨基等。 在某些实施方式中, 所述催化剂可以不催化外消旋体中的第二光学异构体, 使之保持其非电离形式。 “非电离形式”在本申请中是指在水溶液中不会电离, 也不具有带电基团。在某 些实施方式中, 所述非电离形式包含非电离基团, 例如酯(例如外消旋体中的内 酯)、 酰胺、 醚等。 在某些实施方式中, 所述外消旋体具有环结构, 并且所述可水解的官能团可 以在环结构中。 示例性的环结构例如, 内酯、 内酰胺。 这些环内官能团可以发生 反应, 从而开环。在某些实施方式中, 在所述第二光学异构体的非电离形式中所 述环结构是闭环的。在某些实施方式中, 在所述第一光学异构体的可电离形式中 所述环结构是开环的。例如, 当环内官能团发生开环反应后, 形成了可电离的基 团。或者在某些实施方式中, 在所述第二光学异构体的非电离形式中所述环结构 是开环的,和 /或在所述第一光学异构体的可电离形式中所述环结构是闭环的。在 具有环结构的外消旋体中, 其手性中心可以在环原子上, 也可以不在环原子上。 在某些实施方式中, 所述外消旋体是酯。 示例性的外消旋酯包括, 3 -环己烯
-1-甲酸甲酯。在某些实施方式中, 所述外消旋体是内酯。 内酯是指在分子结构中 具有由羧基和羟基脱水生成的分子内部酯键 (-C(O)O)。 通常分子内部的酯键在 环结构中。 内酯的示例例如 DL消旋泛解酸内酯、 P-丁内酯、 Y-丁内酯、 a-羟基- 丁内酯、 P-羟基 t丁内酯、 a-乙酰基 -Y-丁内酯、 正丁基苯酞等。 在某些实施方式中, 所述催化剂包含酶组合物。在某些实施方式中, 所述酶 组合物中含有能够特异性与某种光学异构体反应的酶。 例如, 特异性地与 D-构 型光学异构体反应, 或者特异性地与 L-构型光学异构体反应。 在某些实施方式 中, 所述酶组合物含有酯水解酶。在某些实施方式中, 所述酯水解酶特异性地催 化 D-构型的内酯。示例性的酯水解酶包括,例如, D-泛解酸内酯水解酶、 Novozyme 435脂肪酶、 P-丁内酯水解酶、 Y-丁内酯水解酶、 a-羟基 -Y-丁内酯水解酶、 p_羟基 f丁内酯水解酶、 a-乙酰基 -Y-丁内酯水解酶、 正丁基苯酞水解酶等。 以 D-泛解 酸内酯水解酶为例, 其能够特异性地水解外消旋体中的 D-构型的泛解酸内酯, 使其中的内酯结构水解形成分子内独立的羧基和羟基,其中的羧基在水溶液中能 够电离, 因此可以带电,为可电离形式。但是 D-泛解酸内酯水解酶不能水解外消 旋体中的 L-构型泛解酸内酯, 因此 L-构型泛解酸内酯在催化反应后仍然保持内 酯结构, 为非电离形式。 再例如, Novozyme 435脂肪酶能够特异性地水解外消 旋体中的 R-构型的 3 -环己烯 -1-甲酸甲酯, 使其形成 3 -环己烯 -1-甲酸, 在水溶液 中可电离。 S-构型的 3 -环己烯 -1-甲酸甲酯由于不能被水解, 因此仍然保持非电离 形式。 在某些实施方式中, 所述酶组合物含有内酰胺酶。在某些实施方式中, 所述 内酰胺酶特异性地催化 D-构型的内酰胺。示例性的内酰胺酶例如, P-内酰胺酶、 内酰胺酶。以 P-内酰胺酶为例,其能够特异性地水解外消旋体中的 D-构型的 P- 内酰胺, 使其中的内酰胺结构水解形成分子内独立的羧基和氨基, 其中的羧基在 水溶液中能够电离, 因此可以带电, 为可电离形式。但是 P-内酰胺酶不能水解外 消旋体中的 L-构型的 p-内酰胺, 因此 L-构型的 p-内酰胺在催化反应后仍然保持 内酰胺结构, 为非电离形式。 在某些实施方式中, 本发明所述的外消旋体是 DL-泛解酸内酯, 所述第一光 学异构体是 D-泛解酸内酯, 所述第二光学异构体是 L-泛解酸内酯, 所述第一光 学异构体的可电离形式是 D-泛解酸, 所述第二光学异构体的非电离形式是 L-泛 解酸内酯。 在某些实施方式中, 所述外消旋体是 3 -环己烯 -1-甲酸甲酯, 所述第一光学 异构体是 (R) -3 -环己烯 -1-甲酸甲酯, 所述第二光学异构体是 (S) ..3 -环己烯 -1- 甲酸甲酯, 所述第一光学异构体的可电离形式是(R) -3 -环己烯 -1-甲酸, 所述第 二光学异构体的非电离形式是 (S :) -3 -环己稀 -1-甲酸甲酯。 在某些实施方式中,所述外消旋体是 a-羟基 -Y-丁内酯,所述第一光学异构体 是 (R) -a-羟基 -Y-丁内酯, 所述第二光学异构体是 (S) -a-羟基 -Y-丁内酯, 所述 第一光学异构体的可电离形式是(R) -a-羟基 -Y-丁酸, 所述第二光学异构体的非 电离形式是 (S) -a-羟基于丁内酯。 在某些实施方式中,所述外消旋体是 P-羟基 -Y-丁内酯,所述第一光学异构体 是 (R) -P-轻基于丁内酯, 所述第二光学异构体是 (S) -P-轻基 t丁内酯, 所述 第一光学异构体的可电离形式是(R) -P-羟基 -Y-丁酸, 所述第二光学异构体的非 电离形式是 (S) -P-羟基于丁内酯。 在某些实施方式中,所述外消旋体是 a-乙酰基 -Y-丁内酯,所述第一光学异构 体是(R) -a-乙酰基 -Y-丁内酯,所述第二光学异构体是(S) -a-乙酰基 -Y-丁内酯, 所述第一光学异构体的可电离形式是(R) -a-乙酰基 _Y_丁酸, 所述第二光学异构 体的非电离形式是 (S) -a-乙酰基于丁内酯。 任何对光学异构体具有选择性催化功能的酶的形式都可以使用。在某些实施 方式中, 所述酶组合物可以含有纯化的酶、 表达酶的细胞、 或表达酶的细胞裂解 物。表达酶的细胞可以是任何适当的宿主细胞, 可以是原核细胞例如细菌, 也可 以是真核细胞例如酵母、动物细胞等。细胞裂解物可以是任何含有酶的裂解物成 分, 例如细胞裂解液等。 在某些实施方式中, 所述酶组合物是固定在基底上的。 适用的基底可以包括固定化酶的材料, 例如磁性微粒、 大孔树脂等; 也可以包括 固定化细胞的材料, 例如海藻酸钙、 凝胶等。 在某些实施方式中, 所述步骤 a)在反应过程中维持 pH值在 7.0 ~ 7.5的范围 内, 例如, pH值维持在 7.0、 7.1、 7.2、 7.3、 7.4、 7.5或者以上任意两个数值范 围之间的任意数值。在某些实施方式中,使用 15N NH3 H20滴定维持所述 pH值。 在某些实施方式中, 所述步骤 a)在反应过程中的温度维持在 20°C~40°C之间, 例 如, 2(TC、 21 °C、 22°C、 23°C、 24°C、 25°C、 26°C、 27°C、 28°C、 29°C、 3(TC、 31 °C、 32°C、 33°C、 34°C、 35°C、 36°C、 37°C、 38°C、 39°C、 40°C或者以上任意 两个数值范围之间的任意数值。 在某些实施方式中, 所述步骤 a)的反应时间为 1~10小时, 例如 1小时、 2小时、 3小时、 4小时、 5小时、 6小时、 7小时、 8 小时、 9小时、 10小时或者以上任意两个数值范围之间的任意数值。 在某些实施方式中, 在所述步骤 a) 以后和步骤 b) 之前进一步包括: 去除 所述混合物中所述催化剂的残留物。所述残留物包括细胞碎片、蛋白质等大分子, 本领域技术人员可以根据其实际需要使用常规的分离手段去除所述混合物中所 述催化剂的残留物, 例如, 过滤、 离心、 微滤、 超滤等多种手段中的一种或者多 种。 在某些实施方式中, 所述过滤通过使用滤纸或滤布来实现。本发明中所述的 滤纸或滤布可以是市售的滤纸或滤布, 例如 GE Healthcare Life Sciences公司、仕 比纯公司、 旭化成公司等公司生产的滤纸或滤布。在某些实施方式中, 滤纸或滤 布的孔径为 10~150(j_m,例如 10[j_m、 20(j_m、 30(j_m、 40(j_m、 50(j_m、 60(j_m、 70(j_m、 80(j_m、 90(j_m、 100(j_m、 110(j_m、 120(j_m、 130(j_m、 140(j_m、 150(j_m或者以上任意 两个数值范围之间的任意数值。本领域技术人员可以根据所述催化剂的残留物的 种类和尺寸, 选择合适的滤纸或滤布孔径来去除所述催化剂的残留物。 在某些实施方式中, 所述离心通过使用离心分离器来实现。本发明中所述的 离心分离器可以是市售的离心分离器, 例如广州富一液体分离技术有限公司、烟 台诚博机械科技有限公司、东竞市耀天电气科技有限公司、 TEMA System、 Kyte、 Heinkel、 GEA 等公司生产的离心分离器。 在某些实施方式中, 离心速率为 1000rpm~2000rpm,例如 lOOOrpm、 llOOrpm、 1200rpm、 1300rpm、 1400rpm、 1500rpm、
1600rpm、 1700rpm、 1800rpm、 1900rpm、 2000rpm或者以上任意两个数值范围之 间的任意数值。 在某些实施方式中, 离心时间为 2~15分钟, 例如, 2分钟、 3分 钟、 4分钟、 5分钟、 6分钟、 7分钟、 8分钟、 9分钟、 10分钟、 11分钟、 12分 钟、 13分钟、 14分钟、 15分钟或者以上任意两个数值范围之间的任意数值。 本 领域技术人员可以根据所述催化剂的残留物的种类和尺寸,选择合适的离心速度 和离心时间来去除所述催化剂的残留物。 在某些实施方式中, 所述微滤通过使得所述混合物经过微滤膜来实现。本发 明中所述的微滤膜可以是市售的微滤膜,例如 GE Healthcare Life Sciences公司、 仕比纯公司、旭化成公司等公司生产的微滤中空纤维膜系列。在某些实施方式中, 所述微滤膜的孔径为
Figure imgf000007_0001
〜 0.6(j_m,例如 0.1pm、 0.15(j_m、 0.2(j_m、 0.22(j_m、 0.25(j_m、
0.3(j_m、 0.35(j_m、 0.4(j_m、 0.45(j_m、 0.5(j_m、 0.55(j_m、 0.6(j_m或者以上任意两个数 值范围之间的任意数值。根据所述催化剂的残留物的尺寸, 选择尽可能小的微滤 膜孔径有助于大颗粒残留物的去除。 在某些实施方式中, 所述超滤通过使得所述混合物经过超滤膜来实现。本发 明中所述的超滤膜可以是市售的超滤膜, 例如 GE Healthcare Life Sciences公司、 仕比纯公司、旭化成公司等公司生产的超滤中空纤维膜系列。在某些实施方式中, 所述超滤膜是孔径为 10kD〜 500kD的中空纤维超滤膜,例如孔径为 10kD、20kD、 30kD、 40kD、 50kD、 60kD、 70kD、 80kD、 90kD、 100kD、 150kD、 200kD、 250kD、
300kD、 350kD、 400kD、 450kD、 500kD或者以上任意两个数值范围之间任意数 值的中空纤维超滤膜。本领域技术人员可以根据所述催化剂的残留物的尺寸, 选 择合适的超滤膜孔径来去除所述催化剂的残留物。 在某些实施方式中, 本发明所述的方法进一步包括纯化和 /或浓缩所述分离 的第一光学异构体的可电离形式, 和 /或纯化和 /或浓缩所述分离的第二光学异构 体的非电离形式。 在某些实施方式中, 所述分离的第一光学异构体的可电离形式和 /或第二光 学异构体的非电离形式可以被进一步纯化。例如, 可以通过用适当的溶剂, 萃取 所述第一光学异构体的可电离形式和 /或第二光学异构体的非电离形式。例如,可 以向收集得到的 (R) -3 -环己稀 -1-甲酸中加入有机溶剂 (例如, 乙酸乙酯), 收 集有机相, 以得到纯化的 (R) .3 -环己烯 -1-甲酸。 再例如, 可以进一步向收集得 到的 (S) -3 -环己烯 -1-甲酸甲酯中加入有机溶剂 (例如, 乙酸乙酯), 收集有机 相, 以得到纯化的 (S) -3 -环己稀 -1-甲酸甲酯。 在某些实施方式中, 所述分离的和 /或纯化的第一光学异构体的可电离形式 和 /或第二光学异构体的非电离形式可以进一步被浓缩。在某些实施方式中,所述 浓缩是通过减压实现的,例如,将所述分离的和 /或纯化的第一光学异构体的可电 离形式和 /或所述分离的和 /或纯化的第二光学异构体的非电离形式泵入浓缩设备 进行减压浓缩。 在某些实施方式中,本发明进一步包括将所述第二光学异构体的非电离形式 转化成所述外消旋体。所述第二光学异构体的非电离形式可以是经本申请提供的 方法已经分离的, 或者进一步已经纯化的, 或者进一步已经浓缩的。 例如, 当所 述外消旋体为酯时, 可以将分离的第二光学异构体的非电离形式(即酯)进行消 旋, 得到具有不同手性异构体的外消旋体。通过将拆分的第二光学异构体重新转 化成外消旋体, 可以允许进一步通过本申请提供的方法进行手性拆分, 以得到更 多的第一光学异构体。 在某些实施方式中,本发明进一步包括将所述分离的第一光学异构体的可电 离形式转化成非电离形式。在某些实施方式中,所述分离的(和 /或纯化的或浓缩 的)第一光学异构体的可电离形式可以被进一步反应, 使其中的可电离基团恢复 为可水解的官能团。例如, 在某些实施方式中, 所述分离的第一光学异构体的可 电离形式是 D-泛解酸, 可以将其内酯化以得到 D-泛解酸内酯, 从而将其中的可 电离基团 (即羧基) 恢复为可水解的官能团 (即内酯)。 本领域技术人员可以使用已知的方法和设备进行本发明方法中的电渗析步 骤。 电渗析的设备和方法请参见《中华人民共和国行业标准一电渗析技术 HY/T 034.1~034.5--1994》。 在某些实施方式中, 所述电渗析处理是在电渗析池中进行的, 其中所述电渗 析池具有以离子交换膜分隔的淡化室与浓缩室。在某些实施方式中,所述离子交 换膜是均相膜或异相膜。在电渗析处理过程中, 在外加电场的驱动下, 利用离子 交换膜的选择透过性(例如, 阳离子可以透过阳离子交换膜, 阴离子可以透过阴 离子交换膜), 阴离子和阳离子分别向阳极和阴极移动。 本领域技术人员根据其实际需要可以选择本领域已知的多种离子交换膜来 进行电渗析。在某些实施方式中, 离子交换膜为阴离子交换膜, 例如 Q膜。在某 些实施方式中, 离子交换膜为阳离子交换膜, 例如 S膜。在某些实施方式中, 离 子交换膜为阳离子交换膜和阴离子交换膜。在某些实施方式中, 阳离子交换膜允 许阳离子通过而排斥阻挡阴离子通过。在某些实施方式中, 阴离子交换膜允许阴 离子通过而排斥阻挡阳离子通过。在某些实施方式中,阳离子交换膜与阳极之间、 阴离子交换膜与阴极之间形成的隔室为浓缩室,阳离子膜与阴离子膜之间形成的 隔室为淡化室。在某些实施方式中, 阳离子交换膜和阴离子交换膜是市售的, 例 如, 来自 Novasep公司、 Eurodia公司、 山东天维膜技术有限公司、 浙江千秋环 保水处理有限公司等。 在某些实施方式中,本领域技术人员可以根据其实际需要选择所述均相膜或 异相膜的膜堆尺寸, 例如为 10*20cm、 10*30cm、 20*30cm等。 在某些实施方式 中, 本领域技术人员可以根据其实际需要选择所述均相膜或异相膜的膜对数,例 如, 5对、 10对、 15对、 20对等。 在某些实施方式中, 所述电渗析处理包括将所述混合物置于所述淡化室, 溶 剂置于所述浓缩室, 通过对所述电渗析池通电, 使得所述淡化室中的所述第一光 学异构体的可电离形式迀移到所述浓缩室的所述溶剂中。 在某些实施方式中, 所述电渗析处理过程中调节流量从而调节浓缩室、淡化 室的压力, 使得浓缩室的压力是淡化室的压力的 1倍、 2倍、 3倍、 4倍、 5倍或 者以上任意两个数值范围之间的任意数值。在某些实施方式中, 所述电渗析处理 是在恒定电压的条件下进行的, 直至淡化室电导率小于 30|as/cm、 40|as/cm、 50|as/cm、 60|as/cm、 70|as/cm、 80|as/cm、 90|as/cm、 100|as/cm、 110|as/cm、 120|as/cm、 130|as/cm、 140|as/cm、 150|as/cm等。 在某些实施方式中, 所述恒定电压是 10V、 15V、 20V、 25V、 30V、 35V、 40V、 45V、 50V #o 在某些实施方式中, 所述溶剂包括纯水。 在某些实施方式中, 所述电渗析处理是在一个电渗析池中进行。在某些实施 方式中,在所述电渗析池中可以重复进行本发明所述方法的步骤 b),从而提高分 离效率。例如,可以将浓缩室清液循环泵入所述电渗析装置中电渗析池的淡化室, 以在该电渗析池中重复所述电渗析的步骤。 在某些实施方式中, 所述电渗析处理在多于一个的串联的电渗析池中进行。 例如,将浓缩室清液泵入二级、三级、四级、甚至更多级的电渗析装置的淡化室, 重复本发明所述方法的步骤 b), 从而提高分离效率。在某些实施方式中, 不同电 渗析池之间的浓缩室、 淡化室的压力相同。在某些实施方式中, 不同电渗析池之 间的浓缩室、 淡化室的压力不同。在某些实施方式中, 不同电渗析池之间的电压 相同。 在某些实施方式中, 不同电渗析池之间的电压不同。 使用本发明的方法拆分得到的光学异构体的纯度大于 90%, 例如, 大于
91%、 大于 92%、 大于 93%、 大于 94%、 大于 95%、 大于 96%、 大于 97%、 大 于 98%、 大于 99%、 甚至为 100%。 在某些实施方式中, 使用本发明的方法拆 分得到的光学异构体的纯度用 ee值表示, 本领域技术人员可以根据本领域的常 规技术手段 (例如, HPLC法) 测量或计算 ee值, 例如, 某外消旋体包含 A、 B两个光学异构体, 则 ee值=八% - B%。 本发明与现有技术相比至少具有以下优点:
1. 本发明的优点之一在于采用生物催化(例如, 酶催化)技术与电渗析技术 相结合, 利用酶催化产生的产物电离程度不同, 使用电渗析技术来拆分外 消旋体中的光学异构体, 反应条件温和, 减少了操作步骤;
2. 以电渗析技术代替传统有机溶剂萃取等常规提取手段,大大减少了有机溶 剂的使用量, 降低了生产成本, 减少了环境污染;
3. 提高了产物的提取率, 产品纯度好, 可直接应用, 无需进一步精制, 减少 了工序, 更有成本优势;
4. 工艺简单易行,便于自动化操作,提高运行安全指数,改善工人工作环境。 具体实脑式
下面结合具体实施例对本发明做进一步描述,但本发明的保护范围不限于此。 实施例一
Figure imgf000011_0001
1. 酶转化液的制备: 2 L体系, 加入 600 g消旋泛解酸内酯, 加入含有 D- 泛解酸内酯水解酶的固定化细胞 300 g, pH7.0&30°C, 200rpm机械搅 拌, 15N NH3fl20滴定维持 pH值为 7.0, 反应 3 h;
2. 酶转化液的预处理: 先用滤布过滤, 然后用 0.2|am的微滤膜过滤, 再用
50KD的超滤膜过滤;
3. 电渗析分离: 使用均相膜膜堆 B(尺寸: 10*30cm; 膜对数: 5对), 将超 滤液清液泵入电渗析淡化室,浓缩室放入 2L纯水,调节流量使三室压力 相等, 恒压 10V运行, 直至淡化室电导率<10(Vs/cm; 将浓缩室清液泵入二级电渗析装置的淡化室,浓缩室放入 2L纯水,调节 流量使三室压力相等, 恒压 10V运行, 直至淡化室电导率 < 10(Vs/cm;
4. 浓缩酸化: 将电渗析浓缩室清液打入浓缩设备减压浓缩至约 400ml, 加 硫酸至 pHl左右使其内酯化;
5. 析晶: 浓缩后将酸化液上层移出, 得到 D-泛解酸内酯 259.2 g, 收率为 43.2%(对 DL-泛解酸内酯计), HPLC测定 D-泛解酸内酯的 ee值为 98.9%。 实施例二
Figure imgf000012_0001
DI^-pantokctoBe L-panto3actone D-pantoic acid
1. 酶转化液的制备: 3 L体系, 加入 900 g消旋泛解酸内酯, 加入含有 D- 泛解酸内酯水解酶的细胞 90 g, pH7.0&30°C, 200rpm机械搅拌, 15N
NH3 H2O滴定维持 pH值为 7.0, 反应 5 h;
2. 酶转化液的预处理:先用蝶式离心机离心,然后用 0.4jjm的微滤膜过滤 再用 20KD的超滤膜过滤;
3. 电渗析分离: 使用异相膜膜堆 Z(尺寸: 10*20cm; 膜对数: 10对), 将 超滤液清液泵入电渗析淡化室,浓缩室放入 3L纯水,调节流量使浓缩室 压力是淡化室的 3倍, 恒压 25V运行, 直至淡化室电导率 < 10(Vs/cm; 将浓缩室清液泵入二级电渗析装置的淡化室,浓缩室放入 3L纯水,调节 流量使浓缩室压力是淡化室的 3倍, 恒压 25V运行, 直至淡化室电导率
< 100|as/cm; 将浓缩室清液泵入三级电渗析装置的淡化室,浓缩室放入 3L纯水,调节 流量使浓缩室压力是淡化室的 3倍, 恒压 25V运行, 直至淡化室电导率
< 100|as/cm; 将浓缩室清液泵入四级电渗析装置的淡化室,浓缩室放入 3L纯水,调节 流量使浓缩室压力是淡化室的 3倍, 恒压 25V运行, 直至淡化室电导率
<100|as/cm;
4. 浓缩酸化: 将电渗析浓缩室清液打入浓缩设备减压浓缩至约 500ml, 加 硫酸至 pHl左右使其内酯化;
5. 析晶: 浓缩后将酸化液上层移出, 得到 D-泛解酸内酯 364.5 g, 收率为 40.5% (对 DL-泛解酸内酯计), HPLC测定 D-泛解酸内酯的 ee值为 97.6%。 实施例三
Figure imgf000013_0001
3-cyclohexene- 1-carboxylate (S)-3-cyclohexene- 1 -carboxylate
(R)-3-cyclohexene-l -carboxylic acid
1. 酶转化液的制备: 1 L体系, 加入 20 ml 3 -环己稀 -1-甲酸甲酯, 加入 10 g Novozyme 435脂肪酶, pH7.5 & 35°C, 200rpm机械搅拌, IN NaOH滴 定维持 pH值为 7.5, 反应 5 h;
2. 酶转化液的预处理: 先用滤纸过滤, 然后用 0.4Mm的微滤膜过滤;
3. 电渗析分离: 使用均相膜膜堆 S (尺寸: 10*20cm; 膜对数: 10对), 将 超滤液清液泵入电渗析淡化室, 浓缩室放入 1 L纯水, 调节流量使三室 压力相等, 恒压 14V运行, 直至淡化室电导率降至<100|as/cm; 将浓缩室清液泵入二级电渗析装置的淡化室, 浓缩室放入 1 L纯水, 调 节流量使三室压力相等,恒压 14V运行,直至淡化室电导率< 10(Vs/cm; 将浓缩室清液泵入三级电渗析装置的淡化室, 浓缩室放入 1 L纯水, 调 节流量使三室压力相等,恒压 14V运行,直至淡化室电导率< 10(Vs/cm; 4. 浓缩室清液处理: 将电渗析浓缩室清液打入浓缩设备减压浓缩至约
350ml, 加硫酸至 pH5左右, 加等体积乙酸乙酯萃取, 收集有机相, 减压 蒸馏, 得到 (R ) -3 -环己稀 -1-甲酸, ee值>99%, 收率约为 25.2%;
5. 淡化室清液处理: 将三级淡化室清液合并, 打入浓缩设备减压浓缩至约 400ml, 加等体积乙酸乙酯萃取, 牧集有机相 减压蒸馏 得到 (S) ! 环己烯 -1-甲酸甲酯, ee值为 74.1%, 收率约为 41.2%。

Claims

权 利 要 求 书
1. 一种通过电渗析从外消旋体中拆分光学异构体的方法, 包括: a) 将外消旋体在催化剂的存在下反应形成包括第一光学异构体的可电离形 式和第二光学异构体的非电离形式的混合物; b) 将所述混合物进行电渗析处理, 以允许所述第一光学异构体的可电离形 式和所述第二光学异构体的非电离形式得以分离; 和 c) 收集分离的所述第一光学异构体的可电离形式, 和 /或收集分离的所述第 二光学异构体的非电离形式。
2. 如权利要求 1所述的方法, 其中所述外消旋体具有可水解的官能团。
3. 如权利要求 2所述的方法, 其中所述官能团被水解后生成可电离的基团。
4. 如权利要求 2-3任一所述的方法, 其中所述催化剂特异性水解所述第一光学 异构体中的所述可水解的官能团, 由此生成所述第一光学异构体的可电离形 式。
5. 如权利要求 1-4任一所述的方法, 其中所述外消旋体具有环结构, 并且所述 可水解的官能团在所述环结构中。
6. 如权利要求 5任一所述的方法, 其中所述环结构包含内酯和内酰胺。
7. 如权利要求 5-6任一所述的方法, 其中在所述第一光学异构体的可电离形式 中所述环结构是开环的。
8. 如权利要求 1-7任一所述的方法, 其中所述催化剂包含酶组合物。
9. 如权利要求 8所述的方法, 其中所述酶组合物含有酯水解酶和 /或内酰胺酶。
10.如权利要求 8-9任一所述的方法, 其中所述酶组合物含有纯化的酶、 表达酶 的细胞、 或表达酶的细胞裂解物。
11.如权利要求 8-10任一所述的方法, 其中所述酶组合物是固定在基底上的。
12.如权利要求 1-11任一所述的方法, 其中在所述步骤 a) 以后和步骤 b) 之前 进一步包括: 去除所述混合物中所述催化剂的残留物。
13.如权利要求 1-12 任一所述的方法, 进一步包括纯化和 /或浓缩所述分离的第 一光学异构体的可电离形式,和 /或纯化和 /或浓缩所述分离的第二光学异构体 的非电离形式。
14.如权利要求 1-13任一所述的方法,进一步包括将所述第二光学异构体的非电 离形式转化成所述外消旋体。
15.如权利要求 1-14任一所述的方法,其中所述电渗析处理是在电渗析池中进行 的, 其中所述电渗析池具有以离子交换膜分隔的淡化室与浓缩室。
16.如权利要求 15所述的方法,其中所述电渗析处理包括将所述混合物置于所述 淡化室, 溶剂置于所述浓缩室, 通过对所述电渗析池通电, 使得所述淡化室 中的所述第一光学异构体的可电离形式迀移到所述浓缩室的所述溶剂中。
17.如权利要求 16所述的方法, 其中所述溶剂包括纯水。
18.如权利要求 15-17任一所述的方法,其中所述离子交换膜是均相膜或异相膜。
19.如权利要求 15-18任一所述的方法, 其中所述电渗析处理是在一个电渗析池 中进行, 或者在多于一个的串联的电渗析池中进行。
20.如权利要求 1-19任一所述的方法, 其中所述外消旋体是 DL-泛解酸内酯, 所 述第一光学异构体的可电离形式是 D-泛解酸, 和所述第二光学异构体的非电 离形式是 L-泛解酸内酯。
21.如权利要求 1-19任一所述的方法, 其中所述外消旋体是 3 -环己烯 -1-甲酸甲 酯, 所述第一光学异构体的可电离形式是 (R) -3 -环己烯 -1-甲酸, 和所述第 二光学异构体的非电离形式是 (S) .3 -环己烯 -1-甲酸甲酯。
22.如权利要求 1-19任一所述的方法,其中所述外消旋体是 a-羟基 -Y-丁内酯,所 述第一光学异构体的可电离形式是 (R) -a-羟基 -Y-丁酸, 和所述第二光学异 构体的非电离形式是 (S) -a-羟基 t丁内酯。
23.如权利要求 1-19任一所述的方法, 其中所述外消旋体是 P-羟基 -Y-丁内酯,所 述第一光学异构体的可电离形式是 (R) -P-羟基- Y-丁酸, 和所述第二光学异 构体的非电离形式是 (S) -p-轻基 -Y-丁内酯。
24.如权利要求 1-19任一所述的方法, 其中所述外消旋体是 a-乙酰基 -Y-丁内酯, 所述第一光学异构体的可电离形式是 (R) -a-乙酰基 -Y-丁酸, 和所述第二光 学异构体的非电离形式是 (S) -a-乙酰基 t丁内酯。
25.如权利要求 1-24任一所述的方法, 其中所述的催化剂是 D-泛解酸内酯水解 酶、 Novozyme 435脂肪酶、 a-轻基 -y-丁内酯水解酶、 P_轻基 -y-丁内酯水解酶 或 a-乙酰基 -Y-丁内酯水解酶。
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