Methods for separating optical isomers using electrodialysis

Abstract

This invention provides a method for separating optical isomers from racemic mixtures via electrodialysis. Specifically, this invention applies electrodialysis technology to enzyme resolution processes, primarily for the separation of products after enzyme resolution. Taking the preparation process of D-pantolactone as an example, the key point is to separate D-pantolactone and L-pantolactone from the enzyme resolution solution using electrodialysis, replacing existing organic solvent extraction methods. The process is simple and easy to implement, yields high-purity D-pantolactone, significantly reduces the amount of organic solvent used, lowers production costs, is environmentally friendly, and can greatly improve the working environment and operational safety. The method provided by this invention has great application potential in enzyme resolution processes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the separation of optical isomers from racemic mixtures using biocatalysis and electrodialysis techniques. Background Technology

[0002] Chirality is an essential property of nature, and many biological macromolecules and bioactive substances possess chiral characteristics. Although two or more different configurations of a chiral substance may have identical chemical compositions, their physiological activities often differ. Usually, only one configuration possesses the desired activity, while other configurations have little or no effect, or may even have toxic side effects. For example, pantothenic acid, also known as dextrorotatory pantothenic acid, is a B vitamin and a component of coenzyme A. It participates in the metabolism of proteins, fats, and carbohydrates, playing an important role in metabolism. Its active ingredient is the D-configuration of dextrorotatory pantothenic acid (vitamin B5), but because pantothenic acid is unstable, its commercial form is mainly calcium D-pantothenate.

[0003] Resolution is one of the main methods for obtaining optically pure chiral compounds. Compared with traditional chemical resolution methods, enzymatic resolution does not require expensive resolving reagents, has mild reaction conditions, good optical selectivity, is environmentally friendly, and can also perform some reactions that are impossible with chemical methods. Due to its significant advantages, enzymatic resolution is increasingly favored by researchers worldwide, and there are already many successful industrial-scale applications.

[0004] For example, D-pantolactone is an important chiral intermediate for the production of pantothenic acid series products such as D-calcium pantothenate, D-panthenol, and D-panthioethylamine. Currently, the industrial synthesis of D-pantolactone mostly employs a combination of chemical and enzymatic hydrolysis methods. Specifically, racemic DL-pantolactone is produced chemically, then hydrolyzed using D-pantolactone hydrolase. The supernatant from the hydrolysis reaction is first extracted with an organic solvent to remove L-pantolactone and unreacted D-pantolactone. The aqueous phase (containing D-pantolactone) is then acidified and extracted again with an organic solvent, followed by desalting, decolorization, and purification by recrystallization. For example, in CN1313402A, DL-pantolactone is separated using free or immobilized cells, then extracted with dichloromethane, acidified with hydrochloric acid in the aqueous phase, and extracted again with dichloromethane. After solvent recovery, the crude D-pantolactone is recrystallized in acetone / isopropyl ether to obtain qualified D-pantolactone. This process has areas for improvement. For instance, the extraction and purification of D-pantolactone obtained from the enzymatic reaction requires a large amount of organic solvent, leading to environmental and cost issues. Furthermore, the crude D-pantolactone requires recrystallization for purification, resulting in low yield and high cost.

[0005] Electrodialysis is an electrochemical separation process that uses ion exchange membranes and a direct current electric field to separate electrolyte components from aqueous solutions. Summary of the Invention

[0006] This invention provides a novel method for resolving optical isomers. This method overcomes the shortcomings of existing chiral resolution processes by utilizing the different ionizability of optical isomers in the chiral resolution products and replacing traditional organic solvent extraction processes with electrodialysis technology, thereby improving product yield and quality while reducing production costs.

[0007] This invention provides a method for separating optical isomers from racemates via electrodialysis, comprising:

[0008] a) Reacting the racemic mixture in the presence of a catalyst to form a mixture comprising an ionizable form of the first optical isomer and a non-ionizable form of the second optical isomer;

[0009] b) subjecting the mixture to electrodialysis to allow separation of the ionizable form of the first optical isomer and the non-ionizable form of the second optical isomer; and

[0010] c) Collect the ionizable form of the separated first optical isomer and / or collect the non-ionizable form of the separated second optical isomer.

[0011] In this application, "racemate" refers to a mixture of two or more optical isomers with different optical properties. For example, a compound having a chiral center may have two optical isomers, one with an R-configuration chiral center and the other with an S-configuration chiral center. For this compound, its racemate includes both the R-configuration and the S-configuration optical isomers. In the racemates described in this application, the different optical isomers may exist in equal molar amounts (i.e., optical rotation cancels out) or in unequal molar amounts.

[0012] In some embodiments, the racemic mixture has a hydrolyzable functional group. Hydrolyzable functional groups include, but are not limited to, ester bonds and amide bonds. In some embodiments, the functional group, upon hydrolysis, can generate an ionizable group. An ionizable group refers to a group that ionizes in aqueous solution, such as a carboxyl group or an amino group. After ionization, the ionizable group generates a charged group, such as a negatively charged carboxylate ion or a positively charged ammonium ion. In some embodiments, the chiral center in the racemic mixture may be located within the hydrolyzable functional group, or it may be located near the hydrolyzable functional group, for example, on an atom adjacent to the hydrolyzable functional group, or at a position separated by one, two, or three atoms.

[0013] In the method of this invention, the catalyst can specifically react with a particular optical isomer in the racemate (e.g., by hydrolyzing hydrolyzable functional groups therein) to make it ionizable. "Ionizable form" in this application refers to a form that ionizes in aqueous solution to form a charged group. In some embodiments, the ionizable form may contain ionizable groups, such as carboxyl, amino, etc. In some embodiments, the catalyst may not catalyze the second optical isomer in the racemate, thus maintaining its non-ionizable form. "Non-ionizable form" in this application refers to a form that does not ionize in aqueous solution and does not have a charged group. In some embodiments, the non-ionizable form contains non-ionizable groups, such as esters (e.g., lactones in a racemate), amides, ethers, etc.

[0014] In some embodiments, the racemate has a ring structure, and the hydrolyzable functional group may be within the ring structure. Exemplary ring structures include, for example, lactones and lactams. These intracyclic functional groups can react to open the ring. In some embodiments, the ring structure is closed in the non-ionized form of the second optical isomer. In some embodiments, the ring structure is open in the ionizable form of the first optical isomer. For example, an ionizable group is formed after the intracyclic functional group undergoes a ring-opening reaction. Alternatively, in some embodiments, the ring structure is open in the non-ionized form of the second optical isomer, and / or closed in the ionizable form of the first optical isomer. In a racemate having a ring structure, the chiral center may or may not be on a ring atom.

[0015] In some embodiments, the racemic mixture is an ester. Exemplary racemic esters include methyl 3-cyclohexene-1-carboxylate. In some embodiments, the racemic mixture is a lactone. A lactone is a molecular structure having an intramolecular ester bond (-C(O)O) formed by the dehydration of a carboxyl and a hydroxyl group. Typically, the intramolecular ester bond is in a ring structure. Examples of lactones include DL racemic pantothenic acid lactone, β-butyrolactone, γ-butyrolactone, α-hydroxy-γ-butyrolactone, β-hydroxy-γ-butyrolactone, α-acetyl-γ-butyrolactone, n-butylphthalide, etc.

[0016] In some embodiments, the catalyst comprises an enzyme composition. In some embodiments, the enzyme composition contains an enzyme capable of reacting specifically with a certain optical isomer. For example, specifically reacting with a D-configuration optical isomer or specifically reacting with an L-configuration optical isomer. In some embodiments, the enzyme composition contains an ester hydrolase. In some embodiments, the ester hydrolase specifically catalyzes D-configuration lactones. Exemplary ester hydrolases include, for example, D-pantolactone hydrolase, Novozyme 435 lipase, β-butyrolactone hydrolase, γ-butyrolactone hydrolase, α-hydroxy-γ-butyrolactone hydrolase, β-hydroxy-γ-butyrolactone hydrolase, α-acetyl-γ-butyrolactone hydrolase, n-butylphthalide hydrolase, etc. For example, D-pantolactone hydrolase can specifically hydrolyze the D-configuration of pantolactones in a racemic mixture, causing the lactone structure to break down into intramolecular carboxyl and hydroxyl groups. The carboxyl groups are ionizable in aqueous solution and therefore can carry a charge, making them ionizable. However, D-pantolactone hydrolase cannot hydrolyze the L-configuration of pantolactones in a racemic mixture. Therefore, the L-configuration of pantolactones retains its lactone structure after the catalytic reaction and remains in a non-ionizable form. As another example, Novozyme 435 lipase can specifically hydrolyze the R-configuration of methyl 3-cyclohexene-1-carboxylate in a racemic mixture, forming 3-cyclohexene-1-carboxylic acid, which is ionizable in aqueous solution. The S-configuration of methyl 3-cyclohexene-1-carboxylate cannot be hydrolyzed and therefore remains in a non-ionizable form.

[0017] In some embodiments, the enzyme composition contains a lactamase. In some embodiments, the lactamase specifically catalyzes D-configuration lactams. Exemplary lactamases include, for example, β-lactamases and γ-lactamases. Taking β-lactamase as an example, it can specifically hydrolyze D-configuration β-lactams in a racemic mixture, causing the lactam structure therein to hydrolyze to form intramolecularly independent carboxyl and amino groups. The carboxyl group therein is ionizable in aqueous solution and therefore can be charged, thus being an ionizable form. However, β-lactamase cannot hydrolyze L-configuration β-lactams in a racemic mixture; therefore, L-configuration β-lactams retain their lactam structure after the catalytic reaction and remain in a non-ionizable form.

[0018] In some embodiments, the racemic form of the present invention is DL-indohydrin lactone, the first optical isomer is D-indohydrin lactone, the second optical isomer is L-indohydrin lactone, the ionizable form of the first optical isomer is D-indohydrin, and the non-ionizable form of the second optical isomer is L-indohydrin lactone.

[0019] In some embodiments, the racemic isomer is methyl 3-cyclohexene-1-carboxylate, the first optical isomer is methyl (R)-3-cyclohexene-1-carboxylate, the second optical isomer is methyl (S)-3-cyclohexene-1-carboxylate, the ionizable form of the first optical isomer is (R)-3-cyclohexene-1-carboxylic acid, and the non-ionizable form of the second optical isomer is methyl (S)-3-cyclohexene-1-carboxylate.

[0020] In some embodiments, the racemic mixture is α-hydroxy-γ-butyrolactone, the first optical isomer is (R)-α-hydroxy-γ-butyrolactone, the second optical isomer is (S)-α-hydroxy-γ-butyrolactone, the ionizable form of the first optical isomer is (R)-α-hydroxy-γ-butyric acid, and the non-ionizable form of the second optical isomer is (S)-α-hydroxy-γ-butyrolactone.

[0021] In some embodiments, the racemic mixture is β-hydroxy-γ-butyrolactone, the first optical isomer is (R)-β-hydroxy-γ-butyrolactone, the second optical isomer is (S)-β-hydroxy-γ-butyrolactone, the ionizable form of the first optical isomer is (R)-β-hydroxy-γ-butyric acid, and the non-ionizable form of the second optical isomer is (S)-β-hydroxy-γ-butyrolactone.

[0022] In some embodiments, the racemic mixture is α-acetyl-γ-butyrolactone, the first optical isomer is (R)-α-acetyl-γ-butyrolactone, the second optical isomer is (S)-α-acetyl-γ-butyrolactone, the ionizable form of the first optical isomer is (R)-α-acetyl-γ-butyric acid, and the non-ionizable form of the second optical isomer is (S)-α-acetyl-γ-butyrolactone.

[0023] Any form of enzyme that selectively catalyzes optical isomers can be used. In some embodiments, the enzyme composition may contain purified enzyme, cells expressing the enzyme, or cell lysates expressing the enzyme. The cells expressing the enzyme can be any suitable host cell, such as prokaryotic cells like bacteria, or eukaryotic cells like yeast, animal cells, etc. The cell lysates can be any lysate component containing the enzyme, such as cell lysate. In some embodiments, the enzyme composition is immobilized on a substrate. Suitable substrates may include materials for immobilizing enzymes, such as magnetic microparticles, macroporous resins, etc.; or materials for immobilizing cells, such as calcium alginate, gels, etc.

[0024] In some embodiments, step a) maintains the pH value within the range of 7.0 to 7.5 during the reaction, for example, the pH value is maintained at any value between any two of the ranges of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or above. In some embodiments, the pH value is maintained by titration with 15N NH3·H2O. In some embodiments, step a) maintains the temperature between 20°C and 40°C during the reaction, for example, at any two of the ranges of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or above. In some implementations, the reaction time of step a) is 1 to 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 of the above ranges.

[0025] In some embodiments, the process after step a) and before step b) further includes removing catalyst residues from the mixture. These residues include macromolecules such as cell debris and proteins. Those skilled in the art can remove these catalyst residues from the mixture using conventional separation methods, such as filtration, centrifugation, microfiltration, ultrafiltration, or a combination thereof, as needed.

[0026] In some embodiments, the filtration is achieved using filter paper or filter cloth. The filter paper or filter cloth described in this invention can be commercially available, such as filter paper or filter cloth manufactured by companies like GE Healthcare Life Sciences, Spice, and Asahi Kasei. In some embodiments, the pore size of the filter paper or filter cloth is 10–150 μm, for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, or any value between any two of these ranges. Those skilled in the art can select an appropriate pore size for the filter paper or filter cloth to remove the catalyst residues based on the type and size of the residues.

[0027] In some embodiments, centrifugation is achieved using a centrifuge. The centrifuge described in this invention can be a commercially available centrifuge, such as those manufactured by Guangzhou Fuyi Liquid Separation Technology Co., Ltd., Yantai Chengbo Machinery Technology Co., Ltd., Dongguan Yaotian Electric Technology Co., Ltd., TEMA System, Kyte, Heinkel, GEA, etc. In some embodiments, the centrifugation rate is 1000 rpm to 2000 rpm, for example, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm, 2000 rpm, or any value between any two of these ranges. In some embodiments, the centrifugation time is 2 to 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 of these ranges. Those skilled in the art can select appropriate centrifugation speed and time to remove the catalyst residues based on the type and size of the residues.

[0028] In some embodiments, the microfiltration is achieved by passing the mixture through a microfiltration membrane. The microfiltration membrane described in this invention can be a commercially available microfiltration membrane, such as the microfiltration hollow fiber membrane series manufactured by GE Healthcare Life Sciences, Spice, Asahi Kasei, etc. In some embodiments, the pore size of the microfiltration membrane is 0.1 μm to 0.6 μm, for example, 0.1 μm, 0.15 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, or any value between any two of the above ranges. Selecting the smallest possible microfiltration membrane pore size, depending on the size of the catalyst residue, facilitates the removal of large particulate residues.

[0029] In some embodiments, ultrafiltration is achieved by passing the mixture through an ultrafiltration membrane. The ultrafiltration membrane described in this invention can be a commercially available ultrafiltration membrane, such as the ultrafiltration hollow fiber membrane series manufactured by GE Healthcare Life Sciences, Spice, Asahi Kasei, etc. In some embodiments, the ultrafiltration membrane is a hollow fiber ultrafiltration membrane with a pore size of 10kD to 500kD, for example, a hollow fiber ultrafiltration membrane with a pore size of 10kD, 20kD, 30kD, 40kD, 50kD, 60kD, 70kD, 80kD, 90kD, 100kD, 150kD, 200kD, 250kD, 300kD, 350kD, 400kD, 450kD, 500kD, or any value between any two of the above ranges. Those skilled in the art can select an appropriate ultrafiltration membrane pore size to remove the catalyst residue based on its size.

[0030] In some embodiments, 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 non-ionizable form of the separated second optical isomer.

[0031] In some embodiments, the ionizable form of the separated first optical isomer and / or the non-ionizable form of the second optical isomer can be further purified. For example, the ionizable form of the first optical isomer and / or the non-ionizable form of the second optical isomer can be extracted with a suitable solvent. For example, an organic solvent (e.g., 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-carboxylic acid. As another example, an organic solvent (e.g., ethyl acetate) can be added to the collected methyl (S)-3-cyclohexene-1-carboxylate, and the organic phase can be collected to obtain purified methyl (S)-3-cyclohexene-1-carboxylate.

[0032] In some embodiments, the ionizable form of the separated and / or purified first optical isomer and / or the non-ionizable form of the second optical isomer can be further concentrated. In some embodiments, the concentration is achieved by depressurization, for example, by pumping the ionizable form of the separated and / or purified first optical isomer and / or the non-ionizable form of the separated and / or purified second optical isomer into a concentration apparatus for depressurized concentration.

[0033] In some embodiments, the invention further includes converting the non-ionized form of the second optical isomer into the racemic mixture. The non-ionized form of the second optical isomer may have been separated by the methods provided in this application, or further purified, or further concentrated. For example, when the racemic mixture is an ester, the separated non-ionized form of the second optical isomer (i.e., the ester) can be racemiced to obtain a racemic mixture with different chiral isomers. By converting the separated second optical isomer back into the racemic mixture, further chiral separation by the methods provided in this application can be performed to obtain more first optical isomers.

[0034] In some embodiments, the invention further includes converting the ionizable form of the separated first optical isomer into a non-ionizable form. In some embodiments, the ionizable form of the separated (and / or purified or concentrated) first optical isomer can be further reacted to restore the ionizable groups therein to hydrolyzable functional groups. For example, in some embodiments, the ionizable form of the separated first optical isomer is a D-pantolytic acid, which can be lactone-treated to give D-pantolytic acid lactone, thereby restoring the ionizable groups (i.e., carboxyl groups) therein to hydrolyzable functional groups (i.e., lactones).

[0035] Those skilled in the art can perform the electrodialysis step in the method of this invention using known methods and equipment. For electrodialysis equipment and methods, please refer to "Industry Standard of the People's Republic of China—Electrodialysis Technology HY / T034.1~034.5--1994".

[0036] In some embodiments, the electrodialysis treatment is carried out in an electrodialysis cell having a desalination chamber and a concentration chamber separated by an ion exchange membrane. In some embodiments, the ion exchange membrane is a homogeneous membrane or a heterogeneous membrane. During the electrodialysis treatment, driven by an applied electric field, anions and cations move toward the anode and cathode, respectively, utilizing the selective permeability of the ion exchange membrane (e.g., cations can pass through a cation exchange membrane, and anions can pass through an anion exchange membrane).

[0037] Those skilled in the art can choose from various ion exchange membranes known in the art for electrodialysis according to their actual needs. In some embodiments, 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 combination of a cation exchange membrane and anion exchange membrane. In some embodiments, the cation exchange membrane allows cations to pass through while repelling and blocking anions. In some embodiments, the anion exchange membrane allows anions to pass through while repelling and blocking cations. In some embodiments, the compartment formed between the cation exchange membrane and the anode, and between the anion exchange membrane and the cathode, is a concentration chamber, and the compartment formed between the cation exchange membrane and the anion exchange membrane is a desalination chamber. In some embodiments, the cation exchange membrane and the anion exchange membrane are commercially available, for example, from Novasep, Eurodia, Shandong Tianwei Membrane Technology Co., Ltd., Zhejiang Qianqiu Environmental Water Treatment Co., Ltd., etc.

[0038] In some embodiments, those skilled in the art can select the membrane stack size of the homogeneous or heterogeneous membrane according to their actual needs, for example, 10*20cm, 10*30cm, 20*30cm, etc. In some embodiments, those skilled in the art can select the number of membrane pairs of the homogeneous or heterogeneous membrane according to their actual needs, for example, 5 pairs, 10 pairs, 15 pairs, 20 pairs, etc.

[0039] In some embodiments, the electrodialysis treatment includes placing the mixture in the desalination chamber, placing the solvent in the concentration chamber, and energizing the electrodialysis cell to cause the ionizable form of the first optical isomer in the desalination chamber to migrate into the solvent in the concentration chamber.

[0040] In some embodiments, the flow rate is adjusted during the electrodialysis process to regulate the pressure in the concentration chamber and the desalination chamber, such that the pressure in the concentration chamber is any value within any two ranges of 1, 2, 3, 4, 5, or more times the pressure in the desalination chamber. In some embodiments, the electrodialysis process is carried out under constant voltage conditions until the conductivity of the desalination chamber is less than 30 μs / cm, 40 μs / cm, 50 μs / cm, 60 μs / cm, 70 μs / cm, 80 μs / cm, 90 μs / cm, 100 μs / cm, 110 μs / cm, 120 μs / cm, 130 μs / cm, 140 μs / cm, 150 μs / cm, etc. In some embodiments, the constant voltage is 10V, 15V, 20V, 25V, 30V, 35V, 40V, 45V, 50V, etc.

[0041] In some embodiments, the solvent includes pure water.

[0042] In some embodiments, the electrodialysis treatment is carried out in an electrodialysis cell. In some embodiments, step b) of the method described in this invention can be repeated in the electrodialysis cell to improve separation efficiency. For example, the clarified liquid from the concentration chamber can be pumped into the desalination chamber of the electrodialysis cell in the electrodialysis apparatus to repeat the electrodialysis steps in that electrodialysis cell.

[0043] In some embodiments, the electrodialysis treatment is carried out in more than one electrodialysis cell connected in series. For example, the supernatant from the concentration chamber is pumped into the desalination chamber of a two-, three-, four-, or even more-stage electrodialysis unit, repeating step b) of the method described in this invention, thereby improving separation efficiency. In some embodiments, the pressure in the concentration and desalination chambers is the same across different electrodialysis cells. In some embodiments, the pressure in the concentration and desalination chambers is different across different electrodialysis cells. In some embodiments, the voltage is the same across different electrodialysis cells. In some embodiments, the voltage is different across different electrodialysis cells.

[0044] The purity of the optical isomers obtained by the method of the present invention is greater than 90%, for example, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even 100%. In some embodiments, the purity of the optical isomers obtained by the method of the present invention is expressed as an ee value. Those skilled in the art can measure or calculate the ee value according to conventional techniques in the art (e.g., HPLC). For example, if a racemic mixture contains two optical isomers A and B, then the ee value = A% - B%.

[0045] Compared with the prior art, the present invention has at least the following advantages:

[0046] 1. One of the advantages of this invention is that it combines biocatalysis (e.g., enzyme catalysis) technology with electrodialysis technology. By utilizing the different degrees of ionization of the products produced by enzyme catalysis, electrodialysis technology is used to separate the optical isomers in the racemic mixture. The reaction conditions are mild and the number of operation steps is reduced.

[0047] 2. Replacing conventional extraction methods such as traditional organic solvent extraction with electrodialysis technology greatly reduces the amount of organic solvents used, lowers production costs, and reduces environmental pollution;

[0048] 3. It improves the extraction rate of the product, has good product purity, and can be used directly without further purification, reducing the number of processes and giving it a cost advantage;

[0049] 4. The process is simple and easy to implement, facilitating automated operation, improving operational safety, and enhancing the working environment for workers. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0051] Example 1

[0052]

[0053] 1. Preparation of enzyme conversion solution: 600g racemic indomethacin lactone was added to a 2L system, along with 300g of immobilized cells containing D-indomethacin lactone hydrolase. The mixture was kept at pH 7.0 and 30℃, mechanically stirred at 200rpm, and titrated with 15N NH3·H2O to maintain the pH at 7.0. The reaction was carried out for 3h.

[0054] 2. Pretreatment of enzyme conversion solution: First filter with filter cloth, then filter with a 0.2μm microfiltration membrane, and then filter with a 50KD ultrafiltration membrane.

[0055] 3. Electrodialysis separation: Using homogeneous membrane stack B (size: 10*30cm; number of membrane pairs: 5 pairs), the supernatant of ultrafiltrate is pumped into the electrodialysis desalination chamber, and 2L of pure water is added to the concentration chamber. The flow rate is adjusted to make the pressure of the three chambers equal, and the constant pressure 10V is maintained until the conductivity of the desalination chamber is <100μs / cm.

[0056] Pump the clear liquid from the concentration chamber into the desalination chamber of the secondary electrodialysis device. Add 2L of pure water to the concentration chamber, adjust the flow rate to make the pressure in the three chambers equal, and run at a constant pressure of 10V until the conductivity of the desalination chamber is <100μs / cm.

[0057] 4. Concentration and acidification: The supernatant from the electrodialysis concentration chamber is pumped into the concentration equipment and concentrated under reduced pressure to about 400 ml. Sulfuric acid is added to bring the pH to about 1 to induce lactone formation.

[0058] 5. Crystallization: After concentration, the upper layer of the acidified solution was removed to obtain 259.2 g of D-indomethacin, with a yield of 43.2% (based on DL-indomethacin). The ee value of D-indomethacin was determined by HPLC to be 98.9%.

[0059] Example 2

[0060]

[0061] 1. Preparation of enzyme conversion solution: 900g racemic indomethacin lactone was added to a 3L system, along with 90g of cells containing D-indomethacin lactone hydrolase. The mixture was kept at pH 7.0 and 30℃, mechanically stirred at 200rpm, and titrated with 15NNH3·H2O to maintain the pH at 7.0. The reaction was carried out for 5h.

[0062] 2. Pretreatment of enzyme conversion solution: First, centrifuge with a disc centrifuge, then filter with a 0.4 μm microfiltration membrane, and then filter with a 20 KD ultrafiltration membrane;

[0063] 3. Electrodialysis separation: Using heterogeneous membrane stack Z (size: 10*20cm; number of membrane pairs: 10 pairs), the supernatant of ultrafiltrate is pumped into the electrodialysis desalination chamber, and 3L of pure water is added to the concentration chamber. The flow rate is adjusted so that the pressure in the concentration chamber is 3 times that in the desalination chamber. The system is operated at a constant pressure of 25V until the conductivity of the desalination chamber is <100μs / cm.

[0064] Pump the clarified liquid from the concentration chamber into the desalination chamber of the secondary electrodialysis unit. Add 3L of pure water to the concentration chamber and adjust the flow rate so that the pressure in the concentration chamber is 3 times that in the desalination chamber. Run the system at a constant pressure of 25V until the conductivity of the desalination chamber is <100μs / cm.

[0065] Pump the clarified liquid from the concentration chamber into the desalination chamber of the three-stage electrodialysis device. Add 3L of pure water to the concentration chamber and adjust the flow rate so that the pressure in the concentration chamber is 3 times that in the desalination chamber. Run the device at a constant pressure of 25V until the conductivity of the desalination chamber is <100μs / cm.

[0066] Pump the clarified liquid from the concentration chamber into the desalination chamber of the four-stage electrodialysis device. Add 3L of pure water to the concentration chamber and adjust the flow rate so that the pressure in the concentration chamber is 3 times that in the desalination chamber. Run the device at a constant pressure of 25V until the conductivity of the desalination chamber is <100μs / cm.

[0067] 4. Concentration and acidification: The supernatant from the electrodialysis concentration chamber is pumped into the concentration equipment and concentrated under reduced pressure to about 500 ml. Sulfuric acid is added to bring the pH to about 1 to induce lactone formation.

[0068] 5. Crystallization: After concentration, the upper layer of the acidified solution was removed to obtain 364.5 g of D-indomethacin, with a yield of 40.5% (based on DL-indomethacin). The ee value of D-indomethacin was determined by HPLC to be 97.6%.

[0069] Example 3

[0070]

[0071] 1. Preparation of enzyme conversion solution: In a 1L system, add 20ml of methyl 3-cyclohexene-1-carboxylate and 10g of Novozyme435 lipase. Set the pH to 7.5 and the temperature to 35℃. Stir mechanically at 200rpm and titrate with 1N NaOH to maintain the pH at 7.5. React for 5 hours.

[0072] 2. Pretreatment of enzyme conversion solution: First filter with filter paper, then filter with a 0.4μm microfiltration membrane;

[0073] 3. Electrodialysis separation: Using a homogeneous membrane stack S (size: 10*20cm; number of membrane pairs: 10 pairs), the supernatant of ultrafiltrate is pumped into the electrodialysis desalination chamber, and 1L of pure water is added to the concentration chamber. The flow rate is adjusted to make the pressure of the three chambers equal, and the operation is carried out at a constant pressure of 14V until the conductivity of the desalination chamber drops to <100μs / cm.

[0074] Pump the clear liquid from the concentration chamber into the desalination chamber of the secondary electrodialysis unit. Add 1L of pure water to the concentration chamber, adjust the flow rate to make the pressure in the three chambers equal, and run at a constant pressure of 14V until the conductivity of the desalination chamber is <100μs / cm.

[0075] Pump the clarified liquid from the concentration chamber into the desalination chamber of the three-stage electrodialysis device. Add 1L of pure water to the concentration chamber, adjust the flow rate to make the pressure in the three chambers equal, and run at a constant pressure of 14V until the conductivity of the desalination chamber is <100μs / cm.

[0076] 4. Treatment of the supernatant from the concentration chamber: The supernatant from the electrodialysis concentration chamber is pumped into a concentration device and concentrated under reduced pressure to about 350 ml. Sulfuric acid is added to adjust the pH to about 5, and an equal volume of ethyl acetate is added for extraction. The organic phase is collected and distilled under reduced pressure to obtain (R)-3-cyclohexene-1-carboxylic acid with an ee value > 99% and a yield of about 25.2%.

[0077] 5. Treatment of desalination chamber supernatant: Combine the supernatants from the three desalination chambers, concentrate them under reduced pressure to about 400 ml in a concentration device, extract with an equal volume of ethyl acetate, collect the organic phase, and distill under reduced pressure to obtain methyl (S)-3-cyclohexene-1-carboxylate with an ee value of 74.1% and a yield of about 41.2%.

Claims

1. A method for separating optical isomers from racemates by electrodialysis, comprising: a) Reacting the racemic mixture in the presence of a catalyst to form a mixture comprising an ionizable form of the first optical isomer and a non-ionizable form of the second optical isomer; b) The mixture is subjected to electrodialysis to allow separation of the ionizable form of the first optical isomer and the non-ionizable form of the second optical isomer, wherein the electrodialysis is performed in an electrodialysis cell having a desalination chamber and a concentration chamber separated by an ion exchange membrane, wherein the ion exchange membrane is a homogeneous membrane. The mixture is placed in the desalination chamber, and pure water is placed in the concentration chamber. By energizing the electrodialysis cell, the ionizable form of the first optical isomer in the desalination chamber migrates to the pure water in the concentration chamber. The electrodialysis is performed under constant voltage until the conductivity of the desalination chamber is less than 100 μS / cm. c) Collect the ionizable form of the separated first optical isomer and / or collect the non-ionizable form of the separated second optical isomer; in, The racemic mixture is DL-pantolactone, the ionizable form of the first optical isomer is D-pantolactone, the non-ionizable form of the second optical isomer is L-pantolactone, and the catalyst is D-pantolactone hydrolase; or The racemic mixture is methyl 3-cyclohexene-1-carboxylate, the ionizable form of the first optical isomer is (R)-3-cyclohexene-1-carboxylic acid, the non-ionizable form of the second optical isomer is (S)-3-cyclohexene-1-carboxylate, and the catalyst is Novozyme 435 lipase.

2. The method of claim 1, wherein after step a) and before step b), it further comprises: Remove any residue of the catalyst from the mixture.

3. The method of claim 1, further comprising purifying and / or concentrating the ionizable form of the separated first optical isomer, and / or purifying and / or concentrating the non-ionizable form of the separated second optical isomer.

4. The method of any one of claims 1-3, further comprising converting the non-ionized form of the second optical isomer into the racemic form.

5. The method of claim 4, wherein the electrodialysis treatment is carried out in one electrodialysis cell or in more than one electrodialysis cell connected in series.

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