CN106497996B - Enzymatic preparation method of chiral alcohol - Google Patents
Enzymatic preparation method of chiral alcohol Download PDFInfo
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Abstract
The invention discloses an enzyme catalysis preparation method of chiral alcohol, which comprises the following steps: the prochiral ketone compound is mixed with the ketoreductase and the coenzyme provided by the invention, and the pH and the temperature of a system are controlled to carry out reduction reaction. The ketoreductase provided by the invention is used for preparing chiral alcohol by asymmetric reduction of prochiral ketone compounds, has high conversion rate and stereoselectivity, the purity of the product is higher than 95.0%, and the ee value can be higher than 99%. The enzymatic preparation method of chiral alcohol provided by the invention has the advantages of simple reaction steps, shortened production period and saved production cost; the reaction condition is mild, the yield is high, the process condition is stable, the reaction is safe, the operation is simple, and the method has the capability of large-scale production; is environment-friendly, and reduces the pollution of organic solvent to the environment.
Description
Technical Field
The invention relates to the technical field of biological catalysis, in particular to an enzymatic preparation method of chiral alcohol, and especially relates to an enzymatic preparation method of cyclic chiral alcohol.
Background
Chiral alcohols are optically active compounds with a hydroxyl group attached to a chiral carbon, and are widely used for the synthesis of chiral drugs and other chiral fine chemicals. The synthesis method of the chiral alcohol mainly comprises a physical separation method, a resolution method and an asymmetric reduction method. Wherein, the chemical resolution is carried out for a plurality of times by using resolving agents such as tartaric acid and the like, the chemical purity of the obtained product can reach more than 99 percent, but the product has the defect of extremely low resolution yield which is only less than 10 percent; in addition, the chiral resolving agent is higher in price, so that the production cost is greatly increased; the operation is more complicated, and the post-treatment does not utilize environmental protection. The method for synthesizing chiral alcohol by asymmetric reduction of ketone compounds is an important method for producing chiral alcohol at present, the theoretical yield of the method can reach 100 percent, and the method comprises a chemical asymmetric reduction method and a biological asymmetric reduction method. The chemical asymmetric reduction method mainly utilizes a chiral metal complex as a catalyst for asymmetric reduction of carbonyl, although the chemical method is partially used for industrial production, the reaction process needs high-pressure hydrogenation, the synthesis of the chiral metal complex is complex and expensive, heavy metal residues exist in the product, the product is difficult to separate, the environmental pollution is large, and the application is limited to a certain extent; the biocatalytic asymmetric reduction method has high chemical, regional and stereoselectivity, mild reaction conditions, avoids heavy metal residues in products, is environment-friendly, makes up for the defects of a chemical method, and is a green, efficient and economic method. The biocatalyst for asymmetric reduction mainly comprises microorganism whole cell and oxidoreductase, for example, patent application CN201510026759.1 discloses a carbonyl reductase derived from Burkholderia gladioli ZJB-12126, and wet thallus obtained by fermentation culture of engineering bacteria containing recombinant carbonyl reductase gene is used as catalyst for asymmetric reduction of prochiral carbonyl compound and other chiral alcohols; for example, patent CN201010599376.0 discloses an oxidoreductase derived from Streptomyces coelicolor A3(2) NRRLB-16638 as carbonyl reductase catalyst for asymmetric reduction of prochiral carbonyl compounds to prepare optically active chiral alcohols; the enzymatic reduction has the advantages of higher selectivity, easy reaction treatment and the like compared with the whole-cell catalytic product.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides an enzyme-catalyzed preparation method of chiral alcohol, which comprises the following steps: mixing prochiral ketone compound with ketoreductase and coenzyme, and carrying out reduction reaction to obtain chiral alcohol;
the reaction route is as follows:
wherein R is1Selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heterocycloalkyl, or R1Form a fused ring system with the heterocycle to which it is attached;
R2selected from: h or an amino protecting group;
n is an integer of 0 to 3;
m is an integer from 0 to 5, and n is not equal to m + 1;
preferably, R1Selected from: H. substituted or unsubstituted straight chain alkyl of C1-C8, substituted or unsubstituted cycloalkyl of C5-C10, substituted or unsubstituted aryl of C6-C12, substituted or unsubstituted heterocyclyl of C5-C12, or R1Form a fused ring system with the attached heterocycle; more preferably, R1Selected from: H. substituted or unsubstituted straight chain alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl of C1-C3, or R1Form a fused ring system with the attached heterocycle;
preferably, the amino protecting group is selected from: ms, Ts, formyl, acetyl, haloacetyl such as Tfa, benzoyl, Boc, Cbz, Alloc, PMB, Bn, Trt, Dmb; more preferably from: boc, Cbz, Bn, acetyl, benzoyl;
preferably, n is an integer of 0 to 2, more preferably 0, 1, 2;
preferably, m is an integer from 0 to 3, more preferably 0, 1, 2, 3;
in a preferred embodiment of the present invention, R is1Is H, n is 1, and m is 1 or 2.
In one embodiment of the present invention, said R2The preparation method also comprises a step of protecting on an amino group.
Preferably, the ketoreductase enzyme has:
a) as set forth in SEQ ID No.: 1 or SEQ ID No.: 2; or the like, or, alternatively,
b) and SEQ ID No.: 1 or SEQ ID No.: 2, and having ketoreductase activity, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% identity;
more preferably, the amino acid sequence of b) can be an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of a) or a mutant thereof.
Preferably, the dosage of the ketoreductase is 0.05-1.0g/g prochiral ketone; more preferably 0.10 to 0.60g/g prochiral ketone;
preferably, the coenzyme is used in an amount of 0.001-0.030g/g prochiral ketone; more preferably 0.001-0.020g/g prochiral ketone;
preferably, the pH of the reduction reaction is 6.0-8.0; more preferably 7.0;
preferably, the temperature of the reduction reaction is 20-50 ℃; more preferably 20 to 40 ℃; further preferably 25 to 35 ℃;
preferably, the reaction time is 10-50 h; more preferably 15-40 h;
preferably, the prochiral ketone compound is uniformly dispersed in a buffer; the pH of the buffer solution is preferably 6.0-8.0, more preferably 7.0; more preferably, the dosage of the buffer solution is 10-100mL/g prochiral ketone; in one embodiment of the present invention, the buffer is a phosphate buffer;
preferably, the reaction mixture also comprises a coenzyme regeneration system;
as known to those skilled in the art, the coenzyme regeneration system is used for regenerating a coenzyme from an oxidized state to a reduced state or from a reduced state to an oxidized state, so as to maintain the coenzyme at a certain catalytic dosage level, and mainly includes regeneration systems such as chemical, photochemical, enzymatic and electrochemical regeneration, wherein enzymatic regeneration is commonly used, such as substrate coupling method and enzyme coupling method. The preparation reaction of the chiral alcohol comprises the following steps:
when a substrate coupling method is adopted, a coenzyme regeneration system comprises an auxiliary substrate, and the enzyme II is the ketoreductase, namely the ketoreductase can catalyze the simultaneous conversion of a target substrate and the auxiliary substrate simultaneously to realize the regeneration of the coenzyme; when an enzyme coupling method is adopted, a coenzyme regeneration system comprises an enzyme II and an auxiliary substrate, the enzyme II is different from the ketoreductase, and the enzyme II catalyzes the auxiliary substrate to convert and simultaneously realizes the coenzyme regeneration.
Preferably, the coenzyme regeneration system comprises an enzyme II and a cosubstrate, wherein the enzyme II is the same as or different from the ketoreductase;
in one embodiment of the invention, the enzyme II, which is different from the ketoreductase described above, is selected from the group consisting of: formate dehydrogenase FDH, glucose dehydrogenase GDH, alcohol dehydrogenase ADH; more preferably FDH or GDH; more preferably, the coenzyme regeneration system is selected from the group consisting of: FDH and formic acid, FDH and ammonium formate, GDH and glucose; preferably, the enzyme II is used in an amount of 0.001 to 0.05g/g prochiral ketone, and/or the cosubstrate is used in an amount of 1 to 5mol/mol prochiral ketone, more preferably 1 to 3mol/mol prochiral ketone;
in a preferred embodiment of the present invention, the coenzyme regeneration system comprises glucose and GDH.
In another embodiment of the invention, the enzyme II is the same as the ketoreductase enzyme described above, i.e., the enzyme II is a ketoreductase enzyme described above, which also catalyzes a reaction of a co-substrate, which is an alcohol, preferably selected from: isopropanol, isobutanol; more preferably isopropanol.
The coenzyme required by the reaction is reduced coenzyme, preferably NADH or NADPH; more preferably NADH;
however, a reduced coenzyme such as NADH is poor in stability and expensive, and in one embodiment of the present invention, an oxidized coenzyme such as NAD + is added to the reaction, and the corresponding reduced coenzyme such as NADH is produced by recycling the oxidized coenzyme such as NAD + by a coenzyme regeneration system such as glucose + glucose dehydrogenase, for use in the above-mentioned production reaction of chiral alcohol.
Preferably, the method further comprises the steps of separation and purification: after the reaction is finished, filtering the system by using diatomite, extracting by using an organic solvent, drying an organic phase, filtering, concentrating and purifying to obtain a chiral alcohol product;
preferably, the dosage of the diatomite is 5-50g/g prochiral ketone;
preferably, the organic solvent is an organic solvent commonly used in the extraction in the field, the type of the organic solvent is not limited in the invention, and the amount of the organic solvent is 10-80mL/g prochiral ketone;
preferably, the purification is performed by column chromatography, and more preferably, the amount of silica gel used in the column chromatography is 5-20g/g prochiral ketone, the eluent is preferably ethyl acetate/petroleum ether-1: 20, and the amount of the eluent is preferably 10-80mL/g prochiral ketone.
The invention also provides an application of the preparation method in chemical synthesis.
The ketoreductase provided by the invention is screened from more than 200 ketoreductases and alcohol dehydrogenase, and has higher conversion rate and stereoselectivity when being used for preparing chiral alcohol by asymmetric reduction of prochiral ketone compounds, the purity of the product is higher than 95.0%, and the ee value can be higher than 99%. The enzymatic preparation method of chiral alcohol provided by the invention has the advantages that the reaction steps are simple, the cyclic chiral alcohol compound with a single configuration is obtained in one step, the overall yield is improved, the production period is shortened, the production cost is saved, the reaction conditions are mild, the yield is high, the process conditions are stable, the reaction is safe, the operation is simple, the capacity of large-scale production is realized, the method is environment-friendly, and the pollution of an organic solvent to the environment is reduced.
Detailed Description
Interpretation of terms
In the present invention, Ms is methanesulfonyl
Ts is p-toluenesulfonyl
Boc is tert-butyloxycarbonyl
Cbz is benzyloxycarbonyl
Alloc is allyloxycarbonyl
PMB is p-methoxybenzyl
Bn is benzyl
Tfa is trifluoroacetyl group
Trt is trityl
Dmb is 2, 4-dimethoxybenzyl
The terms "optionally/any" or "optionally/optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, an "optionally substituted alkyl" refers to an "unsubstituted alkyl" (alkyl unsubstituted by a substituent) or a "substituted alkyl" (alkyl substituted by a substituent), as defined below.
As used herein, C1-Cn includes C1-C2, C1-C3, C1-Cn. For example, the "C1-C4" group refers to a moiety having 1-4 carbon atoms, i.e., the group contains 1, 2, 3, or 4 carbon atoms.
The term "alkyl" as used herein, alone or in combination, refers to an optionally substituted straight chain or optionally substituted branched chain aliphatic hydrocarbon. The "alkyl" groups herein may preferably have from 1 to 20 carbon atoms, for example from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms, or from 1 to 4 carbon atoms or from 1 to 3 carbon atoms. The term "alkoxy" as used herein, alone or in combination, refers to an alkyl ether group (O-alkyl), non-limiting examples of which include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.
The terms "halo" or "halogen substituted", as used herein, alone or in combination, refer to an optionally substituted group (e.g., alkyl, alkenyl, and alkynyl) wherein one or more hydrogen atoms are replaced with a fluorine, chlorine, bromine, iodine atom, or combinations thereof.
The term "aryl/aryl" as used herein, alone or in combination, refers to an optionally substituted aromatic hydrocarbon group having from 6 to 20, such as 6-12 or 6-10 ring-forming carbon atoms. It may be a fused aromatic ring or a non-fused aromatic ring.
The term "heteroaryl" as used herein, alone or in combination, refers to an optionally substituted monovalent heteroaryl group comprising from 5 to 20, such as from 5 to 12 or from 5 to 10, backbone ring-forming atoms, wherein one or more (e.g., 1-4, 1-3, 1-2) ring-forming atoms are heteroatoms independently selected from the group consisting of heteroatoms of oxygen, nitrogen, sulfur, phosphorus, silicon, selenium and tin, but is not limited thereto. The ring of the group does not contain two adjacent O or S atoms. Heteroaryl includes monocyclic heteroaryl or polycyclic heteroaryl (e.g., bicyclic heteroaryl, tricyclic heteroaryl, etc.).
The term "heterocycle" or "heterocyclyl" as used herein, alone or in combination, refers to non-aromatic heterocycles, including heterocycloalkyl and heterocycloalkenyl. Wherein one or more (e.g., 1-4, 1-3, 1-2) ring-forming atoms are heteroatoms, such as oxygen, nitrogen or sulfur atoms. The heterocyclic group may include a monocyclic heterocyclic group (heterocyclic group has one ring) or a polycyclic heterocyclic group (for example, a bicyclic heterocyclic group (heterocyclic group has two rings), a tricyclic heterocyclic group, etc.).
The term "carbocyclyl" as used herein, alone or in combination, refers to non-aromatic carbocyclic rings including cycloalkyl and cycloalkenyl groups. The cycloalkyl group can be a monocyclic cycloalkyl group or a polycyclic cycloalkyl group (e.g., having 2, 3, or 4 rings; e.g., bicyclic cycloalkyl), which can be spiro or bridged. Carbocyclyl groups may have 3 to 20 carbon atoms, for example 3-15 ring-forming carbon atoms or 3-10 ring-forming carbon atoms or 3-6 ring-forming carbon atoms, and may have 0, 1, 2 or 3 double bonds and or 0, 1 or 2 triple bonds. For example cycloalkyl having 3 to 8 or 3 to 6 ring-forming carbon atoms.
"halogen" means fluorine, chlorine, bromine, iodine, preferably fluorine, chlorine and bromine. Cyano means "-CN"; hydroxyl refers to "-OH"; mercapto means "-SH"; amino means-NH2”。
The term "substituted" means that one or more hydrogens on a given atom are replaced with the indicated group, and that the substitution results in a stable compound if the normal valency of the indicated atom is not exceeded under the circumstances at hand.
The modified products, functional equivalents, functional fragments or variants thereof obtained by using the ketoreductase with high stereoselectivity of the invention can be used for asymmetric reduction synthesis of alcohol compounds with higher chiral purity, and are also within the protection scope of the invention.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Feeding: to a 5L reaction vessel, 125g of N-benzyloxycarbonyl-3-pyrrolidone as a main raw material, 2.25L of a phosphate buffer (100mmol/L, pH 7.0) and N-benzyloxycarbonyl-3-pyrrolidone uniformly dispersed in the phosphate buffer were added;
(2) ketoreductase addition: to a 5L reactor, 2g of coenzyme NAD was added+290g of glucose, 25g of the ketoreductase master enzyme GcKR and 0.125g of the glucose dehydrogenase GDH, the system pH being 7.0;
(3) reaction: reacting the system at 25 ℃, and stirring for reacting for 36 hours;
(4) and (3) post-treatment: filtering with 1kg diatomaceous earth, extracting with 2L ethyl acetate, standing, separating, drying organic phase, filtering, concentrating to obtain crude product, purifying by column chromatography, eluting with 500g silica gel, 2L eluent ethyl acetate and petroleum ether at a ratio of 1:20, purifying to obtain 106.16g product with high purity, purity of 95.37%, yield of 81%, ee value of 99.2%, MS (M + H)+＝222.10。
Example 2
(1) Feeding: to a 500mL Erlenmeyer flask, 12.5g of N-benzyloxycarbonyl-3-pyrrolidone as a main raw material, 225mL of a phosphate buffer (100mmol/L, pH 7.0) and N-benzyloxycarbonyl-3-pyrrolidone uniformly dispersed in the phosphate buffer were added;
(2) Ketoreductase addition: to a 500mL Erlenmeyer flask, 0.2g coenzyme NAD was added+29g of glucose, 2.5g of the ketoreductase master enzyme KRED (from the sources indicated in the table below) and 12.5mg of glucose dehydrogenase GDH, system pH 7.0;
(3) reaction: reacting the system at 30 ℃, and stirring for reacting for 36 hours;
(4) and (3) post-treatment: the system was filtered through 100g of celite, extracted with 200mL of ethyl acetate, and the resulting mixture was allowed to stand for liquid separation and gas phase detection was performed.
As shown in Table 1, the reaction system using the ketoreductase had a large amount of remaining raw materials, no product was produced, and the conversion rates were all 0, indicating that not all ketoreductases were suitable for the chiral alcohol production reaction of the present invention.
TABLE 1 sources of ketoreductase and conversion results
Example 3
(1) Feeding: to a 200L reaction vessel, 2kg of a main raw material, N-tert-butoxycarbonyl-3-piperidone, and 120L of a phosphate buffer (100mM, pH 7.0) were added, and N-tert-butoxycarbonyl-3-piperidone was uniformly dispersed in the phosphate buffer;
(2) ketoreductase addition: to a 200L reactor, 15.0g of coenzyme NAD was added+2.2kg of glucose, 1kg of the ketoreductase master enzyme ZrKRs and 0.02kg of the glucose dehydrogenase GDH, with a system pH of 7.0;
(3) reaction: reacting the system at 30 ℃, and stirring for 24 hours;
(4) and (3) post-treatment: filtering with 60kg diatomaceous earth, extracting with 120L ethyl acetate, standing, separating, drying organic phase, filtering, concentrating to obtain crude product, purifying by column chromatography, eluting with 20kg silica gel, 100L eluent ethyl acetate and petroleum ether at a ratio of 1:20, purifying to obtain 1.726kg product with high purity, purity of 95.0%, yield of 82%, ee value of 99.5%，MS(M+H)+＝201.14。
Example 4
(1) Feeding: to a 250ml Erlenmeyer flask, 2g of N-t-butoxycarbonyl-3-piperidone as a main raw material and 6ml of isopropanol, 40ml of a phosphate buffer (50mmol/L, pH 7.0) were added to uniformly disperse the substrate in the buffer;
(2) ketoreductase addition: 0.01g of coenzyme NAD was continuously added to the Erlenmeyer flask+0.3g ketoreductase GcKR, system pH 7.0;
(3) reaction: reacting the system in a shaking table at 200rpm, and preserving the temperature for 20 hours at 30 +/-3 ℃;
(4) and (3) post-treatment: the system is filtered by 40g of diatomite, 60ml of dichloromethane is used for extraction, standing and liquid separation are carried out, an organic phase is dried, filtered and concentrated to obtain a crude product, and then, column chromatography purification is carried out to obtain 1.82g of a product (S) -1-tert-butyloxycarbonyl-3-hydroxypiperidine with high purity, wherein the purity is 93.26%, the yield is 84.86%, the ee value is more than 99%, and MS (M + H) + 201.14.
Therefore, the ketoreductase disclosed by the invention can reduce ketone compounds to obtain chiral alcohol products with higher ee values, the ee value can reach 95-99.5%, the yield is higher than 80%, the synthesis method adopts stable process conditions, the reaction conditions are mild, the operation is simple in the whole production process, the pollution is low, and a new thought and method are provided for obtaining cyclic chiral alcohol by asymmetrically reducing ketone compounds.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (9)
1. A method for the enzymatic preparation of a chiral alcohol, the method comprising: mixing prochiral ketone compound with ketoreductase and coenzyme, and carrying out reduction reaction to obtain chiral alcohol;
the reaction route is as follows:
wherein R is1Is H;
R2selected from: h or an amino protecting group;
n is 1;
m is 1 or 2;
the ketoreductase has a sequence as set forth in SEQ ID No.: 1 or SEQ ID No.: 2;
wherein when the ketoreductase is SEQ ID No.: 1, the prochiral ketone compound is N-benzyloxycarbonyl-3-pyrrolidone or N-tert-butoxycarbonyl-3-piperidone; when the ketoreductase is SEQ ID No.: 2, the prochiral ketone compound is N-tert-butoxycarbonyl-3-piperidone.
2. The method of claim 1, wherein the amino protecting group is selected from the group consisting of: methylsulfonyl, p-toluenesulfonyl, formyl, acetyl, haloacetyl, benzoyl, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, p-methoxybenzyl, benzyl, trityl and 2, 4-dimethoxybenzyl.
3. The method according to claim 2, wherein the haloacetyl group is a trifluoroacetyl group.
4. The method of claim 1, wherein R is2The preparation method also comprises a step of protecting on an amino group.
5. The process according to any one of claims 1 to 4, wherein the ketoreductase is used in an amount of 0.05 to 1.0g/g prochiral ketone; and/or the presence of a gas in the gas,
the dosage of the coenzyme is 0.001-0.030g/g prochiral ketone; and/or the presence of a gas in the gas,
the pH value of the reduction reaction is 6.0-8.0; and/or the presence of a gas in the gas,
the temperature of the reduction reaction is 20-50 ℃; and/or the presence of a gas in the gas,
the reaction time is 10-50 h; and/or the presence of a gas in the gas,
the prochiral ketone compound is uniformly dispersed in a buffer solution; and/or the presence of a gas in the gas,
the reaction mixture also comprises a coenzyme regeneration system.
6. A process according to any one of claims 1 to 4 wherein the coenzyme regeneration system comprises an enzyme II and a co-substrate, the enzyme II being the same as or different from the ketoreductase enzyme.
7. The process according to claim 6, wherein the enzyme II is different from the ketoreductase and is selected from the group consisting of: formate dehydrogenase FDH, glucose dehydrogenase GDH, alcohol dehydrogenase ADH; and/or the presence of a gas in the gas,
the dosage of the enzyme II is 0.001-0.05g/g prochiral ketone; and/or the presence of a gas in the gas,
the dosage of the auxiliary substrate is 1-5mol/mol prochiral ketone; or the like, or, alternatively,
the enzyme II is the ketoreductase, and the auxiliary substrate is alcohol.
8. The method according to claim 6, wherein the coenzyme regeneration system comprises glucose and GDH, or isopropanol.
9. The method according to claim 6, wherein the coenzyme is a reduced coenzyme; or, adding oxidized coenzyme in the reaction, and circulating the oxidized coenzyme through a coenzyme regeneration system to generate reduced coenzyme.
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| CN107586798A (en) * | 2017-09-27 | 2018-01-16 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (S) 1 N benzene methoxycarbonyl group 3 |
| CN107586793B (en) * | 2017-10-10 | 2020-10-20 | 吉林凯莱英医药化学有限公司 | Method for preparing alcohol compounds having multiple chiral centers |
| CN109825538B (en) * | 2017-11-23 | 2022-05-24 | 中国科学院天津工业生物技术研究所 | Synthesis method of chiral 2-amino-1-butanol |
| CN109371067A (en) * | 2018-11-30 | 2019-02-22 | 上海合全药业股份有限公司 | The method that biocatalysis prepares (R) -4- decyl alcohol |
| CN109797174B (en) * | 2018-12-27 | 2020-12-29 | 弈柯莱生物科技(上海)股份有限公司 | Preparation method of florfenicol intermediate |
| CN110643556A (en) * | 2019-08-23 | 2020-01-03 | 浙江工业大学 | Recombinant genetic engineering bacterium for co-expressing enal reductase and glucose dehydrogenase and application thereof |
| CN111705099B (en) * | 2020-07-01 | 2022-03-08 | 天津药明康德新药开发有限公司 | Preparation method of (S) -1- (3, 5-dichloropyridine-4-substituted) ethanol |
| CN114369592B (en) * | 2021-12-31 | 2024-05-07 | 南通常佑药业科技有限公司 | Pickering emulsion and method for preparing chiral alcohol compound based on emulsion enzyme catalysis |
| CN114317619A (en) * | 2021-12-31 | 2022-04-12 | 山西大学 | Method for preparing chiral alcohol by ketoreductase continuous reaction |
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| CN103571908A (en) * | 2013-11-22 | 2014-02-12 | 尚科生物医药（上海）有限公司 | Method for preparing chiral N-tert-butyloxycarboryl-3-hydroxypiperidine |
| CN104232696B (en) * | 2014-06-25 | 2017-12-26 | 武汉科技大学 | The method that asymmetric reduction prochiral carbonyl compounds produce chiral alcohol |
| CN105906656B (en) * | 2016-05-17 | 2018-01-23 | 凯莱英医药集团（天津）股份有限公司 | A kind of gram of azoles replaces the synthetic method of Buddhist nun's intermediate |
| CN105861457B (en) * | 2016-05-26 | 2019-10-08 | 无锡佰翱得生物科学有限公司 | A kind of alcohol dehydrogenase enzyme mutant and the preparation method and application thereof that enzyme activity improves |
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