[go: up one dir, main page]

CN113816836B - Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol - Google Patents

Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol Download PDF

Info

Publication number
CN113816836B
CN113816836B CN202111151324.1A CN202111151324A CN113816836B CN 113816836 B CN113816836 B CN 113816836B CN 202111151324 A CN202111151324 A CN 202111151324A CN 113816836 B CN113816836 B CN 113816836B
Authority
CN
China
Prior art keywords
reaction
substrate
mutated
enzyme
carbonyl reductase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111151324.1A
Other languages
Chinese (zh)
Other versions
CN113816836A (en
Inventor
倪国伟
尚中栋
郭元勇
袁培斋
张数
郜宪兵
王咪
石洪运
冯尚上
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong Ruiying Pharmaceutical Group Co ltd
Original Assignee
Shandong Ruiying Pharmaceutical Group Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong Ruiying Pharmaceutical Group Co ltd filed Critical Shandong Ruiying Pharmaceutical Group Co ltd
Priority to CN202111151324.1A priority Critical patent/CN113816836B/en
Publication of CN113816836A publication Critical patent/CN113816836A/en
Application granted granted Critical
Publication of CN113816836B publication Critical patent/CN113816836B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/132—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group
    • C07C29/136—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group of >C=O containing groups, e.g. —COOH
    • C07C29/147—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group of >C=O containing groups, e.g. —COOH of carboxylic acids or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/09—Preparation of carboxylic acids or their salts, halides or anhydrides from carboxylic acid esters or lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/333—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
    • C07C67/343—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01184—Carbonyl reductase (NADPH) (1.1.1.184)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention relates to an enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol, which comprises the following synthetic route: wherein R 1,R2 is independently selected from C1-6 alkyl. The invention screens carbonyl reductase to obtain wild carbonyl reductase WO03 and mutants thereof, and discovers that the carbonyl reductase WO03 has good catalytic activity on chiral reduction of key medical intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol, high enzyme activity, good stereoselectivity and chiral purity of more than 99.6 percent. The method does not need harsh low-temperature conditions, and is beneficial to large-scale industrialized production.

Description

Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol
Technical Field
The invention belongs to the field of organic synthesis, and particularly relates to an enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol.
Background
(S) -1- (4-chlorophenyl) -1, 3-propanediol is a key intermediate used in liver-targeting drugs and has been used in a number of clinical and preclinical research drugs. Such as perafuwei mesylate, which is currently being developed by the western medicine corporation of new general, has completed clinical stage two and is currently being developed in clinical stage three. The key diol intermediates all adopt chemical synthetic routes.
The preparation process is reported in patent document US20030225277, the process route is as follows:
a similar synthetic route is reported in US2003/229225, and in the literature JACS,2004, vol 126, #16, p 5154-5163.
The synthetic route has three limitations in mass production:
(1) The operation condition of low temperature is needed, particularly the ultralow temperature condition of-60 ℃ is needed in the second step of reaction, the requirement on production equipment is higher, and the large-scale production is difficult;
(2) An organic catalyst (+) -DIPCl (CAS NO: 112246-73-8) is needed, the catalyst is expensive, the catalytic process is complex to operate, and the catalyst is difficult to apply to large-scale production.
(3) The borane is used for reducing carboxylic acid, the mass production cost of the reaction is high, and the quenching reaction is violently exothermic, so that a certain safety risk is provided.
(4) The chiral purity of the prepared diol compound 1 is 98%, and in order to meet the requirements of registration and declaration, the diol compound needs to be refined, so that the production cost is further increased.
The 4-point defect leads to higher production cost of the product, and is difficult to meet the requirement of large-scale production. Therefore, a large-scale process for producing (S) -1- (4-chlorophenyl) -1, 3-propanediol with high optical purity on a large scale is highly desired.
Disclosure of Invention
In order to overcome the defects of high cost and low optical purity in the preparation process of a key medical intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol in the prior art, the invention utilizes specific carbonyl reductase and mutants thereof as catalytic active substances to prepare the key chiral alcohol compound (IM 04) with high stereoselectivity, the chiral purity is more than 99.4 percent, and the target product (S) -1- (4-chlorophenyl) -1, 3-propanediol is obtained by continuous reduction.
In order to solve the technical problems, the invention provides the following technical scheme:
An enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol, the synthetic route is as follows:
Wherein R 1,R2 is independently selected from C1-6 alkyl, such as methyl, ethyl, propyl, butyl.
The carbonyl reductase has a sequence corresponding to SEQ ID NO:2 or corresponds to the amino acid sequence corresponding to SEQ ID NO:2 in the presence of one or a combination of more than two of the following mutations: v at position 45 is mutated to I (V45I), K at position 63 is mutated to Q (K63Q), G at position 141 is mutated to A (G141A), G at position 141 is mutated to V (G141V), I at position 195 is mutated to L (I195L), and A at position 204 is mutated to V (A204V).
SEQ ID NO:2, named WO03, novosphingobium aromaticivorans-derived carbonyl reductase NaKRED, reference CN102482648, and the inventors unexpectedly found that mutants obtained by mutating a specific site of WO03 carbonyl reductase to a specific amino acid were found by screening an enzyme library, and when IM03 was prepared using IM02 as a substrate, the enzyme activity was high, the conversion rate was high, and the stereoselectivity was good.
Preferably, the carbonyl reductase mutant is a mutant corresponding to SEQ ID NO:2 in the presence of one of the following mutations:
(i) The G at 141 is mutated to A (G141A), and the I at 195 is mutated to L (I195L), the amino acid sequence of which is shown in SEQ ID No:4 is shown in the figure;
(ii) The G at 141 is mutated to V (G141V), and the I at 195 is mutated to L (I195L), the amino acid sequence of which is shown in SEQ ID No: shown at 6.
(Iii) V at position 45 is mutated to I (V45I); the G at 141 is mutated into V (G141V, I195L), the I at 195 is mutated into L (I195L), and the amino acid sequence is shown in SEQ ID No: shown as 8;
(iv) V at position 45 is mutated into I (V45I), G at position 141 is mutated into V (G141V, I195L), I at position 195 is mutated into L (I195L), A at position 204 is mutated into V (A204V), and the amino acid sequence is as shown in SEQ ID No:10 is shown in the figure;
(v) V at position 45 is mutated to I (V45I), K at position 63 is mutated to Q (K63Q), L at position 118 is mutated to M (L118M), G at position 141 is mutated to V (G141V, I195L), A at position 204 is mutated to V (A204V), and the amino acid sequence thereof is as shown in SEQ ID No: shown at 12.
The carbonyl reductase mutants also include the following ranges: 4, 6, 8, 10 or 12, and performing one or more amino acid substitutions, deletions, alterations, insertions or additions to the amino acid sequence shown in SEQ ID No. 4, 6, 8, 10 or 12 within the range of maintaining enzyme activity; or (b)
4, 6, 8, 10 Or 12, performing insertion of one or more amino acids at the N end or the C end of the sequence within the range of maintaining the enzyme activity, wherein the number of inserted amino acid residues is 1-20; preferably 1 to 10, more preferably 1 to 5.
The carbonyl reductase is prepared by constructing alcohol dehydrogenase or glucose dehydrogenase or formate dehydrogenase which realizes coenzyme regeneration and a target gene on the same plasmid pET28a (+) vector, then introducing into an expression host escherichia coli, and obtaining a thallus containing the target enzyme through induction expression. The thalli can be obtained directly by centrifugation, and the thalli is broken to obtain crude enzyme liquid or crude enzyme powder.
Further, the enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol provided by the invention comprises the following steps:
(S1) performing esterification reaction on m-chlorobenzoic acid and alcohol R 1 OH to obtain m-chlorobenzoic acid alkyl ester IM01;
(S2) condensing the alkyl m-chlorobenzoate (IM 01) with the alkyl acetate CH 3COOR2 under the action of organic base to obtain a product keto ester IM02;
(S3) taking ketoester IM02 as a substrate of an enzyme catalytic reaction, and preparing a product IM03 under the catalysis of carbonyl reductase;
(S4) hydrolyzing the IM03 to obtain IM04;
(S5) IM04 reduction gives the final product GL01, (S) -1- (4-chlorophenyl) -1, 3-propanediol.
Preferably, the conditions for the esterification reaction in step (S1) are well known in the art, such as in the presence of an acid catalyst, and the optional acids include concentrated sulfuric acid, p-toluene sulfonic acid, trifluoromethane sulfonic acid, phosphotungstic acid, cation exchange resins, and the like. The reaction temperature is 60-100 ℃, and the reaction is carried out for 15-20h under the condition of heating reflux. The ratio of m-chlorobenzoic acid to R 1 OH is not particularly limited, and an excess of alcohol R 1 OH is generally selected, and the alcohol serves as both a reactant and a solvent of the reaction system. In one embodiment of the invention, the molar ratio of m-chlorobenzoic acid to R 1 OH is from 0.3 to 0.6:1.
Post-treatment after the esterification reaction in the step (S1) is well known in the art, and after the reaction is finished, excessive alcohol is distilled off, a low-polarity solvent (n-heptane, n-hexane and petroleum ether) is added, and the low-polarity solvent phase is extracted and washed by water, weak base and salt solution respectively, so that the product IM01 is obtained by concentrating the low-polarity solvent phase, and the yield is 88-93%.
Preferably, the organic base in step (S2) is selected from the potassium and/or sodium salts of organic alcohols, such as at least one of potassium tert-amyl alcohol, sodium tert-amyl alcohol, potassium tert-butyl alcohol, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide. The molar ratio of IM01, ester CH 3COOR2 and organic base is 1:1.1-1.7:1.5-2.5, preferably 1:1.3-1.5:1.8-2.2.
The reaction temperature of the step (S2) is-5 to 5 ℃, the reaction time is 1-3 hours, the reaction solvent is THF, acetone, DMSO and DMF, and the water content of the solvent is less than 1000ppm. Adding acid to quench after the reaction is finished, and then post-treating is well known in the art, specifically, after the acid is added to quench, standing and layering, concentrating an upper solvent, extracting a lower water phase by using the solvent, combining the lower water phase with an upper concentrated solution, washing by water, weak base and salt in sequence, and concentrating to obtain the product IM02 with the yield of 85-90%.
Preferably, the ketoester IM02 in the step (S3) is used as a substrate for the enzyme catalytic reaction, and the concentration is 50-200 g/L; more preferably 100 to 150g/L.
Further, the carbonyl reductase is at least one of an enzyme in a free form, an immobilized enzyme, or an enzyme in a bacterial form. The dosage of the carbonyl reductase is 1 to 6 weight percent of the substrate in the reaction system. In one embodiment of the present invention, wet cells of carbonyl reductase are used, the mass ratio of the wet cells to the substrate being 30wt% to 100wt%, preferably 50wt% to 70wt%.
Further, in the reaction system, a co-substrate is also present, the co-substrate being selected from the group consisting of: at least one of isopropanol, glucose, ammonium formate; preferably, the concentration of the co-substrate is 100-200% of the substrate concentration, preferably the concentration of the co-substrate is 140-170% of the substrate concentration.
Further, the step (S3) is performed in a phosphate buffer system during the reaction, the pH is 6-8, preferably 6.5-7.5, more preferably 6.8-7.1; and/or the reaction temperature is 20 ℃ to 50 ℃, preferably 25 ℃ to 40 ℃, more preferably 30 ℃ to 35 ℃; and/or the reaction time is 3 to 20 hours, more preferably 3 to 10 hours.
Further, the coenzyme refers to a coenzyme capable of realizing electron transfer in oxidation-reduction reaction; comprises at least one of reduced coenzyme and oxidized coenzyme; the reduced coenzyme is selected from NADH, NADPH, or a combination thereof; the oxidized coenzyme is selected from NAD +、NADP+ or a combination thereof; further, the ratio of the amount of the coenzyme to the amount of the substrate is 0.01wt% to 1.0wt%, preferably 0.1wt% to 0.5wt%.
Further, the gene of the carbonyl reductase and/or the enzyme for coenzyme regeneration is constructed on an expression vector.
Further, in the reaction system, there is also an enzyme for coenzyme regeneration, specifically selected from alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.
Preferably, the hydrolysis in step (S4) is performed in the presence of a base, which is not particularly limited, and is generally an inorganic base such as sodium hydroxide and/or potassium hydroxide, and the amount of the base is not particularly limited, and the hydrolysis may be performed completely, and the pH is generally adjusted to 12 to 14. Post-treatment operations after hydrolysis are well known in the art, and specifically, the pH is adjusted to 7-9 by acid, organic impurities are removed by extraction with an organic solvent, the pH is adjusted to 1-2, and the mixture is left to stand for delamination, extracted with an organic solvent, washed with water, dried, filtered and concentrated to obtain oily substances. The oily matter is redissolved by ethyl acetate, heated to 35-50 ℃, added with low-polarity organic solvent in a dropwise manner, cooled to-5 ℃ to separate out a large amount of solid, filtered, washed and dried to obtain acicular solid which is the product IM04. The yield of the step (S4) is 70-80%, and the purity of the product is more than 99%.
Preferably, in the step (S5), a reducing agent is added in batches, wherein the reducing agent is sodium borohydride and/or potassium borohydride, the dosage is 5-10% of the mass of IM04, the reaction temperature is between-5 ℃ and 5 ℃, the adding time is 1-2h, after the reducing agent is added, the low temperature is maintained for continuous reaction for 10-30min, then boron trifluoride diethyl ether is slowly added dropwise, the dosage of boron trifluoride diethyl ether is 1-2 times of that of IM04, the dropwise addition is completed within 1-3h, the low temperature is maintained for reaction for 1-3h, then the temperature is raised to 20-30 ℃, the reaction is carried out for 3-5h, then the temperature is reduced to 10-20 ℃, methanol is dropwise added after the dropwise addition is completed within 1-2h, the continuous reaction is carried out for 1-2h, and then the product GL01 is obtained after the post-treatment step. The post-treatment is well known in the art, and in one embodiment of the invention, after the reaction is finished, the system is adjusted to be slightly alkaline, and after the reaction is extracted by an organic solvent, the mixture is washed and concentrated under reduced pressure, so as to obtain light yellow viscous liquid which is a final product GL01, namely (S) -1- (4-chlorophenyl) -1, 3-propanediol.
The invention screens carbonyl reductase to obtain wild carbonyl reductase WO03 and mutants thereof, and discovers that the carbonyl reductase WO03 has good catalytic activity on chiral reduction of key medical intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol, has high enzyme activity and good stereoselectivity, and the chiral purity is more than 99.6%, and the preferred embodiment can reach more than 99.8%. The method does not need harsh low-temperature conditions, and is beneficial to large-scale industrialized production.
Drawings
FIG. 1 is a chiral spectrum of the product IM03 obtained in step (S3) of example 2;
FIG. 2 is a 1 H NMR spectrum of the product IM03 obtained in step (S3) of example 2;
FIG. 3 is a 1 H NMR spectrum of the product GL01 obtained in step (S5) of example 2;
FIG. 4 is a 13 C NMR spectrum of the product GL01 obtained in step (S5) of example 2;
FIG. 5 is an HPLC chemical purity profile of the product GL01 obtained in step (S5) of example 2;
FIG. 6 is an HPLC chiral purity spectrum of the product GL01 obtained in step (S5) of example 2.
Detailed Description
Preparation example
Preparation example of carbonyl reductase engineering bacteria and construction of carbonyl reductase homologous mutation library
Cloning 5 carbonyl reductase enzyme genes from WO03 carbonyl reductase enzyme genes and mutant genes thereof into a pET28a (+) vector, then introducing into host escherichia coli BL21, and obtaining recombinant genetically engineered bacteria of carbonyl reductase through induction expression.
Wild carbonyl reductase WO03 (with the nucleic acid sequence shown as SEQ NO.1 and the amino acid sequence shown as SEQ NO. 2) is used as a template, and a random point mutation kit is usedII Site-Directed Mutagenesis Kit) or mutating the wild-type carbonyl reductase gene by directed evolution to obtain a plasmid library comprising the evolved carbonyl reductase gene. The constructed plasmid library was transferred into E.coli BL21 (DE 3) (cat# kang century CW 0809S) and cultured overnight in an oven at 37℃on LB solid medium containing 50. Mu.g/mL kanamycin. Single colonies were picked into 96-well plates containing 400. Mu.L of LB liquid medium (containing 50. Mu.g/mL kanamycin), cultured at 37℃overnight at 200rpm, and seed solutions were obtained. Then 10. Mu.L of seed solution was transferred to 96-well plates containing 400. Mu.L of fermentation medium (containing 50. Mu.g/mL kanamycin) and incubated at 37℃to an OD600 >0.8. Then isopropyl thiogalactoside (IPTG) with the final concentration of 1mM is added, the temperature is reduced to 28 ℃ to induce the expression of the mutant, and the culture is continued for 20 hours. After the fermentation was completed, the cells were collected by centrifugation at 4000g for 30min, and then resuspended in 200. Mu.L of lysis buffer (0.1M phosphate buffer containing 1000U of lysozyme, pH 7.0) and lysed at 30℃for 1h. After lysis was completed, the supernatant was centrifuged at 4000g at 4℃for 30min and clarified supernatant was used to determine mutant activity. After adding 190. Mu.L of the reaction solution (containing 0.4mM substrate, 1mM NADH, 40. Mu.L of dimethyl sulfoxide) to a new 96-well plate and adding 10. Mu.L of the supernatant, the change in NADH was detected at 340nm, and the amount of NADH consumed was used to react with the level of the mutant enzyme activity, and the carbonyl reductase mutants shown in Table 1 below were obtained by screening.
TABLE 1
Example 1
Screening carbonyl reductase by using the compound IV as a substrate, wherein the screening method and the screening result are as follows;
The detection method of the reaction conversion rate comprises the following steps: phenomenex Gemini C18.6 x 250mm 5 μm); mobile phase A is 10% acetonitrile, mobile phase B is acetonitrile, and gradient elution is carried out according to the following table; the flow rate is 1.0ml per minute; the column temperature is 30 ℃; the detection wavelength was 220nm.
The chiral monitoring method of the compound V comprises the following steps: chromatographic column: macroxylonite IB-3,5 μm, 4.6X250 mm; mobile phase: isopropanol: n-hexane=10:90; the flow rate is 1.0mL/min; run time: 20min; column temperature is 30 ℃; detection wavelength: 220nm. S configuration 10min and R configuration 13min.
The results are shown in Table 2 below:
TABLE 2
Enzyme numbering Conversion rate E.e. value Configuration of
WO03(SEQ ID No.2)[a] 98.9％ 99.80％ S
LSADH[b] 21.3％ 93.78％ R
LK[b] 12.4％ 38.52％ S
Note that: carbonyl reductase LSADH is derived from Leifsonia sp.Strain S749, accession number AB213459, carbonyl reductase LK is derived from Lactobacillus kefir, reference WO 2010/025238.
Reaction conditions (a) 1g IV compound, 0.2g wet cell, 0.001g NADP+,0.1g GDH wet cell, 1.5g glucose and 10ml phosphate buffer (100 mM, pH 7.0) were shake-reacted at 30℃and 220rpm for 24h; (b) 1g of IV compound, 0.2g of wet cells, 0.001g of NAD+,20% isopropyl alcohol and 10ml of phosphate buffer (100 mM, pH 7.0) were subjected to shaking reaction at 30℃and 220rpm for 24 hours.
It can be seen that the carbonyl reductase, except WO03 (the amino acid sequence of which is shown as SEQ ID No. 2, and the encoding gene of which is shown as SEQ ID No. 1), has conversion rate and stereoselectivity which cannot meet the requirements of industrial production, is not suitable for large-scale production of a key intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol, but has insufficient enzyme activity of carbonyl reductase WO03, so that the carbonyl reductase mutant with excellent enzyme activity, conversion rate and stereoselectivity is screened by taking the carbonyl reductase of WO03 as a basis to mutate the carbonyl reductase.
Inoculating recombinant genetically engineered bacteria stored in glycerol in preparation example 1 into LB liquid medium containing 100ug/ml of ammonia-gas, culturing for 12-16h at 37℃ and 220rpm to obtain seed culture medium, inoculating the seed liquid into liquid medium containing 100ug/ml of ammonia-gas resistance according to the proportion of 1.5%, culturing at 37 ℃ and 220rpm to OD 600 value of >2.0, adding lactose with the final concentration of 1.0%, cooling to 25C, continuously culturing for 2h, adding lactose with the final concentration of 0.5%, culturing for 20h, placing in a tank, and centrifuging to obtain a bacterial body used for bioconversion. According to reaction conditions (a) of example 1, namely: compound V was prepared by shaking reaction of 0.1g of IV compound, 0.2g of wet cell, 0.001g of NADP+,0.1g of GDH wet cell, 0.2g of glucose and 10ml of phosphate buffer (100 mM, pH 7.0) at 30℃and 220rpm for 24 hours. The resulting library of carbonyl reductase homologous mutations was screened and the results are shown in table 3 below:
TABLE 3 Table 3
Definition of enzyme Activity: in a 50ml centrifuge tube, weighing 0.5g of substrate, adding 1.5ml of isopropanol, adding 3.5ml of phosphate buffer with pH of 6.0-6.5, weighing 0.025g of coenzyme NAD, preheating the system in a water bath kettle at 30 ℃ for 15min, weighing 0.5g of thalli, adding the reaction system, putting the reaction system in a shaking table at a constant temperature of 30 ℃ for 150rpm, and starting timing for 1h. After completion, 0.1ml of the reaction solution was rapidly removed by a pipette in a shaking-up state, added to a 10ml centrifuge tube, and then 4.9ml of isopropyl alcohol was added and thoroughly mixed by a pipette. Centrifuge for 10min at 3500rpm. Pouring the supernatant into a sampling tube, and detecting the conversion rate, namely the enzyme activity.
Because of the degeneracy of the codons, the nucleic acid sequences of the carbonyl reductase mutants described above are not limited to the nucleic acid sequences listed in table 1. Homologs of the base sequence can be obtained by a person skilled in the art by appropriate introduction of substitutions, deletions, alterations, insertions or additions, which are covered by the present invention as long as the expressed recombinase thereof retains catalytic reduction activity on the compound of IM 02. Homologs of the polynucleotides of the present invention may be obtained by substituting, deleting or adding one or more bases of the base sequence within a range that retains the enzymatic activity.
The invention provides carbonyl reductase mutants with amino acid sequences shown as SEQ ID No. 4,6, 8, 10 or 12, which have obviously improved enzyme activity compared with wild type enzyme WO03, can reduce the use amount of enzyme, and can achieve more than 99% of stereoselectivity on S-configuration products. Therefore, by means of WO03 carbonyl reductase and mutants thereof, the IM02 compound can be effectively reduced into the chiral alcohol compound IM03 with S configuration, the yield, the stereoselectivity and the utilization efficiency of the enzyme are all satisfactory, and the preparation of the key intermediate (S) -1- (4-chlorophenyl) -1, 3-propanediol by means of the catalytic activity of a specific enzyme is expected.
Example 2
(S1)：
1.5Kg of SM1 is put into a 20L reaction kettle, 8.3kg of absolute ethyl alcohol is added, sulfuric acid is added, the temperature is raised to 80 ℃, the temperature is kept for 18h, HPLC detection is carried out until 95% of the product is obtained, the reaction solution starts to be concentrated, and the ethanol is distilled off. Adding 4L of n-heptane into the concentrate, adding 4L of water for washing, washing with 4L of saturated sodium bicarbonate water solution after water washing, washing with 4L of saturated saline water, and concentrating the n-heptane to obtain a product IM 01.6 kg.
(S2)：
7.6Kg of THF (water content < 1000 ppm) was added to a 20L reactor, the temperature was set at-5℃and 1.9kg of potassium tert-butoxide (99.5%) was added thereto and stirred until dissolved. Then 1.6kg of IM01 is added, 0.99kg of anhydrous EA is added dropwise at the temperature below 0 ℃, the mixture is incubated for 2 hours at 5 ℃ after 90 minutes, and HPLC monitoring is carried out. Starting material <1%, quenched by addition of 3N HCl. Followed by static delamination. The upper layer was concentrated in THF and the lower layer was extracted once with 2L EA and combined with the upper concentrate. And diluted with EA. Washing with water, saturated sodium carbonate and saturated NaCl, and concentrating to obtain IM021.67kg.
(S3)：
Adding 1.67kg of IM02 to 1.6L of 0.1mol/LPBS buffer solution, adding 2.2kg of isopropanol and 0.53kg of WO03 wet thalli, then adding 3.2gNAD +, reacting in a 35 ℃ water bath under stirring, testing the pH value of the system at intervals (0.5-1 h) during the reaction, adjusting the pH value of the system to 6.9-7.2 by saturated sodium carbonate, monitoring the residual content of the raw materials by HPLC after 15h of reaction to be less than 0.5%, stopping the reaction, concentrating the isopropanol, heating to 70 ℃ after adding 500g of diatomite, filtering to obtain filtrate, extracting the water layer by ethyl acetate twice (1 x 2L), combining organic layers, washing by saturated sodium chloride, drying by anhydrous sodium sulfate, filtering and concentrating to obtain a light yellow oily product IM03 of 1.47kg, and an ee value of 99.9%, wherein the chiral spectrum and 1 HNMR spectrum are respectively shown in fig. 1 and 2. 1 HNMR and 13 C NMR were ：(600MHz,Chloroform-d)δ7.39(s,1H),7.26(dq,J＝15.9,7.7Hz,3H),5.24–4.94(m,1H),4.18(q,J＝7.1Hz,2H),3.52(s,1H),2.82–2.52(m,2H),J＝7.2Hz,3H).13C NMR(151MHz,Chloroform-d)δ172.20,144.60,134.44,129.82,125.95,123.81,69.65,61.04,43.19,14.13.
(S4)：
In a 5L four-mouth bottle, IM03 (1.47 kg) is put into the bottle, the temperature is reduced to 10-15 ℃, alkaline water (300 g of sodium hydroxide is dissolved in 3L of water) is added dropwise, after 2h of addition, the pH is controlled to be 13-14, the reaction is continued for 3h, HPLC detection is carried out, the raw material is <0.5%, concentrated hydrochloric acid is added to adjust the pH to 8-9, ethyl acetate 1L of an extraction water layer is added to wash out organic impurities, the pH is adjusted to be 1 by concentrated hydrochloric acid, layering is carried out, ethyl acetate extraction water layer (1.5Lx2) is carried out, the organic layers are combined, saturated saline water is washed (1.5L), anhydrous sodium sulfate (200 g) is dried, filtered and concentrated to obtain a viscous oily substance (inclusion solid). Adding ethyl acetate (900 ml) into the oily substance, heating to 40deg.C, dissolving, dripping 3.6L petroleum ether, cooling to 0deg.C, precipitating a large amount of solid, maintaining the temperature for 3 hr, filtering, washing with cold ethyl acetate/petroleum ether (1:4), drying to obtain needle-like solid IM04, 995g, purity of 99.7%, recovering mother liquor 85.5g.[α]D25＝-17.3°(c 1.00,MeOH).ESI-MS(m/z)：199.0[M-H]-.1H NMR(400MHz,Chloroform-d)δ7.40(q,J＝1.4,0.9Hz,1H),7.35–7.22(m,3H),5.14(dd,J＝8.4,4.4Hz,1H),2.88–2.72(m,2H).13C NMR(101MHz,CDCl3)δ176.66,144.08,134.62,129.97,128.18,125.95,123.79,69.52,42.83.
(S5)：
Four-port reaction bottles are protected by nitrogen, IM04 g and tetrahydrofuran 160 g are added, stirring is opened, the temperature is reduced to minus 5 ℃, 6.8 g of sodium borohydride is added in batches at the temperature of minus 5 ℃ to 0 ℃, the time is 1-1.5 hours, in the batch adding process, obvious heat release phenomenon is realized, temperature change is noticed, sodium borohydride is easy to absorb moisture, protection is noticed, reaction is carried out for 20 minutes at the temperature of below 0 ℃, 34 g of boron trifluoride diethyl ether is dropwise added at the temperature of minus 5-0 ℃ for 2.5 hours, after the completion of the dropwise adding, the temperature is controlled at the temperature of minus 3-0 ℃ for 1 hour, the temperature is increased to 5 ℃ for 1 hour, the temperature is controlled at the temperature of 25 ℃ for 4 hours, the temperature is reduced to 15-20 ℃, the methanol is dropwise added for 1-1.5 hours, obvious heat release phenomenon exists in the dropwise adding process, the temperature is required to be controlled, the phenomenon of observing the reaction liquid bubbles is observed, and the temperature is controlled for 1 hour. Concentrating under reduced pressure below 30deg.C to dry, adding water 250, stirring to dissolve, dropwise adding 10% sodium hydroxide solution 50g at 10-12deg.C, regulating pH to 8.5-9.0, stirring for 40 min, repeatedly measuring pH without change, adding dichloromethane 150 g at 12-15deg.C, stirring for 40 min, standing for 40 min to separate lower organic phase, extracting twice with 60 g and 40 g, mixing organic phases, adding saturated saline 50g, regulating neutrality with 6N hydrochloric acid, stirring for 1 hr, standing for 40 min, layering, concentrating under reduced pressure below 30deg.C until no thick liquid is present, concentrating under reduced pressure with oil pump to obtain light yellow viscous liquid 15.5g, HPLC chemical purity of 99.4%, ee value 99.8％.HRMS m/z calcd for C9H11ClNaO2：209.0340,found 209.0347[M+Na]+.1H NMR(600MHz,Chloroform-d)δ7.38(d,J＝2.1Hz,1H),7.33–7.18(m,3H),4.94(dd,J＝8.6,3.8Hz,1H),3.86(dt,J＝7.2,4.2Hz,2H),3.21(s,1H),1.95(dddd,J＝20.3,16.1,8.7,4.7Hz,2H).13C NMR(151MHz,Chloroform-d)δ146.43,134.42,129.79,127.62,125.89,123.79,73.66,61.36,40.33.

Claims (16)

1. An enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol, the synthetic route is as follows:
，
Wherein R 1,R2 is independently selected from C1-6 alkyl;
the mutant of carbonyl reductase is a mutant corresponding to SEQ ID NO:2 in the presence of one of the following mutations:
(i) V at position 45 is mutated into I (V45I), G at position 141 is mutated into V (G141V, I195L), I at position 195 is mutated into L (I195L), A at position 204 is mutated into V (A204V), and the amino acid sequence is as shown in SEQ ID No:10 is shown in the figure;
(ii) V at position 45 is mutated to I (V45I), K at position 63 is mutated to Q (K63Q), L at position 118 is mutated to M (L118M), G at position 141 is mutated to V (G141V, I195L), A at position 204 is mutated to V (A204V), and the amino acid sequence thereof is as shown in SEQ ID No: shown at 12.
2. The production method according to claim 1, characterized by comprising the steps of:
(S1) performing esterification reaction on m-chlorobenzoic acid and alcohol R 1 OH to obtain m-chlorobenzoic acid alkyl ester IM01;
(S2) condensing the alkyl m-chlorobenzoate (IM 01) with the alkyl acetate CH 3COOR2 under the action of organic base to obtain a product keto ester IM02;
(S3) taking ketoester IM02 as a substrate of an enzyme catalytic reaction, and preparing a product IM03 under the catalysis of carbonyl reductase;
(S4) hydrolyzing the IM03 to obtain IM04;
(S5) IM04 reduction gives the final product GL01, (S) -1- (4-chlorophenyl) -1, 3-propanediol.
3. The production method according to claim 2, wherein the esterification reaction in step (S1) is carried out under a heating reflux condition at a reaction temperature of 60 to 100 ℃ under an acid catalyst for 15 to 20 hours.
4. A production method according to claim 3, wherein the molar ratio of m-chlorobenzoic acid to R 1 OH is 0.3-0.6:1.
5. The process according to claim 2, wherein the organic base in step (S2) is selected from potassium and/or sodium salts of organic alcohols; the molar ratio of IM01, ester CH 3COOR2 and organic base is 1:1.1-1.7:1.5-2.5.
6. The process of claim 5, wherein the molar ratio of IM01, ester CH 3COOR2 and organic base is 1:1.3-1.5:1.8-2.2.
7. The production method according to claim 2, wherein the ketoester IM02 in the step (S3) is used as a substrate for the enzyme-catalyzed reaction at a concentration of 50 to 200g/L.
8. The production method according to claim 7, wherein the ketoester IM02 in the step (S3) is used as a substrate for the enzyme-catalyzed reaction at a concentration of 100 to 150g/L.
9. The method according to claim 2, wherein the carbonyl reductase is at least one of an enzyme in a free form, an immobilized enzyme, or an enzyme in a bacterial form, and the amount of the carbonyl reductase is 1wt% to 6wt% of the substrate in the reaction system.
10. The method of claim 2, wherein in step (S3) a co-substrate and a co-enzyme are also present, said co-substrate being selected from the group consisting of: at least one of isopropanol, glucose, ammonium formate; the coenzyme comprises at least one of reduced coenzyme and oxidized coenzyme, and the ratio of the coenzyme to the substrate is 0.01-1.0 wt%.
11. The method according to claim 10, wherein in the step (S3), the concentration of the co-substrate is 100 to 200% of the substrate concentration.
12. The method according to claim 10, wherein in the step (S3), the concentration of the co-substrate is 140 to 170% of the substrate concentration.
13. The method according to claim 10, wherein in the step (S3), the ratio of the amount of the coenzyme to the amount of the substrate is 0.1wt% to 0.5wt%.
14. The method according to claim 10, wherein in the step (S3), an enzyme for coenzyme regeneration is further present.
15. The method according to claim 14, wherein the enzyme for coenzyme regeneration is selected from the group consisting of alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, and combinations thereof.
16. The process according to claim 2, wherein the hydrolysis in step (S4) is carried out in the presence of a base in an amount to adjust the pH of the system to 12-14; and/or
In the step (S5), a reducing agent which is sodium borohydride and/or potassium borohydride is added in batches, the consumption is 5-10% of the mass of IM04, the reaction temperature is between-5 ℃ and 5 ℃, the adding time is 1-2h, after the reducing agent is added, the low temperature is maintained for continuous reaction for 10-30min, then boron trifluoride diethyl ether is slowly added dropwise, the consumption of boron trifluoride diethyl ether is 1-2 times of the mass of IM04, after the dropwise addition is completed in 1-3h, the low temperature is maintained for reaction for 1-3h, then the temperature is raised to 20-30 ℃, the reaction is carried out for 3-5h, then the temperature is reduced to 10-20 ℃, methanol is added dropwise after the dropwise addition is completed in 1-2h, the continuous reaction is carried out for 1-2h, and then the product GL01 is obtained after the post-treatment step.
CN202111151324.1A 2021-09-29 2021-09-29 Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol Active CN113816836B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111151324.1A CN113816836B (en) 2021-09-29 2021-09-29 Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111151324.1A CN113816836B (en) 2021-09-29 2021-09-29 Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol

Publications (2)

Publication Number Publication Date
CN113816836A CN113816836A (en) 2021-12-21
CN113816836B true CN113816836B (en) 2024-05-03

Family

ID=78921732

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111151324.1A Active CN113816836B (en) 2021-09-29 2021-09-29 Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol

Country Status (1)

Country Link
CN (1) CN113816836B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023142153A1 (en) * 2022-01-31 2023-08-03 山东寰酶生物制药有限公司 Carbonyl reductase and use thereof
CN117285413A (en) * 2023-08-09 2023-12-26 重庆恩联生物科技有限公司 Synthesis method of terbutaline key intermediate 3, 5-dihydroxyacetophenone

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006033709A2 (en) * 2004-07-29 2006-03-30 Metabasis Therapeutics, Inc. Novel nucleoside derivatives
CN1997377A (en) * 2004-02-13 2007-07-11 症变治疗公司 New 2'-C-Methyl Nucleoside Derivatives
CN101252839A (en) * 2005-08-12 2008-08-27 默克公司 Novel 2 '-C-methyl and 4' -C-methyl nucleoside derivatives
JP2009073739A (en) * 2007-09-19 2009-04-09 Kaneka Corp Method for producing highly pure optically active 1-aryl-1,3-propanediol acceptable as medicine intermediate
CN102482648A (en) * 2009-06-22 2012-05-30 科德克希思公司 A Ketoreductase-Mediated Stereoselective Pathway to α-Chlorohydrins
CN105153229A (en) * 2015-06-18 2015-12-16 武汉凯特立斯科技有限公司 Chiral tridentate PNN ligand and application of same in asymmetric hydrogenation
CN109022473A (en) * 2018-08-13 2018-12-18 浙江海洋大学 A kind of method that enzyme process prepares orlistat intermediate
CN109182410A (en) * 2018-09-29 2019-01-11 台州学院 A kind of enzymatic preparation method of (S)-N-Boc-3- hydroxy piperidine
CN109735553A (en) * 2018-12-29 2019-05-10 南京诺云生物科技有限公司 A kind of preparation method of anti-AIDS drug atazanavir intermediate
CN109943577A (en) * 2018-12-29 2019-06-28 南京诺云生物科技有限公司 A kind of bioconversion method of anti-AIDS drug atazanavir intermediate
CN109943542A (en) * 2018-12-29 2019-06-28 南京诺云生物科技有限公司 A kind of alcohol dehydrogenase for atazanavir intermediate production
CN112708641A (en) * 2019-10-24 2021-04-27 上海医药工业研究院 Chemical-enzymatic synthesis method of tomoxetine
CN112852769A (en) * 2020-08-14 2021-05-28 中国科学院天津工业生物技术研究所 Method for preparing (S) -1- (2-methoxy-3-bromophenyl) ethanol

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1997377A (en) * 2004-02-13 2007-07-11 症变治疗公司 New 2'-C-Methyl Nucleoside Derivatives
WO2006033709A2 (en) * 2004-07-29 2006-03-30 Metabasis Therapeutics, Inc. Novel nucleoside derivatives
CN101252839A (en) * 2005-08-12 2008-08-27 默克公司 Novel 2 '-C-methyl and 4' -C-methyl nucleoside derivatives
JP2009073739A (en) * 2007-09-19 2009-04-09 Kaneka Corp Method for producing highly pure optically active 1-aryl-1,3-propanediol acceptable as medicine intermediate
CN102482648A (en) * 2009-06-22 2012-05-30 科德克希思公司 A Ketoreductase-Mediated Stereoselective Pathway to α-Chlorohydrins
CN105153229A (en) * 2015-06-18 2015-12-16 武汉凯特立斯科技有限公司 Chiral tridentate PNN ligand and application of same in asymmetric hydrogenation
CN109022473A (en) * 2018-08-13 2018-12-18 浙江海洋大学 A kind of method that enzyme process prepares orlistat intermediate
CN109182410A (en) * 2018-09-29 2019-01-11 台州学院 A kind of enzymatic preparation method of (S)-N-Boc-3- hydroxy piperidine
CN109735553A (en) * 2018-12-29 2019-05-10 南京诺云生物科技有限公司 A kind of preparation method of anti-AIDS drug atazanavir intermediate
CN109943577A (en) * 2018-12-29 2019-06-28 南京诺云生物科技有限公司 A kind of bioconversion method of anti-AIDS drug atazanavir intermediate
CN109943542A (en) * 2018-12-29 2019-06-28 南京诺云生物科技有限公司 A kind of alcohol dehydrogenase for atazanavir intermediate production
CN112708641A (en) * 2019-10-24 2021-04-27 上海医药工业研究院 Chemical-enzymatic synthesis method of tomoxetine
CN112852769A (en) * 2020-08-14 2021-05-28 中国科学院天津工业生物技术研究所 Method for preparing (S) -1- (2-methoxy-3-bromophenyl) ethanol

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Design, synthesis, structure-activity relationships, and docking studies of pyrazole-containing derivatives as a novel series of potent glucagon receptor antagonists;Shuangjie Shu;Bioorganic & Medicinal Chemistry;第24卷;2852–2863 *
Imidazolium-based organoiridium-functionalized periodic mesoporous organosilica boosts enantioselective reduction of α-cyanoacetophenones, α-nitroacetophenones, and β-ketoesters.Journal of Catalysis.2014,第320卷70–76. *
SDR family oxidoreductase [Novosphingobium aromaticivorans].Genbank.2021, *
新型喹诺酮类化合物的设计合成与生物活性研究;高佳娣;中国药物化学杂志;第23卷(第1期);21-25 *

Also Published As

Publication number Publication date
CN113816836A (en) 2021-12-21

Similar Documents

Publication Publication Date Title
CN106701840B (en) Biological preparation method of (1R,2S) -2- (3, 4-difluorophenyl) cyclopropylamine D-mandelate (I)
CN113816836B (en) Enzymatic production method of (S) -1- (4-chlorophenyl) -1, 3-propanediol
CN116676285A (en) Carbonyl reductase mutant for preparing chiral alcohol compound and application thereof
WO2017197604A1 (en) Method for synthesizing crizotinib intermediate
JP2023511025A (en) Use and production method of enzyme in production of orlistat intermediate
CN104450798B (en) A kind of external enzyme reaction generates method and the application of 1,2,4 butantriols
CN112795603A (en) Method for preparing (S) -2- (3-pyridine) -pyrrolidine
CN117844830A (en) Application of transgenic yeast expressing cytochrome oxidase in the preparation of cucurbitacin intermediates
CN110396507A (en) L-pantolactone dehydrogenase from Cnuibacter physcomitrellae
CN110004162B (en) Carbonyl reductase, gene and application of carbonyl reductase in methoxamine hydrochloride key intermediate
CN117802177A (en) Method for synthesizing single chiral 2-aryl substituted nitrogen heterocyclic derivative by imine reductase catalysis and application thereof
CN115927224B (en) A carbonyl reductase mutant and its application
CN117887739A (en) Imine reductase or mutant and application thereof
CN115747240B (en) A recombinant Escherichia coli engineering strain for producing equol and its application
US11535601B2 (en) Method for preparing hexahydrofuro-furanol derivative, intermediate thereof and preparation method thereof
CN115404249B (en) Preparation method and application of (S) -nicotine intermediate
CN110372532B (en) Process for producing hexahydrofurofuranol derivative, intermediate therefor, and process for producing the intermediate
CN115948357A (en) Ketone reductase mutant, preparation method and application thereof, and preparation method of eslicarbazepine
CN111808893B (en) Novel biological preparation method of amino alcohol drug intermediate
CN116555205A (en) Asymmetric reduction method of carbonyl by using carbonyl reductase and mutant thereof
CN119709888A (en) Method for producing (S) -1, 1-bis (4-fluorophenyl) -2-propanol using an enzymatic reaction and use of a ketoreductase
CN119432782B (en) Alkene reductase mutant and application thereof in catalytic preparation of buvaracetam synthesis intermediate
CN118374465B (en) Imine reductase and method for preparing (S)-nicotine using the same
CN120005840A (en) A desaturase TgDH and its application
WO2025060847A1 (en) Cytochrome p450 enzyme, nucleic acid molecule, and glycyrrhetinic acid derivative and use thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20240401

Address after: 274007 No. 1777, Kunming Road, Heze City, Shandong Province

Applicant after: Shandong Ruiying Pharmaceutical Group Co.,Ltd.

Country or region after: China

Address before: 274699 chemical industry park, juancheng County, Heze City, Shandong Province

Applicant before: Shandong Huanjiang biopharmaceutical Co.,Ltd.

Country or region before: China

GR01 Patent grant
GR01 Patent grant