CN117210426A - Carbonyl reductase mutant and application thereof - Google Patents
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- CN117210426A CN117210426A CN202310643036.0A CN202310643036A CN117210426A CN 117210426 A CN117210426 A CN 117210426A CN 202310643036 A CN202310643036 A CN 202310643036A CN 117210426 A CN117210426 A CN 117210426A
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
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- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
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- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a carbonyl reductase mutant and application thereof. The amino acid sequence of the carbonyl reductase mutant comprises one or more of the following site mutations compared with SEQ ID NO: 1: T17V, W82L, F88V, V A, A190V, S193A, Y35201F, N204A, A138 and R142, wherein a138 is mutated to V or L and R142 is mutated to M, A or V. When the carbonyl reductase mutant is used for preparing the 4-hydroxy piperidine compounds, the conversion rate and chiral purity are higher, the reaction time is shorter, the operation is simple, and the carbonyl reductase mutant is suitable for industrial production.
Description
Technical Field
The invention belongs to the field of bioconversion, and particularly relates to a carbonyl reductase mutant and application thereof.
Background
At present, no small-molecule target drug aiming at complement factor B related to chronic kidney disease (CDK) exists in the market, but the eculizumab for immunotherapy is taken as the current first-line drug, the price is high, the healing is poor, anemia occurs in patients, and blood transfusion maintenance is needed, so that a small-molecule oral complement system inhibitor is urgently needed in clinic at present, and the drug is suitable for medical treatment. The small molecule LNP023 is developed by North China and provides a new therapeutic drug for diseases with overactive complement bypass system.
LNP023 is synthesized by using 4-methoxypyridine as starting material, reducing pyridine ring, protecting nitrogen atom by benzyloxycarbonyl group to obtain benzyl 4-carbonyl-3, 4-dihydropyridine-1 (2H) -carboxylate (2), michael-adding (4- (methylester < methoxycarbonyl >) phenyl) boric acid to compound 2 under the action of monovalent rhodium catalyst to obtain benzyl (S) -2- (4- (methylester < methoxycarbonyl >) phenyl) -4-carbonylpiperidine-1-carboxylate (3), selectively reducing benzyl (2S, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate (4) with target configuration by carbonyl reductase, protecting benzyl (2S, 4S) -4- ((tert-butyldimethylsilyl) oxo) -2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate by tert-butyldimethylsilane, replacing benzyl (4-carbonyl >) piperidine-1-carboxylate with poly (6) palladium (4S, 4) carbonyl) phenyl) with hydrogen, hydrogenation debenzyloxycarbonyl protection to obtain methyl 4- ((2S, 4S) -4-ethoxypiperidine-2-yl) benzoate (7), catalytic reductive amination by iridium catalyst to obtain tert-butyl 4- (((2S, 4S) -4-ethoxy-2- (4- (methyl ester < methoxycarbonyl >) phenyl) piperidine-1-yl) methyl) -5-methoxy-7-methyl-1H-indole-1-carboxylate (8), and alkaline hydrolysis to obtain 4- ((2S, 4S) -4-ethoxy-1- ((5-methoxy-7-methyl-1H-indole-4-yl) methyl) piperidine-2-yl) benzoate (1).
The synthetic process of the synthetic routes 2 to 3 is to obtain the compound 3 with better chirality through metal catalysis, but the key of the route is to prepare the chiral alcohol with high chiral purity, namely the compound 4 through catalytic reduction of the compound 3, and then obtain the compound 1 through a series of syntheses.
In patent CN112513025a, an enzymatic reduction method is reported, and KRED-EW124 from hanse is used to perform catalytic reduction of compound 3, which has the disadvantage that the chiral purity of the reduced product is not high, and chiral preparation is required subsequently, thus reducing the atom utilization rate. And secondly, the solubility of 3 in the biological reaction system is poor, so that the dosage of DMSO in the reaction is large, thereby bringing a plurality of inconveniences to post-treatment, reducing the reaction rate and being not suitable for industrial production. Meanwhile, the preparation method of the 4-hydroxy piperidine compound has similar problems.
Therefore, there is an urgent need in the art to develop a method for preparing 4-hydroxypiperidine compounds that is environmentally friendly, efficient, highly stereoselective, and more suitable for industrial production.
Disclosure of Invention
The invention aims to solve the technical problems of low chiral purity and low yield of the 4-hydroxy piperidine compounds prepared in the prior art, and provides a carbonyl reductase mutant and application thereof. When the enzyme mutant is used for preparing the 4-hydroxy piperidine compounds, the conversion rate and chiral purity are higher, the reaction time is shorter, the operation is simple, and the enzyme mutant is suitable for industrial production.
The invention solves the technical problems through the following technical proposal.
In a first aspect, the invention provides a carbonyl reductase mutant, the amino acid sequence of which comprises one or more of the following site mutations compared with SEQ ID NO: 1: T17V, W82L, F V, V121A, A V, S193A, Y F, N A, A of F, N138 and R142;
wherein, A138 is mutated to V or L, R142 is mutated to M, A or V;
the site mutation does not comprise any of the following groups:
(1)A190V/S193A；
(2)R142M/A190V/S193A；
(3)F88V/A138L/R142M/A190V/S193A；
(4)W82L/F88V/A138L/R142M/A190V/S193A；
(5) W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
(6)W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F。
in the present invention, "/" between mutation sites means the relationship of the sum, for example, "A190V/S193A" means A190V and S193A.
In some embodiments of the invention, the amino acid sequence of the carbonyl reductase mutant comprises a site mutation selected from any one of the following groups compared to SEQ ID NO: 1:
(1) R142M, R142A or a138V;
(2)F88V/A138V/R142M/A190V/S193A；
(3) W82L/V121A/A138L/R142V/A190V/S193A; and
(4)T17V/W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F/N204A。
in a second aspect the invention provides an isolated nucleic acid encoding a carbonyl reductase mutant according to the first aspect of the invention.
In a third aspect the invention provides a recombinant expression vector comprising a nucleic acid according to the second aspect of the invention.
In some embodiments of the invention, the recombinant expression vector is a plasmid. The backbone of the plasmid is preferably pET28a (+).
In a fourth aspect, the present invention provides a transformant comprising a nucleic acid according to the second aspect of the present invention or a recombinant expression vector according to the third aspect of the present invention.
In some embodiments of the invention, the transformant is a cell; preferably eukaryotic or prokaryotic cells; the eukaryotic cell is preferably a yeast cell; the prokaryotic cell is preferably an E.coli cell.
In a fifth aspect, the present invention provides a method of preparing an enzyme mutant according to the first aspect of the invention, comprising culturing a transformant according to the fourth aspect under conditions suitable for expression.
In some embodiments of the invention, the culture medium comprises the following components: 5-17 g/L yeast extract, 10-22 g/L tryptone, 3-13 g/L NaCl,10g/L glycerol, 2g/L K 2 HPO 4 ·3H 2 O,0.5g/L MgSO 4 ·7H 2 O and 1-2L of deionized water.
In some embodiments of the invention, the culture medium is LB liquid medium and/or fermentation medium, and the formula of the fermentation medium is 12g/L yeast extract, 12g/L tryptone, 3g/L NaCl,10g/L glycerol, 2g/L K 2 HPO 4 ·3H 2 O,0.5g/L MgSO 4 ·7H 2 O and 1L of deionized water.
In some embodiments of the invention, the medium further comprises an antibiotic, such as kanamycin. The antibiotic may be present in an amount of 50. Mu.g/mL.
In some embodiments of the invention, the temperature of the culture is 25 to 37 ℃.
In some embodiments of the invention, the pH of the culture is 7.
In a sixth aspect, the invention provides the use of a carbonyl reductase in the preparation of a compound of formula VI,
is a single bond or a double bond;
carbon atoms with "×" denote, when chiral, S configuration, R configuration or mixtures thereof;
R 1 h, C of a shape of H, C 6 -C 10 Aryl or by R 1-1 Substituted C 6 -C 10 An aryl group; r is R 1-1 Is cyano orWherein R is 1-2 Is C 1 -C 6 An alkyl group;
R 2 is H or an amino protecting group;
the carbonyl reductase is selected from one or more of the following:
(1) Carbonyl reductase mutants of the first aspect of the invention;
(2) Carbonyl reductase with the amino acid sequence shown as SEQ ID NO. 1; and
(3) An enzyme mutant comprising any one of the following sets of site mutations in the amino acid sequence as set forth in SEQ ID NO. 1:
i.A190V/S193A；
ii.R142M/A190V/S193A；
F88I/A138L/R142M/A190V/S193A or F88V/A138L/R142M/A190V/S193A;
W82L/F88V/A138L/R142M/A190V/S193A or W82L/V121A 138L/A190V/S193A/K206H;
W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
vi.W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F。
in some embodiments of the invention, the C 6 -C 10 Aryl is phenyl.
In some embodiments of the invention, the C 1 -C 6 Alkyl is methyl.
In some embodiments of the invention, the amino protecting group is Boc or Cbz.
In some embodiments of the invention, R 1-1 Is cyano or
In some embodiments of the invention, R 1 Is H,
In some embodiments of the invention, R 2 H, boc or Cbz.
In some embodiments of the invention, whenR is a single bond 1 Is R is 1-1 Substituted C 6 -C 10 Aryl, R 2 Is H or an amino protecting group; when->In the case of double bonds, R 1 Is H, R 2 Is an amino protecting group.
In some embodiments of the present invention, the compound of formula VI is
In some embodiments of the invention, the carbonyl reductase is:
(1) Carbonyl reductase mutants of the first aspect of the invention;
(2) Carbonyl reductase with the amino acid sequence shown as SEQ ID NO. 1; or (b)
(3) An enzyme mutant comprising any one of the following sets of site mutations in the amino acid sequence as set forth in SEQ ID NO. 1:
i.A190V/S193A；
ii.R142M/A190V/S193A；
F88I/A138L/R142M/A190V/S193A or F88V/A138L/R142M/A190V/S193A;
W82L/F88V/A138L/R142M/A190V/S193A or W82L/V121A 138L/A190V/S193A/K206H;
W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
vi.W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F。
in a seventh aspect, the present invention provides a method for preparing a compound of formula VI, comprising the steps of: in a liquid reaction system, in the presence of coenzyme and carbonyl reductase, carrying out a reduction reaction of a compound shown in a formula V to obtain a compound shown in a formula VI;
wherein,the definition of carbon atoms with "×", R1 and R2 are as described in the sixth aspect of the invention;
the carbonyl reductase is as described in the sixth aspect of the invention.
In some embodiments of the present invention, the compound of formula V is
In some embodiments of the invention, the coenzyme is a coenzyme conventional in the art, preferably the coenzyme is a reduced coenzyme and/or an oxidized coenzyme. The oxidative coenzyme is preferably NAD+ and/or NADP+; the reduced coenzyme is preferably NADH and/or NADPH.
In some embodiments of the invention, the amount of said coenzyme is conventional in the art, preferably the mass ratio of said coenzyme to said compound of formula V is (0.001-0.1): 1, preferably (0.01-0.05): 1, e.g. 0.02:1.
In some embodiments of the invention, the carbonyl reductase is added to the reduction reaction in a form conventional in the art, preferably in the form of a free enzyme, immobilized enzyme, bacterial powder or bacterial form, such as bacterial form.
In some embodiments of the invention, the carbonyl reductase is used in amounts conventional in the art for carbonyl reductase, preferably the mass ratio of carbonyl reductase to the compound of formula V is (0.1-5): 1, preferably (0.1-1): 1, e.g., 0.5:1.
In some embodiments of the invention, the liquid reaction system comprises a buffer.
The buffer is preferably a phosphate buffer, for example 0.1M phosphate buffer.
The amount of buffer may be that conventional in the art, and preferably the ratio of the buffer to the compound of formula V is from 5 to 100mL/g, e.g., 25mL/g.
In some embodiments of the invention, the liquid reaction system further comprises a co-solvent.
The cosolvent may be a cosolvent conventional in the art, preferably one or more selected from dimethyl sulfoxide, isopropanol and toluene, for example dimethyl sulfoxide.
The amount of the cosolvent may be conventional in the art, and preferably the ratio of the cosolvent to the compound of formula V is 1-20 mL/g, e.g., 5mL/g.
In some embodiments of the invention, the reaction temperature of the reduction reaction is conventional in the art, preferably from 10 ℃ to 50 ℃, more preferably from 20 ℃ to 35 ℃, for example 25 ℃ or 30 ℃.
In some embodiments of the invention, the reaction time of the reduction reaction is dependent on the reaction temperature and the reaction scale, and is preferably 1 to 48 hours, preferably 2 to 24 hours, more preferably 4 to 14 hours.
In some embodiments of the invention, after the reduction reaction is completed, further comprising post-treatment.
The post-treatment may be conventional in the art, and preferably comprises the steps of: adding organic solvent into the liquid reaction system, and extracting. The organic solvent may be dichloromethane.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: when the enzyme mutant is used for preparing the 4-hydroxy piperidine compounds, the conversion rate is higher, the chiral purity is higher, the reaction time is shorter, the post-treatment is simple, the cost is low, the environment is protected, and the enzyme mutant is suitable for industrial production.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The inventors have conducted extensive and intensive studies and have unexpectedly developed, for the first time, a biocatalytic preparation method of benzyl (2S, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate, which is a novel stereospecific synthesis method. Specifically, the present invention provides a screening modification of a key carbonyl reductase in the existing benzyl (2S, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate production process, namely, stereoselective reduction of a compound of formula V to a compound of formula VI in the presence of a carbonyl reductase and a coenzyme (the production of a compound of formula VI having a stereochemical configuration can be carried out very efficiently even at a high concentration of substrate, for example, 50 to 1000 g/L). Then taking the compound of the formula VI as a substrate to carry out subsequent reaction.
Specifically, the present inventors found that when using the mutant 14 of exobacterium sp.f42-derived carbonyl reductase (WTEA) as a representative carbonyl reductase, the tolerance of the reaction system to an organic solvent and a substrate can be significantly improved, and thus the substrate concentration can be significantly improved, and also a compound of formula vi having a steric conformation (ee value: 98%, 99%, or 99.9%) can be produced and obtained very efficiently, thereby greatly improving the production efficiency and reducing the production cost.
EXAMPLE 1 construction of genetically engineered bacteria expressing recombinant carbonyl reductase and glucose dehydrogenase
The gene of carbonyl reductase WTEA (the nucleotide sequence is SEQ ID NO:2, the amino acid sequence is SEQ ID NO: 1), and the gene of glucose dehydrogenase GDH from bacillus megatherium (Bacillus megaterium) are entrusted to commercial company to carry out total gene synthesis, are cloned into pET28a (+) vectors respectively, are transferred into competent cells of escherichia coli BL21 (DE 3), are selected to be single bacteria, are cultivated by LB, and respectively obtain genetically engineered bacteria pET28a (+) -WTEA capable of inducing to express recombinant carbonyl reductase and genetically engineered bacteria pET28a (+) -GDH capable of expressing recombinant glucose dehydrogenase GDH.
EXAMPLE 2 preparation of recombinant carbonyl reductase and glucose dehydrogenase
Inoculating the genetically engineered bacteria stored in the previous step into LB liquid medium containing 50 mug/mL kanamycin, culturing at 37 ℃ and 220rpm for 14 hours, and obtaining seed culture solution. Seed culture broth was inoculated at a ratio of 0.5% onto LB liquid medium containing 50. Mu.g/mL kanamycin resistance, and then cultured at 37℃to OD at 220rmp 600 Value of>3.0. Adding isopropyl thiogalactoside (IPTG) with final concentration of 0.5mM, cooling to 25deg.C to induce protein expression, culturing for 20 hr, placing in a tank, and centrifuging to obtain thallus, and preparing for bioconversion.
LB liquid medium (g/L): tryptone 10.0, yeast extract 5.0, naCl 10.0, deionized water 1L, pH 7.0.
Fermentation medium (g/L): yeast extraction12.0 of substance, 12.0 of tryptone, 3.0 of NaCl, 10.0 of glycerol and K 2 HPO 4 ·3H 2 O 2.0，MgSO 4 ·7H 2 O0.5, deionized water 1L, pH 7.0.
EXAMPLE 3 construction and screening of carbonyl reductase WTEA mutant
The wild-type carbonyl reductase gene WTEA was mutated by directed evolution to obtain a plasmid library comprising the evolved carbonyl reductase gene. This was then transferred into E.coli BL21 (DE 3) (cat# kang century CW 0809S) and plated on LB solid medium containing 50. Mu.g/mL kanamycin. After 14h incubation in an oven at 37℃single colonies were picked into 96-well plates containing 400. Mu.L LB liquid medium (containing 50. Mu.g/mL kanamycin), incubated at 37℃overnight at 200rpm, and seed solutions were obtained. Then 10. Mu.L of the seed solution was transferred to a 96-well plate containing 400. Mu.L of fermentation medium (fermentation medium, containing 50. Mu.g/mL kanamycin) and cultured at 37℃and 200rpm for 3 hours. Then isopropyl thiogalactoside (IPTG) with a final concentration of 1mM was added, the temperature was reduced to 25 ℃ to induce mutant expression, and the culture was continued for 20h. The pelleted cells were then centrifuged at 4000g for 30min, resuspended in 200. Mu.L lysis buffer (0.1M phosphate buffer with 1000U lysozyme, pH 7.0) and lysed for 1h at 30 ℃. The 96-well plate was then centrifuged at 4000g for 30min at 4℃and the clarified supernatant was used to determine mutant activity. mu.L of the reaction solution (containing 0.4mM substrate, 1mM NADPH, 40. Mu.L of dimethyl sulfoxide) was added to a new 96-well plate, and after 10. Mu.L of the supernatant was added, the change in NADPH was detected at 340 nm. The consumption of NADPH reflects the level of mutant enzyme activity, and the relative activities of the mutants are shown in Table 1.
Table 1 mutant and relative Activity thereof
* The activity of the wild-type carbonyl reductase gene WTEA (SEQ ID NO: 1) was set to 100%.
EXAMPLE 4 use of wild-type carbonyl reductase and carbonyl reductase mutant in the preparation of benzyl (2S, 4S) -4-hydroxy-2- (4- (methylcarbo-nyl) phenyl) piperidine-1-carboxylate
Reaction conditions: the reaction was terminated by dissolving compound V-1 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP+ (0.2 g), adding wild-type carbonyl reductase or enzyme mutant 1-14 (5 g), 25℃and 220rpm, shaking the reaction, and monitoring the reaction conversion by HPLC to > 98%. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid, the yield (conversion), ee value and reaction time were as shown in Table 2.
TABLE 2 catalytic Activity and stereoselectivity of wild-type carbonyl reductase and carbonyl reductase mutants
The results in Table 2 show that both wild type and mutant 1-14 show significant advantages in terms of both conversion and product stereoselectivity in the use of preparing benzyl (2S, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate. When the enzyme mutant 1-14 is used for preparing benzyl (2S, 4S) -4-hydroxy-2- (4- (methyl ester < methoxycarbonyl >) phenyl) piperidine-1-carboxylic ester, the reaction time is short and the conversion efficiency is high.
EXAMPLE 5 biocatalytic preparation of benzyl (2S, 4S) -4-hydroxy-2- (4- (carbomethoxy) phenyl) piperidine-1-carboxylate
Dissolving compound V-1 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 14 cells (5 g) prepared in example 3 were added thereto, and the reaction was carried out at 25℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.5g of a white solid in 94.4% yield and 100% ee.
EXAMPLE 6 biocatalytic preparation of tert-butyl (2S, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate
Compound V-2 (10 g) was dissolved in DMSO (50 ml), 0.1M phosphate buffer (250 ml) was added, NADP+ (0.2 g) was added, the WTEA mutant 14 cells (5 g) prepared in example 3 were added, the reaction was stopped when the reaction was monitored by shaking at 25℃and 220rpm, and the conversion rate of the reaction was >98% by HPLC. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.3g of a white solid in 92.4% yield and 100% ee.
EXAMPLE 7 biocatalytic preparation of benzyl (2R, 4S) -4-hydroxy-2- (4- (carbomethoxy) phenyl) piperidine-1-carboxylate
Dissolving compound V-3 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 14 cells (5 g) prepared in example 3 were added thereto, and the reaction was carried out at 25℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.6g of a white solid in a yield of 95.4% and an ee value of 100%.
EXAMPLE 8 biocatalytic preparation of benzyl (2R, 4S) -4-hydroxy-2- (4- (carbomethoxy) phenyl) piperidine-1-carboxylate
Dissolving compound V-3 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 11 cells (6 g) prepared in example 3 were added, and the reaction was carried out at 30℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.2g of a white solid in 91.5% yield and 100% ee.
EXAMPLE 9 biocatalytic preparation of tert-butyl (2R, 4S) -4-hydroxy-2- (4- (methylester < methoxycarbonyl >) phenyl) piperidine-1-carboxylate
Compound V-4 (10 g) was dissolved in DMSO (50 ml), 0.1M phosphate buffer (250 ml) was added, NADP+ (0.2 g) was added, the WTEA mutant 14 cells (5 g) prepared in example 3 were added, the reaction was stopped when the reaction was monitored by shaking at 25℃and 220rpm, and the conversion rate of the reaction was >98% by HPLC. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.3g of a white solid with a yield of 92.4% and an ee value of 100%
EXAMPLE 10 biocatalytic preparation of benzyl (2S, 4S) -2- (4-cyanophenyl) -4-hydroxypiperidine-1-carboxylate
Dissolving compound V-5 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 14 cells (5 g) prepared in example 3 were added thereto, and the reaction was carried out at 25℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.6g of a white solid in a yield of 95.4% and an ee value of 100%.
EXAMPLE 11 biocatalytic preparation of benzyl (2R, 4S) -2- (4-cyanophenyl) -4-hydroxypiperidine-1-carboxylate
Dissolving compound V-6 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 14 cells (5 g) prepared in example 3 were added thereto, and the reaction was carried out at 25℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged.The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.6g of a white solid in a yield of 95.4% and an ee value of 100%.
EXAMPLE 12 biocatalytic preparation of methyl 4- ((2S, 4S) -4-hydroxypiperidin-2-yl) benzoate
Compound V-7 (10 g) was dissolved in DMSO (50 ml), 0.1M phosphate buffer (250 ml) was added, NADP+ (0.2 g) was added, the WTEA mutant 14 cells (5 g) prepared in example 3 were added, the reaction was stopped when the reaction was monitored by shaking at 25℃and 220rpm, and the conversion rate of the reaction was >98% by HPLC. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.3g of a white solid with a yield of 92.4% and an ee value of 100%
Example 13 biocatalytic preparation of benzyl (S) -4-hydroxy-3, 4-dihydropyridine-1 (2H) -carboxylate
Dissolving compound V-8 (10 g) in DMSO (50 ml), adding 0.1M phosphate buffer (250 ml), adding NADP + (0.2 g), WTEA mutant 14 cells (5 g) prepared in example 3 were added thereto, and the reaction was carried out at 25℃and 220rpm with shaking, and the conversion rate was monitored by HPLC>At 98%, the reaction was terminated. Dichloromethane (100 ml) was added, centrifuged, the supernatant was collected, dichloromethane (100 ml) was partially concentrated, extracted, and centrifuged. The aqueous layer was extracted with an organic solvent (100 ml. Times.2), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give 9.6g of a white solid in a yield of 95.4% and an ee value of 100%.
SEQ ID NO:1：
MKYTVITGASSGIGYETAKLLAGKGKSLVLVARRTSELEKLRDEVKQISPDSDVILKSVDLADNQNVHDLYEGLKELDIETWINNAGFGDFDLVQDIELGKIEKMLRLNIEALTILSSLFVRDHHDIEGTTLVNISSAGGYRIVPNAVTYCATKFYVSAYTEGLAQELQKGGAKLRAKVLAPAATETEFADRSRGEAGFDYSKNVKKYHTAAEMAGFLHQLIESDAIVGIVDGETYEFELRGPLFNYAG*
SEQ ID NO:2：
ATGAAATACACCGTTATCACCGGTGCTTCTTCTGGTATCGGTTACGAAACCGCTAAACTGCTGGCTGGTAAAGGTAAATCTCTGGTTCTGGTTGCTCGTCGTACCTCTGAACTGGAAAAACTGCGTGACGAAGTTAAACAGATCTCTCCGGACTCTGACGTTATCCTGAAATCTGTTGACCTGGCTGACAACCAGAACGTTCACGACCTGTACGAAGGTCTGAAAGAACTGGACATCGAAACCTGGATCAACAACGCTGGTTTCGGTGACTTCGACCTGGTTCAGGACATCGAACTGGGTAAAATCGAAAAAATGCTGCGTCTGAACATCGAAGCTCTGACCATCCTGTCTTCTCTGTTCGTTCGTGACCACCACGACATCGAAGGTACCACCCTGGTTAACATCTCTTCTGCGGGTGGTTACCGTATCGTTCCGAACGCTGTTACCTACTGCGCTACCAAATTCTACGTTTCTGCTTACACCGAAGGTCTGGCTCAGGAACTGCAGAAAGGTGGTGCTAAACTGCGTGCTAAAGTTCTGGCTCCGGCTGCTACCGAAACCGAATTCGCTGACCGTAGCCGTGGTGAAGCTGGTTTCGACTACTCTAAAAACGTTAAAAAATACCACACCGCTGCTGAAATGGCTGGTTTCCTGCACCAGCTGATCGAATCTGACGCTATCGTTGGTATCGTTGACGGTGAAACCTACGAATTCGAACTGCGTGGTCCGCTGTTCAACTACGCTGGTTAA
Claims (10)
1. A carbonyl reductase mutant having an amino acid sequence comprising one or more of the following site mutations compared to SEQ ID NO: 1: T17V, W82L, F V, V121A, A V, S193A, Y F, N A, A of F, N138 and R142;
wherein, A138 is mutated to V or L, R142 is mutated to M, A or V;
the site mutation does not comprise any of the following groups:
(1)A190V/S193A；
(2)R142M/A190V/S193A；
(3)F88V/A138L/R142M/A190V/S193A；
(4)W82L/F88V/A138L/R142M/A190V/S193A；
(5) W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
(6)W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F；
preferably, the amino acid sequence of the carbonyl reductase mutant comprises a site mutation selected from any one of the following groups compared with SEQ ID NO: 1:
(1) R142M, R142A or a138V;
(2)F88V/A138V/R142M/A190V/S193A；
(3) W82L/V121A/A138L/R142V/A190V/S193A; and
(4)T17V/W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F/N204A。
2. an isolated nucleic acid encoding the carbonyl reductase mutant of claim 1.
3. A recombinant expression vector comprising the nucleic acid of claim 1.
4. A transformant comprising the nucleic acid of claim 2 or the recombinant expression vector of claim 3.
5. A method of producing the carbonyl reductase mutant of claim 1, comprising culturing the transformant of claim 4 under conditions suitable for expression.
6. An application of carbonyl reductase in preparing a compound shown in a formula VI,
is a single bond or a double bond;
carbon atoms with "×" denote, when chiral, S configuration, R configuration or mixtures thereof;
R 1 h, C of a shape of H, C 6 -C 10 Aryl or by R 1-1 Substituted C 6 -C 10 An aryl group; r is R 1-1 Is cyano orWherein R is 1-2 Is C 1 -C 6 An alkyl group;
R 2 is H or an amino protecting group;
the carbonyl reductase is selected from one or more of the following:
(1) The carbonyl reductase mutant of claim 1;
(2) Carbonyl reductase with the amino acid sequence shown as SEQ ID NO. 1; and
(3) An enzyme mutant comprising any one of the following sets of site mutations in the amino acid sequence as set forth in SEQ ID NO. 1:
i.A190V/S193A；
ii.R142M/A190V/S193A；
F88I/A138L/R142M/A190V/S193A or F88V/A138L/R142M/A190V/S193A;
W82L/F88V/A138L/R142M/A190V/S193A or W82L/V121A 138L/A190V/S193A/K206H;
W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
vi.W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F。
7. the use according to claim 6, characterized in that it fulfils one or more of the following conditions:
(1) The C is 6 -C 10 Aryl is phenyl;
(2) The C is 1 -C 6 Alkyl is methyl;
(3) The amino protecting group is Boc or Cbz; and
(4) The carbonyl reductase is as follows:
a. the carbonyl reductase mutant of claim 1;
b. carbonyl reductase with the amino acid sequence shown as SEQ ID NO. 1; or (b)
c. An enzyme mutant comprising any one of the following sets of site mutations in the amino acid sequence as set forth in SEQ ID NO. 1:
i.A190V/S193A；
ii.R142M/A190V/S193A；
F88I/A138L/R142M/A190V/S193A or F88V/A138L/R142M/A190V/S193A;
W82L/F88V/A138L/R142M/A190V/S193A or W82L/V121A 138L/A190V/S193A/K206H;
W82L/F88V/V121A/A138L/R142M/A190V/S193A; and
vi.W82L/F88V/V121A/A138L/R142M/A190V/S193A/Y201F。
8. the use according to claim 6, wherein,
when (when)R is a single bond 1 Is R is 1-1 Substituted C 6 -C 10 Aryl (e.g. R 1 Is-> )，R 2 Is H or an amino protecting group; when->In the case of double bonds, R 1 Is H, R 2 Is an amino protecting group;
preferably, the compound shown in the formula VI is
9. The use according to any one of claims 6-8, characterized in that the use comprises the steps of: in a liquid reaction system, in the presence of coenzyme and carbonyl reductase, carrying out a reduction reaction of a compound shown in a formula V to obtain a compound shown in a formula VI;
wherein,with carbon atoms, R 1 And R is 2 Is as defined in any one of claims 6 to 8;
the carbonyl reductase as described in claim 6 or 7.
10. Use according to claim 9, characterized in that it fulfils one or more of the following conditions:
(1) The coenzyme is a reduced coenzyme and/or an oxidized coenzyme; the oxidative coenzyme is preferably NAD+ and/or NADP+; the reduced coenzyme is preferably NADH and/or NADPH;
(2) The mass ratio of the coenzyme to the compound shown in the formula V is (0.001-0.1): 1, preferably (0.01-0.05): 1, for example 0.02:1;
(3) The carbonyl reductase is added to the reduction reaction in the form of free enzyme, immobilized enzyme, bacterial powder or bacterial form, preferably in the form of bacterial form;
(4) The mass ratio of the carbonyl reductase to the compound shown as the formula V is (0.1-5): 1, preferably (0.1-1): 1, for example, 0.5:1;
(5) The liquid reaction system comprises a buffer solution; the buffer is preferably a phosphate buffer, for example 0.1M phosphate buffer; preferably, the volume to mass ratio of the buffer solution to the compound of formula V is 5-100 mL/g, for example 25mL/g;
(6) The liquid reaction system comprises a cosolvent; the cosolvent is preferably selected from one or more of dimethyl sulfoxide, isopropanol and toluene, such as dimethyl sulfoxide; preferably, the volume to mass ratio of the cosolvent to the compound of formula V is 1-20 mL/g, for example 5mL/g;
(7) The reaction temperature of the reduction reaction is 10 ℃ to 50 ℃, preferably 20 ℃ to 35 ℃, such as 25 ℃ or 30 ℃;
(8) The reaction time of the reduction reaction is 1-48h, preferably 2-24h, more preferably 4-14h;
(9) After the reduction reaction is finished, the method further comprises post-treatment; preferably, the post-processing includes the steps of: adding an organic solvent into the liquid reaction system, and extracting; the organic solvent can be dichloromethane; and
(10) The compound shown in the formula V is
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| CN202310643036.0A CN117210426A (en) | 2023-06-01 | 2023-06-01 | Carbonyl reductase mutant and application thereof |
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