CN113249417A - Preparation method of chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound - Google Patents
Preparation method of chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound Download PDFInfo
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Abstract
The invention belongs to the technical field of biological pharmacy, and particularly relates to chiral cis-form-βAryl-β-hydroxy-αA process for producing the amino ester compound. The invention adopts carbonyl reductase to catalyze asymmetric reductionβAryl-β-oxo-α-amino propionate compounds (II) to compounds (I) comprising the steps of: preparing engineering bacteria containing carbonyl reductase gene and engineering bacteria containing glucose dehydrogenase gene; preparing resting cell suspension of two engineering bacteria; preparing cell supernatant containing carbonyl reductase and cell supernatant containing glucose dehydrogenase gene; mixing the two cell supernatants, mixing with compound (II), solvent, hydrogen donor and cofactor, and performing asymmetric reduction reaction to obtain compound (A)I) In that respect The method disclosed by the invention is environment-friendly, simple and convenient to operate, easy for industrial amplification, wide in substrate universality, high in product yield and good in industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of a chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound.
Background
The chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound is a basic skeleton widely existing in the structures of drug molecules such as thiamphenicol, florfenicol, chloramphenicol, droxidopa and eliglutacteoside, and active natural products, and the structural formula of the basic skeleton is shown as (I), wherein R in the formula1Hydrogen, methylsulfonyl, nitro, methylthio, halogen, ether, trifluoromethyl, C1-C6 alkyl or cycloalkyl; r2Is a protective group of tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, benzyl, acetyl, p-methoxyphenyl or trityl derivative; r3Is C1-C8 alkyl or cycloalkyl, mono-or poly-substituted aryl.
There are many reports on chemical methods for synthesizing the compound (I) and structural analogues thereof by dynamic reduction kinetic resolution, but these chemical methods have some defects generally, such as the use of transition metal ruthenium or rhodium is often required, the environment is seriously polluted (org.Lett.2017,19, 4339-. In addition, the biocatalytic dynamic reduction kinetic resolution method has been attracting attention due to its high stereoselectivity and environmental friendliness, but has rarely been used for synthesizing compound (I) and its structural analogs. In earlier studies, Merck reported that catalytic reduction of compound (III) by carbonyl reductase CDX-018 as shown in reaction scheme B selectively gave cis-form compound (IV) with ee > 99%, dr > 99:1, and 92% yield (org. Lett.2013,15, 1342-); recently, Zhang Fu Li and Chen Shaoxing team collaborated to report the preparation of chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compounds (Chinese patent application 201710533399.3; Eur.J.org.chem.2018,5044-5053) including key intermediate (V) for the synthesis of florfenicol by dynamic reductive kinetic resolution catalyzed by carbonyl reductase KRED-02, with high yield of reaction and excellent stereoselectivity. Despite these two reports, there is still a need for a method for preparing chiral cis- β -aryl- β -hydroxy- α -amino ester compounds by dynamic reductive kinetic resolution catalyzed by a carbonyl reductase, particularly suitable for industrial production.
Disclosure of Invention
The invention aims to provide a preparation method of a chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound with wide substrate universality, high substrate concentration, high efficiency, high stereoselectivity and high yield.
The structural formula of the chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound is shown as the following formula (I):
in the formula, R1Hydrogen, methylsulfonyl, nitro, halogen elements, ether groups and trifluoromethyl; r2Is tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, p-methoxyphenyl,A trityl derivative protecting group; r3Is C1-C8 alkyl, mono-substituted or poly-substituted aryl.
The preparation method of the chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound adopts carbonyl reductase from Bacillus thuringiensis (Bacillus thuringiensis) to catalyze and asymmetrically reduce the beta-aryl-beta-oxo-alpha-amino propionate compound (II) to generate the compound (I), and comprises the following specific steps:
(1) preparing a first engineering bacterium containing a carbonyl reductase gene and a second engineering bacterium containing a glucose dehydrogenase gene;
(2) respectively preparing resting cell suspension of two engineering bacteria;
(3) respectively preparing cell supernatant containing carbonyl reductase and cell supernatant containing glucose dehydrogenase genes;
(4) mixing the two cell supernatants, mixing with a compound (II), a solvent, a hydrogen donor and a cofactor, and performing asymmetric reduction reaction to obtain a compound (I); the reaction formula is as follows:
wherein the hydrogen donor is glucose and the cofactor is NADP+NADPH; the corresponding amino acid sequence of the carbonyl reductase (KRED-Bt for short) is shown in SEQ ID NO. 1.
In the step (1) of the invention, the engineering bacteria contain an expression vector pET-28a-KRED-Bt, and the host cell is Escherichia coli BL21(DE 3).
In the step (2), the preparation of the resting cell suspension of the two engineering bacteria comprises the following specific steps: inoculating the first engineering bacterium or the second engineering bacterium to a culture medium containing kanamycin, activating a shaking table, carrying out amplification culture until the OD600 value reaches 0.8-1.2, adding an inducer, continuing culture, centrifugally collecting cells, and carrying out resuspension by using a buffer solution to obtain a first resting cell suspension or a second resting cell suspension.
Further, the inducer is IPTG, and the concentration of the inducer is 0.05mM-0.8 mM; the culture conditions after adding the inducer are as follows: the culture temperature is 15-25 deg.C, and the culture time is 8-24 h.
Further, the buffer solution is phosphate buffer solution, and the concentration is 30-300 mM.
In step (3), the method for preparing two cell supernatants comprises the following specific steps: and carrying out ultrasonic crushing and centrifugation on the first resting cell suspension or the second resting cell suspension to obtain the first cell supernatant or the second cell supernatant.
In the step (4) of the present invention, in the mixture of the two cell supernatants, the volume ratio of the first cell supernatant to the second cell supernatant is 20:1 to 1: 2. More preferably, the volume ratio of the two is 10:1-8: 1.
In the asymmetric redox reaction of step (4) of the present invention, the concentration of the compound (II) is 1.0 to 300g/L, the cell concentration of the first resting cell suspension is 0.1g wet weight/L to 25g wet weight/L, the cell concentration of the second resting cell suspension is 0.1g wet weight/L to 25g wet weight/L, the concentration of the hydrogen donor is 5 to 750g/L, and the concentration of the cofactor is 0 to 0.5 mM.
In the whole asymmetric reduction reaction process, on one hand, the carbonyl reductase KRED-Bt catalyzes and reduces the dynamic reduction kinetic resolution of the compound (II) to generate a stereoisomerically pure compound (I); on the other hand, Glucose Dehydrogenase (GDH) oxidizes glucose to gluconolactone and consumes the oxidized coenzyme NADP+The reduced coenzyme factor NADPH, which will supply hydrogen to the substrate and is itself oxidized to the oxidized coenzyme factor NADP, is regenerated+And a closed loop of coenzyme factor consumption and regeneration is formed to promote the main reaction.
In the asymmetric reduction reaction, the reaction temperature is 20-40 ℃, and the pH value of the reaction buffer solution is 6.0-9.0. More preferably, the reaction temperature is 20 to 25 ℃ and the pH of the reaction buffer is 6.5 to 7.5. The buffer concentration was 100-250 mM.
In the asymmetric reduction reaction, the solvent is a mixed solvent of a phosphate buffer solution and a cosolvent. Wherein the cosolvent is selected from high dielectric constant solvents: dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide, an aromatic solvent: benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, nonpolar solvents: n-hexane, cyclohexane, and polar solvent: one or more of acetonitrile, ethyl acetate, dichloromethane, 1, 2-dichloroethane, methanol, ethanol, isopropanol.
The preferable solvent is a mixed solvent of dimethyl sulfoxide and phosphate buffer solution, and the volume ratio is 10: 90.
In the whole reaction process, after the reaction is carried out until a GC detection substrate is completely consumed, extracting for 3-4 times by using ethyl acetate with the same volume, combining organic phases, washing for 2 times by using saturated sodium bicarbonate, washing for 1 time by using water, washing for 1 time by using saturated salt, drying by using anhydrous sodium sulfate, and removing an organic solvent by reduced pressure distillation to obtain a target product.
Compared with the prior art, the invention has the following effects:
the engineering bacteria containing carbonyl reductase KRED-Bt and glucose dehydrogenase GDH constructed by the invention are applied to catalytic reduction of the compound (II), and a new biological preparation way is provided for the production of the compound (I). Compared with other preparation methods, the engineering bacteria containing the carbonyl reductase KRED-Bt and the glucose dehydrogenase GDH prepared by the method have the advantages of environmental friendliness, simplicity and convenience in operation, easiness in industrial amplification and the like, can obtain the compound (I) with wide substrate universality, high substrate concentration, high efficiency, high stereoselectivity and high yield, and has good industrial application prospects.
Drawings
FIG. 1 is SDS-PAGE electrophoresis picture of KRED-Bt cell supernatant protein induced and expressed by genetically engineered bacteria. M is a protein Marker, and 1 is KRED-Bt cell disruption liquid supernatant expressed by the induction of the genetic engineering bacteria.
FIG. 2 is an HPLC analysis chart of racemic methyl 2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoate (Ia).
FIG. 3 is a HPLC analysis chart of methyl (2S,3R) -2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoate (Ia).
FIG. 4 shows a nuclear magnetic spectrum of methyl (2S,3R) -2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoate (Ia).
FIG. 5 shows a nuclear magnetic carbon spectrum of methyl (2S,3R) -2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoate (Ia).
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 construction and inducible expression of genetically engineered bacteria
The expression host BL21(DE3) was transformed with the plasmid pET28 a-KRED-Bt. BL21(DE3) -pET28a-KRED-Bt is inoculated into 5mL of liquid LB test tube culture medium seeds containing kanamycin resistance, the liquid LB test tube culture medium seeds are placed in a shaking table at 37 ℃ to be activated for 8 hours, the activated culture is transferred into liquid LB shaking flask culture medium containing kanamycin resistance according to the transfer quantity of 1 percent, and the liquid LB shaking flask culture medium is subjected to constant temperature shaking culture for 3 hours in a fermentation culture medium, wherein the culture temperature is 37 ℃, and the rotation speed is 200 rpm. When the bacterial cell density is 0.6-1.0, 0.1mM IPTG (final concentration) is added, induction is carried out for 16h at 18 ℃, cells are collected by centrifugation at 8500rpm for 15min, then suspended by 250mM sodium phosphate buffer solution with pH7.0, disrupted by 55% ultrasonic power for 15min, and centrifuged to obtain cell supernatant containing pET28a-KRED-Bt, and the cell supernatant is stored in a refrigerator at 4 ℃.
EXAMPLE 2 preparation of cis- (2S,3R) -2- [ (tert-Butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoic acid methyl ester (Ia)
17.5mL of KRED-Bt cell supernatant and 5mL of GDH cell supernatant obtained in example 1 were added to a solution containing 0.5mmol of 2- [ (t-butoxycarbonyl) amino group]-3-oxo-3- [4- (methylsulfonyl) phenyl]2.5mL of methyl propionate (IIa) in dimethylsulfoxide, 1mmol of glucose as co-substrate, and a final concentration of 0.025mM NADP+The volume was adjusted to 50mL with 100mM pH7.0 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. Extracting with ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, filtering, and collecting filtrateDry as a white solid in 91% yield by chiral HPLC: (
IB, cyclohexane/ethanol is mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ7.74(d,J＝8.2Hz,2H),7.51(d,J＝8.3Hz,2H),5.43(d,J＝9.6Hz,1H),5.34(s,1H),4.48(dd,J＝9.6,1.8Hz,1H),3.76(s,3H),2.95(s,3H),1.23(s,9H)；13C NMR(100MHz,CDCl3)δ170.8,155.6,146.9,139.3,127.2,127.1,80.2,72.9,59.1,52.8,44.5,28.1。
EXAMPLE 3 preparation of cis-2- [ (tert-Butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methoxy) phenyl ] propionic acid methyl ester (Ib)
17.5mL of KRED-Bt cell supernatant and 5mL of GDH cell supernatant obtained in example 1 were added to a solution containing 2.0mmol of 2- [ (t-butoxycarbonyl) amino group]-3-oxo-3- [4- (methoxy) phenyl]5mL of methyl propionate (IIb) in methanol, 2mmol of glucose as cosubstrate, and a final concentration of 0.125mM NADP+The volume was adjusted to 50mL with 100mM pH7.0 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 70%. Chiral HPLC (
IC, cyclohexane/isopropanol as mobile phase) analysis, ee value is more than or equal to 99 percent, and dr value is 67: 1.1H NMR(400MHz,CDCl3)δ7.24(d,J＝8.6Hz,2H),6.83(d,J＝8.6Hz,2H),5.44(d,J＝9.1Hz,1H),5.11(s,1H),4.44(d,J＝9.2Hz,1H),3.75(s,3H),3.69(s,3H),1.31(s,9H)；13C NMR(100MHz,CDCl3)δ171.6,159.2,155.7,132.1,127.3,113.7,80.0,73.4,59.6,55.3,52.5,28.2。
EXAMPLE 4 preparation of cis-2- [ (tert-Butoxycarbonyl) amino ] -3-hydroxy-3- (4-chlorophenyl) propanoic acid methyl ester (Ic)
20mL of KRED-Bt cell supernatant and 7.5mL of GDH cell supernatant obtained in example 1 were added to a solution containing 1.5mmol of 2- [ (t-butoxycarbonyl) amino group]-3-oxo-3- [ 4-chlorophenyl group]Methyl propionate (IIc) in N, N-dimethylformamide 3mL, 1.5mmol of glucose as cosubstrate, and NADP to a final concentration of 0.1mM+The volume was adjusted to 50mL with 100mM pH 7.5 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 76%. Chiral HPLC (
IC, cyclohexane/isopropanol as mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ7.29(s,4H),5.40(d,J＝9.1Hz,1H),5.19(s,1H),4.48(d,J＝9.2Hz,1H),3.74(s,3H),1.30(s,9H)。
EXAMPLE 5 preparation of cis-2- [ (benzyloxycarbonyl) amino ] -3-hydroxy-3- [4- (methyl) phenyl ] propionic acid methyl ester (Id)
15mL of KRED-Bt cell supernatant and 5mL of GDH cell supernatant obtained in example 1 were added to a solution containing 0.5mmol of 2- [ (benzyloxycarbonyl) amino group]-3-oxo-3- [4- (methyl) phenyl]2mL of methyl propionate (IId) in isopropanol, 1.5mmol of glucose as co-substrate, final concentration of 0.025mM NADP+The volume was adjusted to 50mL with 100mM pH 7.5 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 80%. Chiral HPLC (
IB, cyclohexane/isopropanol as mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ7.33-7.30(m,3H),7.23(d,J＝8.0Hz,4H),7.11(d,J＝8.0Hz,2H),5.93(d,J＝9.3Hz,1H),5.23(d,J＝2.6Hz,1H),4.96(s,2H),4.59(dd,J＝9.3,2.9Hz,1H),3.71(s,3H),2.34(s,3H)。
EXAMPLE 6 preparation of tert-butyl cis-2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (methylsulfonyl) phenyl ] propanoate (Ie)
17.5mL of KRED-Bt cell supernatant and 7.5mL of GDH cell supernatant obtained in example 1 were added with 2- [ (t-butyloxycarbonyl) amino group in an amount of 5mmol]-3-oxo-3- [4- (methylsulfonyl) phenyl]10mL of a solution of tert-butyl propionate (IIe) in isopropanol, 5mmol of glucose as cosubstrate, and a final concentration of 0.5mM NADP+The volume was adjusted to 50mL with 100mM pH 6.5 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be pale yellow solid, with a yield of 73%. Chiral HPLC (
IB, cyclohexane/isopropanol as mobile phase), ee value is more than or equal to 99 percent, and dr value is 32: 1.1H NMR(400MHz,CDCl3)δ7.83(d,J＝8.1Hz,2H),7.55(d,J＝8.2Hz,2H),5.31(d,J＝9.6Hz,1H),5.28(s,1H),4.42(d,J＝9.5Hz,1H),3.49(s,1H),3.00(s,3H),1.46(s,9H),1.28(s,9H)。
EXAMPLE 7 preparation of benzyl cis-2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (trifluoromethyl) phenyl ] propionate (If)
17.5mL of KRED-Bt cell supernatant and 7.5mL of GDH cell supernatant obtained in example 1 were added with 2- [ (t-butyloxycarbonyl) amino group in an amount of 1mmol]-3-oxo-3- [4- (trifluoromethyl) phenyl]Benzyl propionate (IIf) in 5mL of isopropanol, 1.5mmol of co-substrate glucose, final concentration of 0.2mM NADP+The volume was adjusted to 50mL with 100mM pH7.0 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a yellow solid, with a yield of 88%. Chiral HPLC (
IB, cyclohexane/isopropanol as mobile phase), ee value is more than or equal to 99 percent, and dr value is 20: 1.1H NMR(400MHz,CDCl3)δ7.54(d,2H),7.39(s,1H),7.33(d,4H),7.32(d,1H),7.21(d,2H),5.34(s,1H),5.22(d,1H),5.17(s,2H),4.48(d,1H),1.46(s,9H)。
EXAMPLE 8 preparation of cyclohexyl cis-2- [ (acetyl) amino ] -3-hydroxy-3-phenylpropionate (Ig)
17.5mL of KRED-Bt cell supernatant and 7.5mL of GDH cell supernatant obtained in example 1 were added with 5mmol of 2- [ (acetyl) amino group]5mL of a solution of cyclohexyl (IIg) -3-oxo-3-phenylpropionate in isopropanol, 1.5mmol of glucose as cosubstrate and a final concentration of 0.2mM NADP+The volume was adjusted to 50mL with 100mM pH7.0 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 91%. Chiral HPLC (
IC, cyclohexane/isopropanol as mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ8.32(s,1H),7.25(d,3H),7.32(d,2H),5.22(d,1H),5.17(s,1H),4.61(m,1H),4.48(d,1H),1.84(s,3H),1.55-1.43(m,10H)。
EXAMPLE 9 preparation of cis-2- [ (benzyl) amino ] -3-hydroxy-3- [4- (methylthio) phenyl ] propanoic acid ethyl ester (Ih)
15mL of KRED-Bt cell supernatant and 5mL of GDH cell supernatant obtained in example 1 were added to a mixture containing 3mmol of 2- [ (benzyl) amino group]-3-oxo-3- [4- (methylthio) phenyl]Ethyl propionate (IIh) in dimethylsulfoxide 5mL, 3.5mmol of co-substrate glucose, NADP to a final concentration of 0.025mM+The volume was adjusted to 50mL with 100mM pH7.0 sodium phosphate buffer, and the reaction was stirred in a constant temperature water bath at 30 ℃ until the reaction was complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 84%. Chiral HPLC (
IC, cyclohexane/isopropanol as mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ7.35-7.29(m,7H),7.18(m,2H),6.36(s,1H),5.17(s,1H),5.02(d,1H),4.11(m,2H),3.84-3.82(m,3H),2.38(s,3H),1.21(t,3H)。
EXAMPLE 10 preparation of cis-tert-butyl-2- [ (tert-butoxycarbonyl) amino ] -3-hydroxy-3- [4- (nitro) phenyl ] propanoate (Ii)
20mL of KRED-Bt cell supernatant and 10mL of GDH cell supernatant obtained in example 1 were added to a mixture containing 6mmol of 2- [ (t-butyloxycarbonyl) amino group]-3-oxo-3- [4- (nitro) phenyl]5mL of a solution of tert-butyl propionate (IIi) in dimethyl sulfoxide, 8mmol of glucose as cosubstrate, and a final concentration of 0.1mM NADP+The volume was adjusted with 100mM pH7.0 sodium phosphate bufferTo 50mL, and then placing the mixture in a constant-temperature water bath at 30 ℃ to stir the reaction until the reaction is complete. After the reaction was completed, extraction was performed with ethyl acetate, and organic layers were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was spin-dried to be a white solid, with a yield of 85%. Chiral HPLC (
IC, cyclohexane/isopropanol as mobile phase), the ee value is more than or equal to 99 percent, and the dr value is more than or equal to 99: 1.1H NMR(400MHz,CDCl3)δ8.07(d,2H),7.57(d,2H),7.33(s,1H),5.17(s,1H),5.22(d,1H),4.48(m,2H),1.42(s,9H)。
The above description is only for the purpose of illustrating the present invention and the technical idea and features thereof, and it is intended to enable those skilled in the art to understand the present invention and implement the present invention accordingly, and not to limit the protection scope of the present invention accordingly. Any modification, equivalent replacement, improvement and the like made in accordance with the spirit of the present invention shall be covered within the protection scope of the present invention.
Sequence listing
<110> university of Compound Dan
<120> preparation method of chiral cis-beta-aryl-beta-hydroxy-alpha-amino ester compound
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<170> SIPOSequenceListing 1.0
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<211> 253
<212> PRT
<213> KRED-Bt
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Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu
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Thr Ala Leu Ala Phe Ala Ala Arg Gly Lys Asn Leu Ile Leu Ala Ala
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Arg Arg Thr Glu Glu Met Glu Lys Leu Lys Ser Lys Val Ala Asp Ile
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Asn Ala Asp Leu Asp Val Val Ile Arg Thr Val Asp Leu Ser Val Thr
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Ala Asn Val Tyr Glu Phe Tyr Glu Ser Leu Gln Asp Tyr Gln Ile Glu
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Thr Trp Ile Asn Asn Ala Gly Phe Gly Asn Phe Ala Ser Val Gly Glu
85 90 95
Gln Asn Leu Asn Lys Ile Glu Thr Met Leu His Leu Asn Ile Glu Ala
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Leu Thr Val Leu Ser Ser Leu Tyr Val Arg Asp Tyr Gly Asp Val Glu
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Gly Thr Gln Ile Ile Asn Ile Ser Ser Gly Gly Gly Tyr Leu Ile Val
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Ala Asp Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Phe
145 150 155 160
Thr Glu Gly Leu Ala Gln Glu Leu Lys Glu Lys Gly Ala Thr Met Gln
165 170 175
Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ile Lys Arg
180 185 190
Ser Leu Asp Thr Asp Glu Phe Thr Tyr Glu Glu Val Val Pro Lys Phe
195 200 205
His Thr Ala Lys Glu Met Ala Gly Phe Met Leu Asp Leu His Asp Ser
210 215 220
Glu Lys Val Val Gly Ile Val Asp Gly Glu Thr Tyr Asp Phe Glu Leu
225 230 235 240
Arg Asp Pro Ile Tyr Pro Tyr Val Thr Arg Thr Arg Lys
245 250
Claims (10)
1. Chiral cis-form-βAryl-β-hydroxy-αPreparation method of-amino ester compound by catalyzing asymmetric reduction with carbonyl reductaseβAryl-β-oxo-α-amino propionate compound (II) to compound (I), comprising the steps of:
(1) preparing a first engineering bacterium containing a carbonyl reductase gene and a second engineering bacterium containing a glucose dehydrogenase gene;
(2) respectively preparing resting cell suspension of two engineering bacteria;
(3) respectively preparing cell supernatant containing carbonyl reductase and cell supernatant containing glucose dehydrogenase genes;
(4) mixing the two cell supernatants, mixing with a compound (II), a solvent, a hydrogen donor and a cofactor, and performing asymmetric reduction reaction to obtain a compound (I); the reaction formula is as follows:
wherein the hydrogen donor is glucose and the cofactor is NADP+NADPH; the corresponding amino acid sequence of the carbonyl reductase is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the engineered bacterium comprises the expression vector pET-28a-KRED-Bt, and the host cell is Escherichia coli BL21(DE 3).
3. The method according to claim 1, wherein the preparation of the resting cell suspension of the two engineering bacteria in the step (2) comprises the following steps: inoculating the first engineering bacterium or the second engineering bacterium to a culture medium containing kanamycin, activating a shaking table, carrying out amplification culture until the OD600 value reaches 0.8-1.2, adding an inducer, continuing culture, centrifugally collecting cells, and carrying out resuspension by using a buffer solution to obtain a first resting cell suspension or a second resting cell suspension.
4. The method of claim 3, wherein the inducer is IPTG and the concentration of the inducer is 0.05mM to 0.8 mM; the culture conditions after adding the inducer are as follows: the culture temperature is 15-25 ℃, and the culture time is 8-24 h; the buffer solution is phosphate buffer solution with the concentration of 30-300 mM.
5. The method according to claim 4, wherein the two cell supernatants are prepared in step (3) by: and carrying out ultrasonic crushing and centrifugation on the first resting cell suspension or the second resting cell suspension to obtain the first cell supernatant or the second cell supernatant.
6. The method according to claim 5, wherein the volume ratio of the first cell supernatant to the second cell supernatant in the mixture of the two cell supernatants in the step (4) is 20:1 to 1: 2.
7. The method according to claim 6, wherein in the asymmetric redox reaction in the step (4), the concentration of the compound (II) is 1.0 to 300g/L, the cell concentration of the first resting cell suspension is 0.1g wet weight/L to 25g wet weight/L, the cell concentration of the second resting cell suspension is 0.1g wet weight/L to 25g wet weight/L, the concentration of the hydrogen donor is 5 to 750g/L, and the concentration of the cofactor is 0 to 0.5 mM.
8. The method according to claim 7, wherein in the asymmetric redox reaction of the step (4), the reaction temperature is 20 to 40 ℃ and the pH of the reaction buffer is 6.0 to 9.0; the buffer concentration was 100-250 mM.
9. The method according to claim 8, wherein in the asymmetric reduction reaction in the step (4), the solvent is a mixed solvent of a phosphate buffer and a cosolvent; wherein the cosolvent is selected from high dielectric constant solvent, aromatic solvent, nonpolar solvent and polar solvent; the high dielectric constant solvent is dimethyl sulfoxide, N-dimethylformamide or N, N-dimethylacetamide, the aromatic solvent is benzene, toluene, ethylbenzene, chlorobenzene or bromobenzene, the nonpolar solvent is N-hexane or cyclohexane, and the polar solvent is acetonitrile, ethyl acetate, dichloromethane, 1, 2-dichloroethane, methanol, ethanol or isopropanol.
10. The preparation method according to any one of claims 1 to 9, wherein in the whole reaction process, after the reaction is completed until a GC detection substrate is completely consumed, the reaction product is extracted 3 to 4 times by using ethyl acetate with the same volume, organic phases are combined, washed 2 times by using saturated sodium bicarbonate, 1 time by using water, washed 1 time by using saturated salt solution, dried by anhydrous sodium sulfate, and subjected to reduced pressure distillation to remove the organic solvent, so that the target product is obtained.
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| CN102618590A (en) * | 2012-05-07 | 2012-08-01 | 江南大学 | Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase |
| CN109207531A (en) * | 2017-07-03 | 2019-01-15 | 上海医药工业研究院 | The biological preparation method of Thiamphenicol and Florfenicol key intermediate |
| CN111172124A (en) * | 2020-02-26 | 2020-05-19 | 复旦大学 | Carbonyl reductase mutant and application thereof in preparation of (R) -4-chloro-3-hydroxy-butyrate |
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| CN109207531A (en) * | 2017-07-03 | 2019-01-15 | 上海医药工业研究院 | The biological preparation method of Thiamphenicol and Florfenicol key intermediate |
| CN111172124A (en) * | 2020-02-26 | 2020-05-19 | 复旦大学 | Carbonyl reductase mutant and application thereof in preparation of (R) -4-chloro-3-hydroxy-butyrate |
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| CN113789353A (en) * | 2021-09-25 | 2021-12-14 | 复旦大学 | Preparation method of (2S,3S) -2-chloro-3-hydroxy ester compound |
| CN113789353B (en) * | 2021-09-25 | 2024-03-08 | 复旦大学 | Preparation method of (2S, 3S) -2-chloro-3-hydroxy ester compound |
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