A process for the preparation of chiral aryl-substituted alkenyl beta-hydroxy esters

Abstract

The application discloses a method for preparing chiral aryl-substituted alkenyl beta-hydroxy ester. The method constructs a biological catalysis reaction system in a phosphate buffer solution, and comprises a substrate shown in formula B, coenzyme NADP + , glucose, glucose dehydrogenase, and ketoreductase, and performs a stereoselective reduction reaction at 20-40 DEG C, so as to convert the substrate shown in formula B into chiral aryl-substituted alkenyl beta-hydroxy ester shown in formula A. The ketoreductase is selected from RasADH or RasADH-E189D. By using the method, green and efficient synthesis of chiral aryl-substituted alkenyl beta-hydroxy ester can be realized, and the method has important application value in the field of chiral drug synthesis.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and biocatalysis, specifically relating to a chiral aryl-substituted alkenyl β-hydroxy ester, its preparation method, and its application in the synthesis of pitavastatin intermediates. The method involves the asymmetric reduction of aryl-substituted alkenyl 3,5-dicarbonyl esters catalyzed by ketone reductase. In particular, this invention relates to ketone reductase mutants RasADH-E189D and SSCR-QA suitable for this method. Background Technology

[0002] Multifunctional chiral β-hydroxy esters are important structural units widely found in bioactive molecules, and also chiral building blocks with significant applications in asymmetric synthesis. For example, the structures of compounds such as the lipid-lowering drug pitavastatin, the antitumor lead compound epothilone A, and the macrolide immunosuppressant tacrolimus (FK506) all contain key chiral β-hydroxy ester fragments. Some of these compounds are shown below.

[0003]

[0004] Therefore, developing efficient, simple, and highly selective methods for the synthesis of chiral β-hydroxy esters is of great significance for organic synthesis, medicinal chemistry, and the preparation of related fine chemicals. Among these, aryl-substituted alkenyl β-hydroxy esters are particularly noteworthy because they possess both a well-defined chiral hydroxyl center and a conjugated unsaturated group that can be further derivatized. This bifunctional skeleton provides opportunities for subsequent cyclization, functional group transformation, and stereodispersive synthesis, enabling the more concise and efficient acquisition of highly functionalized complex molecular structures.

[0005] Asymmetric reduction of carbonyl compounds is one of the most direct and widely used methods for preparing chiral alcohols. However, in traditional chemical synthesis, for substrates containing multiple carbonyl functional groups, the competitive recognition of catalysts by different functional groups and the differences in the reactivity of each carbonyl group often weaken the reaction selectivity. Therefore, the target transformation usually needs to achieve chemoselectivity, regioselectivity, and stereoselectivity simultaneously in a single operation. Although selectivity can sometimes be improved by adjusting the solvent, temperature, and utilizing steric hindrance differences, these empirical strategies are often substrate-dependent, lack universality, and many methods still require expensive metal catalysts or relatively harsh reaction conditions.

[0006] In contrast, biocatalysis can achieve complex transformations with excellent stereoselectivity, regioselectivity, and chemoselectivity under mild conditions. For example, ketone reductases (KREDs) have been widely used to prepare enantiomeric alcohols, including chiral β-hydroxy esters. However, existing reports typically rely on pre-reduction, protection, or shielding of adjacent carbonyl groups before the reaction, or pre-functionalization of the substrate, to improve site selectivity. Nevertheless, methods for preparing chiral β-hydroxy esters directly from polycarbonyl compounds with well-defined site control without additional substrate pretreatment remain underdeveloped in both synthetic organic chemistry and biocatalysis.

[0007] Meanwhile, compounds with alkenyl units and multicarbonyl skeletons are an important class of synthetic intermediates, widely present in various bioactive molecules, and therefore have significant research and application value. However, these compounds have complex structures and often contain multiple potential reaction sites within the molecule, making it challenging to achieve highly selective asymmetric reduction of specific carbonyl sites without destroying the alkenyl structure. This is especially true for aryl-substituted alkenyl 3,5-dicarbonyl esters, which simultaneously possess multicarbonyl functional groups and conjugated alkenyl structures, making it difficult for existing methods to simultaneously achieve chemoselectivity, regioselectivity, and stereoselectivity. Therefore, there is an urgent need to develop a method that can directly achieve efficient asymmetric reduction of these substrates and prepare chiral β-hydroxy esters without requiring pre-reduction, protection, or pre-functionalization of the substrate. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a highly efficient ketone reductase for reducing aryl-substituted alkenyl 3,5-dicarbonyl esters, a method for preparing chiral aryl-substituted alkenyl β-hydroxy esters, and its application in the synthesis of pitavastatin intermediates. Through screening of ketone reductases and semi-rational enzyme design, a universal biocatalytic platform for the efficient asymmetric reduction of aryl-substituted alkenyl 3,5-dicarbonyl esters is constructed. Pitavastatin drug intermediates are obtained efficiently via a chemical-enzymatic method.

[0009] This invention provides the following technical solutions:

[0010] This invention provides a ketone reductase selected from RasADH, RasADH-E189D, KmCR2, SSCR-QA, or SsCR. This ketone reductase can efficiently and stereoselectively catalyze the asymmetric reduction reaction of aryl-substituted alkenyl 3,5-dicarbonyl esters, thereby preparing high-purity chiral aryl-substituted alkenyl β-hydroxy esters, which has important application value in the field of chiral drug synthesis.

[0011] The amino acid sequence of the ketone reductase RasADH is shown in SEQ ID NO.2. The amino acid sequence of the ketone reductase RasADH-E189D is shown in SEQ ID NO.4. The amino acid sequence of the ketone reductase SSCR-QA is shown in SEQ ID NO.8.

[0012] A gene encoding the ketone reductase RasADH. Specifically, the gene sequence encoding RasADH is shown in SEQ ID NO. 1.

[0013] Another encoding gene encodes the ketone reductase RasADH-E189D. Specifically, the gene sequence encoding RasADH-E189D is shown in SEQ ID NO.3.

[0014] Another encoding gene encodes the ketone reductase SSCR-QA. Specifically, the gene sequence encoding SSCR-QA is shown in SEQ ID NO.7.

[0015] A recombinant expression vector comprising the aforementioned coding gene.

[0016] A host cell comprising the recombinant expression vector, or having the coding gene integrated into its genome.

[0017] The structure of the chiral aryl-substituted alkenyl β-hydroxy ester is shown in Formula A:

[0018]

[0019] Where R2 is methyl, ethyl, or tert-butyl, and R3 is phenyl, methoxyphenyl, benzyl, fluorophenyl, chlorophenyl, bromophenyl, cyanophenyl, trifluoromethylphenyl, pyridyl, naphthyl, or... .

[0020] The methoxyphenyl can be p-methoxyphenyl, m-methoxyphenyl, or o-methoxyphenyl.

[0021] According to specific embodiments of the present invention, the benzyl group is p-benzyl; the fluorophenyl group is p-fluorophenyl; the chlorophenyl group is p-chlorophenyl; the bromophenyl group is p-bromophenyl; the p-cyanophenyl group is cyanophenyl; and the trifluoromethylphenyl group is m-trifluoromethylphenyl.

[0022] Specifically, the structural formula of the chiral aryl-substituted alkenyl β-hydroxy ester is shown below:

[0023]

[0024] .

[0025] A method for preparing a chiral aryl-substituted alkenyl β-hydroxy ester of formula A includes the following steps:

[0026] A biocatalytic reaction system comprising the following components was constructed in phosphate buffer: (a) the substrate shown in Formula B; (b) the coenzyme NADP. + (c) Glucose; (d) Glucose dehydrogenase (GDH); and (e) Ketoreductase;

[0027] The reaction system was subjected to a stereoselective reduction reaction at 20°C to 40°C, thereby converting the substrate shown in Formula B into a chiral aryl-substituted alkenyl β-hydroxy ester as shown in Formula A.

[0028] Wherein, the ketone reductase is RasADH, RasADH-E189D, KmCR2, SSCR-QA, or SsCR; Formula B is shown below:

[0029] ,

[0030] Where R2 is methyl, ethyl, or tert-butyl, and R3 is phenyl, methoxyphenyl, benzyl, fluorophenyl, chlorophenyl, bromophenyl, cyanophenyl, trifluoromethylphenyl, pyridyl, naphthyl, or... .

[0031] Specifically, the glucose dehydrogenase and ketone reductase are added in the form of lyophilized powder, and the mass ratio of the two is 1:1 to 1:3.

[0032] Specifically, the reduction reaction is carried out at 30°C under shaking conditions for 24 hours.

[0033] Preferably, the glucose and NADP + The molar ratio of the substrate shown in Formula B is 20:1:10, the amount of glucose dehydrogenase lyophilized powder used is 80-90 mg / mmol substrate, and the amount of ketone reductase lyophilized powder used is 160-170 mg / mmol substrate.

[0034] Furthermore, after the reduction reaction is completed, the reaction mixture is extracted with ethyl acetate, the organic phases are combined, and the mixture is concentrated to obtain compound A.

[0035] Specifically, the structural formula of the substrate shown in Equation B is as follows:

[0036]

[0037] .

[0038] A method for synthesizing a pitavastatin intermediate includes the following steps:

[0039] Using 1o as a substrate, the product R-2o was obtained by catalytic reaction of ketone reductase SsCR or SSCR-QA, and then R-2o was chemically induced to reduce to pitavastatin intermediate 3o.

[0040] The structural formulas of R-2o and 3o are as follows:

[0041]

[0042] Specifically, the steps of chemically induced reduction of R-2o to pitavastatin intermediate 3o include:

[0043] Under inert gas protection and at a temperature of -78±5℃, the compound shown in R-2o was dissolved in a mixed solvent of ethers and alcohols, and diethylmethoxyborane was added; then, while maintaining the temperature at -78±5℃, sodium borohydride was added to the reaction system; the resulting reaction mixture was quenched and extracted, the combined organic phases were dried and concentrated, and the crude product was purified by silica gel column chromatography to obtain the compound of formula 3o.

[0044] Beneficial effects of this invention:

[0045] 1. This invention constructs a biocatalytic platform centered on ketone reductase (KRED), achieving highly selective asymmetric reduction of the C3 carbonyl group in aryl-substituted alkenyl 3,5-dicarbonyl esters under multifunctional group competition, and establishing an efficient biosynthetic method for chiral aryl-substituted alkenyl β-hydroxy esters. Specifically, RasADH-E189D catalyzes the generation of (S)-configuration products with a conversion rate of 89%-99% and an enantioselectivity of 93%-99%; while KmCR2, SSCR-QA, or SsCR catalyze the generation of the corresponding (R)-configuration products with a maximum conversion rate of 97% and an enantioselectivity greater than 99%.

[0046] 2. The key intermediate R-2o of pitavastatin was prepared by SsCR catalysis, and the pitavastatin intermediate 3o was successfully obtained by a subsequent chemically induced reduction reaction. The method showed good diastereoselectivity with a dr value of 90:10. Attached Figure Description

[0047] Figure 1 This is the active site of RasADH.

[0048] Figure 2 The results are from Rosetta simulations of point mutations.

[0049] Figure 3 A performance comparison between the RasADH mutant and RasADH-WT.

[0050] Figure 4The catalytic effect of the biocatalytic platform on substrates of aryl-substituted alkenyl 3,5-dicarbonyl esters is evaluated.

[0051] Figure 5 This is the H spectrum of compound 1a.

[0052] Figure 6 This is the C spectrum of compound 1a.

[0053] Figure 7 The image shows the HRMS spectrum of compound 1a.

[0054] Figure 8 This is the H spectrum of compound 2a.

[0055] Figure 9 This is the C spectrum of compound 2a.

[0056] Figure 10 The image shows the HRMS spectrum of compound 2a.

[0057] Figure 11 This is the 1H spectrum of compound 1o.

[0058] Figure 12 This is the C spectrum of compound 1o.

[0059] Figure 13 The image shows the F spectrum of compound 1o.

[0060] Figure 14 The image shows the HRMS spectrum of compound 1o.

[0061] Figure 15 This is the HCl spectrum of compound 2o.

[0062] Figure 16 This is the C spectrum of compound 2o.

[0063] Figure 17 This is the F spectrum of compound 2o.

[0064] Figure 18 The image shows the HRMS spectrum of compound 2o.

[0065] Figure 19 This is the HCl spectrum of compound 3o.

[0066] Figure 20 The image shows the HRMS spectrum of compound 3o. Detailed Implementation

[0067] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0068] Example 1

[0069] I. Screening of ketone reductases

[0070] Using 1a as a template substrate, ketone reductases were initially screened to obtain chiral aryl-substituted alkenyl β-hydroxy ester 2a via divergent synthesis. The synthetic route is as follows:

[0071]

[0072] Add 5 mg of GDH lyophilized powder, 10 mg of KREDS lyophilized powder, 0.5 mL of anhydrous glucose (0.12 mmol, 2 equiv.) in phosphate-buffered saline (PBS) to a 50 mL centrifuge tube, and add NADP. + A solution of compound 1a (0.006 mmol, 0.1 equiv.) in PBS (0.5 mL) was added, followed by PBS (3.5 mL), and then a solution of compound 1a (0.06 mmol, 1 equiv.) in methanol (0.5 mL). The solution was incubated at 30°C. o The reaction was carried out at 200 rpm for 24 h under constant temperature and shaking. Afterwards, the mixture was extracted with ethyl acetate (10 mL × 3), and the solvent was removed by rotary evaporation. The conversion (Conv.) and enantioselectivity (ee value) were determined by high-performance liquid chromatography (HPLC) and chiral high-performance liquid chromatography (CHIRAL HPLC). The conversion was calculated as follows: Conversion = 100% - (Residual substrate amount / Substrate feed amount × 100%).

[0073] As shown in Table 1, based on the screening results, RasADH was finally identified as the dominant enzyme for catalyzing the production of the S-configuration product; KmCR2, SsCR, and SSCR-QA were identified as the dominant enzymes for catalyzing the production of the R-configuration product.

[0074] Table 1

[0075]

[0076] a HPLC was used to determine the conversion rate. b The ee value was confirmed using CHIRAL HPLC. c nd indicates that the target product was not detected.

[0077] II. Molecular docking and Rosetta simulated point mutation

[0078] RasADH (PDB ID: 4BMS) was obtained from the Protein Database (PDB). The protein structure was preprocessed using the AutoDock tool: water molecules were removed from the crystal structure, and hydrogen atoms were added to complete the protonation state. A docking grid was set centered on the NADPH binding site, and molecular docking was performed using template substrate 1a as the ligand. Flexible docking was performed using AutoDockVina 1.1.2, with each system independently repeated 50 times. The conformation with the lowest predicted binding free energy (lowest score) was used as the final docking result for subsequent analysis. Amino acid residues within a 5 Å range of the docking pocket were scanned using Discovery Studio 2024 software, and Rosetta was used to simulate point mutations and calculate the changes in binding energy.

[0079] Figure 1 This is for amino acid residues within a 5 Å range of the docking pocket, used for subsequent Rosetta-simulated point mutations; Figure 2 The smaller the calculated ΔE value, the more favorable the mutation, according to Rosetta simulation point mutation results.

[0080] III. Site-directed mutation

[0081] Using a plasmid containing the wild-type RasADH (RasADH-WT) gene as a template, a RasADH mutant was constructed using site-directed PCR mutagenesis. The total volume of the PCR reaction system was 50 μL, containing 50 ng of template plasmid, 1 μL of forward primer (F), 1 μL of reverse primer (R), 25 μL of PrimeSTAR® Max DNA Polymerase Ver. 2, and ddH2O to a final volume of 50 μL. The PCR amplification program was as follows: pre-denaturation at 98 °C for 5 min; followed by 20 cycles of 98 °C for 30 s, 60 °C for 30 s, 68 °C for 90 s, and a final extension at 68 °C for 10 min.

[0082] After PCR amplification, 1 μL of QuickCut Dpn I and 5 μL of 10× QuickCutBuffer were added to the reaction system, and the mixture was incubated at 37 °C for 5 min to digest the methylated parent plasmid. 10 μL of the digestion product was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing kanamycin (50 μg / mL) for selection. Single colonies were picked, plasmids were extracted, and Sanger sequencing was performed to verify the mutation sites. Strains with correct sequencing results were selected for induced expression and protein purification to obtain high-purity RasADH mutant protein. The RasADH mutant screening method was the same as for ketone reductase screening, measuring substrate 1a conversion rate and ee value to evaluate the performance of the RasADH mutant.

[0083] like Figure 3 As shown, compared with RasADH-WT, the optimal mutant RasADH-E189D achieved a transformation rate of 99%, an increase of 1.04 times; and an ee value of 99%, an increase of 1.14 times. The primer pair sequences RasADH-E189D-F / R used to construct the RasADH-E189D mutant are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0084] Based on the preliminary screening results, the R-type selectivity and transformation rate of the SSCR enzyme (PDB ID: 1Y1P) still need to be improved. Referring to the RasADH site-directed mutagenesis method, a two-step PCR method was used to construct the SSCR double mutant SSCR-QA. First, using a plasmid containing the wild-type SSCR (SSCR-WT) gene as a template, the SSCR-W226Q single mutant plasmid was obtained by full-plasmid PCR using primers SSCR-W226Q-F / R (sequences shown in SEQ ID NO.11 and SEQ ID NO.12). Then, using this plasmid as a template, a second round of PCR was performed using primers SSCR-T134A-F / R (sequences shown in SEQ ID NO.13 and SEQ ID NO.14), and sequencing verification yielded a recombinant plasmid containing the SSCR-QA double mutant gene. The verified engineered bacteria were induced to express and purified to obtain the SSCR-QA enzyme. Performance tests show that, compared with SSCR-WT, SSCR-QA improves substrate 1a conversion rate to 45% and ee value to >99% (R-type).

[0085] IV. Substrate Applicability Study

[0086] 1. Substrate synthesis methods

[0087] The synthesis methods for 1a, 1f, 1j, 1k, and 1l are as follows:

[0088]

[0089] Substrate synthesis route 1

[0090] In the series of compounds of the above-mentioned substrate synthesis route one, R1 is hydrogen, para-fluorine or meta-trifluoromethyl, and R2 is methyl, ethyl or tert-butyl.

[0091] Taking 1a as an example: Under nitrogen protection, trans-cinnamoyl chloride (S1) (30 mmol, 1.0 equiv.) and N,O-dimethylhydroxylamine hydrochloride (S2) (39 mmol, 1.3 equiv.) were dissolved in dry chloroform (165 mL) and cooled to 0 °C. Pyridine (69 mmol, 2.3 equiv.) was slowly added, and the reaction system was then brought to room temperature and stirred for 2 h. After the reaction was completed, saturated sodium chloride aqueous solution was added for quenching. After separation, the aqueous phase was extracted with dichloromethane (CH2Cl2, 200 mL × 3). The organic phases were combined and concentrated. The concentrate was dissolved in water and adjusted to a weakly alkaline state (pH≈8-9), and then extracted with CH2Cl2 (100 mL × 3). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude Weinreb amide S3; this crude product was used directly in the next reaction without further purification.

[0092] Under nitrogen protection, methyl acetoacetate (90.9 mmol, 3.0 equiv.) was added to a suspension of 60% NaH (90.9 mmol, 3.0 equiv.) and dry THF (200 mL) stirred at 0 °C. The mixture was stirred at 0 °C for 20 min and then cooled to -10 °C. A 2.5 M n-butyllithium solution in n-hexane (90.9 mmol, 3.0 equiv.) was added, and the resulting mixture was stirred at -10 °C for another 20 min, followed by cooling to -78 °C to generate acetoacetate dianion in situ. The crude product S3 (30.3 mmol, 1.0 equiv.) obtained above was added to the dianion solution at -78 °C, and the mixture was stirred at -78 °C until the reaction was complete (TLC monitoring showed complete disappearance of S3). The reaction was quenched with dilute hydrochloric acid, and the aqueous phase was adjusted to moderate acidity. Extracted with ethyl acetate (EA), the combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target product 1a.

[0093] The synthesis methods for 1b, 1c, 1d, 1e, 1g, 1h, 1i, 1m, and 1n are as follows:

[0094]

[0095] Substrate synthesis route 2

[0096]

[0097] 1m synthesis route

[0098]

[0099] 1n synthesis route

[0100] In the series of compounds of the above-mentioned substrate synthesis route two, R1 is ortho-methoxy, ortho-methoxy, para-methoxy, para-methyl, para-chlorine, para-bromine, or para-cyano, and R2 is methyl.

[0101] The synthesis method is taken as an example of 1b.

[0102] (E)-3-(2-methoxyphenyl)acrylic acid (S4, 11.22 mmol, 1.0 equiv.), N,O-dimethylhydroxylamine hydrochloride (16.84 mmol, 1.5 equiv.), DMAP (2.24 mmol, 0.2 equiv.), and EDCI (16.84 mmol, 1.5 equiv.) were dissolved in DCM (25 mL) and cooled to 0 °C in an ice bath. Triethylamine (16.84 mmol, 1.5 equiv.) was then added, and the mixture was stirred at 0 °C for 10 min, then allowed to rise to room temperature and reacted for another 2 h. The reaction was monitored by TLC. After the reaction was complete, 1 N HCl was added to quench the reaction, and the organic and aqueous phases were separated. The aqueous phase was adjusted to a weakly alkaline state and extracted with EA (15 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product Weinreb amide S3, which was used directly in the next reaction without further purification.

[0103] Under nitrogen protection, methyl acetoacetate (30.26 mmol, 3.0 equiv.) was added to a stirred suspension formed by 60% NaH (30.26 mmol, 3.0 equiv.) in anhydrous THF (30 mL), and the reaction was carried out at 0 °C for 20 min. The system was then cooled to -10 °C, and a 2.5 M n-butyllithium hexane solution (30.26 mmol, 3.0 equiv.) was slowly added dropwise, while stirring was continued at -10 °C for another 20 min. The system was then further cooled to -78 °C, where acetoacetate dianion was generated in situ. The resulting crude S3 (10.09 mmol, 1.0 equiv.) was added to the reaction system at -78 °C, and stirring continued until the reaction was complete. After the reaction was complete, the mixture was quenched with dilute hydrochloric acid, and the aqueous phase was adjusted to a weakly acidic state before extraction with EA (30 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was then purified by silica gel column chromatography to finally obtain the target compound 1b.

[0104] The synthesis method of 1o is as follows:

[0105]

[0106] Synthetic route of 1o

[0107] Under nitrogen protection, diethyl (N-methoxy-N-formylmethyl)phosphate (3.74 mmol, 1.21 equiv.) was dissolved in anhydrous THF (25 mL) and cooled to -78 °C. Then, a 2.5 M n-butyllithium solution in hexane (3.74 mmol, 1.21 equiv.) was slowly added, and the mixture was stirred at -78 °C for 30 min. Next, a THF solution of 2-cyclopropyl-4-(4-fluorophenyl)quinoline-3-carboxaldehyde (3.74 mmol, 1.21 equiv.) was slowly added dropwise, and the reaction was continued at -78 °C for 20 min. The system was then allowed to naturally warm to room temperature over 2–3 h. After the reaction was complete, the mixture was quenched with water in an ice bath and extracted with EA (25 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude amide intermediate S7, which was used directly in the next reaction without further purification.

[0108] Under nitrogen protection, methyl acetoacetate (8.61 mmol, 3.0 equiv.) was added to a stirred suspension formed in 30 mL of THF with 60% NaH (8.61 mmol, 3.0 equiv.) and stirred at 0 °C for 20 min. The reaction system was then cooled to -10 °C, and a 2.5 M hexane solution of n-butyllithium (8.61 mmol, 3.0 equiv.) was added dropwise. Stirring continued at -10 °C for another 20 min, followed by further cooling to -78 °C, resulting in the in-situ formation of acetoacetate dianion. The obtained crude S7 (2.87 mmol, 1.0 equiv.) was added to the reaction system at -78 °C, and stirring continued until the reaction was complete. After the reaction was complete, the solution was quenched with dilute hydrochloric acid, and the aqueous phase was adjusted to a weakly acidic state, followed by extraction with EA (30 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography to finally obtain the target compound 1o.

[0109] 2. Chemical Synthesis of Racemic Products

[0110] The method for synthesizing the racemic product is shown below:

[0111]

[0112] Synthetic route of racemic products

[0113] In the series of compounds synthesized via the racemic product route described above, R1 is ortho-methoxy, ortho-methoxy, para-methoxy, para-methyl, para-chlorine, para-bromine, para-cyano, hydrogen, para-fluorine, or meta-trifluoromethyl, and R2 is methyl, ethyl, or tert-butyl.

[0114] Taking 2a as an example, compound 1a (0.4 mmol, 1.0 equiv.) was dissolved in methanol (MeOH, 4 mL) and cooled to 0 °C, followed by the addition of sodium borohydride (0.44 mmol, 1.1 equiv.) in portions. The reaction system was brought to room temperature and stirred for 2 h. After the reaction was complete, it was quenched with saturated ammonium chloride aqueous solution (NH4Cl) and extracted with CH2Cl2. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain crude product S8; this crude product was used directly in the next reaction without further purification.

[0115] Crude S8 (1.0 equiv.) was dissolved in THF, and DDQ (2.5 equiv.) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated sodium bicarbonate aqueous solution (NaHCO3) and extracted with EA. The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 2a.

[0116] 3. Substrate suitability study for biocatalysis platform

[0117] Using compound 1 as a substrate and RasADH-E189D, KmCR2, SSCR-QA, or SsCR as ketone reductases, the catalytic reaction was carried out. The conversion rate and ee value of compound 1 were determined to evaluate the substrate's applicability. The catalytic reactions of other substrates are described in reference to compound 1a.

[0118] For the suitability of other substrates, refer to the screening process for the reductase of substrate 1a.

[0119] like Figure 4 As shown, the ketone reductase-based biocatalytic platform constructed in this invention exhibits good substrate applicability to aryl-substituted alkenyl 3,5-dicarbonyl ester substrates. This platform is compatible with various aryl substituents with different electronic effects and steric hindrance characteristics, including phenyl, methoxy-substituted phenyl, methyl-substituted phenyl, halophenyl, cyano-substituted phenyl, trifluoromethyl-substituted phenyl, as well as heteroaryl and fused-ring aryl groups; simultaneously, it also shows some tolerance to the ester moiety, making it applicable to different types of substrates such as methyl esters, ethyl esters, and tert-butyl esters. Results show that RasADH-E189D can achieve efficient catalysis for most substrates, yielding the corresponding (S)-configuration products, typically with high conversion rates and excellent enantioselectivity; KmCR2, SSCR-QA, or SsCR can also generate the corresponding stereoconfiguration products, with some substrates achieving high conversion rates and enantioselectivity greater than 99%. For substrates with significant steric hindrance or more complex structures, although the catalytic activity or stereoselectivity may decrease, the substrate applicability can still be significantly expanded through complementary matching between different enzymes. The above results demonstrate that the biocatalytic platform of this invention can achieve highly selective asymmetric reduction of the C3 carbonyl group in a multifunctional group competition context, exhibiting a broad substrate spectrum and good enantiomeric complementarity. It can provide a universal method for the efficient preparation of chiral aryl-substituted alkenyl β-hydroxy esters and their key pitavastatin intermediates.

[0120] 4. Chemical-enzymatic synthesis of pitavastatin intermediates

[0121] Pitavastatin intermediate 3o was synthesized using a chemical-enzymatic method. First, using 1o as a substrate, an enzymatic reaction was conducted to obtain R-2o. Then, using high-optical-purity R-2o obtained through SsCR catalysis, the synthesis of pitavastatin intermediate 3o was further carried out. The overall synthetic route is as follows:

[0122]

[0123] The synthetic route for the chemically induced reduction of R-2o is as follows:

[0124]

[0125] Under nitrogen protection, R-2o (0.185 mmol, 1.0 equiv.) was dissolved in a mixed solvent of anhydrous THF (1.5 mL) and MeOH (0.3 mL). A 1.0 M THF solution of diethylmethoxyborane (0.21 mmol, 1.12 equiv.) was added at -78 °C, and the mixture was stirred for 20 min at this temperature. Sodium borohydride (0.20 mmol, 1.11 equiv.) was then added, and the reaction was continued at -78 °C for 1 h. The reaction was monitored by TLC. After the reaction was complete, an AcOH / EA solution (0.10 mL AcOH, 0.50 mL EA) was added to quench the reaction, and the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into a saturated NaHCO3 aqueous solution, extracted with EA, and the combined organic phases were washed successively with saturated sodium chloride solution, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Finally, the crude product was purified by silica gel column chromatography to obtain pitavastatin intermediate 3o.

[0126] This invention employs a chemical-enzymatic method to efficiently synthesize pitavastatin intermediates, and the resulting product 3o has a dr value of 90:10, exhibiting good diastereoselectivity.

[0127] 5. Structural characterization of the compounds:

[0128] Figure 5-7 The HCl spectrum, C-chromatogram, and HRMS spectrum of compound 1a are shown.

[0129] methyl (E)-3,5-dioxo-7-phenylhept-6-enoate (1a) orange solid, yield: 60%; ¹H NMR (400 MHz, Chloroform-d) δ 14.84 (s, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.56–7.25 (m, 5H), 6.48 (d, J = 15.9 Hz, 1H), 5.76 (s, 1H), 3.76 (s, 3H), 3.46 (s, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 192.66, 176.75, 167.97, 140.64, 134.85, 130.18, 128.97, 128.05, 122.03, 52.49, 46.74. HRMS (ESI + ): calcd. for C14H14O4 [M+Na + : 269.0790, found: 269.0795.

[0130] methyl (E)-7-(2-methoxyphenyl)-3,5-dioxohept-6-enoate (1b). Yellow oily liquid, yield: 35%. 1 H NMR (400 MHz, Chloroform-d) δ 14.93 (s, 1H), 7.94 (d, J =16.0 Hz, 1H), 7.50 (dd, J = 7.7, 1.7 Hz, 1H), 7.37 – 7.31 (m, 1H), 6.96 (t, J= 7.5 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.58 (d, J = 16.0 Hz, 1H), 5.75 (s,1H), 3.89 (s, 3H), 3.76 (s, 3H), 3.45 (s, 2H). 13 C NMR (101 MHz, Chloroform-d)δ 192.22, 177.85, 168.17, 158.43, 136.30, 131.54, 128.78, 123.84, 122.59,120.84, 111.27, 100.65, 55.57, 52.54, 46.74. HRMS (ESI + ): calcd. for C15 H 16 O5[M+Na] + : 299.0895, found: 299.0898.

[0131] methyl (E)-7-(3-methoxyphenyl)-3,5-dioxohept-6-enoate (1c). Yellow oily liquid, yield: 39%. 1 H NMR (400 MHz, Chloroform-d) δ 14.82 (s, 1H), 7.59 (d, J =15.9 Hz, 1H), 7.33 – 7.26 (m, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.04 – 7.01 (m,1H), 6.92 (dd, J = 8.2, 1.6 Hz, 1H), 6.46 (d, J = 15.9 Hz, 1H), 5.75 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.46 (s, 2H). 13 C NMR (101 MHz, Chloroform-d) δ192.80, 176.68, 168.06, 160.02, 140.63, 136.30, 130.02, 122.38, 120.80,116.08, 113.03, 100.96, 55.39, 52.59, 46.84. HRMS (ESI + ): calcd. for C 15 H 16 O5[M+Na] + : 299.0895, found: 299.0896.

[0132] methyl (E)-7-(4-methoxyphenyl)-3,5-dioxohept-6-enoate (1d). Reddish-brown solid, yield: 23%. 1H NMR (400 MHz, Chloroform-d) δ 14.89 (s, 1H), 7.51 (d, J = 15.8Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.27 (d, J = 15.8 Hz, 2H), 1H), 5.64(s, 1H), 3.76(s, 3H), 3.69(s, 3H), 3.37(s, 2H). 13 C NMR(101 MHz, Chloroform-d) δ 191.78, 177.76, 168.10, 161.40, 140.55, 129.77, 127.59, 119.62, 114.44, 100.35, 55.41, 52.47,000. 46.55. HRMS (ESI + ): calcd. forC 15 H 16 O5 [M+Na] + : 299.0895, found:

[0133] methyl (E)-3,5-dioxo-7-(p-tolyl)hept-6-enoate(1e). 1 H NMR (400 MHz, Chloroform-d) δ 14.86 (s, 1H), 7.56 (d, J = 15.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 6.39 (d, J = 15.9Hz, 1H), 5.69(s, 1H), 3.72(s, 3H), 3.41(s, 2H), 2.33(s, 3H). 13 C NMR (101MHz, Chloroform-d) δ 192.28; HRMS (ESI + ): calcd. for C 15 H 16 O4 [M+Na] +:283.0946, found: 283.0948.

[0134] methyl (E)-7-(4-fluorophenyl)-3,5-dioxohept-6-enoate (1f). Yellow solid, yield: 45%. 1 H NMR (400 MHz, Chloroform-d) δ 14.84 (s, 1H), 7.59 (d, J = 15.9 Hz,1H), 7.51 (dd, J = 8.6, 5.5 Hz, 2H), 7.08 (t, J = 8.6 Hz, 2H), 6.40 (d, J =15.8 Hz, 1H), 5.75 (s, 1H), 3.77 (s, 3H), 3.46 (s, 2H). HRMS (ESI + ): calcd.for C 14 H 13 FO4 [M+H] + : 265.0876, found: 265.0878.

[0135] methyl (E)-7-(4-chlorophenyl)-3,5-dioxohept-6-enoate (1 g). Yellow solid, yield: 49.5%. Melting point: 54.5-55.0 °C; 1 H NMR (400 MHz, Chloroform-d) δ 14.78 (s, 1H),7.55 (d, J = 15.8 Hz, 1H), 7.50 – 7.31 (m, 4H), 6.43 (d, J = 15.9 Hz, 1H),5.75 (s, 1H), 3.76 (s, 3H), 3.47 (s, 2H). 13 C NMR (101 MHz, Chloroform-d) δ193.05, 176.17, 167.98, 139.13, 136.07, 133.43, 129.30, 129.22, 122.61,101.12, 52.59, 46.87. HRMS (ESI + ): calcd. for C 14 H 13 ClO4 [M+H] + : 281.0581,found: 281.0583.

[0136] methyl (E)-7-(4-bromophenyl)-3,5-dioxohept-6-enoate (1h). Yellow solid, yield: 52.5%. Melting point: 60.6-61.2 °C; 1 H NMR (400 MHz, Chloroform-d) δ 14.77 (s, 1H),7.61 – 7.31 (m, 5H), 6.45 (d, J = 15.9 Hz, 1H), 5.75 (s, 1H), 3.76 (s, 3H), 3.47 (s, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 193.11, 176.10, 167.98,139.20, 133.86, 132.27, 129.44, 124.44, 122.72, 101.16, 52.61, 46.91. HRMS (ESI + ): calcd. for C 14 H 13 BrO4 [M+H] + : 325.0075, found: 325.0080.

[0137] methyl (E)-7-(4-cyanophenyl)-3,5-dioxohept-6-enoate (1i). Yellow solid, yield: 30.1%. Melting point: 107.3-108.6 °C; 1 H NMR (400 MHz, Chloroform-d) δ 14.60 (s, 1H),7.74 – 7.55 (m, 5H), 6.56 (d, J = 15.9 Hz, 1H), 5.81 (s, 1H), 3.77 (s, 3H), 3.50 (s, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 194.03, 174.51, 167.79,139.24, 137.83, 132.72, 128.31, 125.49, 118.52, 113.03, 101.91, 52.61, 47.08.HRMS (ESI + ): calcd. for C 15 H 13 NO4 [M+Na] +: 294.0742, found: 294.0744.

[0138] methyl (E)-3,5-dioxo-7-(3-(trifluoromethyl)phenyl)hept-6-enoate (1j). Yellow solid, yield: 37.6%. Melting point: 49.5-50.0 ℃; 1 H NMR (400 MHz, Chloroform-d) δ14.69 (s, 1H), 7.79 – 7.49 (m, 5H), 6.54 (d, J = 15.9 Hz, 1H), 5.79 (s, 1H), 3.77 (s, 3H), 3.49 (s, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 199.11, 172.29,142.31, 133.10, 132.28, 129.78, 126.58, 125.14, 64.65, 51.87, 46.31, 40.62. 19 FNMR (376 MHz, Chloroform-d) δ -62.91. HRMS (ESI + ): calcd. for C 14 H 13 F3O4 [M+H] + :315.0844, found: 315.0848.

[0139] ethyl (E)-3,5-dioxo-7-phenylhept-6-enoate (1k). Yellow solid, yield: 67.6%. Melting point: 34.0-34.5℃; 1 H NMR (400 MHz, Chloroform-d) δ 14.86 (s, 1H), 7.62 (d, J= 15.9 Hz, 1H), 7.55 – 7.34 (m, 5H), 6.48 (d, J = 15.9 Hz, 1H), 5.76 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 3.44 (s, 2H), 1.30 (t, J = 7.2 Hz, 3H). 13C NMR (101MHz, Chloroform-d) δ 192.84, 176.70, 167.55, 140.55, 134.87, 130.15, 128.96,128.03, 100.85, 61.50, 47.01, 14.15. HRMS (ESI + ): calcd. for C 15 H 16 O4 [M+Na] + :283.0946, found: 283.0950.

[0140] tert-butyl (E)-3,5-dioxo-7-phenylhept-6-enoate (1 L). Yellow oily liquid, yield: 36.5%. 1 H NMR (400 MHz, Chloroform-d) δ 14.91 (s, 1H), 7.61 (d, J = 15.9Hz, 1H), 7.52 (dd, J = 7.6, 2.1 Hz, 2H), 7.43 – 7.33 (m, 3H), 6.48 (d, J =15.9 Hz, 1H), 5.75 (s, 1H), 3.36 (s, 2H), 1.49 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 193.49, 176.66, 166.89, 140.41, 135.00, 130.16, 129.03,128.09, 122.31, 100.96, 82.07, 48.36, 28.09. HRMS (ESI + ): calcd. for C 17 H 20 O4[M+Na] + : 311.1259, found: 311.1261.

[0141] methyl (E)-3,5-dioxo-7-(pyridin-2-yl)hept-6-enoate (1m). Yellow oily liquid, yield: 20.4%. 1H NMR (400 MHz, Chloroform-d) δ 14.63 (s, 1H), 8.64 (d, J = 4.2Hz, 1H), 7.71 (td, J = 7.7, 1.8 Hz, 1H), 7.59 (d, J = 15.5 Hz, 1H), 7.38 (d,J = 7.9 Hz, 1H), 7.25 (ddd, J = 7.6, 4.8, 1.1 Hz, 1H), 7.05 (d, J = 15.5 Hz, 1H), 5.82 (s, 1H), 3.76 (s, 3H), 3.49 (s, 2H). 13 C NMR (101 MHz, Chloroform-d)δ 194.12, 175.02, 167.84, 153.15, 150.19, 138.69, 136.94, 126.00, 124.84,124.07, 102.02, 52.55, 47.19. HRMS (ESI + ): calcd. for C 13 H 13 NO4 [M+H] + :248.0923, found: 248.0924.

[0142] methyl (E)-7-(naphthalen-2-yl)-3,5-dioxohept-6-enoate (1n). Yellow solid, yield: 55.2%. Melting point: 96.8-97.4 °C; 1 H NMR (400 MHz, Chloroform-d) δ 14.89 (s, 1H),7.88 (s, 1H), 7.85 – 7.73 (m, 4H), 7.63 (d, J = 8.6 Hz, 1H), 7.53 – 7.45 (m,2H), 6.55 (d, J = 15.8 Hz, 1H), 5.76 (s, 1H), 3.76 (s, 3H), 3.46 (s, 2H). 13CNMR (101 MHz, Chloroform-d) δ 192.74, 176.76, 168.07, 140.76, 134.21, 133.42,132.40, 130.02, 128.79, 128.65, 127.86, 127.34, 126.83, 123.47, 122.21,100.96, 52.57, 46.84. HRMS (ESI + ): calcd. for C 18 H 16 O4 [M+Na] + : 319.0946, found: 319.0948.

[0143] methyl(E)-7-(2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)-3,5-dioxohept-6-enoate (1O). Yellow solid, yield: 73.8%. Melting point: 110.7-111.3 ℃. 1 H NMR (400MHz, Chloroform-d) δ 14.63 (s, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.71 (d, J =16.2 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.44 – 7.30 (m, 2H), 1.40 (dd, J = 4.7, 2.6 Hz, 2H), 1.14 –1.04 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 193.19, 175.57, 167.93, 162.64,160.16, 147.38, 145.85, 137.57, 132.46, 131.80, 129.82, 129.56, 127.48,126.37, 125.97, 125.88, 115.82, 101.12, 52.60, 46.83, 16.48, 10.61. 19F NMR (376 MHz, Chloroform-d) δ -113.17. HRMS (ESI + ): calcd. for C 26 H 22 FNO4 [M+H] + :432.1611, found: 432.1612. Figure 11-14 The 1H, 1C, 1F and HRMS spectra of compound 1o are shown.

[0144] Figure 8-10 The HCl spectrum, C-chromatogram, and HRMS spectrum of compound 2a are shown.

[0145] methyl (E)-3-hydroxy-5-oxo-7-phenylhept-6-enoate (2a) is a white oily liquid with a yield of 72.8%. 1H NMR (400 MHz, Chloroform-d) δ 7.58 (d, J = 16.4 Hz, 1H), 7.56 –7.52 (m, 2H), 7.40 (dd, J = 5.1, 1.9 Hz, 3H), 6.74 (d, J = 16.2 Hz, 1H), 4.59(dd, J = 12.5, 6.4 Hz, 1H), 3.72 (s, 3H), 3.65 (d, J = 3.5 Hz, 1H), 2.94 (d,J = 6.4 Hz, 2H), 2.61 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ199.5, 172.4, 143.9, 134.2, 130.9, 129.1, 128.5, 126.2, 64.8, 51.9, 46.2,40.7. HRMS (ESI + ): calcd. for C14H16O4 [M+Na + : 271.0946, found: 271.0951.

[0146] methyl (E)-3-hydroxy-7-(2-methoxyphenyl)-5-oxohept-6-enoate (2b). Yellow oily liquid, yield: 73%. 1H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 16.5 Hz, 1H), 7.54 (dd, J = 7.7, 1.8 Hz, 1H), 7.41 – 7.34 (m, 1H), 6.97 (t, J = 7.5Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 16.4 Hz, 1H), 4.58 (p, J =6.9, 6.3 Hz, 1H), 3.90 (s, 3H), 3.72 (s, 3H), 3.68 (d, J = 10.1 Hz, 1H), 2.95(d, J = 5.6 Hz, 2H), 2.61 (d, J = 6.5 Hz, 2H). 13 C NMR (101 MHz, Chloroform-d)δ 200.17, 172.38, 158.59, 139.26, 132.24, 128.74, 126.82, 123.12, 120.90,111.27, 64.88, 55.60, 51.88, 45.66, 40.79. HRMS (ESI + ): calcd. for C 15 H 18 O5 [M+Na] + 301.1052, found: 301.1055.

[0147] methyl (E)-3-hydroxy-7-(3-methoxyphenyl)-5-oxohept-6-enoate (2c). Yellow oily liquid, yield: 57.1%. 11H NMR (400 MHz, Chloroform-d) δ 7.52 (d, J = 16.2 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.04 (t, J = 2.1 Hz, 1H), 6.94 (dd, J = 8.2, 2.4 Hz, 1H), 6.70 (d, J = 16.2 Hz, 1H), 4.57 (p, J = 6.2 Hz, 1H), 3.81 (s, 3H), 3.70 (s, 3H), 3.62 (s, 1H), 2.92 (d, J = 6.4 Hz, 2H), 2.59 (d, J = 6.4 Hz, 2H). 13 13C NMR (101 MHz, Chloroform-d) δ 199.47, 172.36, 160.01, 143.81, 135.59, 130.08, 126.49, 121.25, 116.79, 113.26, 64.76, 55.40, 51.91, 46.14, 40.73. HRMS (ESI + ): calcd. for C 15 15H 18 O5 [M+Na] + : 301.1052, found: 301.1056.

[0148] methyl (E)-3-hydroxy-7-(4-methoxyphenyl)-5-oxohept-6-enoate (2d). Yellow oily liquid, yield: 50.6%. 1 1H NMR (400 MHz, Chloroform-d) δ 7.55 (d, J = 16.2 Hz, 1H), 7.50 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 16.1 Hz, 1H), 4.67 – 4.52 (m, 1H), 3.84 (s, 3H), 3.72 (s, 3H), 3.70 (d, J = 3.8 Hz, 1H), 2.91 (d, J = 5.5 Hz, 2H), 2.64 – 2.57 (m, 2H). 13C NMR (101 MHz, Chloroform-d) δ 199.48, 172.35, 161.95, 143.74, 130.31, 126.86, 124.01,114.56, 64.89, 55.49, 51.88, 45.94, 40.78. HRMS (ESI + ): calcd. for C 15 H 18 O5 [M+Na] + 301.1052, found: 301.1054.

[0149] methyl (E)-3-hydroxy-5-oxo-7-(p-tolyl)hept-6-enoate (2e). Yellow oily liquid, yield: 85.1%. 1 H NMR (400 MHz, Chloroform-d) δ 7.55 (d, J = 16.2 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 6.68 (d, J = 16.2 Hz, 1H), 4.57 (p, J = 6.3 Hz, 1H), 3.70 (s, 3H), 3.66 (s, 1H), 2.91 (d, J = 6.1 Hz, 2H), 2.59 (d, J = 5.8 Hz, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ199.60, 172.37, 144.02, 141.52, 131.46, 129.84, 128.55, 125.29, 64.83, 51.90, 46.02, 40.76, 21.61. HRMS (ESI + ): calcd. for C 15 H 18 O4 [M+Na] + : 285.1103, found:285.1105.

[0150] methyl (E)-7-(4-fluorophenyl)-3-hydroxy-5-oxohept-6-enoate (2f). Pale yellow oily liquid, yield: 86.3%. 1H NMR (400 MHz, Chloroform-d) δ 7.59 – 7.50 (m, 3H),7.10 (s, 2H), 6.67 (d, J = 16.2 Hz, 1H), 4.59 (p, J = 6.3 Hz, 1H), 3.72 (s,3H), 3.63 (s, 1H), 2.93 (d, J = 6.7 Hz, 2H), 2.61 (d, J = 6.4 Hz, 2H). 13 C NMR(101 MHz, CDCl3) δ 199.26, 172.40, 164.29, 142.55, 130.49, 130.47, 125.94,116.32, 64.78, 51.94, 46.29, 40.72. 19 F NMR (376 MHz, Chloroform-d) δ -108.55.HRMS (ESI + ): calcd. for C 14 H 15 FO4 [M+Na] + : 289.0852, found: 289.0856.

[0151] methyl (E)-7-(4-chlorophenyl)-3-hydroxy-5-oxohept-6-enoate (2 g). White solid, yield: 76.4%. Melting point: 64.8-65.3 °C. 1 H NMR (400 MHz, Chloroform-d) δ 7.53 (d, J= 16.2 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 6.71 (d,J = 16.2 Hz, 1H), 4.59 (td, J = 11.7, 5.5 Hz, 1H), 3.72 (s, 3H), 3.61 (d, J =3.8 Hz, 1H), 2.91 (s, 2H), 2.61 (s, 2H). 13C NMR (101 MHz, Chloroform-d) δ199.14, 172.34, 142.27, 136.79, 132.74, 129.64, 129.37, 126.57, 64.71, 51.91,46.37, 40.69. HRMS (ESI + ): calcd. for C 14 H 15 ClO4 [M+Na] + : 305.0557, found:305.0558.

[0152] methyl (E)-7-(4-bromophenyl)-3-hydroxy-5-oxohept-6-enoate (2h). White solid, yield: 64.6%. Melting point: 62.4-63.5 °C; 1 H NMR (400 MHz, Chloroform-d) δ 7.59 –7.46 (m, 3H), 7.41 (d, J = 8.5 Hz, 2H), 6.72 (d, J = 16.2 Hz, 1H), 4.58 (dd,J = 11.2, 5.0 Hz, 1H), 3.72 (s, 3H), 3.60 (d, J = 3.8 Hz, 1H), 2.98 – 2.89(m, 2H), 2.61 (d, J = 6.3 Hz, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 199.18,172.36, 142.38, 133.17, 132.35, 129.85, 126.65, 125.22, 64.73, 51.94, 46.39,40.69. HRMS (ESI + ): calcd. for C 14 H 15 BrO4 [M+Na] + : 349.0051, found: 349.0047.

[0153] methyl (E)-7-(4-cyanophenyl)-3-hydroxy-5-oxohept-6-enoate (2i). White solid, yield: 74.7%. Melting point: 84.7-85.4 ℃; 11H NMR (400 MHz, Chloroform-d) δ 7.74 –7.62 (m, 4H), 7.57 (d, J = 16.2 Hz, 1H), 6.82 (d, J = 16.2 Hz, 1H), 4.60 (dd,J = 8.9, 5.5 Hz, 1H), 3.73 (s, 3H), 3.54 (d, J = 3.7 Hz, 1H), 3.03 – 2.89 (m,2H), 2.62 (d, J = 6.3 Hz, 2H). 13 13C NMR (101 MHz, Chloroform-d) δ 198.73,172.33, 141.00, 138.62, 132.76, 128.96, 128.77, 118.34, 113.78, 64.60, 51.94,46.77, 40.61. HRMS (ESI + ): calcd. for C 15 H 15 NO4 [M+Na] + : 296.0899, found:296.0901.

[0154] methyl (E)-3-hydroxy-5-oxo-7-(3-(trifluoromethyl)phenyl)hept-6-enoate (2j). Pale yellow oily liquid, yield: 57%. 1 1H NMR (400 MHz, Chloroform-d) δ 7.80 (s, 1H),7.73 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 16.2 Hz,1H), 7.54 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 4.60 (dq, J = 12.5,6.3 Hz, 1H), 3.73 (s, 3H), 3.52 (d, J = 3.8 Hz, 1H), 2.95 (d, J = 4.8 Hz,2H), 2.62 (d, J = 6.3 Hz, 2H). 19 19F NMR (376 MHz, Chloroform-d) δ -62.93. HRMS(ESI + ): calcd. for C15 H 15 F3O4 [M+Na] + : 339.0820, found: 339.0821.

[0155] Ethyl (E)-3-hydroxy-5-oxo-7-phenylhept-6-enoate (2k). Yellow oily liquid, yield: 51%. 1 H NMR (400 MHz, Chloroform-d) δ 7.62 – 7.52 (m, 3H), 7.41 (dd, J =5.0, 1.9 Hz, 3H), 6.74 (d, J = 16.2 Hz, 1H), 4.58 (td, J = 6.3, 2.5 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.57 (d, J = 3.4 Hz, 1H), 2.94 (d, J = 6.0 Hz, 2H), 2.59 (d, J = 6.4 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 199.40, 171.92, 143.78, 134.18, 130.82, 129.02, 128.45,126.21, 64.76, 60.76, 46.14, 40.88, 14.19. HRMS (ESI + ): calcd. for C 15 H 18 O4 [M+Na] + : 285.1103, found: 285.1107.

[0156] tert-butyl (E)-3-hydroxy-5-oxo-7-phenylhept-6-enoate (2l) is a yellow oily liquid with a yield of 58.8%. 1H NMR (400 MHz, Chloroform-d) δ 7.62 – 7.52 (m, 3H), 7.45 –7.36 (m, 3H), 6.75 (d, J = 16.2 Hz, 1H), 4.58 – 4.50 (m, 1H), 3.66 (s, 1H),2.99 – 2.85 (m, 2H), 2.53 – 2.48 (m, 2H), 1.48 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 199.44, 171.48, 143.75, 134.30, 130.86, 129.08, 128.52,126.35, 81.33, 65.00, 46.34, 42.00, 28.20. HRMS (ESI + ): calcd. for C 17 H 22 O4 [M+Na] + 313.1416, found: 313.1417.

[0157] methyl (E)-3-hydroxy-5-oxo-7-(pyridin-2-yl)hept-6-enoate (2m). Yellow oily liquid, yield: 50.7%. 1 H NMR (400 MHz, Chloroform-d) δ 8.66 (d, J = 5.6 Hz, 1H),7.74 (t, J = 8.6 Hz, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.49 (d, J = 8.9 Hz,1H), 7.36 – 7.25 (m, 2H), 7.20 (d, J = 15.9 Hz, 1H), 4.65 – 4.58 (m, 1H), 3.72 (s, 3H), 3.55 (d, J = 9.0 Hz, 1H), 3.00 – 2.95 (m, 2H), 2.61 (d, J = 6.4Hz, 2H). 13C NMR (101 MHz, Chloroform-d) δ 199.60, 172.30, 152.82, 150.27,142.07, 137.01, 129.39, 124.83, 124.68, 64.62, 51.90, 46.87, 40.75. HRMS (ESI + ): calcd. for C 13 H 15 NO4 [M+H] + : 250.1079, found: 250.1081.

[0158] methyl (E)-3-hydroxy-7-(naphthalen-2-yl)-5-oxohept-6-enoate (2n). White solid, yield: 82%. Melting point: 86.6-87.1 °C. 1 H NMR (400 MHz, Chloroform-d) δ 7.96 –7.79 (m, 4H), 7.73 (d, J = 16.1 Hz, 1H), 7.66 (d, J = 10.3 Hz, 1H), 7.55 –7.48 (m, 2H), 6.83 (d, J = 16.1 Hz, 1H), 4.61 (p, J = 6.2 Hz, 1H), 3.73 (s,3H), 3.31 (s, 1H), 2.97 (d, J = 6.1 Hz, 2H), 2.63 (d, J = 6.8 Hz, 2H). 13 C NMR(101 MHz, Chloroform-d) δ 199.38, 172.32, 143.88, 134.48, 133.27, 131.67,130.81, 128.86, 128.67, 127.84, 127.58, 126.87, 126.25, 123.47, 64.77, 51.87,46.19, 40.70. HRMS (ESI + ): calcd. for C 18 H 18 O4 [M+Na] + : 321.1103, found:321.1102.

[0159] methyl(E)-7-(2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)-3-hydroxy-5-oxohept-6-enoate (2o). Yellow oily liquid, yield: 38.6%. 1 H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 8.4 Hz, 1H), 7.72 – 7.61 (m, 2H), 7.40 – 7.32(m, 2H), 7.22 (d, J = 7.9 Hz, 4H), 6.35 (d, J = 16.5 Hz, 1H), 4.46 (p, J =6.3 Hz, 1H), 3.72 (s, 3H), 3.61 (d, J = 14.4 Hz, 1H), 2.70 (d, J = 5.1 Hz, 2H), 2.52 (d, J = 6.4 Hz, 2H), 2.34 (t, J = 4.9 Hz, 1H), 1.40 (dq, J = 6.1,3.4 Hz, 2H), 1.13 – 1.05 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ 198.72, 172.32,162.70, 160.01, 147.52, 146.26, 140.88, 133.52, 132.30, 131.76, 130.05,129.08, 126.90, 126.23, 125.75, 115.86, 64.61, 51.94, 46.29, 40.56, 16.48,10.69. 19 F NMR (376 MHz, Chloroform-d) δ -112.81. HRMS (ESI + ): calcd. forC 26 H 24 FNO4 [M+H] + : 434.1768, found: 434.1768.

[0160] Figure 15-18 The 1H NMR spectrum and HRMS spectrum of compound 2o are shown.

[0161] methyl(E)-7-(2-cyclopropyl-4-(4-fluorophenyl)quinolin-3-yl)-3,5-dihydroxyhept-6-enoate (3o). White solid, yield: 43.75%. 1 H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J = 8.3 Hz, 2H), 7.63 – 7.55 (m, 2H), 7.36 – 7.27(m, 4H), 7.25 – 7.12 (m, 8H), 6.66 (dd, J = 25.0, 16.2 Hz, 2H), 5.62 (ddd, J= 29.9, 16.2, 5.8 Hz, 2H), 4.50 – 4.36 (m, 2H), 4.18 – 3.98 (m, 2H), 3.74 (d,J = 2.9 Hz, 6H), 3.52 (d, J = 80.1 Hz, 2H), 3.05 (d, J = HRMS (ESI) + ): calcd. for C 26 H 26 FNO4 [M+H] + :436.1924, found: 436.1928. Figures 19-20 The 1H NMR spectrum and HRMS spectrum of compound 3o are shown.

[0162] sequence list

[0163] SEQ ID NO.1 (RasADH-WT gene sequence):

[0164] ATGTATCGACTATTAAACAAAACAGCCGTCATAACCGGTGGAAACAGCGGCATTGGCCTCGCCACAGCGAAGCGCTTCGTTGCCGAGGGTGCCTATGTATTCATTGTCGGTCGCCGGCGGAAGGAACTCGAGCAGGCGGCCGCAGAAATCGGTCGGAATGTCACGGCGGTCAAAGCCGATGTGACAAAGCTTGAAGACCTGGACCGACTTTACGCGATTGTGCGTGAGCAACGGGGTAGCATCGACGTACTATTTGCGAATTCCGGCGCAATCGAGCAAAAGACGCTTGAGGAGATTACTCCGGAACACTATGACAGGACTTTCGATGTCAACGTTCGGGGATTGATCTTCACCGTGCAGAAGGCACTTCCTCTGCTGCGAGACGGCGGCAGCGTGATCCTGACAAGCTCGGTAGCCGGCGTCCTAGGATTACAGGCGCACGACACGTATAGTGCCGCCAAGGCAGCGGTAAGGTCGCTCGCGAGGACATGGACCACTGAGTTGAAAGGTCGCAGCATTCGTGTCAACGCGGTAAGCCCAGGGGCGATCGACACGCCTATCATAGAAAACCAGGTCTCTACACAGGAAGAAGCTGACGAGCTGCGTGCGAAATTTGCAGCTGCGACGCCCCTGGGTCGCGTCGGACGACCTGAAGAGCTGGCAGCGGCCGTGTTATTTCTTGCATCGGACGACAGTAGCTACGTAGCCGGCATTGAGCTGTTTGTGGACGGTGGATTGACCCAGGTCTAA

[0165] SEQ ID NO.2 (Amino acid sequence of RasADH-WT):

[0166] MYRLLNKTAVITGGNSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIENQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV

[0167] SEQ ID NO.3 (RasADH - E189D gene sequence):

[0168] ATGTATCGACTATTAAACAAAACAGCCGTCATAACCGGTGGAAACAGCGGCATTGGCCTCGCCACAGCGAAGCGCTTCGTTGCCGAGGGTGCCTATGTATTCATTGTCGGTCGCCGGCGGAAGGAACTCGAGCAGGCGGCCGCAGAAATCGGTCGGAATGTCACGGCGGTCAAAGCCGATGTGACAAAGCTTGAAGACCTGGACCGACTTTACGCGATTGTGCGTGAGCAACGGGGTAGCATCGACGTACTATTTGCGAATTCCGGCGCAATCGAGCAAAAGACGCTTGAGGAGATTACTCCGGAACACTATGACAGGACTTTCGATGTCAACGTTCGGGGATTGATCTTCACCGTGCAGAAGGCACTTCCTCTGCTGCGAGACGGCGGCAGCGTGATCCTGACAAGCTCGGTAGCCGGCGTCCTAGGATTACAGGCGCACGACACGTATAGTGCCGCCAAGGCAGCGGTAAGGTCGCTCGCGAGGACATGGACCACTGAGTTGAAAGGTCGCAGCATTCGTGTCAACGCGGTAAGCCCAGGGGCGATCGACACGCCTATCATAGATAACCAGGTCTCTACACAGGAAGAAGCTGACGAGCTGCGTGCGAAATTTGCAGCTGCGACGCCCCTGGGTCGCGTCGGACGACCTGAAGAGCTGGCAGCGGCCGTGTTATTTCTTGCATCGGACGACAGTAGCTACGTAGCCGGCATTGAGCTGTTTGTGGACGGTGGATTGACCCAGGTCTAA

[0169] SEQ ID NO.4 (Amino acid sequence of RasADH - E189D):

[0170] MYRLLNKTAVITGGSGIGLATAKRFVAEGAYVFIVGRRRKELEQAAAEIGRNVTAVKADVTKLEDLDRLYAIVREQRGSIDVLFANSGAIEQKTLEEITPEHYDRTFDVNVRGLIFTVQKALPLLRDGGSVILTSSVAGVLGLQAHDTYSAAKAAVRSLARTWTTELKGRSIRVNAVSPGAIDTPIIDNQVSTQEEADELRAKFAAATPLGRVGRPEELAAAVLFLASDDSSYVAGIELFVDGGLTQV

[0171] SEQ ID NO.5(SSCR-WT グロンショック:

[0172]

[0173] SEQ ID NO.6 (Amino acid sequence of SSCR-WT):

[0174] MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSFSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPESDPQKSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPALALMPPQYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA

[0175] SEQ ID NO.7 (Gene sequence of SSCR-QA):

[0176]

[0177] SEQ ID NO.8 (SSCR-QA amino acid sequence):

[0178] MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSFSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSAVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPESDPQKSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGQMMSLFNGEVSPALALMPPQYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA

[0179] SEQ ID NO.9 (RasADH-E189D-F primer sequence):

[0180] CACGCCTATCATAGATAACCAGGTCTCTACAC

[0181] SEQ ID NO.10 (RasADH-E189D-R primer sequence):

[0182] GTGTAGAGACCTGGTTATCTATGATAGGCGTG

[0183] SEQ ID NO.11 (SSCR-W226Q-F primer sequence):

[0184] CGGCAGCACAAGCGGTCAGATGATGTCTTTATTC

[0185] SEQ ID NO.12 (SSCR-W226Q-R primer sequence):

[0186] GAATAAAGACATCATCTGACCGCTTGTGCTGCCG

[0187] SEQ ID NO.13 (SSCR-T134A-F primer sequence):

[0188] CGTGCTGACAAGTAGTGCGGTGAGCGCCTTAATTC

[0189] SEQ ID NO.14 (SSCR-T134A-R):

[0190] GAATTAAGGCGCTCACCGCACTACTTGTCAGCACG

Claims

1. A method for preparing chiral aryl-substituted alkenyl β-hydroxy esters, comprising the following steps: In phosphate buffer, a biocatalytic reaction system comprising the following components was constructed: (a) the substrate shown in Formula B; (b) the coenzyme NADP. + (c) Glucose; (d) glucose dehydrogenase (GDH); and (e) ketone reductase; The reaction system was subjected to a stereoselective reduction reaction at 20°C to 40°C, thereby converting the substrate shown in Formula B into a chiral aryl-substituted alkenyl β-hydroxy ester as shown in Formula A. Wherein, equation A is as follows: ； Equation B is shown below: ； In formulas A and B, R2 is methyl, ethyl, or tert-butyl, and R3 is phenyl, methoxyphenyl, benzyl, fluorophenyl, chlorophenyl, bromophenyl, cyanobenzene, trifluoromethylphenyl, pyridyl, naphthyl, or... ; The ketone reductase is RasADH or RasADH-E189D, the amino acid sequence of which is shown in SEQ ID NO.2; the amino acid sequence of which is shown in SEQ ID NO.

4.

2. The method according to claim 1, characterized in that, The structural formula of the chiral aryl-substituted alkenyl β-hydroxy ester is shown below: ； The structural formula of the compound shown in formula B is as follows: 。 3. The method according to claim 1 or 2, characterized in that, The gene sequence encoding RasADH is shown in SEQ ID NO.1; the gene sequence encoding RasADH-E189D is shown in SEQ ID NO.

3.

4. The method according to claim 1 or 2, characterized in that, The reduction reaction was carried out at 30°C under shaking conditions for 24 hours.

5. The method according to claim 1 or 2, characterized in that, The dosage relationship between the components satisfies the following: glucose, NADP + The molar ratio of the substrate shown in Formula B is 20:1:

10. The glucose dehydrogenase and ketone reductase are added in the form of lyophilized powder. The amount of glucose dehydrogenase lyophilized powder used is 80-90 mg / mmol substrate, and the amount of ketone reductase lyophilized powder used is 160-170 mg / mmol substrate.

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