Styrene monooxygenase mutant for preparing ticagrelor intermediate

Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly discloses a styrene monooxygenase mutant with excellent enantioselectivity and high activity. The mutant enzyme can catalyze epoxidation of 3, 4-difluorostyrene to generate (S)-2-(3, 4-difluorophenyl) ethylene oxide, enantiomeric excess is greater than 99%, and the mutant enzyme is an excellent catalyst for synthesizing a ticagrelor intermediate.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a styrene monooxygenase mutant catalyzing the epoxidation of 3,4-difluorostyrene to synthesize the key intermediate (S)-2-(3,4-difluorophenyl)oxirane of ticagrelor. Background Art

[0002] Traditional chemical synthesis methods are facing severe challenges due to their shortcomings such as numerous steps, complex operations, high waste discharge, and difficulty in product separation. Biocatalysis uses natural enzymes or microbial metabolic pathways to achieve efficient synthesis of some compounds under mild conditions. Due to the stereoselective properties of enzymes, they have significant advantages in the synthesis of chiral compounds and can efficiently prepare chiral compounds of single enantiomers.

[0003] Ticagrelor is an oral antiplatelet drug that can be used to prevent acute coronary syndrome and is the first reversible P2Y12 receptor antagonist. It ranks 62nd in the global small molecule drug sales ranking in 2021, with global sales of approximately US$1.5 billion. It has huge market development potential and good prospects. In the existing synthesis route of Ticagrelor, the chiral compound (S)-2-(3,4-difluorophenyl) oxirane is an important intermediate product. The traditional chemical synthesis method is shown in Scheme 1. Borane dimethyl sulfide complex, triethyl phosphoacetate and other compounds and chiral auxiliary (S)-diphenylprolinol are used in the reaction, which is costly and difficult to react (Bioorganic & Medicinal Chemistry Letters, 2012, 22 (11): 3598-3602).

[0004]

[0005] Note: (a) AlCl3, 60℃; (b) BH3-Me2S, (S)-DPPM, B(OCH3)3, toluene; (c) NaOH, H2O, toluene, 40℃Styrene monooxygenase (SMO) can catalyze the epoxidation of styrene and its derivatives to produce the corresponding (S)- or (R)-type epoxides, but there is no report on the epoxidation of 3,4-difluorostyrene catalyzed by SMO to synthesize (S)-2-(3,4-difluorophenyl)oxirane. Summary of the invention

[0006] The present invention uses styrene monooxygenase SeStyA derived from Streptomyces vilmorinianum as the parent, and adopts structure-based semi-rational design to perform molecular modification, thereby obtaining a styrene monooxygenase mutant with improved activity and excellent stereoselectivity. The mutant can efficiently catalyze the epoxidation of 3,4-difluorostyrene to synthesize the key intermediate (S)-2-(3,4-difluorophenyl)oxirane of ticagrelor.

[0007] Specifically, the styrene monooxygenase mutant is based on SeStyA (nucleotide sequence is SEQ ID NO.1; amino acid sequence is SEQ ID NO.2) as the starting sequence, and one amino acid site is mutated to obtain the following single point mutant.

[0008] S178Y: Serine at position 178 mutated to tyrosine.

[0009] According to the common knowledge in the art, vectors and genetically engineered bacteria constructed to express the above mutants also fall within the protection scope of the present invention.

[0010] The mutant of the present invention is obtained as follows:

[0011] First, the enzyme was semi-rationally designed by molecular docking. The protein structure of wild-type SeStyA was molecularly docked with the substrate 3,4-difluorostyrene. According to the docking results, its binding mode was analyzed, and the mutant S178Y was locked in by computer virtual screening after comprehensive consideration of factors such as steric hindrance and amino acid properties. Then a single-point mutant was constructed, and after heterologous expression of the enzyme, it was found through biocatalytic verification that S178Y was a single-point mutant with excellent (S)-selectivity and significantly improved activity. The conversion rate of wild-type SeStyA for 2mmol / L 3,4-difluorostyrene within 2h was 69%, and the product was 2-(3,4-difluorophenyl)ethylene oxide racemate. The conversion rate of mutant SeStyA-S178Y was 95%, and the product was (S)-2-(3,4-difluorophenyl)ethylene oxide, with an enantiomeric excess of >99%ee.

[0012] Advantages of the present invention: The styrene monooxygenase mutant SeStyA-S178Y of the present invention can catalyze the epoxidation of 3,4-difluorostyrene to synthesize the key intermediate (S)-2-(3,4-difluorophenyl) oxirane of ticagrelor, and this is the first time that this type of enzyme is used to synthesize such compounds. The mutant of the present invention can catalyze 2mmol / L of 3,4-difluorostyrene to generate (S)-2-(3,4-difluorophenyl) oxirane within 2 hours, with an enantiomeric excess of >99%ee and a conversion rate of 95%. Compared with the wild type, it has excellent enantioselectivity and catalytic activity, and is an excellent catalyst for preparing the key intermediate (S)-2-(3,4-difluorophenyl) oxirane of ticagrelor. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 , HPLC graphs of epoxidation of 3,4-difluorostyrene catalyzed by wild-type SeStyA and mutant SeStyA-S178Y to produce 2-(3,4-difluorophenyl)ethylene oxide, where A is the standard spectrum, B is the product spectrum after catalysis by wild-type SeStyA, and C is the product spectrum after catalysis by mutant SeStyA-S178Y. DETAILED DESCRIPTION

[0014] Example 1: Rational design of mutation sites and construction of single-point mutants

[0015] The nucleotide sequence of SeStyA is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The predicted structure of SeStyA was obtained by AlphaFold2 and evaluated by SAVER6.0. The molecular docking software AutoDock Vina was used to molecularly dock SeStyA with the substrate 3,4-difluorostyrene. The binding mode between the substrate and the active pocket and the steric hindrance and properties of the surrounding residues were analyzed based on the docking results. Through virtual mutation, it was found that the mutant S178Y would adjust the substrate binding mode and strengthen the prochiral conformation of the generated (S)-type epoxy product. Therefore, it was mutated to tyrosine.

[0016] Since the entire catalytic process requires the participation of the expensive cofactor FADH2, the flavin oxidoreductase StyB was introduced to convert FAD + Reduced to FADH2, realizing the coenzyme cycle. The specific method of constructing the co-expression system of SeStyA, linker and StyB refers to the previous study (Catalysis Science & Technology, 2021, 11(6): 2195-2201).

[0017] The wild-type recombinant plasmid was used as a template for site-directed mutagenesis. The primers were designed as shown in Table 1 , and a single-point mutant was rationally constructed.

[0018] Table 1 Site-directed mutagenesis primers

[0019]

[0020] The PCR reaction system is shown in Table 2, and amplification was performed according to the following conditions: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 8 min, 20 cycles, extension at 72°C for 10 min, and storage at 4°C for future use.

[0021] Table 2 PCR reaction system for site-directed mutagenesis

[0022]

[0023] Add 1 μL DpnI to the PCR product and incubate in a 37°C water bath for 1 hour. Then take 10 μL of the treated PCR product and transfer it into E. coli DH5α competent cells according to conventional chemical methods. Spread it on an LB plate containing 50 μg / mL kanamycin and invert it at 37°C for 12 hours. Pick a single clone and place it in LB liquid culture medium (containing 50 μg / mL kanamycin), incubate it at 37°C for 10 hours, and send the bacterial solution to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0024] Example 2: Heterologous expression of wild-type SeStyA and mutants

[0025] The wild-type and mutant plasmids were transformed into E. coli BL21 (DE3) competent cells for protein expression. Single clones were picked and transferred to 3 mL LB liquid medium (containing kanamycin 50 μg / mL) and cultured at 37°C, 180 r / min for 12 h. 2 mL (1% V / V) was transferred to 200 mL TB liquid medium for expansion culture. 600 When the pH reached about 0.6, IPTG was added to make the final concentration 0.01 mmol / L, and then the culture was continued for 20 h at 20°C and 180 r / min. The cells were collected by refrigerated centrifugation and then washed twice with an equal volume of 0.9% sodium chloride solution for later use.

[0026] Example 3: Biocatalysis of 3,4-difluorostyrene epoxidation by wild-type SeStyA and mutants

[0027] Catalytic epoxidation reaction: The collected wet bacteria were resuspended in potassium phosphate buffer solution (0.1M, pH 7.0). 5mL of the catalytic system includes: bacteria (0.1g / mL), potassium phosphate buffer (0.1M, pH 7.0), 2mmol / L 3,4-difluorostyrene (dissolved in isopropanol to prepare 1mol / L mother solution), placed in a 100mL Erlenmeyer flask, placed in an air bath shaker at 30°C, 200rpm for 2h, extracted twice with an equal volume of ethyl acetate, combined the organic phases and dried with anhydrous sodium sulfate, evaporated under reduced pressure, then redissolved with isopropanol, filtered with a 0.22μm filter membrane, and the substrate conversion rate was detected by GC, and the product ee value was detected by HPLC.

[0028] GC determination of conversion rate: Agilent 7890 was used to detect the conversion rate of 3,4-difluorostyrene epoxidation, and the chromatographic column was CHIRASIL-DEX CB column (Agilent, USA). Procedure: temperature 100°C was maintained for 10 min, carrier gas (nitrogen) flow rate 3 mL / min, split ratio 20:1.

[0029] The ee value of the product was detected by HPLC: chromatographic column Chiralpak IC-H, detection wavelength was 210 nm, column temperature was 30 °C, n-hexane:isopropanol was 99:1, and flow rate was 0.5 mL / min.

[0030] The conversion rate of wild-type SeStyA to 3,4-difluorostyrene was 69% as determined by GC. The product was 2-(3,4-difluorophenyl)oxirane racemate (see the attached manual). Figure 1 , B). The conversion rate of 3,4-difluorostyrene by mutant S178Y was significantly increased to 95%, and the product was (S)-2-(3,4-difluorophenyl)oxirane with an enantiomeric excess of >99%ee (see the attached manual). Figure 1 , C). Therefore, mutant S178Y is an excellent catalyst for catalyzing the preparation of ticagrelor intermediate (S)-2-(3,4-difluorophenyl)oxirane.

Claims

1. A styrene monooxygenase mutant, characterized in that Taking the amino acid sequence SEQ ID NO.2 of styrene monooxygenase SeStyA as the starting sequence, the characteristics after mutation are as follows: the serine at position 178 is mutated to tyrosine.

2. Use of the styrene monooxygenase mutant according to claim 1 in catalyzing the epoxidation of 3,4-difluorostyrene to synthesize (S)-2-(3,4-difluorophenyl)ethylene oxide.

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