A method for the desymmetrization of phenylmethylsulfoxide catalyzed by two enzymes

Abstract

This invention discloses a method for the deracemization of benzyl sulfoxide catalyzed by a dual-enzyme catalyst. The method includes the following steps: a) culturing and expressing genetically engineered bacteria producing cyclohexanone monooxygenase and sulfoxide reductase separately in LB medium, collecting the bacterial cells by centrifugation, and resuspending the cells in PBS buffer to obtain bacterial suspensions; b) mixing the two bacterial suspensions obtained in step a), racemic benzyl sulfoxide, DTT, and D-glucose, and reacting with shaking at 30°C; c) after the reaction is complete, extracting multiple times with an organic solvent, combining the organic phases, drying with anhydrous sodium sulfate, filtering, and recovering the solvent to obtain the target product. This invention achieves the deracemization of benzyl sulfoxide through dual-enzyme catalysis, resulting in a product with high optical purity and low raw material cost. The dynamic kinetic resolution method in aqueous solution is simple to operate, with mild reaction conditions, environmentally friendly operation, high substrate conversion rate, and good stereoselectivity.

Description

Technical Field

[0001] This invention relates to the field of deracemization technology of benzyl sulfoxide, and more specifically to a method for deracemization of benzyl sulfoxide catalyzed by a dual-enzyme approach. Background Technology

[0002] Chiral sulfoxides, as a class of organosulfur compounds with important biological activities, have wide applications in asymmetric synthesis and drug synthesis. Therefore, the preparation of optically pure chiral sulfoxides has become an important research topic. Stereoselective sulfoxide reductases can catalyze the reduction of a substrate with one configuration in racemic sulfoxides, leaving an optically pure sulfoxide with the other configuration. This method has advantages such as high activity, high stereoselectivity, and high substrate tolerance. Furthermore, as a biocatalytic method, compared to most transition metal catalysts and small organic molecule catalysts, it offers advantages such as mild reaction conditions, environmental friendliness, and fewer byproducts. However, as a kinetic resolution strategy, the maximum theoretical yield is only 50%, and the thioether generated by reduction is not the target product. Therefore, by combining a non-selective oxidation reaction to oxidize the generated thioether to sulfoxides, and then using a dynamic kinetic resolution method to convert the racemic sulfoxides into a single chiral product, the theoretical yield can be increased to 100%.

[0003] Currently, dynamic kinetic resolution achieved by methods such as chemical-enzyme catalysis or photo-enzyme catalysis has been reported, but the reaction conditions for chemical catalysis and enzyme catalysis often differ significantly. With the development of enzyme catalysis methods, more and more functional catalytic enzymes have been developed, and efficient dynamic kinetic resolution can be achieved through enzyme cascade catalysis. Therefore, a two-enzyme cascade catalysis method, using racemic sulfoxide as a substrate and selective sulfoxide reductase and selective oxidase as catalysts, to accumulate the desired enantiomer through a continuous cycle of selective reduction and oxidation, is an effective strategy for achieving sulfoxide deracemization.

[0004] Screening for highly efficient selective sulfoxide reductases and oxidases, optimizing the reaction conditions catalyzed by the two enzymes, effectively controlling the catalytic process, and improving the yield and optical purity of the target configuration product are urgent problems to be solved in order to achieve efficient sulfoxide deracemization. Summary of the Invention

[0005] This invention aims to provide a two-enzyme cascade catalytic method for the deracemification of sulfoxides, which achieves high-efficiency, high-yield, and environmentally friendly deracemification of sulfoxides through dynamic kinetic resolution (as shown in chemical equation 1).

[0006]

[0007] Chemical Equation 1 Synthetic Route

[0008] The dual-enzyme cascade catalytic method of this invention uses sulfoxide reductase methionine sulfoxide reductase A from pseudomonas alcaliphila (paMsrA) and cyclohexanone monooxygenase CHMO from Acinetobacter sp. NCIMB 9871 (CHMO). Acineto Using racemic benzene sulfoxide as a raw material, sulfoxide reductase paMsrA can reduce S-configuration benzene sulfoxide to anisole, while cyclohexanone monooxygenase CHMO acts as a catalyst for selective reduction and oxidation, respectively. Acineto The generated anisole can be oxidized to R-configured benzyl sulfoxide. By adding two enzymes to the same reaction system and adjusting the activities of the two enzymes in the system, racemic benzyl sulfoxide can be converted into optically pure R-configured benzyl sulfoxide, achieving deracemicization of benzyl sulfoxide in a one-pot process.

[0009] Specifically, the present invention provides a method for deracemification of sulfoxides catalyzed by a dual-enzyme catalyst, comprising the following steps:

[0010] 1) The genetically engineered bacteria that produce cyclohexanone monooxygenase and sulfoxide reductase are cultured separately, and after induction of expression, the precipitated bacterial cells are collected by centrifugation and resuspended in buffer to obtain bacterial suspension.

[0011] 2) Mix the bacterial suspension of the cyclohexanone monooxygenase-producing genetically engineered bacteria, the bacterial suspension of the sulfoxide reductase-producing genetically engineered bacteria, racemic benzene sulfoxide, dithiothreitol (DTT), and D-glucose obtained in step 1) and react them.

[0012] 3) After the reaction is complete, the product is extracted multiple times with an organic solvent, the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and the solvent is recovered to obtain the target product;

[0013] Preferably, the amino acid sequence of the sulfoxide reductase paMsrA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0014] Preferably, the cyclohexanone monooxygenase CHMO Acineto The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0015] The specific construction method of the genetically engineered bacterium producing sulfoxide reductase described in this invention is as follows: the gene of sulfoxide reductase is synthesized and constructed into a plasmid vector that can express the exogenous gene, then transformed into a host bacterium that can express the exogenous gene, and the genetically engineered bacterium is fermented and cultured to achieve heterologous expression of sulfoxide reductase.

[0016] The specific construction method of the genetically engineered bacterium producing cyclohexanone monooxygenase described in this invention is as follows: the gene of cyclohexanone monooxygenase is synthesized and constructed into a plasmid vector that can express the exogenous gene, then transformed into a host bacterium that can express the exogenous gene, and the genetically engineered bacterium is fermented and cultured to achieve heterologous expression of cyclohexanone monooxygenase.

[0017] In this invention, LB medium is used as the medium for the cultivation and expression of carbon, nitrogen, inorganic and other nutrients in genetically engineered bacteria, and the bacterial cells obtained after centrifuging the medium are used as whole-cell catalysts.

[0018] Preferably, the concentration of the bacterial suspension is 20 g / L.

[0019] Preferably, the buffer solution is a PBS buffer solution of 100 mmol / L with a pH of 6.5 to 9.0, specifically pH 6.5, 7.5, or 9.0, and more preferably pH 7.5.

[0020] Preferably, the bacterial suspension of the genetically engineered bacteria producing cyclohexanone monooxygenase and the bacterial suspension of the genetically engineered bacteria producing sulfoxide reductase are mixed in a volume ratio of 1:1 to 16:1, specifically in the form of 1:1, 2:1, 4:1, 8:1, or 16:1. More preferably, the volume ratio is 4:1.

[0021] Preferably, the final concentration of racemic benzene sulfoxide in the reaction system is 40–48 mmol / L; the final concentration of dithiothreitol in the reaction system is 80–97 mmol / L; and the final concentration of D-glucose in the reaction system is 80–97 mmol / L.

[0022] Preferably, the reaction is carried out by shaking at 30°C for 4 hours.

[0023] The beneficial effects of this invention are as follows: This invention achieves the deracemification of benzyl sulfoxide through a dual-enzyme cascade catalysis method and a one-pot, one-step dynamic kinetic approach. The operation is simple, the reaction conditions are mild, environmentally friendly, and the substrate conversion rate is high with good stereoselectivity. Attached Figure Description

[0024] Figure 1 The mixture was characterized by gas chromatography for racemic benzene sulfoxide.

[0025] Figure 2 It was characterized by gas chromatography for (R)-benzyl sulfoxide. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] Example 1: Construction and Induced Expression of Sulfoxide Reductase Genetically Engineered Bacteria

[0028] The gene for sulfoxide reductase paMsrA (nucleotide sequence shown in SEQ ID NO.2, synthesized by Qingke Biotechnology Co., Ltd.) was ligated into the pET28a vector with NdeI and Xhol restriction sites. This vector was then transformed into *E. coli* BL21(DE3) competent cells, and single colonies were picked to obtain recombinant bacteria. Single colonies from the plate were inoculated into 20 mL of LB medium containing 34 μg / mL kanamycin and cultured for approximately 12 h as a seed culture. This seed culture was then inoculated into 400 mL of LB medium containing 34 μg / mL kanamycin at a 1% v / v inoculation rate and cultured at 37°C with shaking at 200 rpm for approximately 4 h until the OD600 reached 0.5–0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.4 mmol / L to induce expression, and the culture was continued at 16°C with shaking for 8 h. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min. The collected bacterial cells were fully resuspended in PBS buffer (100mM, pH 7.5) at a concentration of 20g / L to obtain a bacterial suspension.

[0029] The amino acid sequence of sulfoxide reductase paMsrA is shown in SEQ ID NO.1, specifically:

[0030] MVLRSQILVNKDVLPTAEQALPGRTEAMPVADTHYVNGNPIKAPFPAGLQQAVFGLGCFWGAERRFWQQPGVFSTAVGYAGGLTPNPTYEEVCSGLTGHTEVVLVVFDP QQTSFEALLKVFWEVHNPTQGMRQGNDQGTQYRSAIYCQDDAQLSAAKASQARFQAELDKAGVGSITTEIAEAPTFYYAETYHQQYLAKNPGGYCGLGGTGVCLPPES*

[0031] The nucleotide sequence is shown in SEQ ID NO.2, specifically as follows:

[0032] .

[0033] Example 2: Construction and Induced Expression of Cyclohexanone Monooxygenase Genetically Engineered Bacteria

[0034] Cyclohexanone monooxygenase CHMO AcinetoThe gene (nucleotide sequence shown in SEQ ID NO.4, synthesized by Qingke Biotechnology Co., Ltd.) was ligated into the pET28a vector with NdeI and Xhol restriction enzyme sites, and then transformed into E. coli BL21(DE3) competent cells. Single colonies were picked to obtain recombinant bacteria. Single colonies on the plate were inoculated into 20 mL of LB medium containing 34 μg / mL kanamycin and cultured for about 12 h as seed culture. The seed culture was then inoculated into 400 mL of LB medium containing 34 μg / mL kanamycin at a 1% v / v inoculation rate and cultured at 37°C with shaking at 200 rpm for about 4 h until the OD600 reached 0.5-0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.4 mmol / L to induce expression, and the culture was continued at 16°C with shaking for 8 h. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min. The collected bacterial cells were fully resuspended in PBS buffer (100mM, pH 7.5) at a concentration of 20g / L to obtain a bacterial suspension.

[0035] Cyclohexanone monooxygenase (CHMO) Acineto The amino acid sequence is shown in SEQ ID NO.3, specifically:

[0036] *

[0037] The nucleotide sequence is shown in SEQ ID NO.4, specifically as follows:

[0038]

[0039] Example 3: Deracemization of benzyl sulfoxide

[0040] Take 1 mL of resuspended bacterial culture expressing sulfoxide reductase paMsrA and cyclohexanone monooxygenase CHMO, respectively. Acineto 4 mL of the resuspended bacterial solution was mixed with 1 mL of 0.4 mol / L racemic benzyl sulfoxide solution, 2 mL of 0.4 mol / L DTT solution, and 2 mL of 0.4 mol / L D-glucose solution. The mixture was thoroughly combined and reacted at 200 rpm and 30 °C for 4 h. After the reaction, the mixture was extracted three times with 5 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain the crude product, which was then analyzed by chiral gas chromatography. The results showed that (R)-benzyl sulfoxide was obtained, with an ee value of 99% and a conversion rate of 97.8%. The gas chromatogram of racemic benzyl sulfoxide is shown in [Figure number missing]. Figure 1 The gas chromatogram of (R)-phenyl sulfoxide is shown in [reference needed]. Figure 2 .

[0041] Example 4: Deracemization of benzyl sulfoxide

[0042] Take 2.5 mL of resuspended bacterial culture expressing sulfoxide reductase paMsrA and cyclohexanone monooxygenase CHMO. Acineto 2.5 mL of the resuspended bacterial solution was added to 1 mL of 0.4 mol / L racemic benzene sulfoxide solution, 2 mL of 0.4 mol / L DTT solution, and 2 mL of 0.4 mol / L D-glucose solution. The mixture was thoroughly mixed and reacted at 200 rpm and 30 °C for 4 h. After the reaction, the mixture was extracted three times with 5 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was detected by chiral gas chromatography. The results showed that (R)-benzene sulfoxide was obtained, with an ee value of 99% and a conversion rate of 91.7%.

[0043] Example 5: Deracemization of benzyl sulfoxide

[0044] Take 2 mL of resuspended bacterial culture expressing sulfoxide reductase paMsrA and cyclohexanone monooxygenase CHMO respectively. Acineto 4 mL of the resuspended bacterial solution was mixed with 1 mL of 0.4 mol / L racemic benzene sulfoxide solution, 2 mL of 0.4 mol / L DTT solution, and 1 mL of 0.8 mol / L D-glucose solution. The mixture was thoroughly combined and reacted at 200 rpm and 30 °C for 4 h. After the reaction, the mixture was extracted three times with 5 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was detected by chiral gas chromatography. The results showed that (R)-benzene sulfoxide was obtained, with an ee value of 99% and a conversion rate of 92.9%.

[0045] Example 6: Deracemization of benzyl sulfoxide

[0046] Take 0.5 mL of resuspended bacterial culture expressing sulfoxide reductase paMsrA and cyclohexanone monooxygenase CHMO, respectively. Acineto 4 mL of the resuspended bacterial solution was mixed with 1 mL of 0.4 mol / L racemic benzene sulfoxide solution, 2 mL of 0.4 mol / L DTT solution, and 1 mL of 0.8 mol / L D-glucose solution. The mixture was thoroughly combined and reacted at 200 rpm and 30 °C for 4 h. After the reaction, the mixture was extracted three times with 5 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was detected by chiral gas chromatography. The results showed that (R)-benzene sulfoxide was obtained, with an ee value of 99% and a conversion rate of 94.1%.

[0047] Example 7: Deracemization of benzyl sulfoxide

[0048] Take 0.25 mL of resuspended bacterial culture expressing sulfoxide reductase paMsrA and cyclohexanone monooxygenase CHMO. Acineto 4 mL of the resuspended bacterial solution was mixed with 1 mL of 0.4 mol / L racemic benzene sulfoxide solution, 2 mL of 0.4 mol / L DTT solution, and 1 mL of 0.8 mol / L D-glucose solution. The mixture was thoroughly combined and reacted at 200 rpm and 30 °C for 4 h. After the reaction, the mixture was extracted three times with 5 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was detected by chiral gas chromatography. The results showed that (R)-benzene sulfoxide was obtained, with an ee value of 93% and a conversion rate of 98.7%.

[0049] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for deracemifying benzyl sulfoxide using a dual-enzyme catalysis, characterized in that, Includes the following steps: 1) The genetically engineered bacteria that produce cyclohexanone monooxygenase and sulfoxide reductase are cultured separately, and after induction of expression, the precipitated bacterial cells are collected by centrifugation and resuspended in buffer to obtain bacterial suspension. 2) Mix the bacterial suspension of the cyclohexanone monooxygenase-producing genetically engineered bacteria, the bacterial suspension of the sulfoxide reductase-producing genetically engineered bacteria obtained in step 1), racemic benzene sulfoxide, dithiothreitol, and D-glucose, and react them. 3) After the reaction is complete, the product is extracted multiple times with an organic solvent, the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and the solvent is recovered to obtain the target product; The amino acid sequence of the sulfoxide reductase is shown in SEQ ID NO.1; the amino acid sequence of the cyclohexanone monooxygenase is shown in SEQ ID NO.

3.

2. The method for deracetamping benzyl sulfoxide catalyzed according to claim 1, characterized in that, The concentration of the bacterial suspension is 20 g / L.

3. The method for deracetamping benzyl sulfoxide catalyzed according to claim 1, characterized in that, The buffer solution is a PBS buffer with a concentration of 100 mmol / L and a pH of 6.5–9.

0.

4. The method for deracetaming of benzyl sulfoxide catalyzed according to claim 3, characterized in that, The buffer solution is a 100 mmol / L PBS buffer with a pH of 7.

5.

5. The method for deracematement of benzyl sulfoxide catalyzed according to claim 1, characterized in that, The bacterial suspensions of genetically engineered bacteria producing cyclohexanone monooxygenase and sulfoxide reductase are prepared by volume ratio. Mix 1:1 to 16:

1.

6. The method for deracetamping benzyl sulfoxide catalyzed according to claim 5, characterized in that, The bacterial suspension of the genetically engineered bacteria producing cyclohexanone monooxygenase and the bacterial suspension of the genetically engineered bacteria producing sulfoxide reductase are mixed at a volume ratio of 4:

1.

7. The method for deracetaming of benzyl sulfoxide catalyzed according to claim 1, characterized in that, The final concentration of the racemic benzene sulfoxide in the reaction system is 40–48 mmol / L; the final concentration of the dithiothreitol in the reaction system is 80–97 mmol / L; and the final concentration of the D-glucose in the reaction system is 80–97 mmol / L.

8. The method for deracetamping benzyl sulfoxide catalyzed according to claim 1, characterized in that, The reaction was carried out under shaking conditions at 30°C for 4 hours.

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