Synthesis method for chiral epoxide catalyzed by styrene monooxygenase

Abstract

A synthesis method for a chiral epoxide catalyzed by styrene monooxygenase. The synthesis method comprises: carrying out an enzyme catalysis reaction on a raw material system comprising oxidase, an olefin compound, reductase, dehydrogenase, a hydrogen donor, FAD, and NAD+ so as to obtain a chiral epoxide. R2 and R3 are each independently selected from substituents such as H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, and a substituted or unsubstituted C2-C6 ester group; and R1 is selected from substituents such as halogen, hydroxy, carboxyl, acetoxy, and substituted or unsubstituted C1-C6 alkyl. The raw material system is used for carrying out the enzyme catalysis reaction, and the oxidase in the raw material system is used so as to obtain the chiral epoxide. The synthesis method for the chiral epoxide catalyzed by the oxidase has the advantages of a short synthesis route, a wide range of substrate types, high selectivity, mild reaction conditions, less three wastes, and low costs.

Description

Process for the synthesis of chiral epoxides catalyzed by styrene monooxygenases

[0001] This application is based on and claims priority to Chinese application No. 202410594631.4, filed on May 14, 2024, the disclosure of which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of synthesis method of chiral epoxides, and in particular, relates to a process for the synthesis of chiral epoxides catalyzed by styrene monooxygenases. BACKGROUND

[0003] Large bite chiral epoxide (in this application refers to a class of compounds in which at least one side of the group on both sides of the three ether structure is larger than methyl) is a very important chiral synthetic segment in organic synthesis, and has important application value in medicine, pesticide, perfume and fine chemical industry. At present, the main method is to use noble metal catalyst and crystalline titanium silicate catalyst with MFI structure as double catalyst to catalyze the epoxidation of olefin substrate, but the chemical catalytic reaction condition is harsh, the selectivity is low, there are many by-products, and the cost of heavy metal catalyst used is high and not environmentally friendly. At present, there are few reports on the synthesis of large bite chiral epoxide, and satisfactory results cannot be achieved. (Jaan Pesti*, Chien-Kuang Chen*, Lori Spangler, Albert J. DelMonte, Serge Benoit, Derek Berglund, Jeffrey Bien, Paul Brodfuehrer, Yeung Chan, Elisabeth Corbett, Carrie Costello, Paul DeMena, Robert P. Discordia, Wendel Doubleday, Zhinong Gao, Stephane Gingras, John Grosso, Oscar Haas, David Kacsur, Chiajen Lai, Simon Leung, Melanie Miller, Jale Muslehiddinoglu, Nina Nguyen, Jun Qiu, Martina Olzog, Emily Reiff, Dominique Thoraval, Michael Totleben, Dale Vanyo, Purushotham Vemishetti, John Wasylak, and Chenkou Wei. The Process Development of Ravuconazole: An Efficient Multikilogram Scale Preparation of an Antifungal Agent. Organic Process Research&Development. 2009, 13(4): 716-728) discloses a synthesis method of large bite chiral epoxide intermediate, but the synthesis route is long, and the active group needs to be protected and deprotected, which leads to the technical problems of harsh reaction condition, poor selectivity and complex process.

[0004] Therefore, the synthesis method of the large steric hindrance chiral epoxide in the prior art has problems of high cost and complex process, and a new synthesis method needs to be provided to replace the current traditional method.

[0005] SUMMARY

[0006] The main purpose of the present application is to provide a synthesis method of chiral epoxide catalyzed by styrene monooxygenase, so as to solve the problems of long synthesis process route, complex synthesis process, poor selectivity, high synthesis cost and large pollution in the synthesis method of chiral epoxide in the prior art.

[0007] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a synthesis method of chiral epoxide catalyzed by styrene monooxygenase is provided, which comprises: carrying out enzyme catalytic reaction on a raw material system comprising an oxidase, an olefin compound, a reductase, a dehydrogenase, a hydrogen donor, FAD, NAD + of the chiral epoxide are as follows:

[0008] R2 and R3 are each independently selected from any one of H, substituted or non-substituted C1-C6 alkyl, substituted or non-substituted C1-C6 heteroalkyl, substituted or non-substituted C2-C6 ester group, substituted or non-substituted C1-C4 amide group, substituted or non-substituted C3-C6 cycloalkyl, substituted or non-substituted C3-C6 cycloalkenyl, and substituted or non-substituted C6-C 12 halogen, hydroxyl, carboxyl, acetoxy, substituted or non-substituted C1-C6 alkyl, and substituted or non-substituted C1-C6 heteroalkyl.

[0009] Further, the heteroatom in the above-mentioned heteroalkyl is selected from any one or more of N, O and S, and R2 and R3 are each independently selected from any one of H, substituted or non-substituted C1-C4 alkyl, substituted or non-substituted C1-C4 alkoxy, substituted or non-substituted C2-C4 ester group, substituted or non-substituted C1-C2 amide group, substituted or non-substituted C3-C5 cycloalkyl, substituted or non-substituted C3-C5 cycloalkenyl, and substituted or non-substituted C6-C 10 aryl.

[0010] Further, each of R2and R3is independently selected from any one or more of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate group, ethyl acetate group, methyl acetate group, carboxamide group, acetyl group, cyclobutyl group, cyclopentyl group, cyclobutadienyl group, cyclopentadienyl group, phenyl group, biphenyl group; when R2and R3have a substituent, the substituent is selected from any one or more of hydroxyl group, carboxyl group, methyl group, ethyl group, nitro group, methylamine group, acetoxy group, five-membered ring heteroaryl group, or six-membered ring heteroaryl group.

[0011] Further, each of R2and R3is independently selected from any one or more of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate group, ethyl acetate group, methyl acetate group, carboxamide group, acetyl group, cyclobutyl group, cyclopentyl group, cyclobutadienyl group, cyclopentadienyl group, phenyl group, biphenyl group; when R2and R3have a substituent, the substituent is selected from any one or more of hydroxyl group, carboxyl group, methyl group, ethyl group, nitro group, methylamine group, acetoxy group, five-membered ring heteroaryl group, or six-membered ring heteroaryl group. Further, each of R2and R3is independently selected from any one or more of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate group, ethyl acetate group, methyl acetate group, carboxamide group, acetyl group, cyclobutyl group, cyclopentyl group, cyclobutadienyl group, cyclopentadienyl group, phenyl group, biphenyl group; when R2and R3have a substituent, the substituent is selected from any one or more of hydroxyl group, carboxyl group, methyl group, ethyl group, nitro group, methylamine group, acetoxy group, five-membered ring heteroaryl group, or six-membered ring heteroaryl group.

[0012] Further, the heteroatom in the above-mentioned heteroalkyl group is selected from any one or more of N, O, S, and R1is selected from any one or more of halogen, hydroxyl group, carboxyl group, substituted or unsubstituted C1-C4 alkyl group, substituted or unsubstituted C1-C4 alkoxy group; when R1has a substituent, the substituent is selected from any one or more of hydroxyl group, carboxyl group, methyl group, ethyl group, nitro group, methylamine group, acetoxy group.

[0013] Further, R1is selected from any one or more of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate group, ethyl acetate group, methyl acetate group, carboxamide group, acetyl group, cyclobutyl group, cyclopentyl group, cyclobutadienyl group, cyclopentadienyl group, phenyl group, biphenyl group.

[0014] Further, R1is selected from any one or more of F, Cl, hydroxyl group, methyl group, ethyl group, methoxy group, hydroxymethyl group, acetoxy group.

[0015] Further, the above-mentioned olefin compound is selected from any one or more of

[0016]

[0017] ​Further, the above-mentioned oxidase is selected from any one or more of an oxidase having a sequence derived from Marinobacterium litorale DSM 23545 and NCBI sequence number WP_027855270.1, an oxidase having a sequence derived from Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, an oxidase having a sequence derived from Streptomyces exfoliatus and NCBI sequence number WP_137992763.1, an oxidase having a sequence derived from Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, an oxidase having a sequence derived from Amycolatopsis albispora and NCBI sequence number WP_113694022.1, an oxidase having a sequence derived from Celeribacter baekdonensis and NCBI sequence number WP_107722930.1, an oxidase having a sequence derived from Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, an oxidase having a sequence derived from Nocardia farcinica and NCBI sequence number MBF6535966.1, an oxidase having a sequence derived from Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, an oxidase having a sequence derived from Pseudomonas oryzae and NCBI sequence number WP_090348206.1, an oxidase having a sequence derived from Pseudomonas sp. SXM-1 and NCBI sequence number WP_134326109.1, an oxidase having a sequence derived from Pseudonocardia sp. SID8383 and NCBI sequence number WP_161151635.1, an oxidase having a sequence derived from Streptomyces sp. WAC01280 and NCBI sequence number WP_125743710.1, an oxidase having a sequence derived from Pseudomonas putida S12 and NCBI sequence number AJA17113.1, and an oxidase having a sequence derived from Variovorax paradoxus EPS and NCBI sequence number ADU39062.1.

[0018] Further, the oxidase is any one or more of the oxidase with sequence origin from Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, the oxidase with sequence origin from Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, the oxidase with sequence origin from Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, the oxidase with sequence origin from Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, and the oxidase with sequence origin from Pseudomonas oryzae and NCBI sequence number WP_090348206.1.

[0019] Further, the temperature of the enzyme catalyzed reaction is 20-40℃, and / or the time of the enzyme catalyzed reaction is 16-72h.

[0020] Further, the mass ratio of the oxidase in the raw material system to the raw material system is 10-50:1; and / or the mass of the oxidase is 20-100mg; and / or the mass ratio of the reductase to the raw material system is 1-10:1; and / or the mass of the reductase is 2-20mg; and / or the mass ratio of the hydrogen donor to the raw material system is 1-10:1; the total mass of FAD and NAD + to the raw material system is 10-1000:1; and / or the reductase is the reductase StyB capable of regenerating FADH2, and / or the dehydrogenase and the hydrogen donor are glucose dehydrogenase and glucose, respectively; and / or the dehydrogenase and the hydrogen donor are alcohol dehydrogenase and isopropanol, respectively; and / or the mass concentration of the olefin compound is 0.5-2mg / mL; and / or the origin of the reductase StyB is any one or more of Pseudomonas sp. LQ26, Bradyrhizobium sp. ORS 375, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas sp. Y2, Rhodococcus sp. ST-5, and Rhodococcus sp. ST-10; and / or the raw material system comprises a buffer solution, the buffer solution is any one or more of a phosphate buffer, a Tris-HCl buffer, and a boric acid buffer, and / or the pH value of the buffer solution is 7.5-10.

[0021] The application has the advantages that the steric hindrance of the ethylene double bond in the above olefin compound is large, but the raw material system is used for enzyme catalytic reaction, and the chiral epoxide is obtained by using the oxidase. Compared with the traditional metal catalyst synthesis method, the above enzyme catalytic synthesis method of the chiral epoxide has the advantages of short synthesis route, wide substrate type, high selectivity, mild reaction condition, less waste, and low cost. DETAILED DESCRIPTION

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0023] As analyzed in the background art, the synthesis method of the chiral epoxide in the prior art has the problems of long synthesis process route, complex synthesis process, poor selectivity, high synthesis cost, and large pollution. In order to solve the problem, the present application provides a synthesis method of a chiral epoxide catalyzed by a styrene monooxygenase.

[0024] In a typical embodiment of the present application, a synthesis method of a chiral epoxide catalyzed by a styrene monooxygenase is provided, which comprises: performing enzyme catalytic reaction on a raw material system comprising an oxidase, an olefin compound, a reductase, a dehydrogenase, a hydrogen donor, FAD, NAD + , to obtain a chiral epoxide; wherein the structural formulas of the olefin compound and the chiral epoxide are as follows:

[0025] R2 and R3 are each independently selected from any one of H, substituted or non-substituted C1-C6 alkyl, substituted or non-substituted C1-C6 heteroalkyl, substituted or non-substituted C2-C6 ester group, substituted or non-substituted C1-C4 amide group, substituted or non-substituted C3-C6 cycloalkyl, substituted or non-substituted C3-C6 cycloalkenyl, and substituted or non-substituted C6-C10 aryl; and R1 is selected from any one of halogen, hydroxyl, carboxyl, acetoxy, substituted or non-substituted C1-C6 alkyl, and substituted or non-substituted C1-C6 heteroalkyl. 12 The steric hindrance of the ethylene double bond in the above olefin compound is large, but the raw material system is used for enzyme catalytic reaction, and the chiral epoxide is obtained by using the oxidase. Compared with the traditional metal catalyst synthesis method, the above enzyme catalytic synthesis method of the chiral epoxide has the advantages of short synthesis route, wide substrate type, high selectivity, mild reaction condition, less waste, and low cost.

[0026] The steric hindrance of the ethylene double bond in the above olefin compound is large, but the raw material system is used for enzyme catalytic reaction, and the chiral epoxide is obtained by using the oxidase. Compared with the traditional metal catalyst synthesis method, the above enzyme catalytic synthesis method of the chiral epoxide has the advantages of short synthesis route, wide substrate type, high selectivity, mild reaction condition, less waste, and low cost.

[0027] In one embodiment of this application, preferably, the heteroatom on the heteroalkyl group is selected from any one or more of N, O, and S, and R2 and R3 are each independently selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, substituted or unsubstituted C2-C4 ester, substituted or unsubstituted C1-C2 amide, substituted or unsubstituted C3-C5 cycloalkyl, substituted or unsubstituted C3-C5 cycloalkenyl, substituted or unsubstituted C6-C 10 Any of the aryl groups further enriches the selectivity of olefin compounds.

[0028] In one embodiment of this application, preferably, R2 and R3 are each independently selected from any one of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate, ethyl acetate, methyl acetate, formamido, acetyl, cyclobutyl, cyclopentyl, cyclobutadienyl, cyclopentadienyl, phenyl, and biphenyl; when R2 and R3 have substituents, the substituents are selected from any one or more of hydroxyl, carboxyl, methyl, ethyl, nitro, methylamino, acetoxy, five-membered ring heteroaryl, or six-membered ring heteroaryl, preferably the five-membered ring heteroaryl is an azaheterocyclic aryl, and preferably the azaheterocyclic aryl is... This further enriches the selectivity of olefin compounds.

[0029] In one embodiment of this application, preferably R2 and R3 are each independently selected from H, methyl, ethyl, phenyl, The presence of any one of the formamido, hydroxymethyl, or cyclopentadienyl groups further enriches the selectivity of olefin compounds.

[0030] In one embodiment of this application, preferably, the heteroatom on the heteroalkyl group is selected from any one or more of N, O, and S, and R1 is selected from any one of halogen, hydroxyl, carboxyl, substituted or unsubstituted C1-C4 alkyl, and substituted or unsubstituted C1-C4 alkoxy. When R1 has a substituent, the substituent is selected from any one or more of hydroxyl, carboxyl, methyl, ethyl, nitro, methylamino, and acetoxy, further enriching the selectivity of olefin compounds.

[0031] In one embodiment of this application, R1 is preferably selected from any one of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethyl formate, ethyl acetate, methyl acetate, formamido, acetyl, cyclobutyl, cyclopentyl, cyclobutadienyl, cyclopentadienyl, phenyl, and biphenyl, further enriching the selectivity of olefin compounds.

[0032] In an embodiment of the present application, it is preferred that R1is selected from the group consisting of F, CI, hydroxyl, methyl, ethyl, methoxy, hydroxymethyl, acetoxy, and any one or more of the group consisting of Further enriches the selectivity of olefin compounds.

[0033] In an embodiment of the present application, it is preferred that the olefin compound is selected from the group consisting of Further enriches the types of chiral epoxides.

[0034] In an embodiment of the application, the above oxidase is selected from any one or more of the oxidases of sequence origin Marinobacterium litorale DSM 23545 and NCBI sequence number WP_027855270.1, Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, Streptomyces exfoliatus and NCBI sequence number WP_137992763.1, Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, Amycolatopsis albispora and NCBI sequence number WP_113694022.1, Celeribacter baekdonensis and NCBI sequence number WP_107722930.1, Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, Nocardia farcinica and NCBI sequence number MBF6535966.1, Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, Pseudomonas oryzae and NCBI sequence number WP_090348206.1, Pseudomonas sp. SXM-1 and NCBI sequence number WP_134326109.1, Pseudonocardia sp. SID8383 and NCBI sequence number WP_161151635.1, Streptomyces sp. WAC01280 and NCBI sequence number WP_125743710.1, Pseudomonas putida S12 and NCBI sequence number AJA17113.1, and Variovorax paradoxus EPS and NCBI sequence number ADU39062.1.

[0035] The preferred above oxidases help facilitate the conversion of the more hindered olefinic compounds of the application to chiral epoxides.

[0036] Further, it is preferred that the above-mentioned oxidase is any one or more of oxidases with sequence source selected from Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, oxidases with sequence source selected from Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, oxidases with sequence source selected from Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, oxidases with sequence source selected from Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, and oxidases with sequence source selected from Pseudomonas oryzae and NCBI sequence number WP_090348206.1, which helps to improve the efficiency of converting the olefin compound into the chiral epoxide.

[0037] In an embodiment of the present application, it is preferred that the temperature of the above-mentioned enzyme catalytic reaction is 20-40°C, preferably 25-35°C, and / or the time of the enzyme catalytic reaction is 16-72h, which helps to improve the efficiency and effect of converting the olefin compound into the chiral epoxide.

[0038] In an embodiment of the present application, the mass ratio of the above-mentioned oxidase to the raw material system is 10-50:1; and / or the mass of the oxidase is 20-100mg; and / or the mass ratio of the reductase to the raw material system is 1-10:1; and / or the mass of the reductase is 2-20mg; and / or the mass ratio of the hydrogen donor to the raw material system is 1-10:1; the total mass of FAD and NAD + ; and / or the reductase is the reductase StyB capable of regenerating FADH2, and / or the dehydrogenase and the hydrogen donor are glucose dehydrogenase and glucose, respectively; and / or the dehydrogenase and the hydrogen donor are alcohol dehydrogenase and isopropanol, respectively; and / or the mass concentration of the olefin compound is 0.5-2mg / mL; and / or the source of the reductase StyB is selected from any one or more of Pseudomonas sp. LQ26, Bradyrhizobium sp. ORS 375, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas sp. Y2, Rhodococcus sp. ST-5, and Rhodococcus sp. ST-10; and / or the raw material system comprises a buffer solution selected from any one or more of phosphate buffer, Tris-HCl buffer, and boric acid buffer, and / or the pH value of the buffer solution is 7.5-10.

[0039] The above preferred conditions further optimize the reaction conditions for the conversion of olefin compounds to chiral epoxides, and under the above reaction conditions, the efficiency of the conversion of olefin compounds to chiral epoxides is further improved.

[0040] The beneficial effects of the present application will be further illustrated below in conjunction with examples.

[0041] The following examples involve the following olefin compounds:

[0042] Example 1

[0043] To solve the problems of long technical route, harsh reaction conditions, poor selectivity, complex process and high cost in the prior art, the inventors attempted to use a biological enzyme method to synthesize a large steric chiral epoxide, but there has been no report in the prior art on a styrene monooxygenase with the ability to synthesize a large steric chiral epoxide. In order to improve this situation, the inventors screened a large number of enzyme libraries of Kayline Company, and screened more than one hundred kinds of oxidoreductases including styrene monooxygenase. Most of the enzymes showed no activity (HPLC did not detect product generation), but a small number of enzymes had certain catalytic activity for the conversion of large steric substrates to chiral epoxides, as follows:

[0044] Screening and verification of large steric olefin substrates by different species of oxidases:

[0045] In this example, more than one hundred kinds of oxidases in the enzyme library of Kayline Company were selected for enzyme screening of olefin substrate 1, and the screening system was as follows: 1 mL of the reaction system included 1 mg of substrate, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μM FAD, 20 mM NAD + , 100 mM phosphate buffer with pH value of 7.0, 30°C reaction for 2 h. After the reaction was completed, 2 mL of acetonitrile was added to terminate the reaction, and after thorough shaking and mixing, centrifugation was performed at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was sent for HPLC detection of conversion rate.

[0046] The results showed that most of the enzymes in the enzyme library had no product generation detected by HPLC, but a small number of enzymes had product generation, with a conversion of about 30% at most, and these enzymes included the following sequences (see Table 1).

[0047] Table 1

[0048] Note: In the above Table 1, * represents a conversion rate less than 1%, ** represents a conversion rate greater than or equal to 1% and less than 10%, *** represents a conversion rate greater than or equal to 10% and less than 20%, **** represents a conversion rate greater than or equal to 20% and less than 30%.

[0049] Example 2

[0050] Optimization of reaction conditions:

[0051] StyA derived from Sphingopyxis fribergensis which is better active to olefin substrate 1 was selected for reaction optimization, different temperatures, pH, substrate concentration, StyB, GDH, FAD, NAD + , glucose were set respectively.

[0052] Reaction condition 1: 1 mL reaction system including 1 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μM FAD, 20 mM NAD + and 100 mM phosphate buffer with pH value of 7.0, respectively, at 20℃, 25℃, 30℃, 35℃ and 40℃ for 16h. After the reaction was completed, 2 mL of acetonitrile was added to terminate the reaction, and after fully shaking and mixing, 8000 rpm centrifugation for 1 min, the supernatant aqueous phase was obtained and sent to HPLC for detection of conversion rate, and the results are as follows in Table 2.

[0053] Table 2

[0054] Note: In the above Table 2, - represents a decrease in conversion rate, + represents an increase in conversion rate, -+ represents no change in conversion rate.

[0055] According to the results in Table 2, 35℃ was selected for the following optimization.

[0056] Reaction condition 2: 1 mL reaction system including 1 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μM FAD and 20 mM NAD + , 100 mM phosphate buffer with different pH was set, and reacted at 30℃ for 16h. After the reaction was completed, 2 mL of acetonitrile was added to terminate the reaction, and after fully shaking and mixing, 8000 rpm centrifugation for 1 min, the supernatant aqueous phase was obtained and sent to HPLC for detection of conversion rate, and the results are as follows in Table 3.

[0057] Table 3

[0058] Note: In the above table, - represents a decrease in conversion, + represents an increase in conversion, and -+ represents no change in conversion.

[0059] Based on the results in Table 3, pH 7.5 was selected for the following optimization.

[0060] Reaction condition 3: 1 mL reaction system including different amounts of olefin substrate, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μΜ FAD, 20 mM NAD + and 100 mM phosphate buffer at pH 7.5, reacted at 35°C for 16 h. After the reaction was completed, 2 mL of acetonitrile was added to the system to terminate the reaction, and after thorough shaking and mixing, centrifugation was performed at 8000 rpm for 1 min, the supernatant aqueous phase was obtained and sent to HPLC to detect the conversion rate, and the results are as shown in Table 4 below.

[0061] Table 4

[0062] Note: In the above table, - represents a decrease in conversion, + represents an increase in conversion, and -+ represents no change in conversion.

[0063] Based on the results in Table 4, the concentration of the olefin substrate was selected as 2 mg / mL for the following optimization.

[0064] Reaction condition 4: 1 mL reaction system including 2 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder from different sources, 5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μΜ FAD, 20 mM NAD + and 100 mM phosphate buffer at pH 7.5, reacted at 35°C for 16 h. After the reaction was completed, 2 mL of acetonitrile was added to the system to terminate the reaction, and after thorough shaking and mixing, centrifugation was performed at 8000 rpm for 1 min, the supernatant aqueous phase was obtained and sent to HPLC to detect the conversion rate, and the results are as shown in Table 5 below.

[0065] Table 5

[0066] Note: In the above table, - represents a decrease in conversion, + represents an increase in conversion, and -+ represents no change in conversion.

[0067] Based on the results in Table 4, StyB from Pseudomonas putida was selected for the following optimization.

[0068] Reaction condition 5: 1 mL reaction system including 2 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, different amounts of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 100 μM FAD, 20 mM NAD + and 100 mM phosphate buffer with pH value of 7.5, and reacting for 16 h at 35 °C. After the reaction was completed, 2 mL of acetonitrile was added to the system to terminate the reaction, and after being fully shaken and mixed, centrifugation was performed at 8000 rpm for 1 min to obtain the supernatant aqueous phase which was sent to HPLC for detection of the conversion rate, and the results are shown in Table 6 below.

[0069] Table 6

[0070] Note: In the above table, - represents a decrease in conversion rate, + represents an increase in conversion rate, and -+ represents no change in conversion rate.

[0071] According to the results in Table 6, 7.5 mg of GDH was selected for the following optimization.

[0072] Reaction condition 6: 1 mL reaction system including 2 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, different amounts of FAD, 20 mM NAD + and 100 mM phosphate buffer with pH value of 7.5, and reacting for 16 h at 35 °C. After the reaction was completed, 2 mL of acetonitrile was added to the system to terminate the reaction, and after being fully shaken and mixed, centrifugation was performed at 8000 rpm for 1 min to obtain the supernatant aqueous phase which was sent to HPLC for detection of the conversion rate, and the results are shown in Table 7 below.

[0073] Table 7

[0074] Note: In the above table, - represents a decrease in conversion rate, + represents an increase in conversion rate, and -+ represents no change in conversion rate.

[0075] According to the results in Table 7, 25 μM of FAD was selected for the following optimization.

[0076] Reaction condition 7: 1 mL reaction system including 2 mg of olefin substrate 1, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 25 μM of FAD, different concentrations of NAD +and 100 mM phosphate buffer at pH 7.5, at 35 °C for 16 h. After the reaction was completed, 2 mL acetonitrile was added to the system to terminate the reaction, and after the mixture was shaken well, centrifugation was performed at 8000 rpm for 1 min, and the supernatant was obtained and sent to HPLC to determine the conversion rate, with the results shown in Table 8 below.

[0077] Table 8

[0078] Note: In the above table, - represents a decrease in the conversion rate, + represents an increase in the conversion rate, and -+ represents no change in the conversion rate.

[0079] According to the results in Table 8, 5 mM NAD + was selected for the next optimization.

[0080] Reaction condition 8: 1 mL of the reaction system included 2 mg of the olefin substrate 1, 50 mg of the styrene monooxygenase StyA enzyme powder, 10 mg of the reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 10 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer at pH 7.5, at 35 °C for 16 h. After the reaction was completed, 2 mL acetonitrile was added to the system to terminate the reaction, and after the mixture was shaken well, centrifugation was performed at 8000 rpm for 1 min, and the supernatant was obtained and sent to HPLC to determine the conversion rate, with the results shown in Table 9 below.

[0081] Table 9

[0082] Note: In the above table, - represents a decrease in the conversion rate, + represents an increase in the conversion rate, and -+ represents no change in the conversion rate.

[0083] According to the results in Table 9, it was found that the glucose in the reaction system could be reduced to 5 mg.

[0084] From the single-factor optimization results of the above reaction conditions, the optimal reaction conditions at the present stage were (1 mL system) 2 mg / mL of the concentration of the olefin substrate, 7.5 mg of GDH, 25 μM of FAD, 5 mM of NAD + , 5 mg of glucose, pH 7.5, and a reaction temperature of 35 °C.

[0085] Example 3

[0086] The catalytic effect of the styrene monooxygenase obtained in Example 1 on the olefin substrate 2 was tested

[0087] * represents a chiral carbon atom

[0088] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 2, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant aqueous phase, which is sent to HPLC and GC for detection of conversion rate and chirality, and the results are shown in Table 10 below. + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant aqueous phase, which is sent to HPLC and GC for detection of conversion rate and chirality, and the results are shown in Table 10 below.

[0089] Table 10

[0090] Note: In the above table, * represents conversion rate less than 1%, ** represents conversion rate greater than or equal to 1% and less than 10%, *** represents conversion rate greater than or equal to 10% and less than 20%, and **** represents conversion rate greater than or equal to 20% and less than 30%.

[0091] Example 4

[0092] Testing the catalytic effect of the styrene monooxygenase obtained in Example 1 on olefin substrate 3

[0093] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 3, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant aqueous phase, which is sent to HPLC and GC for detection of conversion rate and chirality, and the results are shown in Table 11 below. + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, 8000 rpm centrifugation is carried out for 1 min to obtain the supernatant aqueous phase, which is sent to HPLC and GC for detection of conversion rate and chirality, and the results are shown in Table 10 below.

[0094] Table 11

[0095] Note: In the above table, * represents conversion rate less than 1%, ** represents conversion rate greater than or equal to 1% and less than 10%, *** represents conversion rate greater than or equal to 10% and less than 20%, and **** represents conversion rate greater than or equal to 20% and less than 30%.

[0096] Example 5

[0097] Testing the catalytic effect of the styrene monooxygenase obtained in Example 1 on olefin substrate 4

[0098] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 4, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, centrifugation is carried out at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are as shown in Table 12.

[0099] Table 12

[0100] Note: In the above table, * represents a conversion rate less than 1%, ** represents a conversion rate greater than or equal to 1% and less than 10%, *** represents a conversion rate greater than or equal to 10% and less than 20%, and **** represents a conversion rate greater than or equal to 20% and less than 30%.

[0101] Example 6

[0102] Testing the catalysis of the styrene monooxygenase obtained in Example 1 on olefin substrate 5

[0103] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 5, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, centrifugation is carried out at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are as shown in Table 13.

[0104] Table 13

[0105] Note: In the above table, * represents a conversion rate less than 1%, ** represents a conversion rate greater than or equal to 1% and less than 10%, *** represents a conversion rate greater than or equal to 10% and less than 20%, and **** represents a conversion rate greater than or equal to 20% and less than 30%.

[0106] Example 7

[0107] Testing the catalysis of the styrene monooxygenase obtained in Example 1 on olefin substrate 6

[0108] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 6, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, centrifugation is carried out at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are as shown in Table 14.

[0109] Table 14

[0110] Note: In the above table, * represents a conversion rate less than 1%, ** represents a conversion rate greater than or equal to 1% and less than 10%, *** represents a conversion rate greater than or equal to 10% and less than 20%, and **** represents a conversion rate greater than or equal to 20% and less than 30%.

[0111] Example 8

[0112] Testing the catalysis of the styrene monooxygenase obtained in Example 1 on olefin substrate 7

[0113] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 7, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after sufficient shaking and mixing, centrifugation is carried out at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are as shown in Table 15.

[0114] Table 15

[0115] Note: In the above table, * represents a conversion rate less than 1%, ** represents a conversion rate greater than or equal to 1% and less than 10%, *** represents a conversion rate greater than or equal to 10% and less than 20%, and **** represents a conversion rate greater than or equal to 20% and less than 30%.

[0116] Example 9

[0117] Testing the catalysis of the styrene monooxygenase obtained in Example 1 on olefin substrate 8

[0118] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 8, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after being fully shaken and mixed, the system is centrifuged at 8000 rpm for 1 min to obtain the supernatant water phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are shown in Table 16 below. + The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 8, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after being fully shaken and mixed, the system is centrifuged at 8000 rpm for 1 min to obtain the supernatant water phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are shown in Table 16 below.

[0119] Table 16

[0120] Note: In the above table, * represents conversion rate less than 1%, ** represents conversion rate greater than or equal to 1% and less than 10%, *** represents conversion rate greater than or equal to 10% and less than 20%, and **** represents conversion rate greater than or equal to 20% and less than 30%.

[0121] Example 10

[0122] Testing the catalytic effect of the styrene monooxygenase obtained in Example 1 on olefin substrate 9

[0123] The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 9, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after being fully shaken and mixed, the system is centrifuged at 8000 rpm for 1 min to obtain the supernatant water phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are shown in Table 17 below. + The experimental conditions are as follows: 1 mL reaction system includes 2 mg of olefin substrate 9, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD, and 100 mM of phosphate buffer with pH value of 7.5, and the reaction is carried out at 35 °C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to the system to terminate the reaction, and after being fully shaken and mixed, the system is centrifuged at 8000 rpm for 1 min to obtain the supernatant water phase, which is sent for HPLC and GC detection of conversion rate and chirality, and the results are shown in Table 17 below.

[0124] Table 17

[0125] Note: In the above table, * represents conversion rate less than 1%, ** represents conversion rate greater than or equal to 1% and less than 10%, *** represents conversion rate greater than or equal to 10% and less than 20%, and **** represents conversion rate greater than or equal to 20% and less than 30%.

[0126] Example 11

[0127] Testing the catalytic effect of the styrene monooxygenase obtained in Example 1 on olefin substrate 10

[0128] The experimental conditions are as follows: 1 mL of reaction system includes 2 mg of olefin substrate 10, 50 mg of styrene monooxygenase StyA enzyme powder, 10 mg of reductase StyB enzyme powder, 7.5 mg of glucose dehydrogenase (GDH) enzyme powder, 5 mg of glucose, 25 μM of FAD, 5 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 16 h. After the reaction is completed, 2 mL of acetonitrile is added to terminate the reaction, and after sufficient shaking and mixing, centrifugation is carried out at 8000 rpm for 1 min to obtain the supernatant water phase, which is sent to HPLC and GC for detection of conversion rate and chirality, and the results are shown in Table 18.

[0129] Table 18

[0130] Note: In the above table, * represents that the conversion rate is less than 1%, ** represents that the conversion rate is greater than or equal to 1% and less than 10%, *** represents that the conversion rate is greater than or equal to 10% and less than 20%, and **** represents that the conversion rate is greater than or equal to 20% and less than 30%.

[0131] Example 12

[0132] The reaction of the olefin substrate 8 by the oxidase numbered 2 and the oxidase numbered 3 is subjected to product chirality identification, and the experimental conditions are as follows: 100 mL of reaction system includes 200 mg of olefin substrate, 5000 mg of styrene monooxygenase StyA enzyme powder, 1000 mg of reductase StyB enzyme powder, 750 mg of glucose dehydrogenase (GDH) enzyme powder, 500 mg of glucose, 2.5 mM of FAD, 500 mM of NAD + and 100 mM phosphate buffer with pH value of 7.5, and the reaction is carried out at 35°C for 70 h. After the reaction is completed, extraction is carried out using ethyl acetate, column chromatography purification is carried out, and after evaporation to dryness, white powder is obtained, which is redissolved with ethanol and sent to GC for determination of chirality, and the results are shown in Table 19.

[0133] Table 19

[0134] From the above description, it can be seen that the above-mentioned examples of the present application achieve the following technical effects:

[0135] The steric hindrance of the ethylene double bond in the above-structured olefin compound is large, but the present application adopts the above raw material system for enzyme catalytic reaction, and the chiral epoxide is obtained by using the oxidase. Compared with the traditional metal catalyst synthesis method, the above-mentioned synthesis method of chiral epoxide catalyzed by oxidase has the advantages of short synthesis route, wide substrate type, high selectivity, mild reaction condition, less waste, and low cost.

[0136] The above merely describes the preferred embodiments of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the synthesis of chiral epoxides catalyzed by styrene monooxygenases, characterized in that, The synthetic method comprises: A raw material system including an oxidase, an olefinic compound, a reductase, a dehydrogenase, a hydrogen donor, FAD, NAD + is subjected to an enzyme catalytic reaction to obtain the chiral epoxide; The structural formula of the olefin compound and the chiral epoxide are as follows, respectively: R2 and R3 are each independently selected from H, substituted or unsubstituted C1-C6 alkyl groups, substituted or unsubstituted C1-C6 heteroalkyl groups, substituted or unsubstituted C2-C6 ester groups, substituted or unsubstituted C1-C4 amide groups, substituted or unsubstituted C3-C6 cycloalkyl groups, substituted or unsubstituted C3-C6 cycloalkenyl groups, and substituted or unsubstituted C6-C6 groups. 12 Any of the aryl groups; R1is selected from any one of halogen, hydroxyl, carboxyl, acetoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl.

2. The method of synthesis of claim 1, wherein, the heteroatom in said heteroalkyl is selected from any one or more of N, O, S, each of said R2and said R3is independently selected from H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4alkoxy, substituted or unsubstituted C2-C4ester, substituted or unsubstituted C1-C2amide, substituted or unsubstituted C3-C5cycloalkyl, substituted or unsubstituted C3-C5cycloalkenyl, substituted or unsubstituted C6-C10aryl, substituted or unsubstituted C6-C10alkylaryl, and substituted or unsubstituted C6-C10arylalkyl. 10 the heteroatom in said heteroalkyl is selected from any one or more of N, O, S, each of said R2and said R3is independently selected from H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4alkoxy, substituted or unsubstituted C2-C4ester, substituted or unsubstituted C1-C2amide, substituted or unsubstituted C3-C5cycloalkyl, substituted or unsubstituted C3-C5cycloalkenyl, substituted or unsubstituted C6-C10aryl, substituted or unsubstituted C6-C10alkylaryl, and substituted or unsubstituted C6-C10arylalkyl.

3. The method of synthesis according to claim 1 or 2, wherein, R2and R3are each independently selected from any one of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethylformate, ethylacetate, methylacetate, formamide, acetyl, cyclobutyl, cyclopentyl, cyclobutadienyl, cyclopentadienyl, phenyl, biphenyl; When R2and R3have substituents, the substituents are selected from any one or more of hydroxyl, carboxyl, methyl, ethyl, nitro, methylamine, acetoxy, five-membered ring heteroaryl, or six-membered ring heteroaryl.

4. The method of synthesis according to claim 1 or 2, wherein, each of said R2and said R3is independently selected from any one of H, methyl, ethyl, phenyl, formylamido, hydroxymethyl, cyclopentadienyl.

5. The method of synthesis of claim 1 or 2, wherein, The heteroatom in the heteroalkyl is selected from any one or more of N, O, S, R1is selected from any one of halogen, hydroxyl, carboxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; When R1has substituents, the substituents are selected from any one or more of hydroxyl, carboxyl, methyl, ethyl, nitro, methylamine, acetoxy.

6. The method of synthesis of claim 5, wherein, R1is selected from any one of H, methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, hydroxymethyl, ethylformate, ethylacetate, methylacetate, formamide, acetyl, cyclobutyl, cyclopentyl, cyclobutadienyl, cyclopentadienyl, phenyl, biphenyl.

7. The method of synthesis of claim 5, wherein, said R1is selected from any one of F, Cl, hydroxyl, methyl, ethyl, methoxy, hydroxymethyl, acetoxy.

8. The method of synthesis of claim 1 or 2, wherein, said olefinic compound is selected from any one or more of 9. The method of synthesis of claim 1 or 2, wherein, The oxidase is selected from any one or more of an oxidase having sequence origin Marinobacterium litorale DSM 23545 and NCBI sequence number WP_027855270.1, an oxidase having sequence origin Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, an oxidase having sequence origin Streptomyces exfoliatus and NCBI sequence number WP_137992763.1, an oxidase having sequence origin Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, an oxidase having sequence origin Amycolatopsis albispora and NCBI sequence number WP_113694022.1, an oxidase having sequence origin Celeribacter baekdonensis and NCBI sequence number WP_107722930.1, an oxidase having sequence origin Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, an oxidase having sequence origin Nocardia farcinica and NCBI sequence number MBF6535966.1, an oxidase having sequence origin Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, an oxidase having sequence origin Pseudomonas oryzae and NCBI sequence number WP_090348206.1, an oxidase having sequence origin Pseudomonas sp. SXM-1 and NCBI sequence number WP_134326109.1, an oxidase having sequence origin Pseudonocardia sp. SID8383 and NCBI sequence number WP_161151635.1, an oxidase having sequence origin Streptomyces sp. WAC01280 and NCBI sequence number WP_125743710.1, an oxidase having sequence origin Pseudomonas putida S12 and NCBI sequence number AJA17113.1, and an oxidase having sequence origin Variovorax paradoxus EPS and NCBI sequence number ADU39062.

1.

10. The method of synthesis of claim 9, wherein, The oxidase is any one or more of the oxidases with sequence origin selected from Sphingopyxis fribergensis and NCBI sequence number AJA07151.1, Bradyrhizobium sp. ORS 375 and NCBI sequence number WP_009031806.1, Rhodococcus sp. ST-5 and NCBI sequence number BAL04132.1, Pseudomonas aeruginosa and NCBI sequence number WP_121207564.1, Pseudomonas oryzae and NCBI sequence number WP_090348206.

1.

11. The method of synthesis of claim 1 or 2, wherein, The temperature for the enzyme catalyzed reaction is 20-40℃, and / or the time for the enzyme catalyzed reaction is 16-72h.

12. The method of synthesis of claim 1 or 2, wherein, the oxidase enzyme to the mass of the feedstock system is 10 to 50:1; and / or the mass of the oxidase enzyme is 20 to 100 mg; and / or the mass ratio of the reductase enzyme to the mass of the feedstock system is 1 to 10:1; and / or the mass of the reductase enzyme is 2 to 20 mg; and / or the mass ratio of the hydrogen donor to the mass of the feedstock system is 1 to 10:1; the total mass of the FAD and the NAD + to the mass of the feedstock system is 10 to 1000:

1. The reducing enzyme is a reducing enzyme StyB capable of regenerating FADH2, and / or the dehydrogenase and the hydrogen donor are glucose dehydrogenase and glucose, respectively; and / or the dehydrogenase and the hydrogen donor are alcohol dehydrogenase and isopropanol, respectively. The mass concentration of the olefin compound is 0.5-2mg / mL. The source of the reducing enzyme StyB is any one or more of Pseudomonas sp. LQ26, Bradyrhizobium sp. ORS 375, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas sp. Y2, Rhodococcus sp. ST-5, Rhodococcus sp. ST-10. The raw material system comprises a buffer solution selected from any one or more of phosphate buffer, Tris-HCl buffer and boric acid buffer, and / or the pH value of the buffer solution is 7.5-10. ​

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