A method for the preparation of chiral 5-aryl-pentenols catalyzed by a P450 enzyme

Abstract

The application discloses a method for preparing chiral 5-aryl-pentenol compounds by catalysis of P450 enzymes, and the method comprises the following steps: taking 5-aryl-pent-1-en derivative as a substrate, and performing hydroxylation reaction by using oxygen in air under catalysis of a P450 BM3 mutant to obtain (R)-5-aryl-pent-1-en-5-ol shown in formula II or (S)-5-aryl-pent-1-en-3-ol shown in formula III. The method has the advantages of mild reaction condition, green sustainability, high selectivity, wide applicable range of substrates and the like, and has wide application prospect in the field of diversity synthesis of chiral drug intermediates.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and specifically to a method for preparing chiral 5-arylpentenol compounds catalyzed by P450 enzyme. Background Technology

[0002] Chiral polyhydroxy compounds have wide applications in food, medicine, agrochemicals, and fragrances, playing a crucial role as building blocks in the synthesis of chiral drugs. Among them, chiral benzyl alcohols serve as intermediates for many clinical drugs and pesticides, such as the novel hypoglycemic drug PPARα / γ dual agonist, various α- / β-adrenergic drugs, and a series of oxetine-like antidepressants, all of which contain a chiral benzyl alcohol skeleton. Simultaneously, chiral allyl alcohols are also intermediates for various compounds, such as multiple HMG-CoA reductase inhibitors, statins, and eyelash growth serums like Latisse, all of which contain an allyl alcohol skeleton.

[0003] Traditional methods for synthesizing chiral alcohols mainly rely on chemical catalysis, with direct hydroxylation of CH bonds considered one of the most direct and atom-economical routes. However, traditional chemical methods face several bottlenecks in the synthesis of asymmetric aromatic alcohols: on the one hand, they often require noble metal catalysts such as ruthenium, rhodium, palladium, and iridium, or highly toxic heavy metal catalysts, resulting in low economic and environmental benefits; on the other hand, the reaction conditions are usually harsh (high temperature, high pressure, strong acids and bases, etc.), with limited tolerance to functional groups, and precise control of regioselectivity and stereoselectivity remains a significant challenge.

[0004] Cytochrome P450 enzymes are a class of heme-containing monooxygenases widely found in organisms, capable of catalyzing regioselective and stereoselective oxidation reactions of inert CH bonds under mild conditions. Among them, P450 BM3, derived from Bacillus megaterium, is one of the most extensively studied P450 enzymes in protein engineering due to its unique self-sufficiency; its reductase domain is fused with the heme domain within the same polypeptide chain, requiring no additional electron transport proteins. P450 BM3 can catalyze the hydroxylation of CH bonds at the benzylic, allyl, carbonyl α-positions, and heteroatom ortho-positions to generate corresponding chiral hydroxyl compounds, and can precisely control the regioselectivity, chemoselectivity, and stereoselectivity of the catalytic reaction under mild reaction conditions.

[0005] However, existing methods for preparing chiral alcohols using P450 BM3 catalysis still have limitations. Current reports on P450 BM3-catalyzed chiral alcohol synthesis mainly focus on specific substrate types. For example, Neufeld et al. reported the allylic hydroxylation of ω-alkeny acids and esters catalyzed by the P450 BM3 mutant A74G / L188Q, but these substrates all contain carboxyl or ester functional groups, and their applicability to simple olefin substrates without such polar functional groups has not been verified. Similarly, in benzylic hydroxylation, existing research mainly focuses on simple aromatic hydrocarbons such as toluene and ethylbenzene, or their carbonyl / carboxyl derivatives; systematic studies on complex substrates containing both allylic and benzylic potential hydroxylation sites are still lacking.

[0006] Furthermore, existing technologies typically develop specific mutants for a single substrate to obtain a particular hydroxylation product. When multiple oxidizable CH bonds exist simultaneously in a substrate molecule (such as the simultaneous presence of allylic and benzylic positions), there is currently no effective solution for achieving selective switching between different hydroxylation sites on the same substrate series. Summary of the Invention

[0007] The present invention aims to provide a method for preparing chiral 5-arylpentenol compounds by P450 enzyme catalysis. By selecting different P450 BM3 mutants as catalysts, (S)-5-arylpent-1-en-3-ol or (R)-5-arylpent-1-en-5-ol can be obtained on the same substrate 5-aryl-pent-1-ene, thereby achieving flexible control.

[0008] To achieve the above objectives, the first aspect of this application provides the following technical solution: A method for preparing chiral 5-arylpentenol compounds catalyzed by P450 enzyme, characterized in that, using the 5-aryl-pent-1-ene derivative of Formula I as a substrate, a hydroxylation reaction is carried out using oxygen in the air under the catalysis of a P450 BM3 mutant to obtain (R)-5-aryl-pent-1-en-5-ol of Formula II or (S)-5-aryl-pent-1-en-3-ol of Formula III, the general reaction formula of which is shown below: .

[0009] Furthermore, the P450 BM3 mutant is selected from: P450 BM3-P326R, P450 BM3-F81W / A264P / A330W / L437F and F81W / A264P / A330W / T438S.

[0010] As a preferred option, when P450 BM3-P326R is used as a biocatalyst, the (R)-5-aryl-pent-1-en-5-ol shown in Formula II is mainly obtained; When P450 BM3-F81W / A264P / A330W / L437F or P450 BM3-F81W / A264P / A330W / T438S is used as a biocatalyst, the (S)-5-aryl-pent-1-en-3-ol of Formula III is mainly obtained.

[0011] As a preferred embodiment, R is a fluorine, chlorine, bromine, methyl, methoxy, or trifluoromethyl substituted at the ortho, meta, or para positions.

[0012] As a preferred embodiment, R is a 1-substituted naphthalene ring, a 2-substituted naphthalene ring, a thiophene ring, a pyridine ring, or an indole ring.

[0013] Furthermore, the P450 BM3 mutant is provided in the form of a cell suspension.

[0014] The optimized buffer solution is a potassium phosphate buffer with a concentration of 0.1-0.2 M and a pH of 6.0-8.0; the substrate concentration is 1-3 mM; the reaction is carried out at a temperature of 10-25°C for 4-8 h; the glucose solution concentration is 10%-30%, and the amount added is 5%-15% of the total volume of the reaction system.

[0015] As a preferred option, When using P450 BM3-F81W / A264P / A330W / L437F or P450 BM3-F81W / A264P / A330W / T438S as a biocatalyst, the reaction conditions were: cell concentration 10 g cdw / L, buffer pH 6.0, substrate concentration 3 mM, reaction temperature 10℃, and reaction time 8 h.

[0016] Working principle and beneficial effects of the present invention: (1) This invention creatively utilizes oxygen from the air to provide oxygen atoms, and synthesizes (S)-5-aryl-pent-1-en-3-ols and (R)-5-aryl-pent-1-en-5-ols with high selectivity under the catalysis of P450 enzyme. This reaction makes full use of oxygen from the air, and the reaction conditions are mild and green, achieving high selectivity, high greenness and high atom economy.

[0017] (2) The method of the present invention is applicable to 5-aryl-pent-1-ene substrates with various substituents (halogen, methyl, methoxy, trifluoromethyl) at the ortho, meta, and para positions on the benzene ring, as well as various aromatic heterocyclic substituted substrates such as naphthalene, thiophene, pyridine, and indole, and has good substrate versatility.

[0018] (3) By simply replacing the P450 BM3 mutant, the present invention can achieve the selective synthesis of two hydroxylation products at different positions on the same substrate 5-aryl-pent-1-ene. This strategy provides great flexibility for the diverse synthesis of chiral alcohols. Attached Figure Description

[0019] Figure 1 Compound 1a prepared in Example 1 1 H NMR spectrum; Figure 2 Compound 1a prepared in Example 1 13 C NMR spectrum; Figure 3 Compound 2a prepared in Example 2 1 H NMR spectrum; Figure 4 Compound 2a prepared in Example 2 13 C NMR spectrum; Figure 5 This is a single crystal image of compound 2a derived from the compound prepared in Example 2; Figure 6 Compound 3a prepared in Example 3 1 H NMR spectrum; Figure 7 Compound 3a prepared in Example 3 13 C NMR spectrum; Figure 8 Compound 4a prepared in Example 4 1 H NMR spectrum; Figure 9 Compound 4a prepared in Example 4 13 C NMR spectrum; Figure 10 Compound 5a prepared in Example 5 1 H NMR spectrum; Figure 11 Compound 5a prepared in Example 5 13 C NMR spectrum; Figure 12 Compound 6a prepared in Example 6 1 H NMR spectrum; Figure 13 Compound 6a prepared in Example 6 13 C NMR spectrum; Figure 14 Compound 7a prepared in Example 7 1 H NMR spectrum; Figure 15Compound 7a prepared in Example 7 13 C NMR spectrum; Figure 16 The HLLC spectrum of compound 1a prepared in Example 1; Figure 17 The HPLC spectrum of compound 2a prepared in Example 2; Figure 18 The HPLC spectrum of compound 3a prepared in Example 3. Figure 19 The HPLC spectrum of compound 4a prepared in Example 3; Figure 20 The HPLC spectrum of compound 5a prepared in Example 3; Figure 21 The HPLC spectrum of compound 6a prepared in Example 3; Figure 22 The HPLC spectrum of compound 7a prepared in Example 3 is shown.

[0020] Appendix Description Appendix 1 is the DNA sequence of P450 BM3 WT; Appendix 2 contains the DNA sequence of the P450 BM3 mutant F81W / A264P / A330W / L437F; Appendix 3 contains the DNA sequence of the P450 BM3 mutant F81W / A264P / A330W / T438S. Detailed Implementation

[0021] The following detailed description illustrates the specific implementation method: Example 1: 5-Phenylacet-1-ene was used as a substrate and P450 BM3-P326R was used as a biocatalyst to prepare 5-phenyl-pent-1-en-5-ol.

[0022] 1. Strain activation, culture and expression (1) Activation of strain: Take the required self-sufficient P450 BM3 mutant strain out of the -80 ℃ strain storage box, thaw it at room temperature, and streak the inoculation loop sterilized by open flame on the ultra-clean laboratory bench onto the TB solid plate containing Kan resistance. After sealing with sealing film, invert it in a constant temperature incubator at 37 ℃ for 14-16 h. After the appropriate size and round single colony fall, take it out and place it in a 4 ℃ refrigerator for later use.

[0023] (2) Culture of the strain: Add the corresponding volume of Kan antibiotic stock solution (50 mg / mL) to the pre-prepared 5 mL or 50 mL TB liquid medium to make the final concentration 50 μg / mL. Pick a single colony from the above activated TB solid medium plate and inoculate it into the medium containing the resistance, and incubate at 37 ºC and 250 rpm for 7.5 h.

[0024] (3) Induction of bacterial protein expression: Add the corresponding volume of Kan antibiotic stock solution (50 mg / mL) to the pre-prepared 50 mL and 100 mL TB liquid medium to make the final concentration 50 μg / mL. Transfer the primary seed culture into the corresponding volume of TB liquid medium at a volume ratio of 2%, and culture at 37 ºC and 250 rpm for 2.5 h. Then add the corresponding volume of IPTG stock solution (200 mM) to make the final concentration 0.2 mM, and culture at 25 ºC and 250 rpm for 10-12 h.

[0025] 2. Whole-cell biocatalytic reactions Add 300 μL of induced bacterial culture to a quartz cuvette, then add 2.7 mL of dd H2O to dilute it 10-fold. Place the cuvette in a UV-Vis spectrophotometer, using dd H2O as a blank control, and measure its OD value. 600 The absorbance was measured. The required volume of bacterial culture was measured, balanced, and placed in a high-speed refrigerated centrifuge. The centrifuge was carried out at 9000 rpm for 3 min at 4 ºC to collect the bacterial cells. The supernatant was discarded, and the bacterial cells were retained. Based on the OD previously established in our laboratory... 600 The relationship between absorbance and cell concentration is shown in Equation 1. Add the appropriate volume of KP buffer solution (pH 6.0, 0.1 M) and vortex thoroughly to mix the bacterial cells. Pipette 5 mL of the mixed bacterial solution into a 25 mL Erlenmeyer flask with a stopper and ground glass stopper. Add 25 μL of the prepared substrate stock solution (200 mM) to bring the final substrate concentration to 1 mM. Then add 500 μL of the prepared 20% glucose solution to bring the glucose content in the system to 2%. Seal the flask tightly and place it in a constant temperature shaker at 10 ºC and 250 rpm for 8 h.

[0026] OD 600 Formula for calculating the relationship between absorbance and cell concentration: (1) X1: OD 600 absorbance value X2: Centrifuged bacterial culture volume (mL) X3: Required cell concentration (g cdw / L) Y: Volume of buffer solution to be added (mL) 3. Sample preparation and high-performance liquid chromatography analysis Sample preparation: After reacting at 10℃ and 250 rpm for 8 h on a vertical shaker, 1 mL of the reaction solution was transferred to a 2 mL EP tube, and 1 mL of ethyl acetate solution containing 0.5 mM acetophenone was added at a 1:1 ratio for extraction. After thorough mixing, the mixture was extracted at 12000 rpm for 4 hours. o Centrifuge at high speed for 3 min under C conditions to completely separate the aqueous and organic phases. Take the upper ethyl acetate and add it to a 1.5 mL EP tube containing an appropriate amount of anhydrous Na2SO4 to remove water. Filter with a 0.22 μm microporous membrane and inject into a clean liquid chromatography vial.

[0027] Sample Analysis: The prepared samples were analyzed using a high-performance liquid chromatograph (Shimadzu LC-20A). The chromatograms were integrated, and the product concentration was obtained by substituting the peak area of ​​the product and its ratio to the peak area of ​​the internal standard into the prepared standard curve. The yield was then calculated, and the product 1a was calculated by analyzing the partial area of ​​each configuration of the product. ee Value > 99.0%, yield 19%.

[0028] The detection data for product 1a are as follows: 1 H NMR (400 MHz, CDCl3)δ 7.25 (d, J = 8.3 Hz, 2H), 7.14 – 7.09 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.84 (d, J = 7.0 Hz, 1H), 6.80 (d, J = 8.0 Hz, 2H), 6.45(s, 1H), 6.25 (s, 2H), 4.88 (d, J = 5.0 Hz, 1H), 3.85 – 3.82 (m, 1H), 3.80 (s, 3H), 3.66 (s, 6H), 3.63 (s, 3H), 3.54 – 3.47 (m, 2H), 2.23 (s, 3H). 13 C NMR (101 MHz, CDCl3)δ 144.7, 138.7, 128.5, 127.6, 125.9, 115.0,74.0, 38.1, 30.1 Example 2: Using 5-phenyl-pent-1-ene as a substrate and P450 BM3-F81W / A264P / A330W / T438S (WPWS) as a biocatalyst, 5-phenyl-pent-1-en-3-ol was prepared (reaction formula 2).

[0029]

[0030] The P450 BM3 mutant P326R in Example 1 was replaced with WPWS, and the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL). All other conditions remained the same as in Example 1, resulting in 2a (…). ee 97.9%; Yield 65%.

[0031] The detection data for product 2a are as follows: 1 H NMR (400 MHz, CDCl3)δ 7.32 – 7.10 (m, 5H), 5.89 (ddd, J = 16.8,10.3, 6.1 Hz, 1H), 5.23 (d, J = 17.2 Hz, 1H), 5.12 (d, J = 10.5 Hz, 1H), 4.11(q, J = 6.6 Hz, 1H), 2.70 (hept, J = 8.6, 7.5 Hz, 2H), 1.93 – 1.67 (m, 3H). 13 C NMR(101 MHz, CDCl3)δ 141.9, 141.1, 128.5, 128.4, 125.9, 114.9,72.5, 38.6, 31.7. Example 3: Preparation of 2-fluoro-5-phenyl-pent-1-en-3-ol with the following structural formula (Reaction Formula 3)

[0032]

[0033] In Example 1, the P450 BM3 mutant P326R was replaced with WPWF, 5-phenyl-pent-1-ene was replaced with 2-fluoro-5-phenyl-pent-1-ene, the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL), the internal standard was 2 mM 2-nitro-benzyl bromide, and other conditions were the same as in Example 1, finally yielding 3a ( ee 94.5%; Yield 33%).

[0034] The test data for product 3a are as follows: 1 H NMR (400 MHz, CDCl3)δ 7.25 – 7.12 (m, 2H), 7.09 – 6.96 (m, 2H), 5.91 (ddd, J = 16.8, 10.4, 6.1 Hz, 1H), 5.26 (dt, J = 17.3, 1.5 Hz, 1H), 5.14(dd, J = 10.4, 1.5 Hz, 1H), 4.13 (q, J = 6.4 Hz, 1H), 2.75 (dtd, J = 21.8,14.1, 7.8 Hz, 2H), 1.84 (q, J = 7.6 Hz, 2H), 1.73 – 1.59 (m, 1H). 13 C NMR (101 MHz, CDCl3)δ 145.1, 138.0, 131.5, 128.8, 127.3, 124.3,115.4, 115.2, 115.1, 68.0, 37.0, 30.0. Example 4: Preparation of 2-chloro-5-phenyl-pent-1-en-3-ol with the following structural formula (reaction formula 4)

[0035] The P450 BM3 mutant P326R in Example 1 was replaced with WPWS, 5-phenyl-pent-1-ene was replaced with 2-chloro-5-phenyl-pent-1-ene, the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL), and the internal standard was 2 mM acetophenone. All other conditions were the same as in Example 1, ultimately yielding 4a (… ee 99.0%; Yield 21%).

[0036] The detection data for product 4a are as follows: 1H NMR (400 MHz, CDCl3)δ 7.34 (dd, J = 7.7, 1.6 Hz, 1H), 7.27 – 7.22(m, 1H), 7.16 (dtd, J = 18.9, 7.4, 1.7 Hz, 2H), 5.92 (ddd, J = 16.8, 10.4, 6.1 Hz, 1H), 5.28 (dt, J = 17.2, 1.4 Hz, 1H), 5.15 (dt, J = 10.4, 1.4 Hz, 1H), 4.21 – 4.07 (m, 1H), 2.94 – 2.74 (m, 2H), 1.94 – 1.80 (m, 2H), 1.68 –1.60 (m, 1H). 13 C NMR (101 MHz, CDCl3)δ 140.9, 139.5, 134.0, 130.5, 129.5, 127.4,126.8, 115.0, 72.5, 36.8, 29.5. Example 5: Preparation of 2-bromo-5-phenyl-pent-1-en-3-ol with the following structural formula (Reaction Formula 5)

[0037]

[0038] In Example 1, the P450 BM3 mutant P326R was replaced with WPWS, 5-phenyl-pent-1-ene was replaced with 2-bromo-5-phenyl-pent-1-ene, the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL), and the internal standard was 2 mM phenylethanol. All other conditions were the same as in Example 1, ultimately yielding 5a ( ee 96.3%; Yield 24%).

[0039] The test data for product 5a are as follows: 1H NMR (400 MHz, CDCl3)δ 7.52 (dd, J = 7.7, 1.0 Hz, 1H), 7.29 – 7.18 (m, 2H), 7.05 (ddd, J = 8.0, 6.4, 2.6 Hz, 1H), 5.93 (ddd, J = 17.2, 10.4, 6.1Hz, 1H), 5.28 (dt, J = 17.2, 1.4 Hz, 1H), 5.15 (dt, J = 10.4, 1.4 Hz, 1H), 4.22 – 4.11 (m, 1H), 2.95 – 2.74 (m, 2H), 1.91 – 1.76 (m, 2H), 1.71 – 1.61 (m, 1H). 13 C NMR(101 MHz, CDCl3)δ 141.2, 140.8, 132.9, 130.5, 127.7, 127.5,124.5, 115.1, 72.5, 36.9, 32.1. Example 6: Preparation of 2-methyl-5-phenyl-pent-1-en-3-ol with the following structural formula (Reaction Formula 6)

[0040]

[0041] The P450 BM3 mutant P326R in Example 1 was replaced with WPWS, 5-phenyl-pent-1-ene was replaced with 2-methyl-5-phenyl-pent-1-ene, the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL), the internal standard was 2 mM 4-phenyl-2-butanone, and other conditions were the same as in Example 1, finally yielding 6a ( ee 94.6%; Yield 39%).

[0042] The test data for product 6a are as follows: 1H NMR(400 MHz, CDCl3)δ 7.18 – 7.05 (m, 4H), 5.98 – 5.85 (m, 1H), 5.27(dt, J = 17.3, 1.3 Hz, 1H), 5.15 (dt, J = 10.4, 1.3 Hz, 1H), 4.22 – 4.10 (m,1H), 2.81 – 2.58 (m, 2H), 2.31 (d, J = 1.3 Hz, 3H), 1.87 – 1.73 (m, 2H), 1.68– 1.59 (m, 1H). 13 C NMR(101 MHz, CDCl3)δ 141.1, 140.1, 136.0, 130.3, 128.9, 126.0,126.0, 115.0, 73.2, 37.7, 28.4, 20.7. Example 7: Preparation of 2-methyl-5-phenyl-pent-1-en-3-ol with the following structural formula (Reaction Formula 7)

[0043]

[0044] The P450 BM3 mutant P326R in Example 1 was replaced with WPWS, 5-phenyl-pent-1-ene was replaced with 5-thiophene-pent-1-ene, the substrate concentration of 1 mM (25 μL) was replaced with 3 mM (75 μL), the internal standard was 2 mM 4-phenyl-2-butanone, and other conditions were the same as in Example 1, finally yielding 6a ( ee 99.0%; Yield 27%).

[0045] The test data for product 7a are as follows: 1 H NMR (400 MHz, CDCl3)δ 7.28 – 7.22 (m, 1H), 6.95 (d, J = 4.1 Hz, 2H), 5.90 (ddd, J = 16.9, 10.4, 6.2 Hz, 1H), 5.24 (dt, J = 17.2, 1.5 Hz, 1H),5.14 (dt, J = 10.4, 1.3 Hz, 1H), 4.18 – 4.08 (m, 1H), 2.85 – 2.65 (m, 2H),1.91 – 1.77 (m, 2H), 1.64 (ddd, J = 11.8, 5.8, 3.3 Hz, 1H). 13 C NMR(101 MHz, CDCl3)δ 142.1, 141.0, 128.2, 125.4, 120.2, 115.0,72.5, 37.6, 26.1. The single crystal obtained after derivatization of product 2a is as follows: Figure 5 As shown, the specific data is as follows:

[0046] The above descriptions are merely some embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

[0047] Appendix 1 DNA sequence of P450 BM3 WT Appendix 2 DNA sequence of P450 BM3-F81W / A264P / A330W / L437F Appendix 3 DNA sequence of P450 BM3-F81W / A264P / A330W / T438S

Claims

1. A method for preparing chiral 5-arylpentenol compounds catalyzed by P450 enzyme, characterized in that, Using the 5-aryl-pent-1-ene derivative shown in Formula I as a substrate, hydroxylation was carried out with oxygen in the air under the catalysis of the P450 BM3 mutant to obtain (R)-5-aryl-pent-1-en-5-ol shown in Formula II or (S)-5-aryl-pent-1-en-3-ol shown in Formula III, and the general reaction formula is shown below: 。 2. The preparation method according to claim 1, characterized in that, The P450 BM3 mutants are selected from: P450BM3-P326R, P450 BM3-F81W / A264P / A330W / L437F and F81W / A264P / A330W / T438S.

3. The preparation method according to claim 2, characterized in that, When P450 BM3-P326R is used as a biocatalyst, (R)-5-aryl-pent-1-en-5-ol, represented by Formula II, is mainly obtained; when P450 BM3-F81W / A264P / A330W / L437F or P450 BM3-F81W / A264P / A330W / T438S is used as a biocatalyst, (S)-5-aryl-pent-1-en-3-ol, represented by Formula III, is mainly obtained.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The R is a fluorine, chlorine, bromine, methyl, methoxy, or trifluoromethyl substituted at the ortho, meta, or para position.

5. The preparation method according to any one of claims 1 to 3, characterized in that, The R is a 1-substituted naphthalene ring, a 2-substituted naphthalene ring, a thiophene ring, a pyridine ring, or an indole ring.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The P450 BM3 mutant was provided as a cell suspension.

7. The preparation method according to claim 6, characterized in that, The buffer solution is a potassium phosphate buffer with a concentration of 0.1-0.2 M and a pH of 6.0-8.0; the substrate concentration is 1-3 mM; the reaction is carried out at a temperature of 10-25°C for 4-8 hours; the glucose solution concentration is 10%-30%, and the amount added is 5%-15% of the total volume of the reaction system.

8. The preparation method according to claim 7, characterized in that, When using P450 BM3-F81W / A264P / A330W / L437F or P450 BM3-F81W / A264P / A330W / T438S as a biocatalyst, the reaction conditions are: cell concentration 10 g cdw / L, buffer pH 6.0, substrate concentration 3 mM, reaction temperature 10℃, and reaction time 8 h.

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