A method for the synthesis of p-chiral phosphine oxides

Abstract

The application discloses a synthesis method of P-chiral phosphine oxide and belongs to the technical field of enzyme engineering. The synthesis method of the P-chiral phosphine oxide comprises the following steps: dissolving a material containing cytochrome P450 monooxygenase and a racemic substrate in a solvent to obtain a reaction solution; catalyzing the reaction of the reaction solution at 20-40 DEG C for 20-30 hours; centrifuging; collecting supernatant and extracting and concentrating, so as to obtain the P-chiral phosphine oxide. The biological catalysis method provided by the application can solve the problems of low enzyme activity, complicated steps and high cost in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a method for synthesizing P-chiral phosphine oxides. Background Technology

[0002] Compounds containing phosphorus stereocenters (P-chiral centers) are widely found in phosphonates, phosphine oxides, and phosphine ligands, and have significant applications in catalysis, materials science, agricultural chemistry, and drug development. Since the first application of chiral monodentate phosphine ligands in asymmetric hydrogenation reactions, chiral phosphine ligands have become key components in various asymmetric transformation reactions, such as transition metal-catalyzed asymmetric hydrogenation, cross-coupling, transisomeric selective CH bond activation, and organocatalysis. The development of chiral phosphine ligands has provided crucial support for organic synthesis methodologies and drug development.

[0003] Currently, various strategies exist for the synthesis of p-chiral compounds, with the construction of p-chiral phosphine oxides primarily relying on asymmetric synthesis. While chiral organocatalysis and metal catalysis have shown some potential, noble metal catalysis (such as rhodium and palladium) remains dominant in the asymmetric construction of p-chiral phosphine oxides. These methods typically suffer from problems such as high catalyst costs, harsh reaction conditions, and insufficient environmental friendliness.

[0004] Biocatalysis offers advantages such as mild reaction conditions and high atom economy, aligning with the principles of green and sustainable development. Cytochrome P450 enzymes (CYPs / P450) are a superfamily of heme-containing proteins widely distributed in various organisms in nature. They can catalyze a variety of reactions and often exhibit excellent regioselectivity and stereoselectivity, attracting significant attention in the fields of green chemistry and biocatalysis. In the biosynthesis of p-chiral phosphophosphine oxides, studies have reported the use of phosphotriesterases and triacylglycerol hydrolases from Pseudomonas to synthesize p-chiral phosphophosphine oxides. However, these enzyme catalytic systems suffer from narrow substrate spectra, limited stereoselectivity control, and a single enzyme source, making it difficult to meet the synthetic needs of diverse substrates and limiting their widespread application in the synthesis of p-chiral phosphophosphine oxides.

[0005] Therefore, there is an urgent need to develop a method for synthesizing P-chiral phosphine oxides that features mild reaction conditions, high atom economy, excellent stereoselectivity, and broad substrate adaptability. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for synthesizing P-chiral phosphine oxides, thereby overcoming the deficiencies of existing catalysts such as high cost, harsh reaction conditions, and insufficient environmental friendliness, as well as the problems of existing biosynthetic methods such as narrow substrate spectrum, limited stereoselectivity control, and single enzyme source.

[0007] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A method for synthesizing P-chiral phosphine oxide is provided, comprising the following steps: The substance containing cytochrome P450 monooxygenase was co-dissolved with a racemic substrate in a solvent to obtain a reaction solution. The reaction solution was then subjected to a catalytic reaction at 20–40 °C for 20–30 h to prepare P-chiral phosphine oxide. The amino acid sequence of cytochrome P450 monooxygenase is shown in SEQ ID NO.1, and the structural formula of the racemic substrate is shown below.

[0008] Wherein, R is hydrogen, C1-C10 alkyl, C1-C10 alkoxy or halogen; The reaction formula is shown below: .

[0009] The beneficial effects of this invention are as follows: The active center of P450 monooxygenase contains a highly active ferro-oxygen species (commonly known as Compound I). It precisely targets a hydrogen atom on the carbon atom (α-carbon) adjacent to the nitrogen atom in the substrate (such as a tertiary amine). After Compound I "captures" this hydrogen atom, the substrate becomes a carbon-centered free radical, while heme iron becomes a hydroxyl iron species (Fe-OH). The free radical intermediate is extremely unstable and will immediately undergo the next reaction. The generated hydroxyl iron species (Fe-OH) will rapidly "bounce" the hydroxyl group (-OH) back to the carbon free radical, generating an unstable N-hydroxyalkyl intermediate. This intermediate will immediately and spontaneously decompose, eventually cleaving into a secondary amine.

[0010] Further, R can be hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, or halogen.

[0011] Furthermore, the racemic substrates are ethoxy-P-butyl-N-methyl-N-phenylphosphonamide, ethyl-P-butyl-N-(2-fluorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-(3-fluorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-methyl-N-(o-tolyl)phosphonamide ester, ethyl-P-butyl-N-methyl-N-(p-tolyl)phosphonamide ester, ethyl-P-butyl-N-(4-chlorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-(4-methoxyphenyl)-N-methylphosphonamide ester, and ethyl-N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester.

[0012] Furthermore, the substance containing cytochrome P450 monooxygenase is a recombinant cell containing cytochrome P450 monooxygenase; the dry weight concentration of the recombinant cell containing cytochrome P450 monooxygenase in the reaction solution is 10-30 g / L, and the concentration of the racemic substrate is 1-60 mM; preferably, the dry weight concentration of the recombinant cell containing cytochrome P450 monooxygenase in the reaction solution is 15 g / L, and the concentration of the racemic substrate is 60 mM.

[0013] Furthermore, the substance containing cytochrome P450 monooxygenase is frozen stem cells containing cytochrome P450 monooxygenase; the concentration of frozen stem cells containing cytochrome P450 monooxygenase in the reaction solution is 1-4 mg / mL, and the concentration of the racemic substrate is 1-60 mM; preferably, the concentration of frozen stem cells containing cytochrome P450 monooxygenase in the reaction solution is 2 mg / mL, and the concentration of the racemic substrate is 60 mM.

[0014] Further, the substance containing cytochrome P450 monooxygenase is a crude enzyme solution containing cytochrome P450 monooxygenase; the concentration of the crude enzyme solution containing cytochrome P450 monooxygenase in the reaction solution is 0.1~0.41 μM, and the concentration of the racemic substrate is 1~60 mM; preferably, the concentration of the crude enzyme solution containing cytochrome P450 monooxygenase in the reaction solution is 0.1 μM, and the concentration of the racemic substrate is 60 mM.

[0015] Furthermore, the catalytic reaction was carried out at a temperature of 30 °C for 24 h.

[0016] Furthermore, the solvent is 50 mM phosphate buffer with a pH of 7-8.

[0017] The present invention has the following beneficial effects: This invention utilizes cytochrome P450 monooxygenase, a mild and green biocatalyst, to catalyze the synthesis of P-chiral phosphine oxide products containing NC bonds. Using recombinant cells, frozen stem cells, or crude enzyme solutions containing cytochrome P450 monooxygenase prepared according to this invention as catalysts, P-chiral phosphine oxide products are prepared in 50 mM phosphate buffer at pH 7-8. The reaction yield can reach up to 45%, meaning that most of the substrate is successfully converted to the target product during the reaction. Furthermore, the optical purity of the product can reach up to 92% ee (enantiomer excess), indicating that the resulting product has a predominantly single-chiral structure and extremely high stereoselectivity. As is well known, enantioselectivity is particularly important in the preparation of chiral drugs and chiral compounds.

[0018] The biocatalytic reaction employed in this invention not only exhibits extremely high stereoselectivity but also excellent regioselectivity and chemoselectivity. The biocatalytic reaction conditions of this invention are mild, which not only helps protect the structural integrity of the substrate and product but also reduces energy consumption and improves the safety and feasibility of the reaction. The catalytic process of this invention is environmentally friendly, producing no toxic or harmful byproducts, conforming to the principles of green chemistry, and is simple to operate, with the reaction system easily controlled and adjusted. Attached Figure Description

[0019] Figure 1 The liquid phase analysis spectrum is that of the chiral N-phenyl-P-butyl-P-ethylphosphamide ester prepared in Example 2.

[0020] Figure 2 The liquid phase analysis spectrum is that of the chiral ethyl P-butyl-N-(2-fluorophenyl)phosphonamide ester prepared in Example 3.

[0021] Figure 3 The liquid phase analysis spectrum is that of the chiral ethyl P-butyl-N-(3-fluorophenyl)phosphonamide ester prepared in Example 4.

[0022] Figure 4 The liquid phase analysis spectrum of the chiral ethyl p-butyl-N-(o-tolyl)phosphonamide ester prepared in Example 5.

[0023] Figure 5 The liquid phase analysis spectrum is that of the chiral ethyl p-butyl-N-(p-tolyl)phosphonamide ester prepared in Example 6.

[0024] Figure 6 The liquid phase analysis spectrum is that of the chiral ethyl P-butyl-N-(4-chlorophenyl)phosphonamide ester prepared in Example 7.

[0025] Figure 7 The chiral ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester prepared in Example 8 The liquid phase analysis spectrum.

[0026] Figure 8 The liquid phase analysis spectrum of the chiral ethyl N-(4-bromophenyl)-P-butylphosphonamide ester prepared in Example 9. Detailed Implementation

[0027] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1: Expression of cytochrome P450 monooxygenase and preparation of reaction catalyst I. Preparation of cytochrome P450 monooxygenase plasmid transformants The amino acid sequence shown in SEQ ID NO.1 was optimized according to the codon preference of E. coli to make it more suitable for expression in E. coli, and the optimized nucleotide sequence is shown in SEQ ID NO.2. The optimized sequence SEQ ID NO.2 was artificially synthesized as a whole gene and ligated into the E. coli expression vector pET28b to construct the recombinant expression plasmid pET28b-P450.

[0029] The specific steps for transforming cytochrome P450 monooxygenase plasmids are as follows: (1) Gene optimization and synthesis: Based on the codon preference of Escherichia coli, the amino acid sequence shown in SEQ ID NO.1 was optimized to obtain the nucleotide sequence SEQ ID NO.2. The optimized gene sequence SEQ ID NO.2 was synthesized by whole-genome artificial synthesis method.

[0030] (2) Plasmid construction: The synthesized gene sequence of SEQ ID NO.2 was inserted into the Escherichia coli expression vector pET28b to obtain the recombinant expression plasmid pET28b-P450.

[0031] (3) Transformation and screening: The constructed recombinant expression plasmid pET28b-P450 was transformed into Escherichia coli. E.coli In BL21(DE3) competent cells, the transformation solution was spread onto TB plates containing kanamycin and streptomycin, sealed with sealing film, and incubated upside down overnight at 37 °C to screen for resistant colonies.

[0032] (4) Cloning verification: The selected colonies were initially screened by colony PCR to select positive clones. The positive clones were then verified by gene sequencing to confirm the recombinant plasmid pET28b-P450. E.coli Correct insertion and expression in BL21(DE3) yields recombinant Escherichia coli. E.coli BL21(DE3) / pET28b-P450.

[0033] II. Expression of cytochrome P450 monooxygenase The recombinant Escherichia coli obtained above E.coli BL21(DE3) / pET28b-P450 was cultured and induced to express according to the following steps: (1) Seed culture: Recombinant Escherichia coli was inoculated into TB medium containing 50 µg / mL kanamycin and streptomycin and cultured overnight at 37 ℃ and 250 rpm with shaking to allow the bacterial culture to grow fully; the components of TB medium were: 12 g / L peptone, 24 g / L yeast extract, pH 7.0.

[0034] (2) Expanded culture: On the second day, the overnight cultured seed culture was inoculated into 100 mL of TB medium containing 50 µg / mL kanamycin and streptomycin at an inoculation rate of 1% (v / v). The TB medium was placed in a 500 mL Erlenmeyer flask and cultured with shaking at 37°C and 250 rpm until the OD of the culture medium was reached. 600 The value reached 0.6.

[0035] (3) Induced expression: OD 600 When the value reached 0.6, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM was added to the culture medium as an inducer to induce P450 monooxygenase expression. The induced culture system was then cultured with shaking at 25℃ and 250 rpm for 12 h to allow the recombinant protein to be fully expressed.

[0036] (4) Collection of bacterial cells: After protein expression, the culture is collected by centrifugation. The centrifugation conditions are set at 4 ℃, 5000 rpm, and centrifugation time is 10 min. After centrifugation, the supernatant is discarded and the precipitated recombinant bacterial cells are collected, which are the recombinant cells expressing cytochrome P450 monooxygenase.

[0037] III. Preparation of Frozen Stem Cells Containing Cytochrome P450 Monooxygenase and Crude Enzyme Solution of Cytochrome P450 Monooxygenase (1) Preparation of frozen stem cells: The collected recombinant cells expressing cytochrome P450 monooxygenase were added to a freeze-drying tray and pre-frozen overnight at -20 °C for 12 h. Pre-freezing can reduce the damage of ice crystals to the enzyme. The pre-frozen recombinant cells were taken out and placed in a freeze dryer for freeze-drying. The vent was closed and the refrigeration and vacuum pump were turned on. When the temperature dropped to -50 °C and the vacuum degree dropped to 5 Pa, it indicates that the sealing is good and there is no leakage. If the vacuum degree cannot be reduced to the standard value, there may be leakage. The sealing needs to be checked. After freeze-drying for 12~24 h, the refrigeration and vacuum pump were turned off and the vent was slowly opened to prevent the freeze-dried powder from being blown up by too fast venting. The frozen stem cells were collected in a clean container and sealed with sealing film and stored at -20~-80 °C for later use.

[0038] (2) Preparation of crude enzyme solution: Recombinant cells expressing cytochrome P450 monooxygenase were collected in an ice bath and an appropriate volume of bacterial solution was taken and sonicated. The operating conditions were: sonication power 60 kW, sonication for 3 s, stop for 3 s, sonication time 30 min. The lysate was collected and centrifuged at 4 ℃ and 9000 rpm for 30 min. The supernatant was the crude enzyme solution. The crude enzyme solution can be lyophilized by the freeze-drying step in step (1) and stored at -20~-80 ℃ for later use.

[0039] Through the above steps, recombinant cells expressing cytochrome P450 monooxygenase can be successfully cultured and induced. Frozen stem cells and crude enzyme solution were also prepared, providing an efficient catalyst source for the biocatalytic synthesis of bipolar P-chiral phosphine oxides.

[0040] The synthetic pathway for the bipolar P-chiral phosphine oxide product catalyzed by cytochrome P450 monooxygenase is shown in the figure below. Figure 1 As shown, the specific synthesis process is illustrated in the following examples.

[0041] Example 2: Synthesis of N-phenyl-P-butylphosphonamide ethyl ester catalyzed by cytochrome P450 monooxygenase The reaction formula for the synthesis of N-phenyl-p-butylphosphonamide ethyl ester catalyzed by cytochrome P450 monooxygenase is shown below:

[0042] The specific process includes the following steps: I. Preparation from recombinant cells In a 500 mL Erlenmeyer flask, 100 mL of phosphate buffer solution with a concentration of 50 mM and a pH adjusted to 8 was added to ensure a suitable reaction environment. Recombinant cells (prepared in Example 1) were added to the buffer solution with a dry weight concentration (cdw) of 15 g / L. These recombinant cells contain cytochrome P450 monooxygenase, which can catalyze the target reaction. Then, ethoxy-P-butyl-N-methyl-N-phenylphosphineamide with a final concentration of 60 mM was added to the Erlenmeyer flask as a reaction substrate. The substrate was ensured to be evenly distributed in the buffer solution. The Erlenmeyer flask was placed on a constant temperature shaker at 30 °C with a shaking speed of 250 rpm. The reaction was carried out under these conditions with stirring to ensure sufficient oxygen supply and uniform mixing of the reactants. The reaction duration was set to 24 h to ensure that the substrate was fully converted into the target product.

[0043] After the reaction was completed, the reaction solution was removed and centrifuged at 4 ℃, 9000 rpm for 3 min. After centrifugation, the precipitated bacterial cells were discarded, and the supernatant was collected for subsequent product extraction. The supernatant was extracted with 100 mL of ethyl acetate. After each extraction, the mixture was thoroughly shaken and allowed to stand for separation. This process was repeated three times to ensure complete product extraction. The organic phases obtained from each extraction were separated and combined into one container. The combined organic phase was dried with anhydrous sodium sulfate to adsorb the water in the organic phase. After standing for a period of time to ensure complete drying, the organic phase was filtered through filter paper to remove the anhydrous sodium sulfate. The dried filtrate was collected.

[0044] The filtrate obtained after filtration was concentrated by vacuum distillation to remove ethyl acetate solvent and concentrate the target product. Temperature and pressure needed to be controlled during concentration to prevent degradation of the target product. Finally, the target product was obtained by vacuum distillation with a yield of 39% and an optical purity (enantiomer excess, ee value) of 96%, indicating high optical purity. The purity and composition of the prepared chiral N-phenyl-P-butylphosphonic acid ethyl ester were verified by high-performance liquid chromatography (HPLC), and its HPLC chromatogram is shown below. Figure 1 As shown, liquid chromatography analysis can determine the purity and optical purity of the product, ensuring the accuracy of the experimental results.

[0045] II. Preparation from frozen stem cells Frozen stem cells were added to the buffer solution to a final concentration of 2 mg / mL, and the remaining steps were the same as in step one above. The target product, N-phenyl-P-butylphosphonic acid ethyl ester, was obtained by vacuum distillation. The yield of the product was 30%, and the optical purity (enantiomer excess, ee value) was 95%, indicating that the product has high optical purity.

[0046] III. Preparation from crude enzyme solution Crude enzyme solution was added to the buffer solution to bring the final concentration to 0.1 μM, and the remaining steps were the same as in step one above. The target product, N-phenyl-P-butylphosphonic acid ethyl ester, was obtained by vacuum distillation. The yield of the product was 29%, and the optical purity (enantiomer excess, ee value) was 94%, indicating that the product has high optical purity.

[0047] Example 3: Synthesis of ethyl p-butyl-N-(2-fluorophenyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis. The reaction formula for the synthesis of ethyl p-butyl-N-(2-fluorophenyl)phosphonamide ester catalyzed by cytochrome P450 monooxygenase is shown below:

[0048] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl P-butyl-N-(2-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate. The remaining steps were the same as in Example 2. The target product, ethyl P-butyl-N-(2-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 43%, and the optical purity (enantiomer excess, ee value) was 97%, indicating that the product has high optical purity. The purity and composition of the prepared chiral ethyl P-butyl-N-(2-fluorophenyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC). The HPLC chromatogram is shown below. Figure 2 As shown.

[0049] II. Preparation from frozen stem cells Ethyl P-butyl-N-(2-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl P-butyl-N-(2-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 33%, and the optical purity (enantiomer excess, ee value) was 93%, indicating that the product has high optical purity.

[0050] III. Preparation from crude enzyme solution Ethyl p-butyl-N-(2-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(2-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 31%, and the optical purity (enantiomer excess, ee value) was 95%, indicating that the product has high optical purity.

[0051] Example 4: Synthesis of ethyl p-butyl-N-(3-fluorophenyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis. The reaction formula for the synthesis of ethyl p-butyl-N-(3-fluorophenyl)phosphonamide ester catalyzed by cytochrome P450 monooxygenase is shown below:

[0052] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl P-butyl-N-(3-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl P-butyl-N-(3-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 48%, and the optical purity (enantiomer excess, ee value) was 73%, indicating that the product has high optical purity. The purity and composition of the prepared chiral ethyl P-butyl-N-(3-fluorophenyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC), and its HPLC chromatogram is shown below. Figure 3 As shown.

[0053] II. Preparation from frozen stem cells Ethyl p-butyl-N-(3-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(3-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 38%, and the optical purity (enantiomer excess, ee value) was 95%, indicating that the product has high optical purity.

[0054] III. Preparation from crude enzyme solution Ethyl p-butyl-N-(3-fluorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(3-fluorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 35%, and the optical purity (enantiomer excess, ee value) was 96%, indicating that the product has high optical purity.

[0055] Example 5: Synthesis of ethyl p-butyl-N-(o-tolyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis The reaction formula for the synthesis of ethyl p-butyl-N-(o-tolyl)phosphonamide ester catalyzed by cytochrome P450 monooxygenase is as follows:

[0056] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl p-butyl-N-methyl-N-(o-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate. The remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(o-tolyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 37%, and the optical purity (enantiomer excess, ee value) was 89%, indicating high optical purity. The purity and composition of the prepared chiral ethyl p-butyl-N-(o-tolyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC). The HPLC chromatogram is shown below. Figure 4 As shown.

[0057] II. Preparation from frozen stem cells Ethyl p-butyl-N-methyl-N-(o-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(o-tolyl)phosphonamide ester, was obtained by vacuum distillation in a yield of 30% with an optical purity (enantiomer excess, ee value) of 93%, indicating that the product has high optical purity.

[0058] III. Preparation from crude enzyme solution Ethyl p-butyl-N-methyl-N-(o-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(o-tolyl)phosphonamide ester, was obtained by vacuum distillation. The yield of the product was 31%, and the optical purity (enantiomer excess, ee value) was 95%, indicating that the product has high optical purity.

[0059] Example 6: Synthesis of ethyl p-butyl-N-(p-tolyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis The reaction formula for the synthesis of ethyl p-butyl-N-(p-tolyl)phosphonamidate catalyzed by cytochrome P450 monooxygenase is shown below:

[0060] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl p-butyl-N-methyl-N-(p-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(p-tolyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 41%, and the optical purity (enantiomer excess, ee value) was 87%, indicating that the product has high optical purity. The purity and composition of the prepared chiral ethyl p-butyl-N-(p-tolyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC), and its HPLC chromatogram is shown below. Figure 5 As shown.

[0061] II. Preparation from frozen stem cells Ethyl p-butyl-N-methyl-N-(p-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(p-tolyl)phosphonamide ester, was obtained by vacuum distillation in a yield of 35% and an optical purity (enantiomer excess, ee value) of 94%, indicating that the product has high optical purity.

[0062] III. Preparation from crude enzyme solution Ethyl p-butyl-N-methyl-N-(p-tolyl)phosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(p-tolyl)phosphonamide ester, was obtained by vacuum distillation in a yield of 35% and an optical purity (enantiomer excess, ee value) of 96%, indicating that the product has high optical purity.

[0063] Example 7: Synthesis of ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis The reaction formula for the synthesis of ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester catalyzed by cytochrome P450 monooxygenase is shown below:

[0064] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl p-butyl-N-(4-chlorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate. The remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 28%, and its optical purity (enantiomer excess, ee value) was 83%, indicating high optical purity. The purity and composition of the prepared chiral ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC). Its HPLC chromatogram is shown below. Figure 6 As shown.

[0065] II. Preparation from frozen stem cells Ethyl p-butyl-N-(4-chlorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of the product was 31%, and the optical purity (enantiomer excess, ee value) was 93%, indicating that the product has high optical purity.

[0066] III. Preparation from crude enzyme solution Ethyl P-butyl-N-(4-chlorophenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl P-butyl-N-(4-chlorophenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 30%, and the optical purity (enantiomer excess, ee value) was 91%, indicating that the product has high optical purity.

[0067] Example 8: Synthesis of ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester by cytochrome P450 monooxygenase catalysis The reaction formula for the synthesis of ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester by cytochrome P450 monooxygenase is shown below:

[0068] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl p-butyl-N-(4-methoxyphenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate. The remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 16%, and its optical purity (enantiomer excess, ee value) was 84%, indicating high optical purity. The purity and composition of the prepared chiral ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester were verified by high-performance liquid chromatography (HPLC). The HPLC chromatogram is shown below. Figure 7 As shown.

[0069] II. Preparation from frozen stem cells Ethyl p-butyl-N-(4-methoxyphenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester, was obtained by vacuum distillation. The yield of this product was 28%, and the optical purity (enantiomer excess, ee value) was 93%, indicating that the product has high optical purity.

[0070] III. Preparation from crude enzyme solution Ethyl p-butyl-N-(4-methoxyphenyl)-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester, was obtained by vacuum distillation in a yield of 30% with an optical purity (enantiomer excess, ee value) of 94%, indicating that the product has high optical purity.

[0071] Example 9: Synthesis of ethyl N-(4-bromophenyl)-P-butylphosphonamide ester by cytochrome P450 monooxygenase catalysis The reaction formula for the synthesis of ethyl N-(4-bromophenyl)-P-butylphosphonamide ester by cytochrome P450 monooxygenase catalyzed by ethyl N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester is shown below:

[0072] The specific process includes the following steps: I. Preparation from recombinant cells Ethyl N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate. The remaining steps were the same as in Example 2. The target product, ethyl N-(4-bromophenyl)-P-butylphosphonamide ester, was obtained by vacuum distillation. The yield of this product was 16%, and its optical purity (enantiomer excess, ee value) was 82%, indicating high optical purity. The purity and composition of the prepared chiral ethyl N-(4-bromophenyl)-P-butylphosphonamide ester were verified by high-performance liquid chromatography (HPLC). HPLC analysis can determine the purity and optical purity of the product, ensuring the accuracy of the experimental results. The HPLC chromatogram is shown below. Figure 8 As shown.

[0073] II. Preparation from frozen stem cells Ethyl N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl N-(4-bromophenyl)-P-butylphosphonamide ester, was obtained by vacuum distillation. The yield of this product was 29%, and the optical purity (enantiomer excess, ee value) was 89%, indicating that the product has high optical purity.

[0074] III. Preparation from crude enzyme solution Ethyl N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester was added to an Erlenmeyer flask to a final concentration of 60 mM as the reaction substrate, and the remaining steps were the same as in Example 2. The target product, ethyl N-(4-bromophenyl)-P-butylphosphonamide ester, was obtained by vacuum distillation. The yield of this product was 28%, and the optical purity (enantiomer excess, ee value) was 92%, indicating that the product has high optical purity.

[0075] The amino acid sequence of the cytochrome P450 monooxygenase in this invention is shown below:

[0076] The nucleotide sequence encoding cytochrome P450 monooxygenase is shown below:

[0077] Preparation methods of various substrates and racemic products Procedure: Under argon protection, add diethyl butylphosphonate (5 mmol, 1.0 equiv.), ultra-dry dichloromethane (8 mL), and ultra-dry dichloromethane to a dry round-bottom flask in sequence. N , N Dimethylacetamide (DMC, 2 drops); the reaction system was placed in an ice bath, and oxaloyl chloride (15 mmol, 3.0 equiv.) was added dropwise. After the addition was complete, the mixture was stirred at 0 °C for 5 min, and then transferred to room temperature and stirred for another 16 h. After the reaction was completed, the solvent and low-boiling residues in the system were removed by vacuum distillation to obtain crude phosphonoyl chloride, which was used directly in subsequent reactions.

[0078] Under argon protection, ultra-dry dichloromethane (20 mL), various amines (4.7 mmol, 1.0 equiv.), 4-dimethylaminopyridine (DMAP, 0.9 mmol, 0.2 equiv.), and triethylamine (9.4 mmol, 2.0 equiv.) were added sequentially to a dry round-bottom flask. Then, crude phosphonyl chloride (4.7 mmol, 1.0 equiv.) was added dropwise under ice bath conditions. After the addition was complete, the mixture was stirred at 0 °C for 5 min, and then moved to room temperature and stirred for 12 h.

[0079] After the reaction was completed, saturated sodium bicarbonate solution was added to quench the reaction, and the mixture was extracted three times with dichloromethane (DCM). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was purified by column chromatography (PE / EA = 3:1, v / v) to obtain the target substrate or product.

[0080] Characterization of product racemate data Ethyl N-phenyl-P-butylphosphonic acid ester

[0081] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d 7.27 – 7.21 (m, 2H), 7.04 – 6.89 (m,3H), 6.48 (s, 1H), 4.26 – 4.13 (m, 1H), 4.08 – 3.94 (m, 1H), 2.00 – 1.82 (m,2H), 1.74 – 1.47 (m, 2H), 1.40 – 1.28 (m, 5H), 0.84 (t, J = 7.3 Hz, 3H). 13CNMR (100 MHz, CDCl3) d 140.78, 129.55, 121.27, 117.17 (d, J = 6.1 Hz), 60.01 (d, J = 7.0 Hz), 25.69 (d, J = 129.3 Hz), 24.04 (d, J = 4.3 Hz), 23.76 (d, J= 17.4 Hz), 16.32 (d, J = 6.9 Hz), 13.67. 31 p NMR (162 MHz, CDCl3) d 31.36. Ethyl p-butyl-N-(2-fluorophenyl)phosphonamide ester

[0082] Yellow solid. 1 H NMR (400 MHz, CDCl3) d 7.28 – 7.21 (m, 1H), 7.09 – 6.98 (m,2H), 6.93 – 6.83 (m, 1H), 5.23 (s, 1H), 4.28 – 4.15 (m, 1H), 4.11 – 3.97 (m,1H), 1.96 – 1.79 (m, 2H), 1.63 – 1.50 (m, 2H), 1.41 – 1.28 (m, 5H), 0.86 (t,J = 7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d 152.39 (m), 128.75 (d, J = 12.3 Hz), 124.81 (d, J = 3.8 Hz), 121.81 (d, J = 7.2 Hz), 117.65, 115.51 (d, J = 19.3Hz), 60.52 (d, J = 7.0 Hz), 26.25 (d, J = 129.6 Hz), 24.10 (d, J = 4.5 Hz), 23.73 (d, J = 17.4 Hz), 16.35 (d, J = 6.6 Hz), 13.64. 31 p NMR (162 MHz, CDCl3) d 30.70. 19 F NMR (376 MHz, CDCl3) d -132.06. Ethyl p-butyl-N-(3-fluorophenyl)phosphonamide ester

[0083] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d 7.20 – 7.12 (m, 1H), 7.08 (s, 1H), 6.81 – 6.71 (m, 2H), 6.66 – 6.56 (m, 1H), 4.24 – 4.12 (m, 1H), 4.08 – 3.95(m, 1H), 1.96 – 1.82 (m, 2H), 1.72 – 1.48 (m, 2H), 1.41 – 1.28 (m, 5H), 0.84 (t, J = 7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d 163.73 (d, J = 244.4 Hz), 142.76 (d, J = 10.5 Hz), 130.69 (d, J = 9.8 Hz), 112.95 (dd, J = 6.6, 2.8 Hz), 107.90 (d, J = 21.2 Hz), 104.31 (dd, J = 25.4, 5.9 Hz), 60.22 (d, J = 7.0Hz), 25.64 (d, J = 129.2 Hz), 24.02 (d, J = 4.1 Hz), 23.72 (d, J = 17.3 Hz), 16.30 (d, J = 6.8 Hz), 13.67. 31 p NMR (162 MHz, CDCl3) d 31.29. 19 F NMR (376 MHz, CDCl3) d -111.49. Ethyl p-butyl-N-(o-tolyl)phosphonamide ester

[0084] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d7.15 – 7.09 (m, 3H), 6.93 – 6.83 (m,1H), 4.78 (s, 1H), 4.26 – 4.14 (m, 1H), 4.10 – 3.95 (m, 1H), 2.22 (s, 3H),2.03 – 1.76 (m, 2H), 1.72 – 1.44 (m, 2H), 1.44 – 1.25 (m, 5H), 0.85 (t, J =7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d 138.52, 131.02, 127.34, 125.32 (d, J =8.6 Hz), 121.69, 116.03, 60.14 (d, J = 7.0 Hz), 25.79 (d, J = 129.4 Hz), 24.09 (d, J = 4.3 Hz), 23.73 (d, J = 17.5 Hz), 17.94, 16.34 (d, J = 6.8 Hz), 13.65. 31 p NMR (162 MHz, CDCl3) d 31.23. Ethyl p-butyl-N-(p-tolyl)phosphonamide ester

[0085] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d 7.05 – 7.01 (m, 2H), 6.92 – 6.84 (m,2H), 6.04 (s, 1H), 4.24 – 4.11 (m, 1H), 4.08 – 3.93 (m, 1H), 2.27 (s, 3H),2.01 – 1.77 (m, 2H), 1.70 – 1.48 (m, 2H), 1.39 – 1.27 (m, 5H), 0.84 (t, J =7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d138.03, 130.75, 130.08, 117.27 (d, J =5.9 Hz), 59.94 (d, J = 7.0 Hz), 25.71 (d, J = 129.2 Hz), 24.08 (d, J = 4.3Hz), 23.78 (d, J = 17.4 Hz), 20.66, 16.34 (d, J = 6.9 Hz), 13.69. 31 P NMR (162MHz, CDCl3) d 31.42. Ethyl p-butyl-N-(4-chlorophenyl)phosphonamide ester

[0086] Yellow solid. 1 H NMR (400 MHz, CDCl3) d 7.21 – 7.15 (m, 2H), 7.04 (s, 1H), 6.98 – 6.91 (m, 2H), 4.22 – 4.10 (m, 1H), 4.05 – 3.91 (m, 1H), 1.99 – 1.77(m, 2H), 1.68 – 1.45 (m, 2H), 1.42 – 1.27 (m, 5H), 0.83 (t, J = 7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d 139.63, 129.49, 126.22, 118.37 (d, J = 6.2 Hz), 60.13 (d, J = 7.1 Hz), 25.57 (d, J = 129.0 Hz), 24.01 (d, J = 4.1 Hz), 23.73(d, J = 17.3 Hz), 16.32 (d, J = 6.8 Hz), 13.69. 31 p NMR (162 MHz, CDCl3) d 31.44.HRMS (ESI)m / z: calculated for C 12 H 20 NO2PCl [M+H] + : 276.0915, found:276.0924. Ethyl p-butyl-N-(4-methoxyphenyl)phosphonamide ester

[0087] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d 6.95 – 6.88 (m, 2H), 6.83 – 6.75 (m,2H), 6.05 (s, 1H), 4.25 – 4.11 (m, 1H), 4.08 – 3.94 (m, 1H), 3.75 (s, 3H),1.94 – 1.73 (m, 2H), 1.70 – 1.43 (m, 2H), 1.40 – 1.25 (m, 5H), 0.83 (t, J =7.3 Hz, 3H). 13 C NMR (100 MHz, CDCl3) d 154.64, 133.79, 118.96 (d, J = 5.6 Hz), 114.85, 59.87 (d, J = 7.0 Hz), 55.63, 25.59 (d, J = 129.3 Hz), 24.07 (d, J =4.3 Hz), 23.77 (d, J = 17.4Hz), 16.36 (d, J = 6.9 Hz), 13.69. 31 P NMR (162 MHz, CDCl3) d 31.75.HRMS (ESI)m / z: calculated for C 13 H 23 NO3P [M+H] + : 272.1410, found:272.1419. Ethyl N-(4-bromophenyl)-P-butylphosphonamide ester

[0088] Yellow liquid. 1 H NMR (400 MHz, CDCl3) d 7.33 – 7.28 (m, 2H), 6.90 – 6.87 (m,2H), 6.84 (s, 1H), 4.20 – 4.10 (m, 1H), 4.04 – 3.91 (m, 1H), 1.96 – 1.81 (m,2H), 1.67 – 1.44 (m, 2H), 1.36 – 1.27 (m, 5H), 0.83 (t, J = 7.3 Hz, 3H). 13CNMR (100 MHz, CDCl3) d 140.05, 132.42, 118.81 (d, J = 6.1 Hz), 113.63, 60.21 (d, J = 7.0 Hz), 25.63 (d, J = 129.3 Hz), 24.01 (d, J = 4.2 Hz), 23.73 (d, J= 17.3 Hz), 16.32 (d, J = 6.7 Hz), 13.69. 31 p NMR (162 MHz, CDCl3) d 31.32.HRMS(ESI)m / z: calculated for C 12 H 20 NO2PBr [M+H] + : 320.0410, found: 320.0416. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing a p-chiral phosphine oxide, characterized in that, Includes the following steps: The substance containing cytochrome P450 monooxygenase was co-dissolved with a racemic substrate in a solvent to obtain a reaction solution. The reaction solution was then subjected to a catalytic reaction at 20–40 °C for 20–30 h to prepare P-chiral phosphine oxide. The amino acid sequence of cytochrome P450 monooxygenase is shown in SEQ ID NO.1, and the structural formula of the racemic substrate is shown below. Wherein, R is hydrogen, C1-C10 alkyl, C1-C10 alkoxy, or halogen.

2. The method for synthesizing p-chiral phosphine oxide according to claim 1, characterized in that, R can be hydrogen, methyl, ethyl, propyl, methoxy, ethoxy, propoxy, or halogen.

3. The method for synthesizing p-chiral phosphine oxide according to claim 1, characterized in that, The racemic substrates are ethoxy-P-butyl-N-methyl-N-phenylphosphonamide, ethyl-P-butyl-N-(2-fluorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-(3-fluorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-methyl-N-(o-tolyl)phosphonamide ester, ethyl-P-butyl-N-methyl-N-(p-tolyl)phosphonamide ester, ethyl-P-butyl-N-(4-chlorophenyl)-N-methylphosphonamide ester, ethyl-P-butyl-N-(4-methoxyphenyl)-N-methylphosphonamide ester, and ethyl-N-(4-bromophenyl)-P-butyl-N-methylphosphonamide ester.

4. The method for synthesizing p-chiral phosphine oxide according to any one of claims 1 to 3, characterized in that, The substance containing cytochrome P450 monooxygenase is a recombinant cell containing cytochrome P450 monooxygenase, a frozen stem cell containing cytochrome P450 monooxygenase, or a crude enzyme solution containing cytochrome P450 monooxygenase.

5. The method for synthesizing p-chiral phosphine oxide according to claim 4, characterized in that, The dry weight concentration of recombinant cells containing cytochrome P450 monooxygenase in the reaction solution is 10–30 g / L, or the concentration of frozen stem cells containing cytochrome P450 monooxygenase in the reaction solution is 1–4 mg / mL, or the concentration of crude enzyme solution containing cytochrome P450 monooxygenase in the reaction solution is 0.1–0.41 μM; the concentration of racemic substrate is 1–60 mM.

6. The method for synthesizing p-chiral phosphine oxide according to claim 5, characterized in that, The reaction solution contained 15 g / L of recombinant cells with cytochrome P450 monooxygenase and 60 mM of racemic substrate.

7. The method for synthesizing p-chiral phosphine oxide according to claim 5, characterized in that, The reaction solution contained frozen stem cells with a concentration of 2 mg / mL of cytochrome P450 monooxygenase and a racemic substrate concentration of 60 mM.

8. The method for synthesizing p-chiral phosphine oxide according to claim 5, characterized in that, The concentration of the crude enzyme solution containing cytochrome P450 monooxygenase in the reaction solution was 0.1 μM, and the concentration of the racemic substrate was 60 mM.

9. The method for synthesizing the p-chiral phosphine oxide according to any one of claims 1 to 3, characterized in that, The catalytic reaction was carried out at a temperature of 30 °C for 24 h.

10. The method for synthesizing the p-chiral phosphine oxide according to any one of claims 1 to 3, characterized in that, The solvent is 50 mM phosphate buffer, pH 7-8.

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