Chemical-enzymatic coupling synthesis method of oseltamivir phosphate

Abstract

The invention discloses a chemical-enzymatic coupling synthesis method of oseltamivir phosphate, which comprises the following steps: by taking monasic acid OS-01 as a substrate, sequentially carrying out S1 esterification, S2 ketalation, S3 selective ring opening, S4 TEMPO oxidation, S5 primary transaminase reaction, S6 acetylation, S7 secondary transaminase reaction and S8 phosphorylation reaction to obtain the final product oseltamivir phosphate OS-09. According to the method, the step of sodium azide required by a traditional route is avoided, the production safety and the industrialization possibility are enhanced, two different transaminases are used for respectively constructing different chiral centers, the steps of protecting groups and deprotecting groups on the traditional route are not needed, the overall route is short, the used chemical reagents are few, the yield is high, and the method is suitable for industrial production. The industrial requirements of environmental protection and low cost are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biosynthesis, in particular to a chemical-enzymatic synthesis method of oseltamivir phosphate. BACKGROUND

[0002] Oseltamivir phosphate is the most widely used anti-influenza drug in the world. It was developed by Gilead Sciences in 1996 and first marketed in Switzerland in 1999. It was successfully marketed in China in October 2001. Oseltamivir phosphate is a specific inhibitor of neuraminidase. By inhibiting the action of neuraminidase, it can inhibit the release of mature influenza virus from host cells, thereby inhibiting the spread of influenza virus in the human body to treat influenza. There are two main oseltamivir phosphate dosage forms on the domestic market, namely Roche's Tamiflu capsule preparation and Dongyang Pharmaceutical's Wellicon granule preparation.

[0003] The synthesis route of oseltamivir phosphate mainly includes two categories: (1) using chiral natural compounds as raw materials, including quinic acid, shikimic acid, amino acids, sugars, D-tartaric acid diethyl ester, etc.; (2) using non-chiral chemical compounds such as butadiene and benzene as raw materials, through catalytic asymmetric synthesis and chiral resolution. At present, there are more than 30 reported synthesis routes of oseltamivir phosphate. The starting material of the industrial synthesis process of oseltamivir phosphate is still mainly natural compounds. Chiral synthesis is currently only used for laboratory small-scale research, and there is still a certain distance to realize industrial production. For example, Gilead Sciences developed a synthesis route of oseltamivir phosphate using quinic acid as raw material. This route uses quinic acid as raw material to protect the hydroxyl group by ketalization, esterification occurs within the molecule, the hydroxyl group is activated by methylsulfonyl chloride, and a carboxylic acid ester derivative is obtained by rapid dehydration. Then, under the catalysis of perchloric acid, the transformation of the acetone ketal protecting group to the pentanone ketal protecting group is realized, and selective ring-opening reaction is carried out with borane-methyl sulfide complex. The key intermediate epoxide compound is obtained by cyclization reaction. After ring opening, 3-aziridine derivative is obtained by reaction with trimethyl phosphorus. Finally, through sodium azide ring opening, acylation, Raney nickel hydrogenation reduction and phosphoric acid salt formation, the final product oseltamivir phosphate is obtained, but the total yield is only 4.4%. Therefore, the problems of low yield, difficulty in construction of multiple chiral centers, high cost, and environmental pollution still restrict the production of oseltamivir phosphate.

[0004] Some researchers have also developed new processes for chemical-enzyme method, such as using ethyl benzoate as a substrate, through fermentation by a specific E. coli strain E. coli JM109 to obtain cyclohexadiene-cis-diol, and then through a multi-step chemical process to obtain oseltamivir phosphate; patent CN 119219516 A esterifies, cyclizes, and performs Mitsunobu amino substitution, selective ring opening, TEMPO oxidation, transaminase enzyme catalysis, acylation, and deprotection to obtain oseltamivivir, wherein esterification, oxidation, and protection groups are classical reactions, and the combination of enzyme-catalyzed selective amino group and hydrazine hydrate deprotection obtains the target compound. However, the above chemical-enzyme method still has some defects, for example, due to the need to protect the already constructed chiral center, the need for protection and deprotection groups increases the additional steps and reduces the overall yield. SUMMARY

[0005] The purpose of the present application is to provide a chemical-enzyme method for the synthesis of oseltamivir phosphate, which avoids the need for sodium azide in the traditional route, enhances the safety of production and the possibility of industrialization, and uses two different transaminases to construct different chiral centers, without the need for the traditional route of protection and deprotection groups, resulting in a short overall route, the use of fewer chemical reagents, high yield, and better compliance with the needs of green environmental protection and low-cost industrialization.

[0006] The technical solution adopted by the present application to solve its technical problems is: A chemical-enzyme method for the synthesis of oseltamivir phosphate, using OS-01 as a substrate, sequentially esterifying, S2 ketalizing, S3 selectively ring opening, S4 TEMPO oxidizing, S5 transaminase reaction, S6 acetylating, S7 secondary transaminase reaction, and S8 phosphorylating to obtain the final product oseltamivir phosphate OS-09. .

[0007] The present application analyzes the existing preparation route and designs a new synthesis route. Using natural compound shikimic acid as raw material, through esterification, ketalization, selective ring opening, oxidation, transaminase catalysis to synthesize different chiral centers, and phosphorylation, oseltamivir phosphate is obtained. Among them, esterification, oxidation, ketalization, selective ring opening, and phosphorylation are classical reactions.

[0008] In S1 esterification, OS-01 (shikimic acid) is used as the initial material for esterification to obtain intermediate OS-02. The alcohol solvent can be any one of methanol, ethanol, and isopropanol, and the preferred one is ethanol. The acid solvent can be any one of acetic acid, oxalic acid, thionyl chloride, concentrated sulfuric acid, concentrated hydrochloric acid, and phosphoric acid, and the amount of acid solvent is 3%-10% of the amount of alcohol solvent, and the preferred one is thionyl chloride. The reaction temperature is 20-80℃.

[0009] S2 ketone is catalyzed into OS-03 by using a dehydrating agent and a catalyst, preferably trimethyl phosphate (dehydrating agent) and p-toluenesulfonic acid (catalyst).

[0010] S3 selective ring opening: OS-03 becomes OS-04, the solvent used can be any one of dichloromethane, acetonitrile, tetrahydrofuran and methyl tert-butyl ether, and the catalyst used can be any one of zinc dichloride, aluminum trichloride, iron trichloride, titanium tetrachloride and boron trifluoride etherate, and the amount of catalyst used ranges from 1 to 1.5 equivalents.

[0011] S4 TEMPO oxidation: both chiral hydroxyl groups of OS-04 are oxidized into ketones by using an oxidizing agent to obtain OS-05.

[0012] The innovation of the present application lies in the use of two different transaminases to selectively catalyze the amino groups of the ketones at different sites to obtain chiral compounds, thereby eliminating the additional step of protecting groups in the traditional process and avoiding the step of sodium azide in the traditional route, thereby enhancing the safety of production and the possibility of industrialization. Therefore, the overall process of the present application is simple to operate, reduces the use of expensive chemical reagents, and is more in line with the industrialization needs of green environmental protection and safe production. Moreover, due to the reduction in the number of steps in the overall process route, the overall yield will be more stable, and it has good industrial application prospects.

[0013] As a preferred embodiment, the transaminase-1 is used for enzyme catalysis in the S5 one-step transaminase reaction, and the amino acid sequence of the transaminase-1 is shown in SEQ ID NO: 2. Enzymes with the amino acid sequence substituted, deleted or added with several amino acids and having the function of catalyzing the intermediate OS-05 are also within the protection scope of the present application. In the field, when substituting with similar or similar amino acids, the function of the protein will not be changed. At the same time, adding one or several amino acids at the C-terminal or N-terminal will not change the function of the protein.

[0014] As a preferred embodiment, the reaction system of the S5 one-step transaminase reaction comprises: The concentration of the substrate OS-05 is 50-200 g / L, the wet bacterial mass of the recombinant genetically engineered bacteria expressing the transaminase-1 is 25-100 g / L, the pyridoxal phosphate is 0.5-2.0 mM / L, the isopropylamine is used in an amount of 1-2 times the molar amount of the substrate OS-05, and the reaction medium is a phosphate buffer with a pH of 8.0-8.5.

[0015] As a preferred embodiment, the reaction temperature of the S5 one-step transaminase reaction is 5-40℃, and the reaction time is 2-24 hours.

[0016] As preferred, in the acetylation reaction of S6, the molar ratio of compound OS-06: acetylation reagent = 1:2-4, and the reaction temperature is 110-115℃.

[0017] The acetylation reagent is one or several of acetic anhydride, ethyl formate, and acetyl chloride.

[0018] As preferred, the secondary transaminase reaction of S7 is catalyzed by transaminase-2, and the amino acid sequence of transaminase-2 is shown in SEQ ID NO: 4. Enzymes with several amino acids substituted, deleted, or added in the amino acid sequence and having the function of catalyzing intermediate OS-05 are also within the protection scope of the present application. In the art, when amino acids with similar or similar functions are substituted, the function of the protein is usually not changed. Meanwhile, adding one or several amino acids at the C-terminal or N-terminal usually does not change the function of the protein.

[0019] As preferred, the reaction system of the secondary transaminase reaction of S7 comprises: The concentration of substrate OS-07 is 50-200 g / L, the wet bacterial mass of recombinant genetically engineered bacteria expressing transaminase-1 is 25-100 g / L, pyridoxal phosphate is 0.5-2.0 mM / L, isopropylamine is used in an amount of 1-2 times the molar amount of substrate OS-07, and the reaction medium is a phosphate buffer with pH 8.0-8.5.

[0020] As preferred, the reaction temperature of the secondary transaminase reaction of S7 is 5-40℃, and the reaction time is 2-24 hours.

[0021] The transaminase-1 is added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet bacterial mass of recombinant genetically engineered bacteria; and the transaminase-2 is added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet bacterial mass of recombinant genetically engineered bacteria.

[0022] The present application has the following beneficial effects: The synthesis route of the present application avoids the step of using sodium azide in the traditional route, enhances the safety of production and the possibility of industrialization, and uses two different transaminases to construct different chiral centers, without the steps of protecting groups and deprotecting groups in the traditional route, so that the overall route is short, the use of chemical reagents is less, the yield is high, and it is more in line with the needs of green environmental protection and low cost industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the product confirmation spectrum of oseltamivir phosphate OS-09. DETAILED DESCRIPTION

[0024] The technical solutions of the present application are further specifically described below through specific examples.

[0025] In the present application, the raw materials and equipment used, unless specified otherwise, can be purchased from the market or commonly used in the art. The methods in the following examples, unless specified otherwise, are conventional methods in the art.

[0026] The catalytic process of the present application is shown as follows: .

[0027] The present application uses inexpensive shikimic acid as a starting material, which is esterified by thionyl chloride, ketal, selective ring opening, TEMPO oxidation, enzyme catalysis by transaminase, acylation and phosphorylation to obtain oseltamivir phosphate.

[0028] The preparation of transaminase-1 and transaminase-2 used in the present application is a conventional biological operation, and the protein expression host microorganism can be selected from Escherichia coli BL21 (DE3) Escherichia coli The molecular biology operations involved in the operation steps are conventional experimental operation methods well known in the biological field, including gene acquisition (PCR), splicing of plasmids and target genes (i.e. vector construction), introduction of plasmids containing target gene fragments into bacterial cells (i.e. transformation), cultivation of bacteria in culture medium and enzyme production (i.e. fermentation). In the present application, the formula of LB liquid culture medium is: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; the formula of LB solid culture medium is: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L.

[0029] Preparation of intermediate OS-02 in Example 1 Shikimic acid 1000 g and anhydrous ethanol 1000 mL were added to a reaction bottle, and stirring was started under nitrogen protection. A constant pressure dropping funnel was installed, and thionyl chloride 80 g was weighed and slowly added to the reaction solution. During the addition process, the reaction was exothermic, and the temperature rose to 40℃. After the addition was completed, the temperature was raised to 80℃ and refluxed for 4h. The reaction solution was initially milky white, and after 30min of reflux, the reaction solution gradually clarified. After 4h of reaction, the reaction solution was cooled to 25℃, and then concentrated under reduced pressure to obtain an oily product OS-02 with a mass of 1102.7g and a yield of 95%.

[0030] Preparation of intermediate OS-03 in Example 2 Some reported pure chemical routes, this step usually need to protect the multiple hydroxyl groups of OS-02, through multi-step steps to get the epoxide compound, and then through the ring opening process to get oseltamivir phosphate; There are great differences between different routes, such as the earliest gilead company uses epoxide compound as raw material, and synthesizes oseltamivir phosphate through 5 steps, which uses sodium azide, and the industrial production has the risk of explosion, and the yield is only 4.4%; Roche company developed a route without azide, using epoxide compound as raw material, synthesizing oseltamivir phosphate through 7 steps, which is the route for completing industrial synthesis at present, but the total yield is only 35%, which still has shortcomings in atom utilization rate and green environmental protection. The process of the present application uses two different transaminases to synthesize chiral hydroxyl groups in turn, so it does not need to synthesize epoxide compounds for chiral protection, can omit the process steps of multi-step protection, and avoid the use of various expensive reagents and unsafe reagents such as sodium azide.

[0031] The OS-02 obtained in Example 1 was used for the preparation of the next step, and the flow chart was as follows: 1200 g of triethyl orthoformate, 600 g of 3-pentanone, 280 mL of anhydrous ethanol and 2 g of p-toluenesulfonic acid were added to a reaction bottle, and stirred at room temperature for 3 hours under nitrogen protection. After the reaction was completed, OS-02 prepared in Example 1 was added, and stirring was continued at room temperature for 3 hours. After the reaction was completed, 3 L of dichloromethane and 1 L of pure water were added to the reaction bottle, and after stirring for 15-30 minutes, the liquid was separated. The organic phase was washed with saturated NaHCO3 solution and tap water respectively, each time stirring for 15-30 minutes and separating the liquid layer. All the aqueous phases were combined and back-extracted with dichloromethane, and the combined organic phase was collected. Anhydrous sodium sulfate was added to the organic phase, dried, stirred and filtered, and the obtained filtrate was concentrated under reduced pressure to obtain oil OS-03 1378.6 g, with a yield of 94%.

[0032] Synthesis of intermediate OS-04 in Example 3 A reaction bottle was added with 1378.6 g of OS-03 (the product of the previous step) and 10 L of dichloromethane, and after stirring uniformly, 500 g of triethylsilane was added, then the reaction system was cooled to 5℃, and nitrogen protection was maintained. Another reaction bottle was prepared by dissolving 800 g of titanium tetrachloride in 3.5 L of dichloromethane, and was transferred to a constant pressure dropping funnel for standby use. At 5℃, the above titanium tetrachloride solution was slowly added to the reaction system, and after dripping, the reaction was continued to stir at the same temperature for 1 h. After the reaction was completed, 1.4 L of ice water was slowly added at 2℃ to quench the reaction, and after stirring, the liquid was separated and extracted. The organic phase was washed with saturated NaHCO3 solution, and after separation, the organic phase was collected and concentrated under reduced pressure to obtain OS-04 1249.9 g, with a yield of 90%.

[0033] Synthesis of intermediate OS-05 in Example 4 To the reaction bottle, 1249.9 g of the product OS-04 from the previous step and 15 L of dichloromethane were added, and after stirring and dissolving, 1113 g of tetramethylpiperidine oxide was added. The reaction system was cooled and maintained at 0°C, and stirred under nitrogen protection. While maintaining the temperature, 800 g of trichloroisocyanuric acid was added in batches, and after addition, the reaction was continued to stir at the same temperature for 2 h. After the reaction was completed, the reaction liquid was filtered, and the filter cake was washed with 2.5 L of dichloromethane. The filtrate and the washing liquid were combined, and 5% sodium sulfite solution was added to quench the reaction, and after stirring, the liquid was separated. The organic phase was collected and concentrated under reduced pressure to obtain 1062.4 g of the product OS-05, with a yield of 85%.

[0034] Example 5 Preparation of recombinant cells of transaminase-1 and transaminase-2 (1) The expression vector was synthesized externally (Shanghai Generay Biotech Co., Ltd.), and the target gene fragment was inserted into pET28 to complete the construction of the expression vector using the commercial plasmid pET28. Gene sequence: transaminase-1: SEQ ID NO: 1, transaminase-2: SEQ ID NO: 3.

[0035] (2) The enzyme protein was expressed using Escherichia coli BL21 (DE3) (BL21 (DE3)). Escherichia coli The plasmid constructed externally was transformed into E. coli cells in turn, and LB plate culture medium (added with kanamycin 50 mg / L) was coated. The specific implementation is as follows: First, the plasmid pET28 was added to the competent cells of BL21 (DE3), and then it was placed on ice for 30 min, and then it was heat shocked at 42°C for 90 s. An appropriate amount of LB liquid medium (without adding antibiotics) was added, and then it was recovered and grown at 37°C for 1 h. The bacterial cells were coated on LB plate culture medium containing kanamycin resistance (kanamycin 50 mg / L), and incubated at 37°C for 12 hours. The single colonies grown on the plate were verified by sequencing, and the recombinant bacteria of transaminase-1 and transaminase-2 were obtained.

[0036] (3) Scale-up culture: The recombinant bacteria of transaminase-1 and transaminase-2 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C overnight. Then, 2% (v / v) of the inoculum was inoculated into LB liquid medium containing 50 μg / mL kanamycin, and incubated at 37°C, 150 rpm until the bacterial concentration reached OD 600 =0.6. IPTG was added at a final concentration of 0.1 mM, and the culture was induced at 28°C for 12 h. The bacterial cells were collected by centrifugation at 4°C, 12000 rpm for 10 min. The wet bacterial cells were washed with 0.85% physiological saline, and stored at -20°C for use.

[0037] Example 6 Transaminase-1 catalyzed preparation of OS-05 to OS-06 The chemical-enzymatic process provided by the present application still has a large difference from other reported enzymatic routes. For example, patent CN 119219516 A prepares oseltamivir through esterification with dichlorosulfoxide, cyclization, amino substitution by Mitsunobu, selective ring opening, TEMPO oxidation, transaminase enzyme catalysis, acylation, and deprotection. This route has some similarity with the route of the present application, but it only uses one transaminase to synthesize one chiral center, and still cannot avoid the step of chiral protection, which is one more step than the route provided by the present application.

[0038] The actual operation of the enzymatic step of the present application is listed below. The transaminase-1 recombinant genetically engineered bacteria prepared in Example 5 are used as catalysts to perform the catalytic reaction of OS-06, and the specific reaction conditions are as follows: The transaminase-1 wet bacteria are resuspended with 21 L of phosphate buffer (0.1 M, pH 8.0) to a final concentration of 50 g / L, then 1 mM / L of coenzyme pyridoxal phosphate and 50 g / L of substrate are added, and 2 equivalents of isopropylamine are used as the amino donor, and the reaction is performed at 35°C for 22 h. The pH is controlled to maintain 8.0 during the reaction, and the conversion result is determined by periodic sampling analysis. The results show that the conversion rate is 96%, the chirality of the product OS-06 meets the standard, and the OS-06 is extracted by conventional extraction operation. After multiple extractions, the organic phase is combined and concentrated under reduced pressure to obtain 934.2 g of intermediate OS-06, with a yield of 89%.

[0039] Based on the method of the present example, different parameters are changed for implementation: The reaction temperature is changed to 5°C, and the reaction is performed for 24 hours. The yield of intermediate OS-06 is about 50-60%; The reaction temperature is changed to 40°C, and the reaction is performed for 2 hours. The yield of intermediate OS-06 is about 25-30%; The concentration of the substrate is set to 50 g / L, the amount of transaminase-1 wet cells is set to 25 g / L, the concentration of pyridoxal phosphate is set to 0.5 mM / L, and the amount of isopropylamine is set to 1 equivalent. The yield of OS-06 is 60-65%; The concentration of the substrate is set to 200 g / L, the amount of transaminase-1 wet cells is set to 100 g / L, the concentration of pyridoxal phosphate is set to 2 mM / L, and the amount of isopropylamine is set to 2 equivalents. The yield of OS-06 is 45-48%; The concentration of the substrate is set to 100 g / L, the amount of transaminase-1 wet cells is set to 50 g / L, the concentration of pyridoxal phosphate is set to 1 mM / L, and the amount of isopropylamine is set to 1.5 equivalents. The yield of OS-06 is 52-55%.

[0040] Example 7 Acetylation reaction to prepare OS-07 The product OS-06 (934.2 g) from the previous step was added to the reaction flask, and stirring was started. Acetic anhydride (1400 g) and anhydrous sodium acetate (165 g) were added sequentially. Under nitrogen protection, the reaction system was heated to 110 °C and maintained at this temperature for 4 h. After the reaction was complete, the system was cooled to 45 °C, and 2.5 L of ethyl acetate was added. The mixture was stirred for 30 min. Then, 1.25 L of water was added, and the mixture was further cooled to room temperature for liquid-liquid extraction. The organic phase was collected and concentrated under reduced pressure to obtain 992.7 g of product OS-07, with a yield of 92%.

[0041] Based on the method of this embodiment, different parameters are changed for implementation: The reaction system was heated to 115℃ and maintained at this temperature for 4 hours. The yield of product OS-07 was 89%.

[0042] Example 8: Preparation of OS-08 from OS-07 catalyzed by transaminase-2 The recombinant genetically engineered transaminase-2 bacteria prepared in Example 5 were used as catalysts to catalyze the OS-08 reaction. The specific reaction conditions were as follows: transaminase-2 wet cells (final concentration 50 g / L) were resuspended in 25 L of phosphate buffer (0.1 M, pH 8.0). Then, pyridoxal coenzyme phosphate and substrate (final concentration 40 g / L) were added, and isopropylamine (2 equivalents) was used as the amino donor. The reaction was carried out at 35 °C for 22 h. During the reaction, the pH was maintained at 8.0. Samples were taken periodically to analyze the transformation results. The results showed that the conversion rate reached 95%, and the chirality of the product OS-08 met the standard. OS-08 was extracted by conventional extraction. The organic phases after multiple extractions were combined and concentrated under reduced pressure to obtain 853.7 g of intermediate OS-08, with a yield of 86%.

[0043] Based on the method of this embodiment, different parameters are changed for implementation: By changing the reaction temperature to 5℃ and reacting for 24 hours, the yield of intermediate OS-08 was 43-46%. The reaction temperature was changed to 40℃, and the reaction time was 2 hours. The yield of intermediate OS-08 was 21-25%. With the substrate concentration set at 50 g / L, the amount of transaminase-2 wet cells at 25 g / L, pyridoxal phosphate at 0.5 mM / L, and isopropylamine at 1 equivalent, the OS-08 yield was 54-57%. With the substrate concentration set at 200 g / L, the amount of transaminase-2 wet cells at 100 g / L, pyridoxal phosphate at 2 mM / L, and isopropylamine at 2 equivalents, the OS-08 yield was 37-41%. The concentration of the substrate was set at 100 g / L, the amount of transaminase-2 wet cells was 50 g / L, pyridoxal phosphate was 1 mM / L, isopropylamine was 1.5 times the equivalent amount, and the yield of OS-08 was 42-45%.

[0044] Example 9 Preparation of OS-09 OS-08 was phosphorylated, and the process was as follows: 853.7 g of intermediate OS-08 was dissolved in 8 L of acetone at 40°C with stirring, then 1.1 equivalents of a 45 wt% phosphoric acid acetone mixed solution was added dropwise, stirring was maintained at 45°C for 2 h, then the temperature was slowly reduced to 5°C, stirring was continued for 2 h, then filtration was performed, the filter cake was washed with acetone three times, then the material was dried under reduced pressure for 12-14 h, and 1030.5 g of the final product OS-09 was obtained, with a total yield of 43.5% for the eight steps. The confirmation spectrum of OS-09 is shown in Figure 1 .

[0045] SEQ ID NO: 1 SEQ ID NO: 2 MQKQRTTSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDSEGNKIIDGMAGAWCVNVGYGRKDFAEAARRQMEELSFMHTADGITHPAVVELSSLLAEVTPAGFDRVFYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLSGMKYMHEQGDLPIPGVAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQGAGGAIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPIGAVSVGKRVAEGLIAGGDFNHGHTTSGHPVCAAVAHANVAALRDEGIVQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMVAAFTLVKNKAKRELFPDFGEIGTLCRDIFFRNNLIMRICGDHIVAAPPLVMTRAEVDEMLAVAERCLEELEQSLKARGLAHHHHHH; SEQ ID NO: 3 ATGGCGTCTATGGACAAAGTGTTCGCTGGTTACGCTGCTCGTCAGGCTATTCTGGAATCTACCGAAACCACCAATCCGTTCGCGAAAGGTATCGCTTGGGTTGAAGGTGAACTGGTTCCACTTGCAGAAGCTCGTATCCCACTGCTGGATCAGGGTTTCATGCACTCTGACCTGACCTACGACGTTCCGTCTGTTTGGGATGGTCGTTTCTTCCGTCTGGATGACCACATCACTCGTCTGGAAGCGAGCTGCACCAAACTTCGTCTGCGTCTGCCATTGCCACGTGATCAGGTGAAACAGATTCTGGTTGAAATGGTTGCGAAATCCGGTATCCGTGACGCATTCGTTGAACTGATCGTTACTCGTGGTCTGAAAGGTGTTCGTGGTACTCGTCCAGAGGACATCGTTAACAACCTGTATATGTTCGTTCAGCCATACGTTTGGGTTATGGAACCGGACATGCAGCGTGTTGGTGGTTCTGCTGTTGTTGCACGTACCGTTCGTCGTGTACCACCAGGTGCAATCGATCCAACCGTTAAGAACCTGCAATGGGGTGATCTTGTGCGTGGTATGTTCGAGGCTGCTGATCGTGGTGCAACCTATCCGTTTCTGACTGACGGTGATGCTCACCTGACCGAAGGTTCTGGTTTCAACATCGTTCTGGTTAAAGACGGTGTTCTGTACACTCCAGACCGTGGTGTTCTTCAGGGTGTTACCCGTAAATCCGTTATCAACGCTGCTGAAGCGTTCGGTATCGAAGTTCGTGTTGAGTTCGTTCCGGTTGAACTGGCTTACCGTTGCGACGAAATCTTCATGTGCACTACCGCTGGTGGTATCATGCCGATCACTACTCTGGACGGTATGCCGGTTAACGGTGGTCAGATCGGTCCGATCACCAAGAAAATCTGGGACGGTTACTGGGCTATGCACTACGACGCAGCGTACAGCTTCGAGATCGACTACAACGAACGTAACCACCACCATCACCACCACTAA SEQ ID NO:4 MASMDKVFAGYAARQAILESTETTNPFAKGIAWVEGELVPLAEARIPLLDQGFMHSDLTYDVPSVWDGRFFRLDDHITRLEASCTKLRLRLPLPRDQVKQILVEMVAKSGIRDAFVELIVTRGLKGVRGTRPEDIVNNLYMFVQPYVWVMEPDMQRVGGSAVVARTVRRVPPGAIDPTVKNLQWGDLVRGMFEAADRGATYPFLTDGDAHLTEGSGFNIVLVKDGVLYTPDRGVLQGVTRKSVINAAEAFGIEVRVEFVPVELAYRCDEIFMCTTAGGIMPITTLDGMPVNGGQIGPITKKIWDGYWAMHYDAAYSFEIDYNERNHHHHHH.

[0046] The above-described embodiments are merely preferred embodiments of the present application, and are not intended to limit the present application in any form. Other variations and modifications can be made without departing from the technical scope of the present application as recited in the claims.

Claims

1. A chemo-enzymatic coupling method of oseltamivir phosphate characterized in that, The final product, oseltamivir phosphate OS-09, is obtained by sequentially subjecting the grass acid OS-01 to S1 esterification, S2 ketalization, S3 selective ring opening, S4 TEMPO oxidation, S5 one transaminase reaction, S6 acetylation, S7 two transaminase reactions, and S8 phosphorylation; 。 2. The chemo-enzymatic conjugation method according to claim 1, characterized in that, The S5 one transaminase reaction is an enzyme catalysis reaction using transaminase-1, and the amino acid sequence of the transaminase-1 is shown in SEQ ID NO:

2.

3. The chemo-enzymatic conjugation method according to claim 1, characterized in that, The reaction system of the S5 one transaminase reaction comprises: The substrate OS-05 has a concentration of 50-200 g / L, the recombinant genetically engineered bacteria expressing the transaminase-1 have a wet bacterial body of 25-100 g / L, pyridoxal phosphate has a concentration of 0.5-2.0 mM / L, isopropylamine has a dosage of 1-2 times the molar amount of the substrate OS-05, and the reaction medium is a phosphate buffer with a pH of 8.0-8.

5.

4. The chemo-enzymatic conjugation method according to claim 1 or 2 or 3, characterized in that, The reaction temperature of the S5 one transaminase reaction is 5-40 °C, and the reaction time is 2-24 hours.

5. The chemo-enzymatic conjugation method according to claim 1, wherein, In the S6 acetylation reaction, the molar ratio of the compound OS-06 to the acetylation reagent is 1:2-4, and the reaction temperature is 110-115 °C.

6. The chemo-enzymatic conjugation method according to claim 5, characterized in that, The acetylation reagent is one or more of acetic anhydride, ethyl formate, and acetyl chloride.

7. The chemo-enzymatic conjugation method according to claim 1, wherein, The S7 two transaminase reactions are enzyme catalysis reactions using transaminase-2, and the amino acid sequence of the transaminase-2 is shown in SEQ ID NO:

4.

8. The chemo-enzymatic conjugation method according to claim 1, wherein, The reaction system of the S7 two transaminase reactions comprises: The substrate OS-07 has a concentration of 50-200 g / L, the recombinant genetically engineered bacteria expressing the transaminase-1 have a wet bacterial body of 25-100 g / L, pyridoxal phosphate has a concentration of 0.5-2.0 mM / L, isopropylamine has a dosage of 1-2 times the molar amount of the substrate OS-07, and the reaction medium is a phosphate buffer with a pH of 8.0-8.

5.

9. The chemo-enzymatic conjugation method according to claim 1 or 7 or 8, characterized in that, The reaction temperature of the S7 two transaminase reactions is 5-40 °C, and the reaction time is 2-24 hours.

10. The chemo-enzymatic conjugation method according to claim 2 or 7, characterized in that, The transaminase-1 is added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet bacterial body of recombinant genetically engineered bacteria, and the transaminase-2 is added in the form of pure enzyme, immobilized enzyme, crude enzyme solution, or wet bacterial body of recombinant genetically engineered bacteria.

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