Process for preparing (S)-3-aminobutyronitrile through transaminase catalysis

Abstract

The invention provides transaminase, the amino acid sequence of the transaminase is an amino acid sequence obtained by mutation of an amino acid sequence shown as SEQ ID NO.2, and the mutation at least comprises one of the following mutation sites: the 418th site and the 419th site; the glycine at the 418th site is mutated into methionine, and the threonine at the 419th site is mutated into proline or serine. The transaminase can be used for synthesizing (S)-3-aminobutyronitrile, methyl acetoacetate serves as an initial substrate, (S)-3-aminobutanamide is synthesized through an enzyme method and a chemical method, and finally the (S)-3-aminobutyronitrile and derivatives are prepared through dehydration, the process does not involve extremely toxic substances such as methylsulfonyl chloride, sodium cyanide and the like, post-treatment is simple and efficient, and the method is suitable for industrial production. Finally, a high-purity product is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical synthesis, and specifically relates to a process for preparing (S)-3-aminobutyronitrile by using transaminase catalysis. Background Art

[0002] (S)-3-aminobutyronitrile is an important intermediate for drug molecules, especially a key starting material for the synthesis of Orforglipron, the first oral non-peptide GLP-1 receptor agonist developed by Eli Lilly and Company, and plays a role in constructing the chirality of the methyl group on the piperidine pyrazole structure of its parent core. Orforglipron was discovered by Chugai Pharmaceutical and licensed to Eli Lilly in 2018. It has the function of lowering blood sugar and is suitable for development into a weight loss drug or a drug for the treatment of type 2 diabetes. The results of a phase 2 clinical study showed that obese or overweight adults lost an average of 14.7% of their weight after 36 weeks of orforglipron treatment.

[0003] Patent CN117069743 reports that the synthesis of (S)-3-aminobutyronitrile is based on (S)-3-aminopropanol as raw material, di-tert-butyl dicarbonate to protect the amino group, and then react with methanesulfonyl chloride to generate (S)-2-((tert-butyloxycarbonyl)amino)propyl methanesulfonate, and then react with sodium cyanide to generate (S)-(1-cyanopropane-2-yl)carbamic acid tert-butyl ester, and then remove the Boc protection under methanesulfonic acid conditions. This route has the following technical problems: 1) The reaction raw materials involve a variety of highly toxic chemicals such as sodium cyanide and methanesulfonyl chloride. 2) The reaction route involves protection and deprotection on the amino group, and the route is cumbersome. 3) Based on (S)-3-aminopropanol, the reaction yield is less than 40%. From the perspective of process production, the cost is high, and the steps such as recycling are complicated, which is not conducive to production.

[0004] Therefore, in view of the problems existing in the existing synthesis of aminonitrile compounds, it is of great significance to develop a new method that is safe, simple to operate and low in cost. Summary of the invention

[0005] In view of the above problems, the present invention provides a process for preparing (S)-3-aminobutyronitrile by transaminase catalysis, which avoids the addition of highly toxic chemicals by combining chemical method and enzymatic method, and the operation is also simplified.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: A transaminase, wherein the amino acid sequence of the transaminase is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO.2, wherein the mutation includes at least one of the following mutation sites: position 418 and position 419; the glycine at position 418 is mutated to methionine, and the threonine at position 419 is mutated to proline or serine.

[0007] Furthermore, the amino acid sequence of the transaminase is SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0008] A DNA molecule, which encodes the above-mentioned transaminase.

[0009] Furthermore, the sequence of the DNA molecule is SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.7.

[0010] An application of the above-mentioned transaminase in producing chiral amines from ketone compounds.

[0011] A method for synthesizing an enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof, wherein in the compound of formula (1), R represents one or more substituents on an aliphatic chain, selected from hydrogen, alkyl, phenyl, optionally substituted cyclic or non-cyclic aliphatic groups having 1 to 10 carbon atoms, optionally substituted aliphatic heterocyclic groups having 1 to 10 carbon atoms, optionally substituted (hetero)aryl groups, optionally substituted alkoxy groups having 1 to 10 carbon atoms, halogen atoms, amine groups, nitro groups, carboxylic acids, carboxylic acid esters, carboxylic acid amides, nitrile or trifluoromethyl groups; The compound of formula (1) is prepared by dehydrating the compound of formula (2), The compound of formula (2) is prepared by esterification and amidation of the compound of formula (3), The ketone compound of the formula (3) is obtained by hydrolysis catalyzed by transaminase, and the transaminase is the transaminase with the amino acid sequence of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8. The definition of R in the compound of formula (2) and the compound of formula (3) is the same as the definition of R in the compound of formula (1). Furthermore, the compound of formula (1) is (S)-3-aminobutyronitrile, and the compound of formula (2) is (S)-3-aminobutyramide. The (S)-3-aminobutyramide is added to a solvent, and phosphorus oxychloride is added dropwise. After the addition is completed, it is separated and purified to obtain (S)-3-aminobutyronitrile; The compound of formula (3) is (S)-3-aminobutyric acid, which is prepared by an enzyme-catalyzed reaction of methyl acetoacetate under the action of transaminase, followed by strong acid hydrolysis and resin purification.

[0012] Furthermore, the (S)-3-aminobutyric acid is subjected to esterification and amidation to obtain (S)-3-aminobutyramide, and the specific steps are: subjecting (S)-3-aminobutyric acid and thionyl chloride to an esterification reaction in a sterically hindered alcohol, cooling after the reaction to precipitate a solid, and filtering to obtain (S)-3-aminobutyric acid ester; (S)-3-aminobutyric acid ester is added with ammonia water to carry out aminolysis reaction. After the aminolysis reaction is completed, the solvent is removed to obtain (S)-3-aminobutyramide.

[0013] Furthermore, the alcohol with large steric hindrance is isopropanol, the reaction temperature of (S)-3-aminobutyric acid and thionyl chloride is 0-50°C, and after the reaction is completed, the temperature during the cooling process of the solid is -10-10°C; The concentration of the aqueous ammonia is 25%, the temperature of the ammonolysis reaction is 0-50°C, and the reaction time is 2-3h.

[0014] Furthermore, the concentration of methyl acetoacetate is 20-100 g / L, the enzyme-catalyzed reaction also requires the addition of pyridoxal phosphate, an amine donor, and a 0.1 M potassium phosphate solution, the pH of the enzyme-catalyzed reaction system is 8, and the reaction temperature is 20-50° C.; preferably, the amine donor includes isopropylamine, phenethylamine or alanine.

[0015] Beneficial effects: 1. The present invention provides a process for preparing (S)-3-aminobutyronitrile by transaminase catalysis, which starts from bulk chemical raw materials, synthesizes (S)-3-aminobutyramide by enzymatic and chemical methods, and finally dehydrates to prepare (S)-3-aminobutyronitrile and derivatives. The process does not involve the use of highly toxic substances such as methylsulfonyl chloride, sodium cyanide, etc., and the post-treatment is simple and efficient, and a high-purity product is finally prepared.

[0016] 2. The invention provides a process for preparing (S)-3-aminobutyronitrile by transaminase catalysis, which avoids the protection and deprotection process, has a simpler route, and the mother liquor can be reused repeatedly, which simplifies the post-treatment process and meets the purpose of modern clean production. In the synthesis of (S)-3-aminobutyramide and its derivatives, the introduction of large steric hindered alcohols to generate corresponding large steric hindered esters is not only easy to separate, but also conducive to the aminolysis reaction, greatly shortening the aminolysis reaction time.

[0017] 3. The present invention provides a process for preparing (S)-3-aminobutyronitrile by catalysis of a transaminase. Compared with the existing process, the enzymatic preparation process has a greatly improved substrate concentration. The transaminase disclosed in the present invention has excellent selectivity for the catalyzed reaction. The activity of wild-type transaminases is generally low and cannot be suitable for industrial scale-up production. However, through today's mature directed evolution technology, the wild-type transaminase can be engineered to achieve the purpose of improving its catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The reaction process route of (S)-3-aminobutyronitrile of the present invention; Figure 2 The electrophoresis diagram of the transaminase SEQ ID NO.2 and SEQ ID NO.4 of Example 2 of the present invention; Figure 3 The (S)-3-aminobutyronitrile synthesis component derivatization chromatogram and chiral derivatization chromatogram of Example 8 of the present invention; Figure 4 This is the NMR image of (S)-3-aminobutyronitrile of Example 9 of the present invention. DETAILED DESCRIPTION

[0019] The present invention provides a synthesis process of (S)-3-aminobutyronitrile, the specific route is as follows Figure 1 As shown, (S)-3-aminobutyronitrile is obtained by dehydrating (S)-3-aminobutyramide, and the steps involve the dehydration reagent phosphorus oxychloride. Preferably, the dehydration solvent is an aprotic solvent, preferably acetonitrile; the molar ratio of (S)-3-aminobutyric acid:phosphorus oxychloride is: 1:0.6eq~4.0eq, preferably 1:1.0eq~2.0eq; after the dehydration is completed, it is concentrated and recrystallized to obtain, and the recrystallization solvent is an alcohol, preferably methanol and ethanol; the product (S)-3-aminobutyronitrile has a purity of ≥99% and an ee value of ≥99%.

[0020] During the amide dehydration process, the carbonyl group of (S)-3-aminobutyramide attacks phosphorus oxychloride to dehydrate and simultaneously forms a bond with the chiral amino group to form a six-membered ring intermediate state, and then reacts with a proton-type reagent to return to the amino group. During the entire process, phosphorus oxychloride acts as a dehydrating agent and also as a chiral protecting agent. The optical purity ee value of the final product (S)-3-aminobutyronitrile is greater than 99%.

[0021] The (S)-3-aminobutyramide involved in the above reaction has a synthesis method as follows: (S)-3-aminobutyric acid and thionyl chloride are subjected to an esterification reaction in a large sterically hindered alcohol, and after the reaction, the solid is precipitated by cooling, and the (S)-3-aminobutyric acid ester is obtained by filtration. (S)-3-aminobutyric acid ester is added with ammonia water for aminolysis reaction, and after the aminolysis reaction, the solvent is removed to obtain (S)-3-aminobutyramide. The obtained (S)-3-aminobutyramide is added to a solvent, and phosphorus oxychloride is added dropwise. After the addition is completed, the (S)-3-aminobutyronitrile is obtained by separation and purification.

[0022] In the amide synthesis, a large sterically hindered alcohol is selected as a solvent, (S)-3-aminobutyric acid reacts with thionyl chloride to generate (S)-3-aminobutyric acid ester, 3-aminobutyric acid ester is precipitated by cooling and crystallization, the mother liquor is used for replication, and the (S)-3-aminobutyric acid ester solid reacts with ammonia water to generate (S)-3-aminobutyramide. Compared with the common methyl ester, this large sterically hindered ester has a faster rate of aminolysis in ammonia water, and the reaction can be carried out at room temperature and pressure; Preferably, the sterically hindered alcohol selected for the esterification of (S)-3-aminobutyric acid is any one of isopropanol, isobutyl alcohol, tert-butyl alcohol, isopentanol, tert-pentanol, and isooctyl alcohol, preferably isopropyl alcohol; the molar ratio of (S)-3-aminobutyric acid to thionyl chloride is 1:1.0eq~2.5eq, preferably 1:1.05eq~1.2eq; the reaction temperature with thionyl chloride is 0~50°C, preferably 25°C; after the reaction, the temperature is lowered to -10~10°C for crystallization, and the crystallization temperature is preferably 0°C; the precipitated solid material is directly put into ammonia water for aminolysis reaction, and the mother liquor is used for the next batch; Preferably, the molar ratio of (S)-3-aminobutyric acid: ammonia water free ammonia is 1:1.0-5.0, preferably 1:1.5-2.0; the concentration of ammonia water is 15%-35%, preferably 25% (mass fraction); the reaction temperature is 0-50°C, preferably 25°C; after the aminolysis reaction is completed, the (S)-3-aminobutyramide is obtained by desolvation.

[0023] The present invention also provides a transaminase, which is derived from a wild-type transaminase of Pseudomonas fluorescens (its gene sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2), and a transaminase mutant is obtained by amino acid mutation of SEQ ID NO.2, wherein the amino acid mutation is any combination of the following three mutations: (a) T419P, (b) G418M or (c) T419S. The transaminase mutant can catalyze the following reaction of converting a keto group to an amino group, wherein R in the substrate represents one or more substituents on a fatty chain, selected from hydrogen, alkyl, phenyl, optionally substituted cyclic or non-cyclic aliphatic groups with 1 to 10 carbon atoms, optionally substituted aliphatic heterocyclic groups with 1 to 10 carbon atoms, optionally substituted (hetero)aryl groups, optionally substituted alkoxy groups with 1 to 10 carbon atoms, halogen atoms, amine groups, nitro groups, carboxylic acids, carboxylic acid esters, carboxylic acid amides, nitrile groups or trifluoromethyl groups.

[0024] Due to its non-natural chirality, the preparation of (S)-3-aminobutyric acid needs to be completed by an engineered (S)-selective transaminase. The starting material involved in the reaction is a large and easily available starting material, namely methyl acetoacetate. The amine donors involved in the reaction include but are not limited to isopropylamine, phenethylamine, alanine and other amine donors.

[0025] The above transaminase catalytic reaction is carried out in a buffer system with a pH of 7-9, a temperature range of 25-50 degrees Celsius, a substrate concentration range of 20-100g / L, and an amine donor of 1-2 molar equivalents. After the reaction is completed, the product is hydrolyzed by strong acids such as hydrochloric acid, sulfuric acid, and benzenesulfonic acid and purified by resin. The optional amino resins are: Amberlite IR-120, HZ-3B, HZ-12B, HZ016, HZD-2, D185, HD-8, etc. Its optimal reaction conditions are: the substrate is preferably 100g / L of methyl acetoacetate, the amine donor is preferably 51g / L of isopropylamine, the amount of pyridoxal phosphate is 0.05g / L, the reaction temperature is 30 degrees Celsius, pH=8.0, and the reaction time is 24 hours. The reaction is treated to obtain a total product yield of 81%, and the ee value of (S)-3-aminobutyric acid is 99.12%.

[0026] As described above, the present invention relates to a process for synthesizing (S)-3-aminobutyramide from bulk chemical raw materials by enzymatic and chemical methods, and finally dehydrating to prepare (S)-3-aminobutyronitrile and derivatives. The process does not involve the use of highly toxic substances such as methylsulfonyl chloride, sodium cyanide, etc., and the post-treatment is simple and efficient, and a high-purity product is finally prepared.

[0027] As described above, the present invention relates to the chemical synthesis of (S)-3-aminobutyronitrile, which avoids the protection and deprotection process, has a simpler route, and the mother liquor is repeatedly used, which simplifies the post-treatment process and meets the purpose of modern clean production. In the synthesis of (S)-3-aminobutyramide and its derivatives, a large steric hindered alcohol is introduced to generate a corresponding large steric hindered ester, which is not only easy to separate, but also conducive to the aminolysis reaction, and greatly shortens the aminolysis reaction time.

[0028] The enzymatic preparation process of (S)-3-aminobutyric acid according to the present invention has a greatly improved substrate concentration compared to the reported cases, and the transaminase disclosed in the present invention has excellent selectivity for the catalyzed reaction. The activity of wild-type transaminase is generally low and cannot be applied to industrial scale-up production. However, through the current mature directed evolution technology, wild-type transaminase can be engineered to improve its catalytic activity.

[0029] The present invention is further illustrated below with examples, and the technical scheme of the present invention is clearly and completely described, but the present invention is not limited thereto, and the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In addition, the experimental methods involved in the following embodiments but not indicating specific conditions are usually in accordance with the conditions conventional in the field of biochemical technology or recommended by the manufacturer, and the reagents and equipment involved but not particularly indicated are all commercially available.

[0030] Example 1: Discovery of wild-type transaminase, construction of engineered enzyme variants and expression system In order to obtain Figure 1 The enzyme that converts the keto group in the methyl acetoacetate substrate into amino group first screened the wild-type transaminase and obtained a better wild-type transaminase, the amino acid sequence of which is SEQ ID NO.2.

[0031] In order to obtain catalytic Figure 1 The inventors used the wild-type transaminase as a starting template, the nucleotide sequence of which is SEQ ID NO.1, and developed three engineered enzyme mutants with higher performance through a creative directed evolution process, the amino acid sequences of which are shown in SEQ ID No.4, 6, and 8 (shown in Table 1 below). For an introduction to directed evolution technology, please refer to "Directed Evolution: Bringing New Chemistry to Life" Frances H. Arnold, Angewandte Chemie, November 28, 2017.

[0032] Table 1 Engineered enzyme variants Amino acid sequence number Corresponding wild-type aminotransferase template The difference in amino acid residues relative to the template SEQ ID NO.4 SEQ ID NO.2 T419P SEQ ID NO.6 SEQ ID NO.2 G418M SEQ ID NO.8 SEQ ID NO.2 T419S The wild-type and mutated amino acid corresponding gene sequences were synthesized by General Biotechnology (Anhui) Co., Ltd. and cloned into the expression vector pACYCDuet-1. The recombinant expression plasmid was transformed into the competent cells of E.coilBL21 (DE3) under the conditions of 42°C heat shock for 90s, ice bath for 5min, and then LB culture medium was added to recover in a 37°C shaking incubator for 1h, and finally coated on an LB plate containing chloramphenicol resistance and inverted culture at 37°C overnight to obtain the recombinant transformant.

[0033] Example 2: Preparation of transaminase A single microbial colony of Escherichia coli BL21 (DE3) containing the expression plasmid carrying the target transaminase gene was inoculated into a 250 mL conical flask containing 50 mL LB medium (containing 30 μg / mL chloramphenicol) and cultured overnight in a shaking incubator at 30°C. When the OD of the culture solution reached 600 When the cell density reached 2, the inoculum was inoculated into a 1000 mL conical flask containing 250 mL TB medium at a 5% (v / v) inoculum, and IPTG with a final concentration of 1 mM was added to induce the expression of transaminase, and the cells were placed in a shaker at 30°C. After 20 hours of shaking culture, the culture solution was centrifuged at 8000 rpm, and the supernatant was discarded after 10 minutes. The cells were collected to obtain wet cells. The wet cells successfully expressed soluble transaminase (specifically, Figure 2 The electrophoresis diagram (SDS-PAGE) of aminotransferase SEQ ID No. 2 and SEQ ID No. 4 is shown, in which the number "1" represents the protein detection band of the supernatant after the wet bacteria are homogenized and broken, and the number "2" represents the protein detection band of the lower precipitate after the wet bacteria are homogenized and broken). The wet bacteria can be directly used for enzyme catalysis reaction, or can be frozen and stored at -20°C for future use.

[0034] Example 3: Transaminase reaction screening Weigh 2.5mg pyridoxal phosphate in a 25mL glass bottle, add 4.5mL 0.1M phosphate buffer and drop 250mg substrate methyl acetoacetate, 127mg isopropylamine, and adjust the pH to 8.0 with hydrochloric acid. Weigh 250mg of wet bacteria of transaminase and add them to the reaction solution, and add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30°C, the stirring speed to 400rpm, and quench with an equal volume of pure acetonitrile after 24h. Take the supernatant of the reaction solution, and use the chiral derivatization agent NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide for derivatization treatment for chiral derivatization detection. Screening determines the optimal sequence SEQ IDNO.4 conversion rate 81%, ee value 99.12%.

[0035] The LC-MS instrument used to detect the conversion rate of the above reaction is Agilent G6470B, the chromatographic column used is Aligent AQ-C18150×4.6mm(ID)5μm; the mobile phase is 5mM ammonium acetate (0.1% formic acid) buffer: acetonitrile = 99:1; the flow rate is 0.5mL / min; the DAD detector, the detection wavelength is 200nm; the column temperature is 25℃, the ion source temperature is 300℃, the cleavage voltage is 20V; the injection volume is 5μL; the total running time of the detection reaction sample is 30min. The substrate and the product are tested with different concentrations of standard samples, and the standard curve is drawn to determine the yield of the product.

[0036] The chirality of the above-mentioned product was derivatized with a chiral derivatizing agent NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide for chiral derivatization detection.

[0037] The HPLC instrument used to detect the chirality of the above reaction products is a commercially available Agilent 1100 chromatograph, a PDA detector, an Agilent ZORBAX SB-C18 4.6*150mm*5μm chromatographic column, an aqueous solution containing one thousandth of formic acid (phase A) and pure acetonitrile (phase B), a flow rate of 0.9mL / min, a column temperature of 30°C, a wavelength of 340nm, and an injection volume of 10μL. The mobile phase for product chirality detection is 56% phase A and 44% phase B for isocratic elution, and the total running time is 15min. The results of each product are shown in Table 2.

[0038] Table 2 The number of the DNA sequence used for recombinant expression Amino acid sequence number Conversion Rate % ee% SEQ ID NO.1 SEQ ID NO.2 35％ 95.71％ SEQ ID NO.3 SEQ ID NO.4 81％ 99.12％ SEQ ID NO.5 SEQ ID NO.6 62％ <![CDATA[96 . 86%]]> SEQ ID NO.7 SEQ ID NO.8 50％ 97.97％ Example 4: Preparation of (S)-3-aminobutyric acid Weigh 0.5g of pyridoxal phosphate in a 2L round-bottom flask, add 850mL of 0.1M phosphate buffer and add 100g of methyl acetoacetate and 51g of isopropylamine respectively, and adjust the pH to 8.5 with hydrochloric acid. Weigh 25g of wet bacteria expressing SEQ ID NO.4 transaminase and add them to the reaction solution, add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30°C, and the stirring speed to 400rpm. During the reaction, add hydrochloric acid dropwise to maintain the pH to the mid-control point of 80% conversion rate, adjust the pH to 1-2 with hydrochloric acid and heat quench at 60 degrees Celsius. After cooling to room temperature, remove the bacteria with diatomaceous earth filtration. Centrifuge the reaction solution, set the speed to 8000 rpm, time 30 minutes, pour out the supernatant, the volume is about 1.5L. Resin pre-activation; weigh 4.5kg of resin, prepare 13.5L (5% to 10%) hydrochloric acid to soak the resin for 1h, and build a resin device; weigh 4.5kg of resin and soak it in 13.5L saturated brine for 18 to 20h, soak it in 5% HCl for 4 to 8h, drain the acid and rinse it with pure water until it is neutral, soak it in 2% to 4% NaOH for 4 to 8h, drain the alkali, rinse it to neutrality, and build a resin device. After adjusting the base of 1.5L filtrate, repeatedly load it on the column until (S)-3-aminobutyric acid is not detected in the effluent. Then wash the resin with pure water until the effluent is neutral. Then repeatedly elute the resin with dilute ammonia water until (S)-3-aminobutyric acid is no longer detected in the effluent. Collect the eluate and concentrate it to dryness to obtain (S)-3-aminobutyric acid. After post-treatment, 55.2 g of (S)-3-aminobutyric acid was obtained. The total yield is shown in Table 3. The chirality of each resin after post-treatment was maintained at ee≥99.00%.

[0039] Table 3 Separation and purification of (S)-3-aminobutyric acid by different resins Resin Model Total yield HZD-2 79％ HZ-12B 77％ HZ016 74％ Amberlite IR-120 72％ D185 69％ HZ-3B 66％ HD-8 60％ Example 5 (S)-Phenylglycine Preparation Weigh 0.5g of pyridoxal phosphate in a 2L round-bottom flask, add 850mL of 0.1M phosphate buffer and add 100g of methyl benzoylformate and 31g of isopropylamine respectively, and adjust the pH to 8.5 with hydrochloric acid. Weigh 25g of wet bacteria expressing the aminotransferase of SEQ ID NO.4 and add them to the reaction solution, and add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30°C, and the stirring speed to 400rpm. During the reaction, hydrochloric acid was added dropwise to maintain the pH to 80% conversion at the mid-control point, and 48.9g of (S)-phenylglycine was obtained after post-treatment with the HZD-2 resin described in Example 4, with chiral ee ≥ 99.00%.

[0040] Example 6: Preparation of (S)-3-aminobutyric acid to (S)-3-aminobutyronitrile hydrochloride In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 1443.68g (S)-3-aminobutyric acid and 3365.60g isopropanol at room temperature, and gradually dissolve under stirring. After dissolving, keep the internal temperature at 25±2℃ and add 1748.66g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 25±2℃ and continue the reaction until there is no (S)-3-aminobutyric acid left. Slowly cool to 0±2℃, and a large amount of solid will precipitate. Centrifuge to obtain (S)-3-aminobutyric acid ester solid.

[0041] 1904 g of 25% aqueous ammonia solution was added to the above (S)-3-aminobutyric acid ester solid. After the addition, the internal temperature was maintained at 25±2°C for 2.5 h, and the solvent was removed to obtain a white solid, namely (S)-3-aminobutyramide.

[0042] Add 4300g of acetonitrile to the above white solid, raise the temperature, and add 3863.66g of phosphorus oxychloride dropwise in batches while maintaining reflux. The total time is 2.5h. After the addition, continue to react for 2h. Slowly cool to 15°C and centrifuge to obtain a crude product.

[0043] The crude product was added to 7900g of anhydrous ethanol, heated to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, centrifuged, and the filter cake was rinsed with 1000g of ethanol. The wet product was vacuum dried at 50°C to obtain 1434.90g of white solid, which was (S)-3-aminobutyronitrile hydrochloride, with a total yield of 85%, a product ee value of ≥99.90%, and a product purity of >99%.

[0044] Example 7: Preparation of (S)-3-aminobutyric acid to (S)-3-aminobutyronitrile hydrochloride In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 1443.68g (S)-3-aminobutyric acid and 1794.24g isopropanol at room temperature, and gradually dissolve under stirring. After dissolving, keep the internal temperature at 0±2℃ and add 1748.66g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 0±2℃ and continue to react until there is no (S)-3-aminobutyric acid left. Slowly cool to -10±2℃, and a large amount of solid will precipitate. Centrifuge to obtain (S)-3-aminobutyric acid ester solid.

[0045] The above (S)-3-aminobutyric acid ester solid was added with 3173 g of 15% ammonia solution, and after the addition, the internal temperature was maintained at 0±2°C and the reaction was continued for 3 hours until the reaction was complete. The solvent was removed, and after the removal of solvent, a white solid was obtained, namely (S)-3-aminobutyramide.

[0046] Add 4900g of acetonitrile to the above white solid, raise the temperature, and add 2360g of phosphorus oxychloride dropwise in batches while maintaining reflux. The total time is 2h. Continue to react for 2.5h after the addition. At this time, a white solid precipitates in the system. Slowly cool to 15°C and centrifuge to obtain a crude product.

[0047] The crude product was added to 7900g of anhydrous ethanol, heated to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, centrifuged, and the wet solid was vacuum dried at 50°C to obtain 1414.64g of white solid, which was (S)-3-aminobutyronitrile hydrochloride. The total yield was 83.8%, the product ee value was ≥99.90%, and the product purity was >99%.

[0048] Example 8: Preparation of (S)-3-aminobutyric acid to (S)-3-aminobutyronitrile hydrochloride In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 1443.68g (S)-3-aminobutyric acid and 3365.60g isopropanol at room temperature, and heat to gradually dissolve while stirring. After dissolving, keep the internal temperature at 10±2℃ and add 1998.70g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 10±2℃ and continue to react until there is no (S)-3-aminobutyric acid left. Slowly cool to 0±2℃ to precipitate a large amount of solid. Centrifuge to obtain (S)-3-aminobutyric acid ester solid.

[0049] The above (S)-3-aminobutyric acid ester solid was added with 1428g of 25% ammonia solution, heated under stirring, and the internal temperature was maintained at 50±2°C for 2h to continue the reaction. The solvent was removed, and after the removal of solvent, a free yellow viscous liquid was obtained, which was (S)-3-aminobutyramide.

[0050] 4302.17g of acetonitrile was added to the yellow viscous liquid, and the temperature was raised to reflux. 3863.66g of phosphorus oxychloride was added dropwise in batches while maintaining the reflux state. The total time of the addition was 2.5h. After the addition was completed, the reflux was maintained and the reaction continued for 2.5h. At this time, a white solid precipitated in the system. The temperature was slowly lowered to 15°C and centrifuged to obtain a crude product.

[0051] The crude product was added to 4485.60 g of anhydrous methanol, and the temperature was raised to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, and the wet product was obtained by centrifugation, and vacuum dried at 50°C to obtain 1228.20 g of a white solid, which was (S)-3-aminobutyronitrile hydrochloride.

[0052] The total yield is 81.8%, the product ee value is ≥99.90%, and the product purity is >99%.

[0053] Example 9: Preparation of (S)-3-aminobutyric acid to (S)-3-aminobutyronitrile hydrochloride In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 1443.68g (S)-3-aminobutyric acid and 3365.60g isopropanol at room temperature, and heat to dissolve while stirring. After dissolving, keep the internal temperature at 50±2℃ and add 1748.66g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 50±2℃ and continue the reaction until there is no (S)-3-aminobutyric acid left. Slowly cool to 10±2℃ to precipitate a large amount of solid. Centrifuge to obtain (S)-3-aminobutyric acid ester solid.

[0054] The above (S)-3-aminobutyric acid ester solid was added to 1360g of 35% ammonia solution precooled to 0±2°C, and the internal temperature was maintained at 10±2°C for 3h to complete the reaction. The solvent was removed, and a white solid was obtained after the removal of solvent, namely (S)-3-aminobutyramide.

[0055] 4302.17g of acetonitrile was added to the above white solid, and the temperature was raised to reflux. 4125.00g of phosphorus oxychloride was added dropwise in batches while maintaining the reflux state. The total time of dropwise addition was 2.5h. The reaction was continued for 2h after the dropwise addition. At this time, a white solid precipitated in the system. The temperature was slowly lowered to 15°C and centrifuged to obtain a crude product.

[0056] The crude product was added to 7900g of anhydrous ethanol, heated to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, centrifuged, and the filter cake was rinsed with 1000g of ethanol. The solid wet product was vacuum dried at 50°C to obtain 1399.45g of white solid, which was (S)-3-aminobutyronitrile hydrochloride. The total yield was 82.9%, the chirality was 99.97%, and the product purity was >99%.

[0057] Example 9: Preparation of (S)-3-aminobutyric acid to (S)-3-aminobutyronitrile In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 1443.68g (S)-3-aminobutyric acid and 4150.83g isobutanol at room temperature, and heat to gradually dissolve while stirring. After dissolving, keep the internal temperature at 30±2℃ and add 1748.66g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 30±2℃ and continue the reaction until there is no (S)-3-aminobutyric acid left. Slowly cool to 10±2℃ to precipitate a large amount of solid. Centrifuge to obtain (S)-3-aminobutyric acid ester solid.

[0058] The above (S)-3-aminobutyric acid ester solid was added with 1428g of 25% ammonia solution, and the reaction was continued for 2h at an internal temperature of 20±2°C until the reaction was complete. The solvent was removed, and after the removal of solvent, a free yellow viscous liquid was obtained, which was (S)-3-aminobutyramide.

[0059] 4302.17g of acetonitrile was added to the yellow viscous liquid, and the temperature was raised to reflux. 2576g of phosphorus oxychloride was added dropwise in batches while maintaining the reflux state. The total time of dropwise addition was 2.5h. The reaction was continued for 2h after the dropwise addition. At this time, a white solid precipitated in the system. The temperature was slowly lowered to 15°C and centrifuged to obtain a crude product.

[0060] The crude product was added to 6728.40 g of anhydrous methanol, and the temperature was raised to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, and centrifuged. The wet solid was dried at 50°C in a vacuum to obtain 1190 g of a white solid, which was (S)-3-aminobutyronitrile hydrochloride.

[0061] The total yield is 70.5%, the chirality is 99.97%, and the product purity is >99%. The chirality test results are as follows: Figure 3 shown, specific Figure 3 3a in the figure indicates the sample detection spectrum; Figure 3 b is the detection spectrum of mixed standard.

[0062] Figure 4 This is the NMR spectrum of the (S)-3-aminobutyronitrile of the sample of Example 9, 1H-NMR (500 Hz, DMSO-d6): 1.31-1.32 (3H, d), 2.89-3.07 (2H, m), 3.53-3.69 (1H, m), 8.57 (3H, s).

[0063] The above reaction conversion rate and purity were derivatized by benzoic anhydride for chiral derivatization detection. The chromatographic column was AQ-C18 250×4.6 mm (ID), 5 μm, the mobile phase consisted of mobile phase A 0.1% phosphoric acid and mobile phase B acetonitrile, the flow rate was 1 mL / min, gradient elution, column temperature was 30°C, wavelength was 230 nm, and the total running time was 32 min. The solvent was 50% acetonitrile, the injection volume was 5 μL, and the retention time of (S)-3-aminobutyronitrile was 16.1 min.

[0064] The chirality of the above product is derivatized by a chiral derivatizing agent NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide for chiral derivatization detection.

[0065] The chromatographic column was Agilent ZORBAX SB-C18 4.6*150mm*5μm, the mobile phase was pure acetonitrile and aqueous solution containing 1 / 1000 formic acid, the flow rate was 0.9mL / min, 36% acetonitrile was eluted isocratically, the wavelength was 340nm, and the total running time was 23min. The solvent was 50% acetonitrile, the injection volume was 10μL, the retention time of (S)-3-aminobutyronitrile was 12.9min, and the retention time of (R)-3-aminobutyronitrile was 16.8min.

[0066] Example 10: Preparation of (S)-aminophenylacetonitrile from (S)-phenylglycine In a 10L clean, dry, well-sealed glass reactor, with nitrogen micro-positive pressure protection, add 90.18g (S)-(S)-phenylglycine and 2593.75g isobutanol at room temperature, and heat to gradually dissolve while stirring. After dissolving, keep the internal temperature at 30±2℃ and add 1800g thionyl chloride dropwise in batches. After the addition is completed, keep the internal temperature at 30±2℃ and continue to react until there is no (S)-3-aminobutyric acid left. Slowly cool to 10±2℃ to precipitate a large amount of solid. Centrifuge to obtain (S)-phenylglycine ester solid.

[0067] Add 1400g of 25% ammonia solution to the above (S)-phenylglycine ester solid. After the addition, keep the internal temperature at 20±2℃ and continue to react for 2h. The reaction is complete. Start to remove the solvent. After the removal of solvent, a free yellow viscous liquid is obtained. Add 3990g of acetonitrile to the above yellow viscous liquid and heat it to reflux. Keep reflux state and add 2400g of phosphorus oxychloride in batches. The total time of dropwise addition is 2.5h. After the dropwise addition, continue to react for 2h. At this time, a white solid precipitates in the system. Slowly cool to 15℃ and centrifuge to obtain a crude product.

[0068] The crude product was added to 6690 g of anhydrous methanol, and the temperature was raised to reflux, and the solid gradually dissolved. The temperature was slowly lowered to 15°C, centrifuged, and the wet solid was vacuum dried at 50°C to obtain 1190 g of white solid, which was (S)-aminophenylacetonitrile hydrochloride. The total yield was 70%, the chirality was 99.98%, and the product purity was >99%.

Claims

1. A transaminase, characterized in that The amino acid sequence of the transaminase is obtained by mutation of the amino acid sequence shown in SEQ ID NO.2, and the mutation includes at least one of the following mutation sites: position 418 and position 419; the threonine at position 419 is mutated to proline or serine, and the glycine at position 418 is mutated to methionine.

2. The transaminase according to claim 1, characterized in that The amino acid sequence of the transaminase is SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.

8.

3. A DNA molecule, characterized in that The DNA molecule encodes the transaminase according to claim 1 or 2.

4. The DNA molecule according to claim 3, characterized in that The sequence of the DNA molecule is SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.

7.

5. Use of the transaminase according to claim 1 or 2 in producing chiral amines from ketone compounds.

6. A method for synthesizing an enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof, characterized in that: In the compound of formula (1), R represents one or more substituents on the aliphatic chain, selected from hydrogen, alkyl, phenyl, optionally substituted cyclic or non-cyclic aliphatic groups having 1 to 10 carbon atoms, optionally substituted aliphatic heterocyclic groups having 1 to 10 carbon atoms, optionally substituted (hetero)aryl groups, optionally substituted alkoxy groups having 1 to 10 carbon atoms, halogen atoms, amine groups, nitro groups, carboxylic acids, carboxylic acid esters, carboxylic acid amides, nitrile or trifluoromethyl groups; (1) The compound of formula (1) is prepared by dehydrating the compound of formula (2), (2) The compound of formula (2) is prepared by esterifying and amidating the compound of formula (3), (3) The compound of formula (3) is obtained by hydrolyzing a ketone compound catalyzed by transaminase, wherein the transaminase is the transaminase according to claim 1 or 2, and the definition of R in the compound of formula (2) and the compound of formula (3) is the same as the definition of R in the compound of formula (1).

7. The method for synthesizing enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof according to claim 6, characterized in that: The compound of formula (1) is (S)-3-aminobutyronitrile, the compound of formula (2) is (S)-3-aminobutyramide, and the preparation method of (S)-3-aminobutyronitrile is: (S)-3-aminobutyramide is added to a solvent, phosphorus oxychloride is added dropwise, and after the addition is completed, (S)-3-aminobutyronitrile is obtained by separation and purification; The compound of formula (3) is (S)-3-aminobutyric acid, which is prepared by an enzyme-catalyzed reaction of methyl acetoacetate under the action of transaminase, followed by strong acid hydrolysis and resin purification.

8. The method for synthesizing enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof according to claim 7, characterized in that: The (S)-3-aminobutyric acid is subjected to esterification and amidation to obtain (S)-3-aminobutyramide. The specific preparation method is as follows: (S)-3-aminobutyric acid and thionyl chloride are subjected to esterification reaction in a sterically hindered alcohol, after the reaction is completed, the temperature is lowered to precipitate a solid, and the (S)-3-aminobutyric acid ester is obtained by filtering; (S)-3-aminobutyric acid ester is added with ammonia water to carry out aminolysis reaction. After the aminolysis reaction is completed, the solvent is removed to obtain (S)-3-aminobutyramide.

9. The method for synthesizing enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof according to claim 8, characterized in that: The alcohol with large steric hindrance is isopropanol, and the reaction temperature of (S)-3-aminobutyric acid and thionyl chloride is 0-50°C. After the reaction is completed, the temperature during the cooling process of the solid is -10-10°C. The concentration of the aqueous ammonia is 25%, the temperature of the ammonolysis reaction is 0-50°C, and the reaction time is 2-3h.

10. The method for synthesizing enantiomerically enriched optionally substituted 3-aminobutyronitrile of formula (1) or a salt thereof according to claim 7, characterized in that: The concentration of methyl acetoacetate is 20-100 g / L, and the enzyme-catalyzed reaction also requires the addition of pyridoxal phosphate, an amine donor, and a 0.1 M potassium phosphate solution. The pH of the enzyme-catalyzed reaction system is 8, and the reaction temperature is 20-50° C.; Preferably, the amine donor comprises isopropylamine, phenethylamine or alanine.

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