Synthesis method of (S)-3-aminobutyronitrile

Abstract

The invention provides a synthesis method of (S)-3-aminobutyronitrile, which specifically comprises the following steps: (1) addition of diketene and ammonia to prepare acetoacetamide; (2) performing an enzyme catalytic reaction on the acetoacetamide obtained in the step (1) in the presence of transaminase, and purifying after the reaction is completed to obtain (S)-3-aminobutanamide; and (3) dehydrating the (S)-3-aminobutanamide obtained in the step (2) to obtain the (S)-3-aminobutyronitrile. According to the synthesis method, the synthesis route is simplified, the production efficiency of the (S)-3-aminobutyronitrile is improved, industrial three wastes are reduced, the use of highly toxic substances is avoided, the cost reduction and benefit improvement in the industrial amplified production are facilitated, and the whole route embodies the value of green chemical synthesis.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical synthesis, and particularly relates to a method for synthesizing (S)-3-aminobutyronitrile. Background Art

[0002] (S)-3-Aminobutyronitrile, also known as (S)-β-aminobutyronitrile, is one of the raw materials for the preparation of GLP-1 receptor agonists, a class of drugs that lower blood sugar levels by activating insulin release and inhibiting glucagon release. They are used to treat type 2 diabetes. These drugs can also reduce appetite and help control weight.

[0003] Patent CN117069743A reports that (S)-3-aminobutyronitrile is synthesized using (S)-2-aminopropanol as a raw material, di-tert-butyl dicarbonate to protect the amino group, and then reacting with methanesulfonyl chloride to produce (S)-2-((tert-butyloxycarbonyl)amino)propyl methanesulfonate, and then reacting with sodium cyanide to produce (S)-(1-cyanopropane-2-yl)carbamic acid tert-butyl ester, and then removing the Boc protection under methanesulfonic acid conditions.

[0004] This route has the following technical problems: 1) The raw materials involved in the reaction include a variety of highly toxic chemicals such as sodium cyanide and methanesulfonyl chloride. 2) The reaction route involves the protection and deprotection of the amino group, which is cumbersome. 3) The reaction yield is less than 40% based on (S)-3-aminopropanol. From the perspective of process production, the cost is high, and the recycling and other steps are complicated, which is not conducive to production.

[0005] Therefore, it is of great significance to develop a new method with simple route and low cost to address the problems existing in the current synthesis of aminonitrile compounds. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for synthesizing (S)-3-aminobutyronitrile, which avoids the protection and deprotection processes by combining chemical methods with enzyme methods, has a simpler route, high atom utilization rate and low production cost.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for synthesizing (S)-3-aminobutyronitrile, specifically comprising the following steps: (1) Addition of diketene and ammonia to prepare acetoacetamide; (2) subjecting the acetoacetamide obtained in step (1) to an enzyme-catalyzed reaction in the presence of a transaminase, and purifying after the reaction is complete to obtain (S)-3-aminobutyramide; (3) Dehydrating the (S)-3-aminobutyramide obtained in step (2) to obtain (S)-3-aminobutyronitrile.

[0008] The specific synthetic route of (S)-3-aminobutyronitrile is as follows Figure 1 shown.

[0009] Furthermore, the specific reaction process of step (1) is: With diketene and ammonia as substrate raw materials and ether compounds as reaction solvents, an addition reaction is carried out through a microchannel reactor to obtain the product acetoacetamide; the equivalent ratio of diketene and ammonia in the microchannel reactor is 1:1.0-3.0, the mass equivalent ratio of diketene and ether solvent is 1:0.5-10, the ether solvent is isopropyl ether or methyl tert-butyl ether, the temperature of the microchannel addition reaction is 30-60°C, and the residence time of each substance molecule participating in the reaction in the microchannel reactor is 10s-120s; Preferably, the equivalent ratio of diketene to ammonia in the microchannel reactor is 1:1.1-1.5; Preferably, the temperature of the microchannel addition reaction is 40-50°C; Preferably, the residence time of each substance molecule participating in the reaction in the microchannel reactor is 20-35s; Preferably, the ether solvent is methyl tert-butyl ether, and the mass equivalent ratio of diketene to methyl tert-butyl ether is 1:1-2.

[0010] Furthermore, the specific process of step (2) is: acetoacetamide and isopropylamine are used as raw materials to synthesize (S)-3-aminobutyramide through a transaminase aqueous phase reaction; the concentration of acetoacetamide is 50g / L-130g / L, the concentration of the transaminase is 10g / L, the initial concentration of isopropylamine added during the reaction is 1.5-2M, preferably 2M; the reaction temperature is 30-45°C; Preferably, the concentration of acetoacetamide is 130 g / L: Preferably, the initial concentration of isopropylamine added during the reaction is 2M; Preferably, the reaction temperature is 45°C.

[0011] Furthermore, the product obtained by the enzyme catalytic reaction in step (2) is purified by resin, and the resin used includes HZ-3B, HZ-12B, HZ016, Amberlite IR-120, HZD-2, D185, HD-8; Preferably, the resin is HZ-3B.

[0012] Furthermore, the transaminase may be selected from the amino acid sequences shown in (i), (ii) or (iii) below: (i) the transaminase has an amino acid sequence as shown in SEQ ID NO.2; (ii) the conserved amino acid sites in the enzyme catalytic center of the amino acid sequence of the transaminase have a sequence identity of more than 65% with all the amino acid sequences described in (i); (iii) the amino acid sequence of the transaminase is obtained by single mutation or multiple mutation at positions 58 and 154 of the amino acid sequence described in (i) and (ii); Preferably, the enzyme catalytic center of the transaminase has a conserved amino acid site that has a sequence identity of more than 75% with all the amino acid sequences described in (i); Preferably, the enzyme catalytic center of the transaminase has a conserved amino acid site that has a sequence identity of more than 85% with all the amino acid sequences described in (i); More preferably, the transaminase has an amino acid sequence as shown in SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58; More preferably, the transaminase has a transaminase mutant amino acid sequence as shown in SEQ ID NO.60, 62, or 64.

[0013] Furthermore, the specific process of step (3) is: using (S)-3-aminobutyramide as a substrate raw material, adding a solvent, dripping thionyl chloride as a reaction dehydration reagent, and purifying to prepare the product (S)-3-aminobutyronitrile; the equivalent ratio of the (S)-3-aminobutyramide and thionyl chloride is 1:1.0-4.0, the solvent is isobutyronitrile, acetonitrile or tetrahydrofuran, the dripping time of thionyl chloride is 1-5h, and the dripping temperature of thionyl chloride is 60-80°C; Preferably, the equivalent ratio of (S)-3-aminobutanamide and thionyl chloride is 1:1.2-2.0; Preferably, the solvent is isobutyronitrile; Preferably, the dripping time of thionyl chloride is 2 to 3 hours; Preferably, the temperature for adding thionyl chloride dropwise is 70-75°C.

[0014] Furthermore, after the dehydration reaction in step (3) is completed, the product (S)-3-aminobutyronitrile is obtained by concentration, cooling, crystallization, filtration, washing and drying. After the mother liquor is filtered and distilled, the solvent can be continuously applied to the next batch of reactions.

[0015] A method for preparing enantiomer-enriched (S)-3-aminobutyramide or its salt by enzyme catalysis, which can be prepared by subjecting a ketone compound to a transaminase-catalyzed reaction, wherein the transaminase is the same as the transaminase described in step (2) of the above-mentioned (S)-3-aminobutyronitrile preparation method; in formula (1), R represents one or more substituents on a fatty chain, and the R group is selected from hydrogen, alkyl such as methyl, an optionally substituted cyclic or non-cyclic aliphatic group having 1 to 10 carbon atoms, an optionally substituted aliphatic heterocyclic group having 1 to 10 carbon atoms, an optionally substituted (hetero)aryl group, an optionally substituted alkoxy group having 1 to 10 carbon atoms, a halogen atom, an amine group, a nitro group or a trifluoromethyl group; The ketone compound is prepared by amidation of the corresponding methyl ester or diketene. The specific reaction route is as follows Figure 2 shown.

[0016] The product obtained by subjecting ketone compounds to a transaminase-catalyzed reaction exists in the form of a free yellow viscous liquid and a white solid salt. The compound is prepared by an enzyme catalyst, an enzymatic reaction, and a post-treatment process that are first created in the present application.

[0017] Furthermore, the transaminase catalyzed reaction is carried out in a 50 mM phosphate buffer system at pH = 7-9, a temperature range of 25-50° C., a substrate loading range of 20-150 g / L, and 1-2 molar equivalents of the amine donor.

[0018] Furthermore, after the transaminase catalyzed reaction is completed, the final product can be purified by resin, and the resin is Amberlite IR-120, HZ-3B, HZ-12B, HZ016, HZD-2, D185 or HD-8.

[0019] Beneficial effects: 1. The present invention provides a method for synthesizing (S)-3-aminobutyronitrile, develops a new process for synthesizing (S)-3-aminobutyronitrile, simplifies the synthesis route, improves the production efficiency of (S)-3-aminobutyronitrile, reduces industrial waste, avoids the use of highly toxic substances, and is conducive to reducing costs and improving benefits in industrialized scale-up production. The overall route embodies the value of green chemical synthesis.

[0020] 2. The present invention provides a method for synthesizing (S)-3-aminobutyronitrile, which uses a microchannel synthesis method to prepare acetoacetamide. By controlling the temperature and reaction time of the microchannel reactor heat exchanger, not only the generation of polymer impurities is reduced, but also the preparation efficiency and product purity are significantly improved, thereby ensuring the safety and continuity of the production process.

[0021] 3. The present invention provides a method for synthesizing (S)-3-aminobutyronitrile, which uses thionyl chloride as a dehydrating agent and isobutyronitrile as a reaction solvent. A qualified product can be directly obtained by concentration and cooling. The filtrate can be applied after distillation, which greatly simplifies the post-processing operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The reaction process route of (S)-3-aminobutyronitrile of the present invention; Figure 2 The present invention provides an enzyme-catalyzed reaction process for (S)-3-aminobutanamide or its salt; Figure 3 This is the SDS protein electrophoresis diagram of SEQ ID NO.2 in Example 3 of the present invention; Figure 4 is the NMR image of (S)-3-aminobutanamide in Example 8 of the present invention; Figure 5 This is the chiral derivatization chromatogram of the (S)-3-aminobutanamide standard sample in Example 4 of the present invention; Figure 6 This is the NMR image of (S)-3-aminobutyronitrile in Example 9 of the present invention; Figure 7 The derivatization chromatogram and chiral derivatization chromatogram of the (S)-3-aminobutyronitrile synthesis component in Example 9 of the present invention are shown.

[0023] The definitions of specific terms in this invention are explained as follows: Conversion rate = molar amount of substrate consumed / molar amount of substrate fed x 100% Total yield = molar amount of product obtained from post-treatment / molar amount of substrate input x 100% ee = [S-type product concentration - R-type product concentration] / [[S-type product concentration + R-type product concentration] Enzyme catalytic center: refers to the region of the enzyme involved in substrate binding and catalytic reaction, which is usually composed of its key conserved amino acid sites to form a specific spatial structure.

[0024] Conserved amino acid sites: Conserved amino acid sites refer to highly similar or identical amino acid sequence regions that are preserved in different sequences, usually indicating that these regions play a key role in substrate binding or catalytic reaction in structure or function. DETAILED DESCRIPTION

[0025] 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 embodiments of the present invention, rather than all 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 premise belong to 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 according to 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, and the acetoacetamide and acetoacetamide in the embodiments are the same compound.

[0026] Example 1: Preparation of acetoacetamide from diketene At room temperature, 252.21 g of diketene and 252.21 g of methyl tert-butyl ether were stirred and mixed evenly, and the feed flow rates of the methyl tert-butyl ether solution of diketene and liquid ammonia were controlled separately by double-plunger feed pumps. A back-pressure valve was added after the liquid ammonia feed pump to ensure accurate metering of liquid ammonia and liquid feeding. After stable operation, the product was collected at the discharge port.

[0027] The microchannel temperature was set to 50°C. After the preparation work was completed, the two feed pumps were switched on and off at the same time. The feed mass ratio of diketene methyl tert-butyl ether solution and liquid ammonia was controlled to be about 6.6:1. The flow rate was adjusted to input it into the microchannel reactor for addition reaction, so that the material stayed in the microchannel reactor for 29 seconds. The received material was distilled and concentrated at a vacuum degree of not less than -0.095MPa, and 299.40g of acetoacetamide was obtained after there was no fraction. Yield: 98.7%, purity>99%.

[0028] Example 2: Discovery of wild-type transaminase, construction of engineered enzyme variants and expression system In order to obtain a transaminase that can catalyze the synthesis of (S)-3-aminobutyramide from acetoacetamide, the inventors first screened a large number of enzymes through a theoretical calculation model and further experimentally verified them. Some of the screened wild-type transaminases are shown in Table 1, and a better wild-type transaminase (amino acid sequence number is SEQ ID NO.2) was further determined.

[0029] Table 1 Recombinant expression gene sequence number Amino acid sequence number source SEQ ID No.1 SEQ ID No.2 Aliidongia sp. SEQ ID No.3 SEQ ID No.4 Acinetobacter baumannii SEQ ID NO.5 SEQ ID NO.6 Variovorax paradoxus SEQ ID NO.7 SEQ ID NO.8 Halomonas elongata Furthermore, the inventors also screened some wild-type transaminases with more than 65%, more than 75% or more than 85% sequence identity with SEQ ID NO. 2. These wild-type transaminases are conserved in the catalytic center sequence of the enzyme, and their differences are more reflected in the amino acid sites outside the catalytic active center of the enzyme. Among them, the wild-type transaminase is derived from the amino acid sequence shown in SEQ ID NO.10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 in Table 2, and its corresponding nucleotide sequence is shown in SEQ ID NO.9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, and any one of the 25 transaminases shown can effectively catalyze Figure 2 The reactions shown, however, did not yield high enough conversions.

[0030] Table 2 source Amino acid sequence number Nucleotide sequence number Aquamicrobium defluvii SEQ ID NO.10 SEQ ID NO.9 Tistrella mobilis SEQ ID NO.12 SEQ ID NO.11 Aminobacter sp.HY435 SEQ ID NO.14 SEQ ID NO.13 Pseudochrobactrum sp.sp1633 SEQ ID NO.16 SEQ ID NO.15 Mesorhizobium sp. SEQ ID NO.18 SEQ ID NO.17 Fulvimarina sp.2208YS6-2-32 SEQ ID NO.20 SEQ ID NO.19 Amorphus coralli SEQ ID NO.22 SEQ ID NO.21 Sedimentitalea xiamensis SEQ ID NO.24 SEQ ID NO.23 Roseovariusnubinhibens ISM SEQ ID NO.26 SEQ ID NO.25 Ensifer sp.Root31 SEQ ID NO.28 SEQ ID NO.27 Phyllobacteriaceae bacteria SEQ ID NO.30 SEQ ID NO.29 Bartonella SEQ ID NO.32 SEQ ID NO.31 Mesorhizobium sp.Root157 SEQ ID NO.34 SEQ ID NO.33 Agrobacterium pusense SEQ ID NO.36 SEQ ID NO.35 Sedimentitalea todarodis SEQ ID NO.38 SEQ ID NO.37 Brucella cytisi SEQ ID NO.40 SEQ ID NO.39 Magnetovibrio sp. SEQ ID NO.42 SEQ ID NO.41 Sinorhizobium sp.BG8 SEQ ID NO.44 SEQ ID NO.43 Mesorhizobium sp. SEQ ID NO.46 SEQ ID NO.45 Aliiroseovarius crassostreae SEQ ID NO.48 SEQ ID NO.47 Pseudochelatococcus lubricantis SEQ ID NO.50 SEQ ID NO.49 Jiella sp.LLJ827 SEQ ID NO.52 SEQ ID NO.51 Pseudomonadota bacterium SEQ ID NO.54 SEQ ID NO.53 Braayrhizobium liaoningense SEQ ID NO.56 SEQ ID NO.55 Nitratireductor soli SEQ ID NO.58 SEQ ID NO.57 In order to obtain the ability to catalyze Figure 1 In order to achieve the biocatalyst of the reaction shown in the figure and meet the industrial production requirements, the inventors used the wild-type aminotransferase SEQ ID NO.2 as the starting template and developed three engineered enzyme mutants with higher performance through a creative directed evolution process, whose amino acid sequences are shown in SEQ ID No.60, 62, and 64 (as shown in Table 3). For an introduction to directed evolution technology, please refer to "Directed Evolution: Bringing New Chemistry to Life" Frances H. Arnold, Angewandte Chemie, November 28, 201 7.

[0031] Table 3 Engineered enzyme mutants Amino acid sequence number Corresponding wild-type aminotransferase template The difference in amino acid residues relative to the template SEQ ID NO.60 SEQ ID NO.2 W58H, V154A SEQ ID NO.62 SEQ ID NO.2 V154S SEQ ID NO.64 SEQ ID NO.2 V154A 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 to obtain a recombinant expression plasmid. The recombinant expression plasmid was transformed into 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 at 37°C overnight to obtain a recombinant transformant.

[0032] Example 3. 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 it reaches 2, press 5% (v / v) The inoculum was inoculated into a 1000mL conical flask containing 250mL TB medium, 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 8000rpm, and the supernatant was discarded after 10 minutes. The cells were collected to obtain wet cells. The wet cells successfully expressed soluble transaminase (specifically, the SDS protein electrophoresis of SEQ ID NO.2 is shown in Figure 2). Figure 3 As shown in the figure, 1 represents the protein band of the supernatant of wet bacterial homogenization and 2 represents the protein band of the precipitate after wet bacterial homogenization and rupture). The wet bacterial cells can be directly used for enzyme catalysis reaction, or can be frozen and stored at -20°C until use.

[0033] Example 4: Transaminase reaction screening Weigh 2.5 mg of pyridoxal phosphate in a 25 mL glass bottle, add 4.5 mL of 0.1 M phosphate buffer and drop 250 mg of substrate acetoacetamide and 191 mg of isopropylamine, and adjust the pH to 7.5 with sulfuric acid. Weigh 250 mg 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 400 rpm, and quench with an equal volume of pure acetonitrile after 24 hours. 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 determined the preferred sequence SEQ ID NO.60 conversion rate 96%, ee value 99.95%.

[0034] 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; DAD detector, the detection wavelength is 200nm; the column temperature is 25℃, the ion source temperature is 300℃, and the cleavage voltage is 20V; the injection volume is 5μL; the total running time is 15min. 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. The chiral derivatization chromatogram of the specific (S)-3-aminobutyramide standard sample is shown in the figure. Figure 5 shown.

[0035] The chirality of the above product was derivatized by a chiral derivatizing agent NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide for chiral derivatization detection. The HPLC instrument used to detect the above reaction was a commercially available Agilent 1100 chromatograph, a PDA detector, an Agilent ZORBAX SB-C184.6*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 10 μL. The mobile phase for the chiral detection of the product was 44% B phase isocratic elution for 1min, 44% to 90% B phase gradient elution for 1min, 90% B phase isocratic elution for 2min, 90% to 44% B phase gradient elution for 1min, 44% B phase isocratic elution for 10min, and the total running time was 15min. The specific test results are shown in Table 3.

[0036] Table 3 DNA sequence number Amino acid sequence number Conversion rate ee SEQ ID NO.1 SEQ ID NO.2 40％ 99.01％ SEQ ID NO.3 SEQ ID NO.4 25％ 94.32％ SEQ ID NO.5 SEQ ID NO.6 35％ 97.99％ SEQ ID NO.7 SEQ ID NO.8 32％ 98.72％ SEQ ID NO.59 SEQ ID NO.60 96％ 99.95％ SEQ ID NO.61 SEQ ID NO.62 70％ 99.35％ SEQ ID NO.63 SEQ ID NO.64 87％ 99.85％ Example 5 Weigh 2.5 mg of pyridoxal phosphate in a 25 mL glass bottle, add 4.5 mL of 0.1 M phosphate buffer and drop 250 mg of substrate 3-carbonyl valeramide and 190 mg of isopropylamine, and adjust the pH to 7.5 with sulfuric acid. Weigh 250 mg of wet bacteria of transaminase SEQ ID NO.60 and add them to the reaction solution, add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30 ° C, the stirring speed to 400 rpm, and quench with an equal volume of pure acetonitrile after 24 hours. 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 determined that the preferred sequence SEQ ID NO.64 had a conversion rate of 90% and an ee value of 99.88%.

[0037] Example 6 Weigh 2.5 mg of pyridoxal phosphate in a 25 mL glass bottle, add 4.5 mL of 0.1 M phosphate buffer and drop 250 mg of substrate 3-oxo-3-phenylpropionamide and 118 mg of isopropylamine, and adjust the pH to 7.5 with sulfuric acid. Weigh 250 mg of wet bacteria of transaminase SEQ ID NO.60 and add them to the reaction solution, add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 30 ° C, the stirring speed to 400 rpm, and quench with an equal volume of pure acetonitrile after 24 hours. 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 determined that the preferred sequence SEQ ID NO.62 had a conversion rate of 89% and an ee value of 99.50%.

[0038] Example 7. Preparation of (S)-3-aminobutyramide Weigh 1.25g of pyridoxal phosphate in a 2L round-bottom flask, add 1.93L of 0.1M phosphate buffer and add 250g of acetoacetamide and 295g of isopropylamine respectively, and adjust the pH to 7.5. Weigh 25g of wet bacteria expressing the aminotransferase of SEQ ID NO.60 and add them to the reaction solution, and add a magnetic stirring rotor to the glass bottle, set the reaction temperature to 40°C, and the stirring speed to 400rpm. During the reaction, add hydrochloric acid dropwise to maintain the pH for 22 hours.

[0039] SEQ ID NO. 2 was used under the same conditions as above, and the conversion rate was 19%.

[0040] Example 8. Aminobutanamide resin purification The reaction liquid of the transaminase catalytic reaction of SEQ ID NO.60 in Example 7 was filtered with diatomaceous earth to remove the bacterial cells, and the reaction liquid was centrifuged at a speed of 8000 rpm for 30 minutes, and the supernatant was poured out, with a volume of about 1.5 L. Resin pre-activation: Weigh 4.5 kg of resin, prepare 13.5 L (5% to 10%) hydrochloric acid to soak the resin for 1 hour, and build a resin device; Weigh 4.5 kg of resin and soak it in 13.5 L saturated salt water for 18 to 20 hours, soak it in 5% HCl for 4 to 8 hours, drain the acid and rinse it with pure water to neutral, soak it in 2% to 4% NaOH for 4 to 8 hours, drain the alkali, rinse it to neutral, and build a resin device (Table 4). The resin was rinsed with pure water until neutral, and 2.5L of the centrifuged clear liquid was repeatedly rinsed until no (S)-3-aminobutyramide flowed out, and then rinsed with pure water until no acetyl acetamide flowed out, and the effluent was collected with 3 / 1 ammonia water, and samples without substrate residue were selected and combined, and the ammonia water was concentrated at 30-45°C, and the pH value was adjusted to 7 with dilute hydrochloric acid, and the sample was repeatedly loaded on the resin until no 2(S)-3-aminobutyramide flowed out, and the ammonia water was rinsed with pure water until no impurities flowed out, and the resin was rinsed with 3 / 1 ammonia water, and the product was received, and the ammonia water was concentrated under reduced pressure at 30-45°C to obtain a light yellow oily liquid with a total yield of 65%-85% and a chirality of 99.97%. Among them, 204.28g of the product was obtained by purification with HZ-3B resin, and the purity was ≥96%. The NMR spectrum of (S)-3-aminobutyramide is shown as follows Figure 4 shown.

[0041] Table 4 Resin Model Total yield HZ-3B 85％ HZ-12B 82％ HZ016 77％ Amberlite IR-120 75％ HZD-2 72％ D185 70％ HD-8 65％ Example 9. Preparation of (S)-3-aminobutyronitrile hydrochloride from (S)-3-aminobutyramide In a 2L clean and dry three-necked flask, add 204.28g (S)-3-aminobutyramide and 817.12g isobutyronitrile prepared in Example 8 at room temperature, stir and heat to 75°C, keep the internal temperature not lower than 75°C, slowly add 356.91g thionyl chloride dropwise for 3h, and after the addition is completed, heat to reflux and continue the reaction for 2h.

[0042] Set the oil bath temperature to 70°C, maintain the vacuum degree not less than -0.09MPa, and perform vacuum distillation and concentration. Stop distillation when solids are just precipitated, slowly cool down to crystallize, cool to 10-15°C, continue to grow crystals for 1h, filter, and rinse the filter cake with 102.14g of isobutyronitrile at 10-15°C. Dry under vacuum at 70°C to obtain 232.72g of (S)-3-aminobutyronitrile hydrochloride, yield: 96.5%, purity>99%, chiral purity greater than 99.5%. The specific (S)-3-aminobutyronitrile NMR is as follows Figure 6 shown.

[0043] The above reaction conversion rate and purity were derivatized with 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 30°C, wavelength 230 nm, and 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. The derivatization chromatogram of the synthetic components of (S)-3-aminobutyronitrile and the chiral derivatization chromatogram are shown in the figure. Figure 7 As shown, Figure 7 The retention times of S-3-aminobutyramide, S-3-aminobutyric acid, S-3-aminobutyronitrile, the hydrolyzate of the derivatizing agent, benzoic acid, S-3-aminobutyric acid methyl ester, and the derivatizing agent benzoic anhydride under this analytical method are marked in sequence in the chromatogram. Figure 7 The horizontal axis of the peak graph is the time (min) axis.

[0044] The chirality of the above product was derivatized by chiral derivatization agent NA-(2,4-dinitro-5-fluorophenyl)-L-valinamide for chiral derivatization detection. The chromatographic column was Agilent ZORBAX SB-C184.6*150mm*5μm, the mobile phase was pure acetonitrile and aqueous solution containing one thousandth of 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 10 μL, the retention time of (S)-3-aminobutyronitrile was 12.9min, and the retention time of (R)-3-aminobutyronitrile was 16.8min.

Claims

1. A method for synthesizing (S)-3-aminobutyronitrile, characterized in that: The synthesis method specifically comprises the following steps: (1) Addition of diketene and ammonia to prepare acetoacetamide; (2) The acetoacetamide obtained in step (1) is subjected to an enzyme-catalyzed reaction in the presence of a transaminase, and after the reaction is complete, (S)-3-aminobutyramide is purified; (3) The (S)-3-aminobutyronitrile is prepared by dehydrating the (S)-3-aminobutyramide obtained in step (2).

2. The synthesis method according to claim 1, characterized in that The specific reaction process of step (1) is as follows: With diketene and ammonia as substrate raw materials and ether compounds as reaction solvents, an addition reaction is carried out through a microchannel reactor to obtain the product acetoacetamide; the equivalent ratio of diketene and ammonia in the microchannel reactor is 1:1.0-3.0, the mass equivalent ratio of diketene and ether solvent is 1:0.5-10, the ether solvent is isopropyl ether or methyl tert-butyl ether, the temperature of the microchannel addition reaction is 30-60°C, and the residence time of each substance molecule participating in the reaction in the microchannel reactor is 10s-120s; Preferably, the equivalent ratio of diketene to ammonia in the microchannel reactor is 1:1.1-1.5; Preferably, the temperature of the microchannel addition reaction is 40-50°C; Preferably, the residence time of each substance molecule participating in the reaction in the microchannel reactor is 20-35s; Preferably, the ether solvent is methyl tert-butyl ether, and the mass equivalent ratio of diketene to methyl tert-butyl ether is 1:1-2.

3. The synthesis method according to claim 1, characterized in that The specific process of step (2) is as follows: acetoacetamide and isopropylamine are used as raw materials, and (S)-3-aminobutyramide is obtained by purification after the aminotransferase aqueous phase reaction is completed; the concentration of acetoacetamide is 50g / L-130g / L, the aminotransferase concentration is 10g / L, the initial concentration of isopropylamine added during the reaction is 1.5-2M, preferably 2M; the reaction temperature is 30-45°C; Preferably, the concentration of acetoacetamide is 130 g / L; Preferably, the initial concentration of isopropylamine added during the reaction is 2M; Preferably, the reaction temperature is 45°C.

4. The synthesis method according to claim 3, characterized in that The purification of the transaminase after the aqueous phase reaction is completed is performed by resin, and the resin used includes HZ-3B, HZ-12B, HZ016, Amberlite IR-120, HZD-2, D185 or HD-8; Preferably, the resin is HZ-3B.

5. The synthesis method according to claim 1, characterized in that The aminotransferase in step (2) can be selected from the following amino acid sequences (i), (ii) or (iii): (i) the transaminase has an amino acid sequence as shown in SEQ ID NO.2; (ii) the conserved amino acid sites in the enzyme catalytic center of the amino acid sequence of the transaminase have a sequence identity of more than 65% with all the amino acid sequences described in (i); (iii) the amino acid sequence of the transaminase is obtained by single mutation or multiple mutation at positions 58 and 154 of the amino acid sequence described in i) and ii); Preferably, the enzyme catalytic center of the transaminase has a conserved amino acid site that has a sequence identity of more than 75% with all the amino acid sequences described in (i); Preferably, the enzyme catalytic center of the transaminase has a conserved amino acid site that has more than 85% sequence identity with all the amino acid sequences described in (i); More preferably, the transaminase has an amino acid sequence as shown in SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58; More preferably, the transaminase has the amino acid sequence of a transaminase mutant as shown in SEQ ID NO.60, 62, or 64.

6. The synthesis method according to claim 1, characterized in that The specific process of step (3) is as follows: (S)-3-aminobutyramide is used as a substrate raw material, a solvent is added, thionyl chloride is added dropwise as a reaction dehydration reagent, and the product (S)-3-aminobutyronitrile is prepared by purification; the equivalent ratio of (S)-3-aminobutyramide to thionyl chloride is 1:1.0-4.0, the solvent is isobutyronitrile, acetonitrile or tetrahydrofuran, the thionyl chloride is added dropwise for 1-5 hours, and the thionyl chloride is added dropwise at a temperature of 60-80°C; Preferably, the equivalent ratio of (S)-3-aminobutanamide and thionyl chloride is 1:1.2-2.0; Preferably, the solvent is isobutyronitrile; Preferably, the dripping time of thionyl chloride is 2 to 3 hours; Preferably, the temperature for adding thionyl chloride dropwise is 70-75°C.

7. The synthesis method according to claim 1, characterized in that The method for purifying and preparing the product (S)-3-aminobutyronitrile is: the product (S)-3-aminobutyronitrile is obtained by concentration, cooling and crystallization, filtering, washing and drying. After the mother liquor is filtered and distilled, the solvent can be continuously applied to the next batch of reactions.

8. A method for preparing enantiomerically enriched (S)-3-aminobutyramide of formula (1) or its salt by enzyme catalysis, characterized in that: The (S)-3-aminobutyramide or its salt can be prepared by subjecting a ketone compound to a transaminase-catalyzed reaction, wherein the amino acid sequence of the transaminase is selected from one of the amino acid sequences of the transaminase described in claim 5; in formula (1), R represents one or more substituents on an aliphatic chain, and the R group is selected from hydrogen, alkyl such as methyl, an optionally substituted cyclic or non-cyclic aliphatic group having 1 to 10 carbon atoms, an optionally substituted aliphatic heterocyclic group having 1 to 10 carbon atoms, an optionally substituted (hetero) aromatic group, an optionally substituted alkoxy group having 1 to 10 carbon atoms, a halogen atom, an amine group, a nitro group or a trifluoromethyl group; （1）， The ketone compound is prepared by amidation of the corresponding methyl ester or diketene.

9. The method of (S)-3-aminobutanamide or a salt thereof according to claim 8, characterized in that The transaminase catalyzed reaction is carried out in a 50 mM phosphate buffer system with a pH of 7-9, a temperature range of 25-50° C., a substrate loading range of 20-150 g / L, and an amine donor of 1-2 molar equivalents.

10. The method of (S)-3-aminobutanamide or a salt thereof according to claim 9, characterized in that After the transaminase catalyzed reaction is completed, the final product can be purified by resin, and the resin is Amberlite IR-120, HZ-3B, HZ-12B, HZ016, HZD-2, D185 or HD-8.

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