Preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester

Abstract

The invention provides a preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester. The preparation method comprises the following steps: (1) fermenting genetically engineered bacteria to prepare a fermentation product (S)-2-amino-4-cyanobutyric acid; (2) carrying out decarboxylation cyanidation on the fermentation product to obtain (S)-N-Boc-3-amino glutaronitrile; (3) catalyzing the (S)-N-Boc-3-amino glutarinitrile by adopting enzyme-light synergistic DKR (Dipotassium Ketone Receptor), so as to obtain a chiral diol intermediate; and (4) cyclizing and synthesizing a product by a one-pot method: dissolving the chiral diol intermediate in tetrahydrofuran, adding boron trifluoride diethyl etherate and triisopropyl silane, reacting for 2 hours, cooling to 0 DEG C, adding potassium tert-butoxide, and reacting for 1 hour to obtain the product. According to the preparation method disclosed by the invention, high-yield and low-cost preparation of the (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester is realized.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and in particular to a method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate. Background Art

[0002] Tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate (CAS 2212021-56-0) is an important pharmaceutical intermediate, available as a white or off-white solid powder. It is used in the synthesis of bioactive molecules such as antibiotics and anti-inflammatory drugs, and is particularly widely used in the design of drugs targeting central nervous system receptors or enzymes. It is also a key intermediate for GLP-1 receptor agonists and is used in the synthesis of diabetes treatments such as oglerone.

[0003] In the prior art, (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester is synthesized by the following methods: (1) using (S)-3-aminobutyronitrile hydrochloride as a starting material, condensing with tert-butyl cyanoacetate at 80°C for 24 hours, and then cyclizing with potassium tert-butoxide at 0-5°C to obtain the product; (2) using (S)-3-aminobutyronitrile hydrochloride as a starting material, condensing with ethyl cyanoacetate at 80°C for 24 hours, hydrolyzing with aqueous NaOH solution, Boc protection, and finally cyclizing with potassium tert-butoxide at 60°C to obtain the product; (3) using chemoenzymatic dynamic kinetic resolution (DKR) technology, using immobilized lipase CAL-B to catalyze an asymmetric reaction to obtain the product. The above synthetic methods all rely on specific chiral amine raw materials and have the problems of expensive raw materials, high energy consumption, complex operation, and low yield (45-52%). Summary of the Invention

[0004] The present application provides a method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate to solve the above-mentioned problems mentioned in the background technology.

[0005] The present application provides a method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, which comprises the following steps: (1) Fermentation of (S)-2-amino-4-cyanobutyric acid by genetically engineered bacteria: Ferment the recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 in a fermentation medium for 48-50 hours to obtain a fermentation broth, and extract the fermentation product (S)-2-amino-4-cyanobutyric acid from the fermentation broth; (2) Photocatalytic decarboxylation cyanation: (S)-2-amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, replaced with nitrogen three times, and [Ru(bpy)3]Cl2 and TMSCN were added. After stirring at 22-28°C for 12 hours under blue light irradiation, Boc2O was added and reacted at -5-5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile. (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and enzyme were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, and [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester were added. After secondary blue light irradiation, the reaction was continued at 30°C for 16-20 hours to obtain a chiral diol intermediate. (4) One-pot cyclization synthesis of the product: The chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester.

[0006] Optionally, the fermentation medium includes 50 g / L D-glucose, 10 g / L ammonium sulfate, 1 g / L MgSO4·7H2O and 0.1 g / L CaCl2.

[0007] Optionally, during the fermentation of recombinant E. coli EC-AMN-001, when the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 25-30°C, pH 6.8-7.2, and dissolved oxygen 28-35%.

[0008] Optionally, extracting the fermentation product (S)-2-amino-4-cyanobutyric acid in step (1) specifically includes: The fermentation broth was centrifuged, and the supernatant was collected. The pH value of the supernatant was adjusted to 3.0 with acid, and crystallized. The crystals were filtered to obtain crystals. The crystals were washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid.

[0009] Optionally, the centrifugal treatment conditions are a rotation speed of 8000-9000 rpm and a time of 15-20 min.

[0010] Optionally, in the photocatalytic decarboxylation cyanation step, the molar ratio of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O and TMSCN is 1:1:1.1:2; The weight-to-volume ratio of the sum of the masses of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O, and TMSCN to the acetonitrile-water mixed solvent is 240-245 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:1.

[0011] Optionally, in the enzyme-photosynergistic DKR catalysis step, the molar ratio of [Ir(ppy)2(dtbbpy)]PF6, (S)-N-Boc-3-aminoglutaronitrile, and Hantzsch ester added is 1:1:1; The weight ratio of the added enzyme to (S)-N-Boc-3-aminoglutaronitrile was 25:1; The weight-to-volume ratio of the sum of the masses of (S)-N-Boc-3-aminoglutaronitrile, enzyme, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester to methyl tert-butyl ether is 105-120 g / 500 mL.

[0012] Optionally, the enzyme is selected from marine fungus lipase MF-01.

[0013] Optionally, in the one-pot cyclization synthesis step, the weight-to-volume ratios of the chiral diol intermediate to tetrahydrofuran, boron trifluoride etherate, and triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively.

[0014] Optionally, it is characterized in that the conditions for the first blue light irradiation are 450 nm and 30W, and the conditions for the second blue light irradiation are 455 nm and 20W.

[0015] The preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester provided in this application achieves high yield and low cost preparation of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester, and has the following beneficial effects compared to the prior art: (1) By using (S)-2-amino-4-cyanobutyric acid, a fermentation product of recombinant Escherichia coli EC-AMN-001, as the starting material for the synthesis product, the cost of the raw materials used is greatly reduced compared to the existing technology. At the same time, by combining genetically engineered bacterial fermentation with photocatalytic technology, the maximum temperature used in the synthesis process is 60°C, achieving mild conditions for the synthesis of the product. At the same time, the total yield is increased to above 68.6%.

[0016] (2) The preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester provided in this application complies with the principles of green chemistry, and the E factor is reduced to 4.2 (existing process>25), which enables enterprises to have better profits.

[0017] (3) This application solves the core problems of the prior art, namely, high raw material cost, high preparation temperature, difficulty in low-temperature operation and low yield, and has significant industrialization prospects and market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 This is a process flow chart of the preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester provided in one embodiment of the present application. DETAILED DESCRIPTION

[0020] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts also fall within the scope of protection of this application.

[0021] like Figure 1 As shown, the present application provides a method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, which comprises the following steps: (1) Fermentation of (S)-2-amino-4-cyanobutyric acid by genetically engineered bacteria: Ferment the recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 in a fermentation medium for 48-50 hours to obtain a fermentation broth, and extract the fermentation product (S)-2-amino-4-cyanobutyric acid from the fermentation broth; The recombinant Escherichia coli EC-AMN-001 was deposited in the China General Microbiological Culture Collection with the accession number CGMCC No. 28765.

[0022] (2) Photocatalytic decarboxylation cyanation: (S)-2-amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, and the atmosphere was replaced with nitrogen three times. [Ru(bpy)3]Cl2 (ruthenium(II) terpyridine complex) and TMSCN (trimethylsilyl cyanide) were added. After stirring at 22-28°C for 12 hours under blue light irradiation, Boc2O (di-tert-butyl dicarbonate) was added and the mixture was reacted at -5-5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile. (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and enzyme were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, and [Ir(ppy)2(dtbbpy)]PF6 (CAS: 676525-77-2) and Hantzsch ester (CAS: 1149-23-1) were added. The reaction was irradiated with blue light for a second time and reacted at 30°C for 16-20 hours to obtain a chiral diol intermediate. (4) One-pot cyclization synthesis of the product: The chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester.

[0023] Specifically, by fermenting recombinant Escherichia coli EC-AMN-001, (S)-2-amino-4-cyanobutyric acid in the fermentation product is extracted as the starting material for the synthesis of the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester, and the yield of the fermentation product (S)-2-amino-4-cyanobutyric acid is 80-86%.

[0024] (S)-2-amino-4-cyanobutyric acid was then decarboxylated and cyanated under a single blue light irradiation. During the photocatalytic decarboxylation and cyanation, Boc2O (di-tert-butyl dicarbonate) was added to introduce a tert-butyloxycarbonyl protecting group for amino acid protection, thereby obtaining (S)-N-Boc-3-aminoglutaronitrile.

[0025] After the photocatalytic decarboxylation cyanation reaction, the reactants were concentrated under reduced pressure and then purified again via column chromatography (petroleum ether / ethyl acetate = 3:1 (volume ratio)) to obtain a white solid, (S)-N-Boc-3-aminoglutaronitrile. The yield of (S)-N-Boc-3-aminoglutaronitrile was 90-92%, and the purity was >99%.

[0026] By synergistically catalyzing (S)-N-Boc-3-aminoglutaronitrile under enzyme and secondary blue light, a chiral diol intermediate is obtained, and [Ir(ppy)2(dtbbpy)]PF6 is used as a photocatalyst to promote the efficient reaction.

[0027] After the enzyme-photosynergistic DKR reaction is completed, the reaction solution is filtered through diatomaceous earth to collect the solid phase to recover the enzyme. The filtrate is then concentrated to obtain an oily product, which is the chiral diol intermediate. The yield of the chiral diol intermediate is 90-92%, and the ee value (enantiomeric excess) is >99.5%.

[0028] Finally, the chiral diol intermediate is dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane are added. The reaction is monitored by TLC (thin layer chromatography). Potassium tert-butoxide is then added and the reaction is continued with stirring to obtain a reaction solution. The reaction solution is purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester in a yield of 89-92%. The total yield of the final product, (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester, is 68.6-75%.

[0029] Through the above-mentioned scheme, this application achieves high-yield, low-cost preparation of tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate. By using (S)-2-amino-4-cyanobutyric acid, a fermentation product of recombinant Escherichia coli EC-AMN-001, as the starting material for the synthesis product, the cost of the raw materials used is significantly reduced compared to existing technologies. Furthermore, by combining genetically engineered bacterial fermentation with photocatalytic technology, the maximum temperature used during the synthesis process is 60°C, achieving mild conditions for the product synthesis. The overall yield is also increased to over 68.6%. Furthermore, in line with the principles of green chemistry, the E-factor is reduced to 4.2 (compared to >25 in existing processes), resulting in higher returns for the company.

[0030] Optionally, the fermentation medium includes 50 g / L D-glucose, 10 g / L ammonium sulfate, 1 g / L MgSO4·7H2O and 0.1 g / L CaCl2.

[0031] Specifically, during the fermentation process of recombinant Escherichia coli EC-AMN-001, D-glucose was used as the carbon source, ammonium sulfate was used as the nitrogen source, and MgSO4·7H2O and CaCl2 provided trace elements, which were beneficial to the fermentation of recombinant Escherichia coli EC-AMN-001.

[0032] At the same time, the concentration and ratio of carbon and nitrogen sources in the culture medium should be controlled. Excessive nitrogen sources will lead to excessive bacterial growth, resulting in a high specific growth rate and a negative impact on the accumulation of metabolites. Insufficient nitrogen sources will result in low bacterial reproduction, and nitrogen starvation will affect metabolic flux and product yield. Trace elements in the culture medium have a certain promoting effect on bacterial growth, but excessive addition can inhibit bacterial growth or affect enzyme activity and metabolic flux.

[0033] Optionally, during the fermentation of recombinant E. coli EC-AMN-001, when the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 25-30°C, pH 6.8-7.2, and dissolved oxygen 28-35%.

[0034] Specifically, when the OD 600=15, 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was used for induction. IPTG efficiently induces the expression of exogenous proteins, and the expressed protein product is stable, resulting in increased expression and product stability, preventing the accumulation of metabolic byproducts that interfere with protein synthesis, while ensuring sufficient bacterial biomass. Premature induction can lead to restricted bacterial growth, while late induction may reduce target product yield due to decreased cell viability. Simultaneously controlling the temperature, pH, and dissolved oxygen during fermentation facilitates stable fermentation of recombinant E. coli EC-AMN-001 and promotes product stability.

[0035] Optionally, extracting the fermentation product (S)-2-amino-4-cyanobutyric acid in step (1) specifically includes: The fermentation broth was centrifuged, and the supernatant was collected. The pH value of the supernatant was adjusted to 3.0 with acid, and crystallized. The crystals were filtered to obtain crystals. The crystals were washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid.

[0036] Specifically, the supernatant is adjusted to a pH of 3.0 using acetic acid or hydrochloric acid at a concentration of 0.1-0.5 mol / L, allowing (S)-2-amino-4-cyanobutyric acid in the supernatant to crystallize at this pH. After filtration, the crystals are washed at least three times with water and then vacuum-dried to constant weight. The resulting white crystals are the fermentation product, (S)-2-amino-4-cyanobutyric acid. The yield of (S)-2-amino-4-cyanobutyric acid is 80-86% (based on D-glucose), with a purity of >99.5% and an ee (enantiomeric excess) of >99.9%.

[0037] The vacuum drying conditions are 50-60°C and -0.07 to -0.15 MPa.

[0038] Optionally, the centrifugal treatment conditions are a rotation speed of 8000-9000 rpm and a time of 15-20 min.

[0039] Optionally, in the photocatalytic decarboxylation cyanation step, the molar ratio of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O and TMSCN is 1:1:1.1:2; The weight-to-volume ratio of the sum of the masses of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O, and TMSCN to the acetonitrile-water mixed solvent is 240-245 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:1.

[0040] Specifically, optionally, in the enzyme-light synergistic DKR catalysis step, the molar ratio of the added amounts of [Ir(ppy)2(dtbbpy)]PF6, (S)-N-Boc-3-aminoglutaronitrile, and Hantzsch ester is 1:1:1; The weight ratio of the added enzyme to (S)-N-Boc-3-aminoglutaronitrile was 25:1; The weight-to-volume ratio of the sum of the masses of (S)-N-Boc-3-aminoglutaronitrile, enzyme, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester to methyl tert-butyl ether is 105-120 g / 500 mL.

[0041] Optionally, the enzyme is selected from marine fungus lipase MF-01.

[0042] Alternatively, in the one-pot cyclization step, the weight-to-volume ratios of the chiral diol intermediate to tetrahydrofuran, boron trifluoride etherate, and triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively. Triisopropylsilane serves as a protective agent, and boron trifluoride etherate serves as a catalyst.

[0043] Optionally, the conditions for the first blue light irradiation are 450 nm and 30 W, and the conditions for the second blue light irradiation are 455 nm and 20 W.

[0044] The technical solution of this application is described in detail below with reference to specific embodiments.

[0045] Example 1 A method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, comprising the following steps: (1) Preparation of (S)-2-amino-4-cyanobutyric acid by fermentation using genetically engineered bacteria: Recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 was fermented in a fermentation medium for 48 h to obtain a fermentation broth. The fermentation broth was centrifuged and the supernatant was collected. The pH value of the supernatant was adjusted to 3.0 with acid, and crystallized. The crystals were filtered to obtain crystals, which were washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid. The yield of the fermentation product (S)-2-amino-4-cyanobutyric acid was 80%.

[0046] When the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 25°C, pH 6.8, and dissolved oxygen 28%.

[0047] The fermentation medium included 50 g / L D-glucose, 10 g / L ammonium sulfate, 1 g / L MgSO4·7H2O and 0.1 g / L CaCl2.

[0048] (2) Photocatalytic decarboxylation cyanation: (S)-2-amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, and the atmosphere was replaced with nitrogen three times. [Ru(bpy)3]Cl2 and TMSCN were added. After stirring at 22°C for 12 hours under blue light irradiation, Boc2O was added and the reaction was carried out at -5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile. The yield of (S)-N-Boc-3-aminoglutaronitrile was 90%. The molar ratio of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O and TMSCN added was 1:1:1.1:2; The weight-to-volume ratio of the sum of the masses of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O, and TMSCN to the acetonitrile-water mixed solvent is 240 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:1.

[0049] (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and marine fungal lipase MF-01 were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, and [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester were added. The mixture was irradiated with blue light for a second time and reacted at 30°C for 16 hours to obtain a chiral diol intermediate. The yield of the chiral diol intermediate was 90%.

[0050] The molar ratio of [Ir(ppy)2(dtbbpy)]PF6, (S)-N-Boc-3-aminoglutaronitrile, and Hantzsch ester added was 1:1:1; The weight ratio of the added amount of marine fungal lipase MF-01 to (S)-N-Boc-3-aminoglutaronitrile was 25:1; The weight-to-volume ratio of the sum of the masses of (S)-N-Boc-3-aminoglutaronitrile, marine fungal lipase MF-01, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester to methyl tert-butyl ether is 105 g / 500 mL.

[0051] (4) One-pot cyclization synthesis of the product: The chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester in a yield of 89%. The total yield of the final product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester was 68.6%.

[0052] The weight-to-volume ratios of the chiral diol intermediate to tetrahydrofuran, boron trifluoride etherate, and triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively.

[0053] Example 2 A method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, comprising the following steps: (1) Preparation of (S)-2-amino-4-cyanobutyric acid by fermentation using genetically engineered bacteria: Recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 was fermented in a fermentation medium for 49 h to obtain a fermentation broth. The fermentation broth was centrifuged and the supernatant was collected. The pH value of the supernatant was adjusted to 3.0 with acid, and crystallized. The crystals were filtered to obtain crystals, which were washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid. The yield of the fermentation product (S)-2-amino-4-cyanobutyric acid was 83%.

[0054] When the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 25°C, pH 7.0, and dissolved oxygen 28%.

[0055] The fermentation medium included 50 g / L D-glucose, 10 g / L ammonium sulfate, 1 g / L MgSO4·7H2O and 0.1 g / L CaCl2.

[0056] (2) Photocatalytic decarboxylation cyanation: (S)-2-amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, replaced with nitrogen three times, and [Ru(bpy)3]Cl2 and TMSCN were added. After stirring at 22-28°C for 12 hours under blue light irradiation, Boc2O was added and the reaction was carried out at -5-5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile; the yield of (S)-N-Boc-3-aminoglutaronitrile was 90-92%. The molar ratio of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O and TMSCN added was 1:1:1.1:2; The weight-to-volume ratio of the sum of the masses of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O, and TMSCN to the acetonitrile-water mixed solvent is 242 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:1.

[0057] (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and marine fungal lipase MF-01 were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, and [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester were added. The mixture was irradiated with blue light for a second time and reacted at 30°C for 16-20 hours to obtain a chiral diol intermediate. The yield of the chiral diol intermediate was 91.5%.

[0058] The molar ratio of [Ir(ppy)2(dtbbpy)]PF6, (S)-N-Boc-3-aminoglutaronitrile, and Hantzsch ester added was 1:1:1; The weight ratio of the added amount of marine fungal lipase MF-01 to (S)-N-Boc-3-aminoglutaronitrile was 25:1; The weight-to-volume ratio of the sum of the masses of (S)-N-Boc-3-aminoglutaronitrile, marine fungal lipase MF-01, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester to methyl tert-butyl ether is 115 g / 500 mL.

[0059] (4) One-pot cyclization synthesis of the product: The chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester in a yield of 92%. The total yield of the final product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester was 75%.

[0060] The weight-to-volume ratios of the chiral diol intermediate to tetrahydrofuran, boron trifluoride etherate, and triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively.

[0061] Example 3 A method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, comprising the following steps: (1) Preparation of (S)-2-amino-4-cyanobutyric acid by fermentation using genetically engineered bacteria: Recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 was fermented in a fermentation medium for 50 h to obtain a fermentation broth. The fermentation broth was centrifuged and the supernatant was collected. The pH value of the supernatant was adjusted to 3.0 with acid, and crystallized. The crystals were filtered to obtain crystals, which were washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid. The yield of the fermentation product (S)-2-amino-4-cyanobutyric acid was 86%.

[0062] When the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 30°C, pH 7.2, and dissolved oxygen 35%.

[0063] The fermentation medium included 50 g / L D-glucose, 10 g / L ammonium sulfate, 1 g / L MgSO4·7H2O and 0.1 g / L CaCl2.

[0064] (2) Photocatalytic decarboxylation cyanation: (S)-2-Amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, replaced with nitrogen three times, and [Ru(bpy)3]Cl2 and TMSCN were added. After stirring at 28°C for 12 hours under blue light irradiation, Boc2O was added and the reaction was carried out at 5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile; the yield of (S)-N-Boc-3-aminoglutaronitrile was 92%.

[0065] The molar ratio of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O and TMSCN added was 1:1:1.1:2; The weight-to-volume ratio of the sum of the masses of [Ru(bpy)3]Cl2, (S)-2-amino-4-cyanobutyric acid, Boc2O, and TMSCN to the acetonitrile-water mixed solvent is 245 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:1.

[0066] (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and marine fungal lipase MF-01 were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, and [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester were added. The mixture was irradiated with blue light for a second time and reacted at 30°C for 16-20 hours to obtain a chiral diol intermediate. The yield of the chiral diol intermediate was 90.5%.

[0067] The molar ratio of [Ir(ppy)2(dtbbpy)]PF6, (S)-N-Boc-3-aminoglutaronitrile, and Hantzsch ester added was 1:1:1; The weight ratio of the added amount of marine fungal lipase MF-01 to (S)-N-Boc-3-aminoglutaronitrile was 25:1; The weight-to-volume ratio of the sum of the masses of (S)-N-Boc-3-aminoglutaronitrile, marine fungal lipase MF-01, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester to methyl tert-butyl ether is 120 g / 500 mL.

[0068] (4) One-pot cyclization synthesis of the product: The chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester in a yield of 90%. The total yield of the final product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester was 69.8%.

[0069] The weight-to-volume ratios of the chiral diol intermediate to tetrahydrofuran, boron trifluoride etherate, and triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively.

[0070] Through the methods provided in the above examples, the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester was synthesized under mild conditions. At the same time, the yield of the product (greater than or equal to 69.8%) was significantly improved compared to the prior art (45-52%), and the cost of the raw materials used was greatly reduced compared to the prior art ((S)-3-aminobutyronitrile hydrochloride).

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate, characterized in that: The steps include: (1) Fermentation of (S)-2-amino-4-cyanobutyric acid by genetically engineered bacteria: Ferment the recombinant Escherichia coli EC-AMN-001 with a preservation number of CGMCC No. 28765 in a fermentation medium for 48-50 hours to obtain a fermentation broth, and extract the fermentation product (S)-2-amino-4-cyanobutyric acid from the fermentation broth; (2) Photocatalytic decarboxylation cyanation: (S)-2-amino-4-cyanobutyric acid was dissolved in an acetonitrile-water mixed solvent, and the atmosphere was replaced with nitrogen three times. [Ru(bpy)3]Cl2 and TMSCN were added. The mixture was stirred at 22-28°C for 12 hours under blue light irradiation. Then, Boc2O was added and the mixture was reacted at -5-5°C for 2 hours to obtain (S)-N-Boc-3-aminoglutaronitrile. (3) Enzyme-photosynergistic DKR catalysis: (S)-N-Boc-3-aminoglutaronitrile and enzyme were added to methyl tert-butyl ether, stirred at 30°C for 30 minutes, [Ir(ppy)2(dtbbpy)]PF6 and Hantzsch ester were added, and blue light was irradiated for a second time. The reaction was carried out at 30°C for 16-20 hours to obtain a chiral diol intermediate; (4) One-pot cyclization synthesis of the product: the chiral diol intermediate was dissolved in tetrahydrofuran, and boron trifluoride etherate and triisopropylsilane were added. The mixture was stirred at 60°C for 2 h, cooled to 0°C, and potassium tert-butoxide was added. The mixture was stirred at 25°C for 1 h to obtain a reaction solution. The reaction solution was purified to obtain the product (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester.

2. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 1, wherein The fermentation medium includes 50 g / L of D-glucose, 10 g / L of ammonium sulfate, 1 g / L of MgSO4·7H2O and 0.1 g / L of CaCl2.

3. The preparation method of (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylic acid tert-butyl ester according to claim 1, wherein During the fermentation of the recombinant Escherichia coli EC-AMN-001, when the OD 600 = 15, induced with 0.5 mM IPTG; The fermentation parameters were set as follows: temperature 25-30°C, pH 6.8-7.2, and dissolved oxygen 28-35%.

4. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 1, wherein Extracting the fermentation product (S)-2-amino-4-cyanobutyric acid in step (1) specifically includes: The fermentation broth is centrifuged, the supernatant is collected, the pH value of the supernatant is adjusted to 3.0 with acid, crystallized, filtered to obtain crystals, the crystals are washed with water and then vacuum-dried to obtain the fermentation product (S)-2-amino-4-cyanobutyric acid.

5. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 4, wherein The centrifugal treatment conditions are a rotation speed of 8000-9000 rpm and a time of 15-20 min.

6. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 1, wherein In the photocatalytic decarboxylation cyanation step, the molar ratio of the added amounts of [Ru(bpy)3]Cl2, the (S)-2-amino-4-cyanobutyric acid, the Boc2O and the TMSCN is 1:1:1.1:2; The weight-to-volume ratio of the sum of the mass of the [Ru(bpy)3]Cl2, the (S)-2-amino-4-cyanobutyric acid, the Boc2O, and the TMSCN to the acetonitrile-water mixed solvent is 240-245 g / 500 mL; The volume ratio of acetonitrile to water in the acetonitrile-water mixed solvent is 9:

1.

7. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 1, wherein In the enzyme-photosynergistic DKR catalysis step, the molar ratio of the added amounts of [Ir(ppy)2(dtbbpy)]PF6, the (S)-N-Boc-3-aminoglutaronitrile, and the Hantzsch ester is 1:1:1; The weight ratio of the added amount of the enzyme to the (S)-N-Boc-3-aminoglutaronitrile is 25:1; The weight-to-volume ratio of the sum of the masses of the (S)-N-Boc-3-aminoglutaronitrile, the enzyme, the [Ir(ppy)2(dtbbpy)]PF6 and the Hantzsch ester to the methyl tert-butyl ether is 105-120 g / 500 mL.

8. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 7, wherein The enzyme is selected from marine fungus lipase MF-01.

9. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to claim 1, wherein In the one-pot cyclization synthesis step, the weight-to-volume ratios of the chiral diol intermediate to the tetrahydrofuran, the boron trifluoride etherate, and the triisopropylsilane are 40 g / 500 mL, 40 g / 0.5 mL, and 40 g / 0.5 mL, respectively.

10. The method for preparing tert-butyl (2S)-3-cyano-2-methyl-4-oxopiperidine-1-carboxylate according to any one of claims 1 to 9, wherein: The conditions for the primary blue light irradiation are 450 nm and 30 W, and the conditions for the secondary blue light irradiation are 455 nm and 20 W.

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