Synthesis method of chiral amine

Abstract

The present invention provides a method for synthesizing chiral amines. The method for synthesizing chiral amines provided by the present invention comprises: subjecting a carbonyl substrate and an amine donor to a reductive amination reaction under the catalytic action of a catalyst to obtain a chiral amine, wherein the catalyst is an imine reductase or a reductive aminase. In the present invention, imine reductase or a reductive aminase is used as a catalyst for the synthesis of chiral amines, and a non-keto acid ketone compound substrate and an amino donor can be synthesized into a chiral amine under mild reaction conditions, which not only has a wide substrate range, but also is simple and economical to operate, and can directly synthesize a variety of chiral amine compounds, and can be suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of chiral amine synthesis, and in particular to a method for synthesizing a chiral amine. Background Art

[0002] Chiral amines are an important structural unit that are widely present in the structure of pharmaceutical intermediates. It is reported that approximately 40% of the structures of small molecule drug compounds approved by the FDA contain chiral amine modules.

[0003] At present, the synthesis methods of chiral amines mainly include chemical catalytic synthesis and enzyme catalytic synthesis. Chemical catalysis has caused great environmental pressure and has increasingly revealed its limitations due to the use of transition metal complexes, harsh conditions, over-alkylation and large amounts of waste generation. Enzymatic catalytic synthesis has the advantages of mild reaction conditions, high selectivity, and greenness and pollution-free, and has gradually been widely used in the synthesis of chiral amines.

[0004] In addition to the chiral resolution mediated by various hydrolases to prepare chiral amines, ω-transaminase, amino acid dehydrogenase, and crown alkaloid dehydrogenase can catalyze the reductive amination of carbonyl substrates with amine donors to obtain chiral amines. However, ω-transaminase can only complete the transfer of -NH2 with the help of pyridoxal phosphate, which limits its application range; crown alkaloid dehydrogenase is similar to amino acid dehydrogenase, and its substrate is limited to keto acid as the carbonyl substrate when catalyzing the formation of chiral amines.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The main purpose of the present invention is to provide a method for synthesizing chiral amines to solve the technical problems that in the prior art, ω-aminotransferase can only complete the transfer of -NH2 with the help of pyridoxal phosphate, and its application range is limited; and that opine dehydrogenase is similar to amino acid dehydrogenase, and its substrate is limited to keto acid as the carbonyl substrate when catalyzing the formation of chiral amines.

[0007] In order to achieve the above object, according to one aspect of the present invention, a method for synthesizing a chiral amine is provided, the method comprising: subjecting a carbonyl substrate and an amine donor to a reductive amination reaction under the catalysis of an imine reductase or a reductive aminase to obtain a chiral amine; wherein the carbonyl substrate is a ketone compound, and the schematic structural formula of the ketone compound is as follows: ) as shown:

[0008]

[0009] Among them, the formula ( ), R1 represents H, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted C5-C10 heteroarylene; R2 represents substituted or unsubstituted C1-C5 alkylene, or substituted or unsubstituted C1-C4 heteroalkyl; wherein the substituents in the substituted aryl, substituted arylene, substituted heteroarylene and substituted alkylene are each independently selected from halogen, methoxy, hydroxyl, amino, -S-CH3, and the heteroatoms in the heteroarylene and heteroalkyl are each independently selected from N, O or S;

[0010] The amine donor is an amino acid or an amino acid ester, and the schematic structural formula of the amine donor is as follows: ) as shown:

[0011]

[0012] Mode( ), R3 represents H, -CH3, -CH2OH, -CH2CH3, -S-CH3, -CH(OH)CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH2SCH3, -CH2COOH, -CH2CONH2, -CH2CH2COOH, -CH2CH2CONH2, -CH2CH2CH2CH2NH2, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 alkyl; R4 represents H, -CH3, -CH2CH3, tert-butyl, substituted or unsubstituted C6-C10 aryl or substituted or unsubstituted C5-C10 alkyl.

[0013] Further, the imine reductase or reductive aminase is derived from Streptomyces ipomoeae, NCBI reference number: WP_009330409.1, Madurella mycetomatis, NCBI reference number: KXX80955.1, Streptosporangium roseum, NCBI reference number: ACZ83929.1, Myxococcus stipitatus, NCBI reference number: AGC43099.1, Streptomyces sp. CNS615, NCBI reference number: WP_108908932.1, Kribbellaflavida DSM 17836, Variovorax paradoxus, NCBI Reference Number: KPU97715.1, NCBI Reference Number: WP_012921542.1, Cystobacter ferrugineus, NCBI Reference Number: WP_071905000.1, Rhizobium sullae, NCBI Reference Number: WP_342352985.1, Streptomyces sp. TOR3209, WP_019324525.1, Streptomyces, WP_003986432, Mesorhizobium sp. L2C084A000: WP_023809753.1 or mutants thereof.

[0014] Further, the imine reductase or reductive aminase is selected from Kribbella flavida DSM 17836, NCBI reference sequence number: WP_012921542.1, Variovorax paradoxus, NCBI reference sequence number: KPU97715.1, Cystobacter ferrugineus, NCBI reference sequence number: WP_071905000.1, Rhizobium sullae: NCBI reference sequence number: WP_342352985.1, Streptomyces sp. TOR3209, WP_019324525.1, Streptomyces, WP_003986432 or Mesorhizobium sp. L2C084A000: One of WP_023809753.1 or a mutant of the above enzyme.

[0015] Furthermore, the temperature of the reductive amination reaction is 20-40° C., and the time is 12-20 h.

[0016] Furthermore, the synthesis method of chiral amine comprises: reacting imine reductase or reductive amination enzyme, carbonyl substrate, amino donor, glucose dehydrogenase, glucose, coenzyme and coenzymes Adding it into a Tris-HCl solution to carry out the reductive amination reaction to obtain a chiral amine reaction solution, and purifying the chiral amine reaction solution to obtain a chiral amine.

[0017] Furthermore, the mass ratio of the imine reductase or reductive aminase to the carbonyl substrate is (8-15):100; and / or the molar ratio of the amine donor to the carbonyl substrate is (1-15):1; and / or the mass ratio of the glucose dehydrogenase to the carbonyl substrate is (10-100):100; and / or the molar ratio of glucose to the carbonyl substrate is (1-5):1.

[0018] Furthermore, coenzyme The molar ratio of the coenzyme to the carbonyl substrate is (5-50):100; and / or The molar ratio to the carbonyl substrate is (5-50):100.

[0019] Furthermore, the carbonyl substrate is selected from at least one of cyclohexanone, 5-methoxy-2-naphthalenone, β-tetralone, and 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone.

[0020] Furthermore, the amine donor is selected from at least one of L-methionine, L-leucine, L-norvaline, L-valine, L-alanine, glycine, L-threonine, L-aspartic acid, L-tyrosine, L-phenylalanine, L-norvaline methyl ester, and L-norvaline tert-butyl ester.

[0021] Further, the carbonyl substrate is 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone, the amine donor is L-norvaline tert-butyl ester, and the imine reductase or reductive aminase is selected from Mesorhizobium sp. L2C084A000: WP_023809753.1, Variovorax paradoxus: KPU97715.1, Streptomyces, WP_003986432, Cystobacter ferrugineus, NCBI reference sequence number: WP_071905000.1, KXX80955.1 or mutants of the above enzymes.

[0022] By applying the technical solution of the present application, in the present application, imine reductase or reductive aminase is used as a catalyst for the synthesis of chiral amines, and non-keto acid ketone compound substrates and amino donors can be used to synthesize chiral amines under mild reaction conditions. Not only is the substrate range wide, but the operation is simple and economical, and a variety of chiral amine compounds can be directly synthesized, which can be suitable for industrial production. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] As analyzed in the background technology of this application, chiral amine is an important structural unit. In the current method of synthesizing chiral amines by enzyme catalysis, ω-aminotransferase can only complete the transfer of -NH2 with the help of pyridoxal phosphate, and its application scope is limited; opine dehydrogenase is similar to amino acid dehydrogenase, and its substrate is limited to keto acid as carbonyl substrate when catalyzing the formation of chiral amines. In order to solve this problem, this application provides a method for synthesizing chiral amines.

[0025] In the first embodiment of the present application, a method for synthesizing a chiral amine is provided, the method comprising: subjecting a carbonyl substrate and an amine donor to a reductive amination reaction under the catalysis of an imine reductase or a reductive aminase to obtain a chiral amine; wherein the carbonyl substrate is a ketone compound, and the schematic structural formula of the ketone compound is as follows: ) as shown:

[0026]

[0027] Mode( ), R1 represents H, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 arylene, or substituted or unsubstituted C5-C10 heteroarylene; R2 represents substituted or unsubstituted C1-C5 alkylene, or substituted or unsubstituted C1-C4 heteroalkyl; wherein the substituents in the substituted aryl, substituted arylene, substituted heteroarylene and substituted alkylene are each independently selected from halogen, methoxy, hydroxyl, amino, -S-CH3, and the heteroatoms in the heteroarylene and heteroalkyl are each independently selected from N, O or S;

[0028] The amine donor is an amino acid or an amino acid ester, and the schematic structural formula of the amine donor is as follows: ) as shown:

[0029]

[0030] Mode( ), wherein R3 represents H, -CH3, -CH2OH, -CH2CH3, -S-CH3, -CH(OH)CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH2SCH3, -CH2COOH, -CH2CONH2, -CH2CH2COOH, -CH2CH2CONH2, -CH2CH2CH2CH2NH2, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 alkyl; R4 represents H, -CH3, -CH2CH3, tert-butyl, substituted or unsubstituted C6-C10 aryl, or substituted or unsubstituted C5~C10 alkyl.

[0031] In the present application, the term "C6-C10 aryl" refers to a 6-10 membered monocyclic, bicyclic or tricyclic aromatic ring system, including phenyl, biphenyl, etc. The term "C5-C10 heteroarylene" refers to a 5-10 membered monocyclic, bicyclic or tricyclic aromatic ring system having 1-4 heteroatoms, such as furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, etc. The heteroarylene group may include a monovalent or divalent heteroarylene group. The term "halogen" or "halo" refers to one or more of fluorine, chlorine, bromine and iodine, especially fluorine or chlorine; "C1-C5 alkylene" means an alkylene group having 1 to 5, especially up to 4 carbon atoms, which group is straight chain or branched with single or multiple branches, for example butylene, such as n-butylene, sec-butylene, isobutylene, tert-butylene; propylene, for example n-propylene or isopropylene; ethylene or methylene; more especially methylene, isopropylene or tert-butylene.

[0032] The present application provides a method for synthesizing chiral amines, wherein a carbonyl substrate and an amine donor undergo a reductive amination reaction under the action of an imine reductase or a reductive aminase, the reaction conditions are mild, and the substrate is a ketone compound other than a keto acid. The method is not only applicable to a wide range of substrates, but also simple and economical to operate, and can directly synthesize a variety of chiral amine compounds, and can be suitable for industrial production.

[0033] In some embodiments of the present application, the imine reductase or reductive aminase is derived from Streptomyces ipomoeae, NCBI reference sequence number: WP_009330409.1, Madurella mycetomatis, NCBI reference sequence number: KXX80955.1, Streptosporangium roseum, NCBI reference sequence number: ACZ83929.1, Myxococcus stipitatus, NCBI reference sequence number: AGC43099.1, Streptomyces sp. CNS615, NCBI reference sequence number: WP_108908932.1, Kribbella flavida DSM 17836, NCBI reference sequence number: WP_012921542.1, Variovorax paradoxus, NCBI reference number: KPU97715.1, Cystobacter ferrugineus, NCBI reference number: WP_071905000.1, Rhizobium sullae: NCBI reference number: WP_342352985.1, Streptomyces sp. TOR3209, WP_019324525.1, Streptomyces, WP_003986432, Mesorhizobium sp. L2C084A000, NCBI sequence number: WP_023809753.1 or mutants of the above enzymes.

[0034] In some embodiments, the imine reductase or reductive amination enzyme has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0035] Among them, the sequence of SEQ ID NO:1 is: MKLSITVIGTGRMGSALAGSLLQSGYPTTVWNRTRQKTDPLARLGAIAASSVEEAVNAGEIIIVNVSDSEATKALLHSDAIASAIRGKLIVELTSGTPSGAREAAEWCTKHGANYLDGAIMATPDYIGTDAGTILLAGPREAFDTNRDVFRALGGNVQHVGEEPGRANALDSALLAIMlGAPFGTLHAIAVSQAEEIELGELARQWSATAPVIDGLVTDLIKRTSAGRFASDLETLSSISAHHGAMQHLLELMQFRGIDRSIVDGYDAIFKRAIAAGHLHDDFAALSHFLATGK。

[0036] The sequence of SEQ ID NO:2 is: MSSKQKITVIGLGAMGATIARLYLEQGHEVTIWNRSADKAAPLVAQGAVLADSAAAAVRASRVVLMCVYDDRAADAILGAEGVAAAMDGRLLVQLTTGSPRDARDAQAWAQRHGATFLEGAIQAAPEQMGKGDTPILMSGDEQVFRAVEPLLAVLGGGIVYLGEKISNAAAMDPATLSTLYGTMLGFLHGARVAESEGFDVAEFGRIVAGIMPTAASFLQHEGAVIQSGDFKISQSPMLISVEATQRILQTARESGINSEFPAFAAGLFQRADAAGLGGEELAALIKLLRAPA。

[0037] The sequence of SEQ ID NO:3 is:

[0038] MKRSITVLGTGRMGSALARALLHAGHRTTVWNRTIQKAEPLAALGATVAPSVLEAVNAAEIIIVNVSDLQATAAIMRNEAIASAVRGKLIVELTSGTPHGAREAAEFWAEHGAIYLDGAIMATPDFIGTDAGTILVSGSSQAFDANEDMFRALGGNVQHIGEESGRANALDSALLALMWGALFGTLHAIAVCQAEEIDLGELAQQWNATAPVVEGLVADLIKRTNAGRFASDDETLSSLSAHYGAFQHLLELMEAREIDRSVVLGYDAIFQRAIAAGQLHEDFAALSQFLGKSA。

[0039] The sequence of SEQ ID NO:4 is:

[0040] MATITSIGIGNMGAALATALLKSSSPPMNVTIWNRTASRPQVQSLISAGAIFEPSLAAALASSEVILLCLLDYPAISSVFSQVDASAKPLAGKTILNLTNGTPKQARDMEAFFKSLGAAVYFDGAVMVTPQLVGTPAAFVVLSGETEQAYNERLANAGLLSPVGAVLYIAEDPGAASLVDCAALAAMYGMFIGAFTGIGLLKRQKHERDGEAAGAKAMVDKVMVPVLTALVPYVGLLALQVDKEAWMDDLGNPLAMQAEGVRNIMQSCEDEGVDGTGLKFLSKLMEKGVKEGFGPGGVAVVAKYLMK。

[0041] SEQ ID The sequence of NO:5 is: MTGGSPVTVVIGLGLMGQALAAAFLKGGHPTTVWNRSADKATDLVSSGAVLADTPDEAVSAADLIVVCVLDDDALHGVLDPVAEKLRGRVVANLTSGSSDQARANAAWSAEWGFTYLDGAIMTVPAGVGAPESVFFYAGPQKA YDRHAEALALLGGGTTYLGEDHALAPLIDVALLGMMWGTLNSFLQGAALLATADVKASEFLPWAIKWVDAVKMFATDYAGQIDAGDGAYPARDAPMDVHAGALKHLLHESESQGVNAELPRFVQGLMQRTIAAGFGQNSYASMIELFRAPEK.

[0042] In order to further reduce energy consumption, the temperature of the reductive amination reaction is preferably 20-40°C and the time is 12-20 hours. The reaction conditions are mild and the reaction can be carried out at room temperature, which greatly reduces the difficulty of operation and is more conducive to reducing costs.

[0043] Typically but not limiting, when imine reductase or reductive aminase is used as a catalyst for chiral amine synthesis, the temperature of the reductive amination reaction is 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C or a range consisting of any two values; the time is 12h, 15h, 18h, 20h or a range consisting of any two values.

[0044] In order to further improve the conversion rate of the substrate, in some preferred embodiments, the method for synthesizing the chiral amine comprises: reacting an imine reductase or a reductive aminase, a carbonyl substrate, an amine donor, glucose dehydrogenase (GDH), glucose (GLu), a coenzyme (NAD + ) and coenzymes (NADP + ) is added to a Tris-HCl solution, reacted at 20-40° C. for 12-20 h to obtain a chiral amine reaction solution, and the chiral amine reaction solution is purified to obtain a chiral amine.

[0045] In this embodiment, the carbonyl substrate, the amine donor, the catalyst, the coenzyme , coenzyme Under the action of glucose dehydrogenase and glucose, it is more conducive to improving the efficiency of the reductive amination reaction and further improving the conversion efficiency of chiral amines.

[0046] In addition, in order to facilitate the termination of the reaction, in the above embodiment, after the reductive amination reaction reaches a corresponding reaction time, a reaction terminator is added to terminate the reaction to obtain a chiral amine reaction solution, and the reaction terminator is preferably acetonitrile.

[0047] In order to further improve the efficiency of chiral amine synthesis, the concentration of the Tris-HCl solution is preferably 80-120 mM, and the pH is 6.0-9.5, such as the molar concentration is 80 mM, 90 mM, 100 mM, 105 mM, 110 mM, 120 mM or a range consisting of any two values, and the pH is 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.2, 8.5, 9.0, 9.5 or a range consisting of any two values.

[0048] In order to further improve the conversion rate of the carbonyl substrate as a substrate, the mass ratio of imine reductase or reductive amination enzyme to carbonyl substrate is preferably (8-15):100, such as 8:100, 10:100, 12:100, 15:100 or a range consisting of any two values.

[0049] In order to further improve the yield of chiral amine, preferably in some embodiments, the molar ratio of the amine donor to the carbonyl substrate is (1-15):1, such as 1:1, 2:1, 5:1, 8:1, 10:1, 12:1, 15:1 or a range consisting of any two values; and / or the mass ratio of glucose dehydrogenase to the carbonyl substrate is (10-100):100, such as 10:100, 20:100, 50:100, 80:100, 100:100 or a range consisting of any two values; and / or the molar ratio of glucose to the carbonyl substrate is (1-5):1, such as 1:1, 2:1, 3:1, 4:1, 5:1 or a range consisting of any two values; coenzyme The molar ratio of coenzyme to carbonyl substrate is (5-50):100, such as 5:100, 10:100, 20:100, 30:100, 40:100, 50:100 or a range of any two values; The molar ratio to the carbonyl substrate is (5~50):100, such as 5:100, 10:100, 20:100, 30:100, 40:100, 50:100 or a range value consisting of any two values.

[0050] The "substituted" in the above "substituted or unsubstituted" means that a hydrogen atom in a functional group is replaced by another atom or group (i.e., a substituent). Each of the substituents is independently selected from at least one of the following groups: deuterium, halogen, C1-C10 alkyl, C1-C10 alkoxy, C6-C10 aryl, C5-C10 heteroaryl, C5-C10 heteroaryl substituted by C6-C10 aryl, benzimidazolyl, C3-C10 cycloalkyl, C5-C7 heterocycloalkyl, carboxyl, nitro, and hydroxyl.

[0051] It should be noted that the amino acid serving as the amine group donor is a natural amino acid or a non-natural amino acid.

[0052] In some embodiments, the carbonyl substrate is selected from any one of cyclohexanone, 5-methoxy-2-naphthalenone, β-tetralone, and 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone, and the amine donor is selected from any one of L-methionine, L-leucine, L-norvaline, L-alanine, glycine, L-threonine, L-aspartic acid, L-tyrosine, L-phenylalanine, L-norvaline methyl ester, and L-norvaline tert-butyl ester.

[0053] In some specific embodiments, the carbonyl substrate is 6,8-dichloro-3,4-dihydro-1H-2-naphthone, the amine donor is L-norvaline, and the imine reductase or reductive aminase used as a catalyst is selected from one of WP_023809753.1, KPU97715.1, WP_071905000.1, KXX80955.1, WP_108908932.1 or mutants of the above enzymes.

[0054] The beneficial effects of the present application will be further illustrated below in combination with embodiments and comparative examples.

[0055] Example 1: Reductive amination reaction of cyclohexanone and L-methionine

[0056] This embodiment provides a method for synthesizing chiral amines using cyclohexanone as a carbonyl substrate and L-methionine as an amine donor, wherein the structural formula of cyclohexanone is The structural formula of L-methionine is .

[0057] The synthesis method comprises the following steps:

[0058] (1) In a 10 mL reaction bottle, add a catalyst in an amount of 10% of the mass of the carbonyl substrate, then add 5.48 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 3.92 mg (20 mmol) cyclohexanone, 59.6 mg (200 mmol) L-methionine, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0059] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12,000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0060] The sources of the specific catalyst are shown in Table 1 below, and the conversion rate of the substrate cyclohexanone is also shown in Table 1.

[0061] Table 1

[0062]

[0063] Example 2: Reductive amination reaction of cyclohexanone and L-isoleucine

[0064] This embodiment provides a method for synthesizing chiral amines using cyclohexanone as a carbonyl substrate, L-leucine as an amine donor, and imine reductase or reductive amination enzyme as a catalyst, wherein the structural formula of cyclo-L-leucine is .

[0065] The synthesis method comprises the following steps:

[0066] (1) In a 10 mL reaction bottle, add 10% of the mass of the carbonyl substrate as catalyst, then add 5.48 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 3.92 mg (20 mmol) cyclohexanone, 52 mg (200 mmol) L-isoleucine, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0067] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0068] The specific sources of imine reductase or reductive amination enzyme are shown in Table 1 below, and the conversion rate of substrate cyclohexanone is also shown in Table 2.

[0069] Table 2

[0070]

[0071] Example 3: Reductive amination reaction of cyclohexanone with various amino acids

[0072] This embodiment provides a synthesis method for preparing chiral amines using cyclohexanone as a carbonyl substrate (substrate), L-norvaline, L-valine, L-alanine, glycine, L-leucine, L-threonine, L-valine, and L-aspartic acid as amine donors, and imine reductase or reductive amination enzyme as a catalyst, wherein the specific structure of the amine donor, the source of the imine reductase or reductive amination enzyme, and the substrate conversion rate are shown in Table 3 below.

[0073] The synthesis method comprises the following steps:

[0074] (1) In a 10 mL reaction bottle, add a catalyst with a mass that is 10% of the carbonyl substrate, and then add 3.92 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 3.92 mg (20 mmol) cyclohexanone, 200 mmol amino acid as amine donor, 100 mM Tris-HCl pH 8.0 to 2 mL, shake reaction at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0075] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0076] Table 3

[0077]

[0078]

[0079] Note: * indicates 0<conversion rate ≤ 5%; ** indicates 5%<conversion rate ≤ 20%; *** indicates 20%<conversion rate ≤ 40%; *** indicates 40%<conversion rate ≤ 60%; **** indicates 60%<conversion rate ≤ 80%; ***** indicates 80%<conversion rate ≤ 100%.

[0080] Example 4: Reductive amination reaction of 5-methoxy-2-tetralone with L-tyrosine

[0081] This embodiment provides a method for preparing a sterically hindered 5-methoxy-2-tetralinone ( ) as the carbonyl substrate, imine reductase (reductive amination enzyme) as the catalyst, L-tyrosine ( ) is an amine donor for synthesizing chiral amines, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 4 below.

[0082] The synthesis method comprises the following steps:

[0083] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 7.04 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 7.04 mg (20 mmol) 5-methoxy-2-tetralone, 72 mg (200 mmol) L-tyrosine, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0084] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0085] Table 4

[0086]

[0087] Example 5: Reductive amination reaction of 5-methoxy-2-tetralone with L-phenylalanine

[0088] This embodiment provides a method for preparing a sterically hindered 5-methoxy-2-tetralinone ( ) as the carbonyl substrate, imine reductase (reductive amination enzyme) as the catalyst, L-phenylalanine ( ) is an amine donor for synthesizing chiral amines, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 5 below.

[0089] The synthesis method comprises the following steps:

[0090] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 7.04 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP +), 7.04 mg (20 mmol) 5-methoxy-2-tetralone, 66 mg (200 mmol) L-phenylalanine, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0091] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0092] Table 5

[0093]

[0094] Example 6: Reductive amination reaction of 5-methoxy-5-tetralone with L-norvaline methyl ester

[0095] This embodiment provides a method for preparing a sterically hindered 5-methoxy-2-tetralinone ( ) as the carbonyl substrate, imine reductase (reductive amination enzyme) as the catalyst, and L-norvaline methyl ester ( ) is an amine donor for synthesizing chiral amines, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 6 below.

[0096] The synthesis method comprises the following steps:

[0097] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 7.04 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 7.04 mg (20 mmol) 5-methoxy-2-tetralone, 52 mg (200 mmol) L-norvaline methyl ester, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0098] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0099] Table 6

[0100]

[0101] Example 7: Reductive amination reaction of 5-methoxy-5-tetralone with L-norvaline tert-butyl ester

[0102] This embodiment provides a method for preparing a novel tert-butyl alcohol containing 5-methoxy-2-tetralinone as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-norvaline tert-butyl ester ( ) is an amine donor for synthesizing chiral amines, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 7 below.

[0103] (1) In a 10 mL reaction bottle, add 10% of the catalyst as the carbonyl substrate, then add 7.04 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 7.04 mg (20 mmol) 5-methoxy-2-naphthalene ketone, 69.2 mg (200 mmol) L-norvaline tert-butyl ester, 100 mM Tris-HCl pH 8.0 to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0104] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0105] Table 7

[0106]

[0107] Example 8: Reductive amination reaction of β-tetralone with L-norvaline methyl ester

[0108] This embodiment provides a method for preparing a sterically hindered β-tetralone ( ) as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-norvaline methyl ester as an amine donor to synthesize chiral amines, wherein the source of the imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 8 below.

[0109] The synthesis method comprises the following steps:

[0110] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 5.48 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 5.84 mg (20 mmol) β-tetralone, 52 mg (200 mmol) L-norvaline methyl ester, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0111] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0112] Table 8

[0113]

[0114] Example 9: Reductive amination reaction of β-tetralone with L-norvaline tert-butyl ester

[0115] This embodiment provides a method for preparing a sterically hindered β-tetralone ( ) as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-norvaline tert-butyl ester as an amine donor to synthesize chiral amines, wherein the source of the imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 9 below.

[0116] The synthesis method comprises the following steps:

[0117] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 7.04 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 5.84 mg (20 mmol) β-tetralone, 69.2 mg (200 mmol) L-norvaline tert-butyl ester, 100 mM Tris-HCl pH 8.0 to make up to 2 mL, shake at 30 ° C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0118] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0119] Table 9

[0120]

[0121] Example 10: Reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone with L-norvaline

[0122] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone ( ) as a carbonyl substrate, imine reductase (reductive aminase) as a catalyst, and L-norvaline as an amine donor to synthesize chiral amines, wherein the source of the imine reductase or reductive aminase and the substrate conversion rate are shown in Table 10 below.

[0123] The synthesis method comprises the following steps:

[0124] (1) In a 10 mL reaction bottle, add 8.56 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53 mg coenzyme (NADP + ), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 46.8 mg (200 mmol) L-norvaline, 100 mMTris-HCl pH 8.0 to 2 mL, shake at 30°C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0125] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0126] Table 10

[0127]

[0128] Example 11: Reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone with L-norvaline methyl ester

[0129] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone ( ) as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-norvaline methyl ester as an amine donor to synthesize chiral amines, wherein the source of the imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 11 below.

[0130] The synthesis method comprises the following steps:

[0131] (1) In a 10 mL reaction bottle, add a catalyst that is 10% of the mass of the carbonyl substrate, then add 8.56 mg of glucose dehydrogenase (GDH), 21.6 mg of glucose (GLu), and 1.33 mg of coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 52 mg (200 mmol) L-norvaline methyl ester, 100 mMTris-HCl pH 8.0 to make up to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0132] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile solution, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0133] Table 11

[0134]

[0135] Example 12: Reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone with L-norvaline tert-butyl ester

[0136] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone ( ) as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-norvaline tert-butyl ester as an amine donor to synthesize chiral amines, wherein the source of the imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 12 below.

[0137] The synthesis method comprises the following steps:

[0138] (1) In a 10 mL reaction bottle, add 10% of the mass of the carbonyl substrate as catalyst, then add 8.56 mg GDH, 21.6 mg glucose dehydrogenase (GDH), 1.33 mg coenzyme (NAD + ), 1.53mg coenzyme (NADP + ), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 69.2 mg (200 mmol) L-norvaline tert-butyl ester, 100 mMTris-HCl pH 8.0 to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain a chiral amine reaction solution;

[0139] (2) Dilute the chiral amine reaction solution 5 times with 50% acetonitrile, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0140] Table 12

[0141]

[0142] Embodiment 13

[0143] The difference between this embodiment and embodiment 1 is that the amount of the catalyst used is 8% of the amount of the carbonyl substrate.

[0144] Embodiment 14

[0145] The difference between this embodiment and embodiment 1 is that the amount of the catalyst used is 15% of the amount of the carbonyl substrate.

[0146] Embodiment 15

[0147] The difference between this embodiment and embodiment 1 is that the amount of the catalyst used is 5% of the amount of the carbonyl substrate.

[0148] Example 16

[0149] The difference between this embodiment and embodiment 1 is that the amount of catalyst used is 20% of the mass of the carbonyl substrate.

[0150] The catalyst sources and substrate conversion rates in Examples 13-16 are shown in Table 13 below.

[0151] Table 13

[0152]

[0153] Embodiment 17

[0154] The difference between this embodiment and embodiment 1 is that in step (1), the reaction condition of shaking reaction at 30° C. for 16 h is changed to shaking reaction at 20° C. for 20 h.

[0155] Embodiment 18

[0156] The difference between this embodiment and embodiment 1 is that the reaction condition of shaking reaction at 30° C. for 16 h in step (1) is changed to shaking reaction at 40° C. for 12 h.

[0157] Embodiment 19

[0158] The difference between this embodiment and embodiment 1 is that the reaction condition of shaking reaction at 30° C. for 16 h in step (1) is changed to shaking reaction at 15° C. for 10 h.

[0159] Embodiment 20

[0160] The difference between this embodiment and embodiment 1 is that the reaction condition of shaking reaction at 30° C. for 16 h in step (1) is changed to shaking reaction at 10° C. for 16 h.

[0161] Embodiment 21

[0162] The difference between this embodiment and embodiment 1 is that the reaction condition of shaking reaction at 30° C. for 16 h in step (1) is changed to shaking reaction at 50° C. for 16 h.

[0163] The catalyst sources and substrate conversion rates in Examples 17-21 are shown in Table 14 below.

[0164] Table 14

[0165]

[0166] Embodiment 22

[0167] Further, error-prone PCR mutations were performed for enzymes that catalyze the reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone with greater steric hindrance and L-norvaline tert-butyl ester. After screening more than 5,000 mutants, 5 mutants with higher conversion rates than their respective parent were obtained as shown in the table below. Among them, SEQ ID NO: 1 is a mutant of WP_023809753.1, SEQ ID NO: 2 is a mutant of KPU97715.1, SEQ ID NO: 3 is a mutant of WP_342352985.1, SEQ ID NO: 4 is a mutant of KXX80955.1, and SEQ ID NO: 5 is a mutant of WP_108908932.1. The specific sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 are as described in the previous specific implementation method section and will not be repeated here.

[0168] Table 15

[0169]

[0170] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention uses imine reductase or reductive amination enzyme or a mutant of the enzyme as a catalyst for the synthesis of chiral amines, and can synthesize chiral amines from non-keto acid ketone compound substrates and amino donors under mild reaction conditions. It not only has a wide substrate range, but also is simple and economical to operate, and can directly synthesize a variety of chiral amine compounds, and can be suitable for industrial production.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for synthesizing a chiral amine, characterized in that: The synthesis method of the chiral amine comprises: subjecting a carbonyl substrate and an amine donor to a reductive amination reaction under the catalytic action of an imine reductase or a reductive aminase to obtain the chiral amine; wherein the imine reductase or the reductive aminase is selected from one of NCBI reference sequence numbers: KXX80955.1, KPU97715.1, WP_003986432, a mutant of KPU97715.1 shown in SEQ ID NO: 2, and a mutant of KXX80955.1 shown in SEQ ID NO: 4; When the imine reductase or reductive aminase is KXX80955.1, the carbonyl substrate is β-tetralone, and the amine donor is L-norvaline tert-butyl ester; or, the carbonyl substrate is 6,8-dihalo-3,4-dihydro-1H-2-naphthalenone, and the amine donor is L-norvaline or L-norvaline tert-butyl ester; When the imine reductase or reductive aminase is KPU97715.1, the carbonyl substrate is β-tetralone, and the amine donor is L-norvaline tert-butyl ester; or, the carbonyl substrate is 6,8-dihalo-3,4-dihydro-1H-2-naphthalenone, and the amine donor is L-norvaline methyl ester or L-norvaline tert-butyl ester; When the imine reductase or reductive aminase is WP_003986432, the carbonyl substrate is cyclohexanone, and the amine donor is L-leucine or L-threonine; or, the carbonyl substrate is 5-methoxy-2-tetralone, and the amine donor is L-norvaline methyl ester; or, the carbonyl substrate is β-tetralone, and the amine donor is L-norvaline methyl ester; or, the carbonyl substrate is 6,8-dihalo-3,4-dihydro-1H-2-naphthone, and the amine donor is L-norvaline or L-norvaline methyl ester; When the imine reductase or reductive aminase is a mutant of KPU97715.1 shown in SEQ ID NO: 2 or a mutant of KXX80955.1 shown in SEQ ID NO: 4; the carbonyl substrate is 6,8-dihalo-3,4-dihydro-1H-2-naphthalenone, and the amine donor is L-norvaline tert-butyl ester.

2. The method for synthesizing chiral amine according to claim 1, characterized in that: The temperature of the reductive amination reaction is 20-40° C. and the time is 12-20 h.

3. The method for synthesizing chiral amine according to claim 1, characterized in that: The synthesis method comprises: adding the imine reductase or the reductive amination enzyme, the carbonyl substrate, the amine donor, glucose dehydrogenase, glucose, coenzyme I and coenzyme II into a Tris-HCl solution to carry out the reductive amination reaction to obtain a chiral amine reaction liquid, and purifying the chiral amine reaction liquid to obtain the chiral amine.

4. The method for synthesizing chiral amine according to claim 3, characterized in that: The mass ratio of the imine reductase or the reductive aminase to the carbonyl substrate is (8-15):100; and / or, the molar ratio of the amine donor to the carbonyl substrate is (1-15):1; and / or, the mass ratio of glucose dehydrogenase to the carbonyl substrate is (10-100):100; And / or, the molar ratio of the glucose to the carbonyl substrate is (1-5):

1.

5. The method for synthesizing chiral amine according to claim 3, characterized in that: The molar ratio of the coenzyme I to the carbonyl substrate is (5-50):100; And / or, the molar ratio of the coenzyme II to the carbonyl substrate is (5-50):

100.

6. The synthesis method according to any one of claims 1 to 3, characterized in that The carbonyl substrate is 6,8-dichloro-3,4-dihydro-1H-2-naphthalenone, and the amine donor is L-norvaline tert-butyl ester.

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