Synthesis method for chiral amine

Abstract

Provided in the present application is a synthesis method for a chiral amine. The synthesis method for a chiral amine provided in the present application comprises: subjecting a carbonyl substrate and an amino donor to a reductive amination reaction under the catalysis of a catalyst to obtain a chiral amine, wherein the catalyst is an imine reductase or a reductive aminase. In the present application, using the imine reductase or the reductive aminase as a catalyst for chiral amine synthesis enables the synthesis of a chiral amine from a non-keto-acid ketone compound substrate and an amino donor under mild reaction conditions. Said method not only has a wide substrate range, but is also simple and economical to operate, and can directly synthesize a variety of chiral amine compounds, making it suitable for industrial production.

Description

Synthesis methods of chiral amines

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024114593472, filed on October 18, 2024, entitled “Method for Synthesizing Chiral Amines”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of chiral amine synthesis technology, and more specifically, to a method for synthesizing chiral amines. Background Technology

[0004] Chiral amines are important structural units that are widely found in the structures of pharmaceutical intermediates. It has been reported that approximately 40% of the small molecule drug compounds approved by the FDA contain chiral amine modules.

[0005] Currently, the main methods for synthesizing chiral amines include chemical catalytic synthesis and enzyme catalytic synthesis. Chemical catalysis has become increasingly limited due to the use of transition metal complexes, harsh conditions, excessive alkylation, and the generation of large amounts of waste, which have caused significant environmental pressure. Enzymatic catalytic synthesis, on the other hand, has advantages such as mild reaction conditions, high selectivity, and being green and pollution-free, and is gradually being widely used in the synthesis of chiral amines.

[0006] Besides the preparation of chiral amines through chiral resolution mediated by various hydrolases, ω-transaminases, amino acid dehydrogenases, and crown gall dehydrogenases can all catalyze the reductive amination of carbonyl substrates with amine donors to obtain chiral amines. However, ω-transaminases can only complete the transfer of -NH2 with the help of pyridoxal phosphate, limiting their application range; similar to amino acid dehydrogenases, crown gall dehydrogenases are limited to using keto acids as carbonyl substrates when catalyzing the formation of chiral amines.

[0007] In view of the above, this application is hereby submitted. Summary of the Invention

[0008] The main objective of this application is to provide a method for synthesizing chiral amines, in order to solve the technical problem that ω-transaminase can only complete the transfer of -NH2 with the help of pyridoxal phosphate, which limits its application scope; and that crown gall dehydrogenase, similar to amino acid dehydrogenase, is limited to keto acids as carbonyl substrates when catalyzing the formation of chiral amines.

[0009] To achieve the above objectives, according to one aspect of this application, a method for synthesizing a chiral amine is provided. The method comprises: performing a reductive amination reaction on a carbonyl substrate and an amine donor under the catalysis of an imine reductase or a reductive amination enzyme to obtain a chiral amine; wherein the carbonyl substrate is a ketone compound, and the structural schematic diagram of the ketone compound is shown in formula (I) below:

[0010] In formula (I), R1 represents H, a substituted or unsubstituted C6-C10 aryl, a substituted or unsubstituted C6-C10 arylene, or a substituted or unsubstituted C5-C10 heteroarylene; R2 represents a substituted or unsubstituted C1-C5 alkylene, or a 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;

[0011] The amino group donor is an amino acid or an amino acid ester. The structural diagram of the amino group donor is shown in formula (II) below:

[0012] In formula (II), 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 reductoaminerase is selected from imine reductases or reductoaminerases having the amino acid sequences shown in SEQ ID NOs: 1-12, or imine reductases or reductoaminerases having more than 70% homology with the amino acid sequences shown in SEQ ID NOs: 1-12.

[0014] Further, the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having the amino acid sequences shown in SEQ ID NOs: 6-12, or imine reductases or reductive amination enzymes having more than 70% homology with the amino acid sequences shown in SEQ ID NOs: 6-12.

[0015] Furthermore, the reductive amination reaction is carried out at a temperature of 20-40℃ for 12-20 hours.

[0016] Furthermore, the method for synthesizing chiral amines includes: adding imine reductase or reductive amination enzyme, carbonyl substrate, amino donor, glucose dehydrogenase, glucose, coenzyme I and coenzyme II into Tris-HCl solution to carry out the reductive amination reaction, obtaining a chiral amine reaction solution, and purifying the chiral amine reaction solution to obtain a chiral amine.

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

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

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

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

[0021] Further, the carbonyl substrate is 6,8-dichloro-3,4-dihydro-1H-2-naphthone, the amino donor is L-valine tert-butyl ester, and the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

[0022] An imine reductase or reductase that has more than 70% homology with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17;

[0023] Preferably, the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having the amino acid sequences shown in SEQ ID NOs: 13-17.

[0024] By applying the technical solution of this application, imine reductase or reductive amination enzyme is used as the catalyst for the synthesis of chiral amines. This allows for the synthesis of chiral amines from non-keto acid ketone compounds and amino donors under mild reaction conditions. The substrate range is broad, the operation is simple and economical, and a variety of chiral amine compounds can be directly synthesized, making it suitable for industrial production. Detailed Implementation

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present application will now be described in detail with reference to the embodiments.

[0026] As analyzed in the background section of this application, chiral amines are an important structural unit. Currently, in enzymatic methods for synthesizing chiral amines, ω-transaminases can only complete the -NH2 transfer with the help of pyridoxal phosphate, limiting their application. Crown gall base dehydrogenases, similar to amino acid dehydrogenases, are limited to using keto acids as carbonyl substrates when catalyzing the formation of chiral amines. To address this issue, this application provides a method for synthesizing chiral amines.

[0027] In a first embodiment of this application, a method for synthesizing a chiral amine is provided. The method includes: performing a reductive amination reaction on a carbonyl substrate and an amine donor under the catalysis of an imine reductase or a reductive amination enzyme to obtain a chiral amine; wherein the carbonyl substrate is a ketone compound, and the structural schematic diagram of the ketone compound is shown in formula (I) below:

[0028] In formula (I), R1 represents H, a substituted or unsubstituted C6-C10 aryl, a substituted or unsubstituted C6-C10 arylene, or a substituted or unsubstituted C5-C10 heteroarylene; R2 represents a substituted or unsubstituted C1-C5 alkylene, or a 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;

[0029] The amino group donor is an amino acid or an amino acid ester. The structural diagram of the amino group donor is shown in formula (II) below:

[0030] In formula (II), 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 this application, the term "C6-C10 aryl" refers to 6-10 membered monocyclic, bicyclic, or tricyclic aromatic ring systems, including phenyl, biphenyl, etc. The term "C5-C10 heteroarylene" refers to 5-10 membered monocyclic, bicyclic, or tricyclic aromatic ring systems having 1-4 heteroatoms, such as furanyl, thiophene, pyrrole, imidazolyl, pyrazolyl, thiazolyl, etc. Heteroarylene groups can include monovalent or divalent heteroarylene groups. The term "halogen" or "halogen" 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, said group being a straight chain or a branched chain having one or more branches, such as butylene, such as n-butylene, sec-butylene, isobutylene, tert-butylene; propylene, such as n-propylene or isopropylene; ethylene or methylene; more particularly, methylene, isopropylene, or tert-butylene.

[0032] The method for synthesizing chiral amines provided in this application involves a reductive amination reaction of a carbonyl substrate and an amine donor under the action of an imine reductase or a reductive amination enzyme. The reaction conditions are mild, and the substrate is a non-keto acid ketone compound. This method is not only applicable to a wide range of substrates, but also simple and economical to operate. It can directly synthesize a variety of chiral amine compounds and is suitable for industrial production.

[0033] In some embodiments of this application, the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having amino acid sequences as shown in SEQ ID NOs: 1-12, or imine reductases or reductive amination enzymes having more than 70% homology with amino acid sequences shown in SEQ ID NOs: 1-12.

[0034] In some embodiments of this application, the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having amino acid sequences as shown in SEQ ID NOs: 6-12, or imine reductases or reductive amination enzymes having more than 70% homology with amino acid sequences shown in SEQ ID NOs: 6-12.

[0035] SEQ ID NO: 1 (Streptomyces ipomoeae, NCBI Reference Serial Number: WP_009330409.1): msntkaaqapvsviglglmgqalaaaflkaghpttvwnrtaakadqlvgegaalagstadaiaasplvvvcvtdytavrelld plagalkgkvlvnlttgtstqaretaewaadkeityldgaimaippdiatdaavllysgpkaafdeheatlralgaagttyldtdhglsalydmsl lgimwgilngflhgaallgtaevkattfaplantminvvteyvtayapqidegkypagdatmtvhqdalehlaeesetlginaemprffkalv drsvaaghaesgyaalieqfrkpav.

[0036] SEQ ID NO: 2 (Madurella mycetomatis, NCBI reference sequence number: KXX80955.1): matitsigignmgaalatallksssppmnvtiwnrtasrpqvqslisagaifepslaaalassevillclldypaissvfsqvdasa kplagktilnltngtpkqardmeaffkslgaavyfdggvmvtpqlvgtpaafvvlsgeteqaynerlanagllspvgavlyiapdpgaaslvd caalaamygmfigaftgigllkrqkherdgeaagakamvdkvmvpvltalvpyvgllaeqvdkeawmddlgnplamqaegvrnimq scedegvdgtglkflsklmekgvkegfgpggvavvakylmk。

[0037] SEQ ID NO: 3 (Streptosporangium roseum, NCBI reference sequence number: ACZ83929.1): mrdtdvtvlglglmgqalagaflkdghattvwnrsegkagqlaeqgavlassardaaeasplvvvcvsdhaavravldplgd vlagrvlvnltsgtseqarataewaaergityldgaimaipqvvgtadafllysgpeaayeaheptlrslgagttylgadhglsslydvallgim wgtlnsflhgaallgtakveattfapfanrwieavtgfvsayagqvdqgaypaldatidthvatvdhliheseaagvntelprlvrtladralagg qgglgyaamieqfrspsa。

[0038] SEQ ID NO: 4 (Myxococcus stipitatus, NCBI reference sequence number: AGC43099.1): mkptltvigagrmgsalikaflqsgytttvwnrtkakseplaklgahladtvrdavkrsdiivvnvldydtsdqllrqdevtrelrgkllvq ltsgspalareqetwarqhgidyldgaimatpdfigqaecallysgsaalfekhravlnvlggatshvgedvghasaldsallfqmwgtlfgtl qalaisraegiplekttafikltepvtqgavadvltrvqqnrltadaqtlasleahnvafqhllalceernihrgvadamysvireavkaghgkdd failtrflk。

[0039] SEQ ID NO: 5 (Streptomyces sp. CNS615, NCBI reference sequence number: WP_108908932.1): mtgngrspvtviglglmgqalaaaflkgghpttvwnrsadkatdlvssgavladtpdeavsaadlivvcvsdddalhgvldpva eklrgrvvanltsgssdqaranaawsaewgfdyldgaimtvpagvgapesvffyagpqkaydrhaealallgggttylgedhalaplydval lgmmwgtlnsflqgaallatadvkaseflpwaikwvdavkmfatdyagqidagdgaypardatmdvhagalkhllhesesqgvnaelpr fvqglmqrtiaagfgqnsyasmielfrapek。

[0040] SEQ ID NO: 6 (Kribbella flavida (Desert Kribbella bacterium) DSM 17836, NCBI reference sequence number: WP_012921542.1): mpptdrtpvtliglgpmgqamtrallaaghpvtvwnrtparaagvvadgavlaaspveaveagdlvilsltd yqamydvlepatgslagrtvvnlssdtpdrtraaadwatehgatfltggvmipapmvgteeayvyysgpaevfekhrttltvigaprylgedt glaqlmyqaqldvflttlsslmhatallgtagvsaaesmpeligmlrtvpamleaggenpgadidadkhpgdlstitmmgatadhivgaset agidlalpravqahyrraienghggdnwtriidgirspr.

[0041] SEQ ID NO: 7 (Variovorax paradoxus (Debatable Phagobacterium), NCBI reference sequence number: KPU97715.1): msskqkitviglgamgatiarlyleqghevtiwnrsadkaaplvaqgavladsaaaavrasrvvlmcvydyraadailgaegvaa amdgrllvqlttgsprdardaqawaqrhgatflegaiqaapeqmgkgdtpilmsgdeqvfravepllavlgggivylgekisnaaamdlatl stiygtmlgflhgarvaesegfdvaefgrivagimptfasflqhegaviqsgdfkisqspmrisveatqrilqtaresginsefpafaaglfqrad aaglggeelaalikllrapa.

[0042] SEQ ID NO: 8 (Cystobacter ferrugineus, NCBI reference sequence number: WP_071905000.1): mkpgisvlgtgrmgsalvgaflkqgynvavwnrtkskcaplaalgarvattvrdavadaevvvvnvndyvtseallrqddvt kglrgklivqltsgsprqaremaawarqhelqyldgaimgtpnfigepggtilysgpgalfekykpvllvlggnslhvgsdvghasaldsall sflwgsmfgvlqavsvceaeglplgaymeyvqatkpmvdgavtdfvkriqtgrfagdektlatveahhgalrhlielceehgihhavpaaf gqlfqaalqaghaqddfavlnkfmk。

[0043] SEQ ID NO: 9 (Rhizobium sullae, NCBI reference sequence number: WP_342352985.1): mttyaqvrivrrpfqhtstksqqkravlrkliwrvsmkrsitvlgtgrmgsalarallhaghrttvwnrtiqkaeplaalgatvapsvleavnaaeiiivnvsdyqataaimrndaiasavrgkliveltsgtphgareaaefwaehgasyldgaimatpdfigtdagtilvsgssqafdanedmfralggnvqhigeesgranaldsallalmwgalfgtlhaiavcqaeeidlgelaqqwnatapvveglvadlikrtnagrfasddetlssisahygafqhllelmeareidrsvvlgydaifqraiaagqlhedfaalsqflgksa。

[0044] SEQ ID NO: 10 (Streptomyces sp. (Streptomyces) TOR3209, NCBI reference sequence number: WP_019324525.1): mhtdlqavtviglgsmgsalaaalldrghpltvwnrspgkarplvekgarladtpeeaiaaspvtltcvfdydvlrtllgpat galagrdlinltsgsaeqareldswlrlhgaghldggimttppgvgdpammflysgspsvldshrqvlealgdpvylgndpglaslydaallg lmwstltgwlhgaalvgadgveagaftpiavrwltavsgfvttysaqvdaaeypgddatvdvqiatidhlihaaqargidtglvallkatmqr akaaghgsdsyasvievlrkpsdha.

[0045] SEQ ID NO: 11 (Streptomyces sp. (Streptomyces), NCBI reference sequence number: WP_003986432): mrsepaavtvlglgsmgtalagallkgghattvwnrsphkakplaergatvaatpeeavaasplliacvldyaalhtvldpvadslagktlvnltsgspeqaaeaaawarahgahyldgaimttppgvgspemmflysgertvfdthrpvlaslgdplhlgtdpglaslydaallglmwaamtgwlhgtalvgaegtpataftpvairwlsavtgflttyapqvdaghypgddatvdvqiaaidhlihaaaargvdnalpellkatmertraaghgsssyasvietlrkaagnr.

[0046] SEQ ID NO: 12 (Mesorhizobium sp. (Bradyrhizobium) L2C084A000NCBI Serial Number: WP_023809753.1): mklsitvigtgrmgsalagsllqsgypttvwnrtrqktdplarlgaiaassveeavnageiiivnvsdyeatkallhsdaias airgkliveltsgtpsgareaaewctkhganyldgaimatpdyigtdagtillagpreafdtnrdvfralggnvqhvgeepgranaldsallai mwgalfgtlhaiavsqaeeielgelarqwsatapvidglvtdlikrtsagrfasdnetlssisahhgamqhllelmqfrgidrsivdgydaifkr aiaaghlhddfaalshflatgk.

[0047] In this specification, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0048] Proteins with over 70% homology and the same function, including those with over 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, and even over 99.9%, have an active site, active pocket, active mechanism, protein structure, etc. that are highly likely to be the same as the protein provided by sequence (a), and are homologous proteins obtained through amino acid mutation.

[0049] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0050] Substitution and replacement rules generally apply to amino acids with similar properties; substituting one another will produce similar effects. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0051] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0052] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0053] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0054] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0055] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0056] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model capable of predicting the conformation of protein complexes. Its predictions of protein three-dimensional structures closely approximate the levels observed in real-world experiments using equipment such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thereby guiding the investigation of protein structure and activity.

[0057] To further reduce energy consumption, the preferred temperature for the reductive amination reaction is 20–40°C, and the preferred time is 12–20 h. 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.

[0058] Typical, but not limiting, when using imine reductase or reductive amination enzyme as a catalyst for the synthesis of chiral amines, the temperature of the reductive amination reaction is such as 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C or any combination of two values; the time is such as 12h, 15h, 18h, 20h or any combination of two values.

[0059] To further improve the conversion rate of the substrate, in some preferred embodiments, the method for synthesizing the chiral amine includes: imine reductase or reductive amination enzyme, carbonyl substrate, amine donor, glucose dehydrogenase (GDH), glucose (GLu), and coenzyme I (NAD).+ ) and coenzyme II (NADP) + Add the chiral amine to a Tris-HCl solution and react at 20-40℃ for 12-20 hours to obtain a chiral amine reaction solution. Purify the chiral amine reaction solution to obtain a chiral amine.

[0060] In this embodiment, the carbonyl substrate and amine donor, under the action of catalyst, coenzyme I, coenzyme II, glucose dehydrogenase and glucose, are more conducive to improving the efficiency of the reductive amination reaction, and further improving the conversion efficiency of chiral amines.

[0061] Furthermore, to facilitate the termination of the reaction, it is preferable in the above embodiments that after the reductive amination reaction has reached the corresponding reaction time, the reaction is terminated by adding a reaction terminator to obtain a chiral amine reaction solution. The preferred reaction terminator is acetonitrile.

[0062] To further improve the efficiency of chiral amine synthesis, the preferred concentration of the Tris-HCl solution is 80–120 mM, and the pH is 6.0–9.5, such as a molar concentration of 80 mM, 90 mM, 100 mM, 105 mM, 110 mM, 120 mM, or any combination of two values, and a pH of 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.2, 8.5, 9.0, 9.5, or any combination of two values.

[0063] To further improve the conversion rate of the carbonyl substrate, the preferred mass ratio of imine reductase or reductive amination enzyme to the carbonyl substrate is (8-15):100, such as 8:100, 10:100, 12:100, 15:100 or any range of two values.

[0064] To further improve the yield of chiral amines, in some preferred 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 any range of 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 any range of two values; and / or, the molar ratio of glucose to the carbonyl substrate is (1-5). The molar ratio of coenzyme I to carbonyl substrate is (5-50):100, such as 5:100, 10:100, 20:100, 30:100, 40:100, 50:100, or any two values; the molar ratio of coenzyme II to carbonyl substrate is (5-50):100, such as 5:100, 10:100, 20:100, 30:100, 40:100, 50:100, or any two values.

[0065] The term "substitution" in "[substituted or unsubstituted]" indicates that a hydrogen atom in a functional group is replaced by another atom or group (i.e., a substituent). Each of these 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 with a C6-C10 aryl, benzimidazolyl, C3-C10 cycloalkyl, C5-C7 heterocycloalkyl, carboxyl, nitro, and hydroxyl.

[0066] It should be noted that the amino acids used as amine donors can be either natural or non-natural.

[0067] In some embodiments, the carbonyl base is selected from any one of cyclohexanone, 5-methoxy-2-naphthoanone, β-tetrahydronaphthoanone, and 6,8-dichloro-3,4-dihydro-1H-2-naphthoanone, and the amino donor is selected from any one of L-methionine, L-leucine, L-isoleucine, L-valine, L-alanine, glycine, L-threonine, L-aspartic acid, L-tyrosine, L-phenylalanine, L-valine methyl ester, and L-valine tert-butyl ester.

[0068] In some specific embodiments, the carbonyl substrate is 6,8-dichloro-3,4-dihydro-1H-2-naphthone, the amino donor is L-valine tert-butyl ester, and the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

[0069] An imine reductase or reductase that has more than 70% homology with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17;

[0070] Preferably, the imine reductase or reductive amination enzyme is selected from imine reductases or reductive amination enzymes having the amino acid sequences shown in SEQ ID NOs: 13-17.

[0071] The beneficial effects of this application will be further illustrated below with reference to embodiments and comparative examples.

[0072] Example 1: Reductive amination reaction of cyclohexanone with L-methionine

[0073] This embodiment provides a method for synthesizing chiral amines using cyclohexanone as a carbonyl substrate and L-methionine as an amino donor, wherein the structural formula of cyclohexanone is as follows: The structural formula of L-methionine is:

[0074] The synthesis method includes the following steps:

[0075] (1) In a 10 mL reaction flask, add catalyst at a concentration equal to 10% of the carbonyl substrate mass, then add 5.48 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) +), 3.92 mg (20 mmol) cyclohexanone, 59.6 mg (200 mmol) L-methionine, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0076] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0077] The specific sources of the catalysts are shown in Table 1 below, as are the conversion rates of the substrate cyclohexanone.

[0078] Table 1

[0079] Example 2: Reductive amination reaction of cyclohexanone with L-isoleucine

[0080] This embodiment provides a method for synthesizing chiral amines using cyclohexanone as a carbonyl substrate, L-isoleucine as an amine donor, and imine reductase or reductive amination enzyme as a catalyst. The structural formula of L-isoleucine is as follows:

[0081] The synthesis method includes the following steps:

[0082] (1) In a 10 mL reaction flask, add catalyst at 10% of the mass of the carbonyl substrate, then add 5.48 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 3.92 mg (20 mmol) cyclohexanone, 52 mg (200 mmol) L-isoleucine, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0083] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0084] The specific sources of imine reductase or reductive amination enzyme are shown in Table 1 below, and the conversion rate of the substrate cyclohexanone is also shown in Table 2.

[0085] Table 2

[0086] Example 3: Reductive amination reaction of cyclohexanone with various amino acids

[0087] This embodiment provides a synthetic method for preparing chiral amines using cyclohexanone as a carbonyl substrate, L-valine, L-alanine, glycine, L-leucine, L-threonine, L-valine, and L-aspartic acid as amino group donors, and imine reductase or reductive amination enzyme as a catalyst. The specific structures of the amino group donors, the sources of imine reductase or reductive amination enzyme, and the substrate conversion rates are shown in Table 3 below.

[0088] The synthesis method includes the following steps:

[0089] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 3.92 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 3.92 mg (20 mmol) cyclohexanone, 200 mmol amino acid as amine donor, 100 mM Tris-HCl pH 8.0 added to 2 mL, shaken at 30 °C for 16 h, the reaction was terminated by adding 2 mL acetonitrile to obtain chiral amine reaction solution;

[0090] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0091] Table 3

[0092] 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%.

[0093] Example 4: Reductive amination reaction of 5-methoxy-2-naphthone with L-tyrosine

[0094] This embodiment provides sterically hindered 5-methoxy-2-naphthone. As a carbonyl substrate, using imine reductase (reductive amination enzyme) as a catalyst, and L-tyrosine... The method for synthesizing chiral amines using amine donors, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 4 below.

[0095] The synthesis method includes the following steps:

[0096] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 7.04 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 7.04 mg (20 mmol) 5-methoxy-2-naphthone, 72 mg (200 mmol) L-tyrosine, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0097] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile with a mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0098] Table 4

[0099] Example 5: Reductive amination reaction of 5-methoxy-2-naphthone with L-phenylalanine

[0100] This embodiment provides sterically hindered 5-methoxy-2-naphthone. As a carbonyl substrate, using imine reductase (reductive amination enzyme) as a catalyst, and L-phenylalanine... The method for synthesizing chiral amines using amine donors, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 5 below.

[0101] The synthesis method includes the following steps:

[0102] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 7.04 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 7.04 mg (20 mmol) 5-methoxy-2-naphthone, 66 mg (200 mmol) L-phenylalanine, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0103] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0104] Table 5

[0105] Example 6: Reductive amination reaction of 5-methoxy-5-naphthone with L-n-valine methyl ester

[0106] This embodiment provides sterically hindered 5-methoxy-2-naphthone. As a carbonyl substrate, using imine reductase (reductive amination enzyme) as a catalyst, and L-valine methyl ester... The method for synthesizing chiral amines using amine group donors, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 6 below.

[0107] The synthesis method includes the following steps:

[0108] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 7.04 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 7.04 mg (20 mmol) 5-methoxy-2-naphthone, 52 mg (200 mmol) L-n-valine methyl ester, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain the chiral amine reaction solution.

[0109] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0110] Table 6

[0111] Example 7: Reductive amination reaction of 5-methoxy-5-naphthone with L-n-valine tert-butyl ester

[0112] This embodiment provides a method using sterically hindered 5-methoxy-2-naphthone as a carbonyl substrate, imine reductase (reductive amination enzyme) as a catalyst, and L-valine tert-butyl ester. A method for synthesizing chiral amines using amine donors, wherein the source of imine reductase or reductive amination enzyme and the substrate conversion rate are shown in Table 7 below.

[0113] (1) In a 10 mL reaction flask, add a catalyst at 10% of the carbonyl base, then add 7.04 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) +), 7.04 mg (20 mmol) 5-methoxy-2-naphthone, 69.2 mg (200 mmol) L-n-valine tert-butyl ester, 100 mM Tris-HCl pH 8.0, add to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0114] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0115] Table 7

[0116] Example 8: Reductive amination reaction of β-tetrahydronaphthone with L-n-valine methyl ester

[0117] This embodiment provides a sterically hindered β-tetrahydronaphthone. A method for synthesizing chiral amines using carbonyl substrate, imine reductase (reductoaminease) as catalyst, and L-valine methyl ester as amine donor, wherein the source of imine reductase or reductoaminease and substrate conversion rate are shown in Table 8 below.

[0118] The synthesis method includes the following steps:

[0119] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 5.48 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 5.84 mg (20 mmol) β-tetrahydronaphthone, 52 mg (200 mmol) L-n-valine methyl ester, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0120] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0121] Table 8

[0122] Example 9: Reductive amination reaction of β-tetrahydronaphthone with L-n-valine tert-butyl ester

[0123] This embodiment provides a sterically hindered β-tetrahydronaphthone. A method for synthesizing chiral amines using carbonyl substrate, imine reductase (reductoaminease) as catalyst, and L-valine tert-butyl ester as amine donor, wherein the source of imine reductase or reductoaminease and substrate conversion rate are shown in Table 9 below.

[0124] The synthesis method includes the following steps:

[0125] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 7.04 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 5.84 mg (20 mmol) β-tetrahydronaphthol, 69.2 mg (200 mmol) L-n-valine tert-butyl ester, 100 mM Tris-HCl pH 8.0, add to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain chiral amine reaction solution;

[0126] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0127] Table 9

[0128] Example 10: Reductive amination of 6,8-dichloro-3,4-dihydro-1H-2-naphthone with L-n-valine

[0129] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthone. A method for synthesizing chiral amines using carbonyl substrate, imine reductase (reductoaminease) as catalyst, and L-valine as amine donor is described in Table 10 below.

[0130] The synthesis method includes the following steps:

[0131] (1) In a 10 mL reaction flask, add catalyst at 10% of the mass of the carbonyl base, then add 8.56 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) +), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 46.8 mg (200 mmol) L-valine, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain the chiral amine reaction solution;

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

[0133] Table 10

[0134] Example 11: Reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthone with L-n-valine methyl ester

[0135] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthone. A method for synthesizing chiral amines using carbonyl substrate, imine reductase (reductoaminease) as catalyst, and L-valine methyl ester as amine donor, wherein the source of imine reductase or reductoaminease and substrate conversion rate are shown in Table 11 below.

[0136] The synthesis method includes the following steps:

[0137] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 8.56 mg glucose dehydrogenase (GDH), 21.6 mg glucose (GLu), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 52 mg (200 mmol) L-n-valine methyl ester, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain the chiral amine reaction solution;

[0138] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile solution of mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0139] Table 11

[0140] Example 12: Reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthone with L-n-valine tert-butyl ester

[0141] This embodiment provides a sterically hindered 6,8-dichloro-3,4-dihydro-1H-2-naphthone. This method describes the synthesis of chiral amines using carbonyl substrate, imine reductase (reductoaminease) as catalyst, and L-valine tert-butyl ester as amine donor. The sources of imine reductase or reductoaminease and the substrate conversion rates are shown in Table 12 below.

[0142] The synthesis method includes the following steps:

[0143] (1) In a 10 mL reaction flask, add a catalyst at 10% of the mass of the carbonyl base, then add 8.56 mg GDH, 21.6 mg glucose dehydrogenase (GDH), and 1.33 mg coenzyme I (NAD). + ), 1.53 mg coenzyme II (NADP) + ), 8.56 mg (20 mmol) 6,8-dichloro-3,4-dihydro-1H-2-naphthone, 69.2 mg (200 mmol) L-valine tert-butyl ester, 100 mM Tris-HCl pH 8.0, bring to 2 mL, shake at 30 °C for 16 h, add 2 mL acetonitrile to terminate the reaction, and obtain the chiral amine reaction solution;

[0144] (2) Dilute the chiral amine reaction solution 5 times with acetonitrile with a mass concentration of 50%, centrifuge at 12000 rpm for 10 min, and take the supernatant to detect the substrate conversion rate.

[0145] Table 12

[0146] Example 13

[0147] The difference between this embodiment and Example 1 is that the amount of catalyst used is 8% of the mass of the carbonyl substrate.

[0148] Example 14

[0149] The difference between this embodiment and Example 1 is that the amount of catalyst used is 15% of the mass of the carbonyl substrate.

[0150] Example 15

[0151] The difference between this embodiment and Example 1 is that the amount of catalyst used is 5% of the mass of the carbonyl substrate.

[0152] Example 16

[0153] The difference between this embodiment and Example 1 is that the amount of catalyst used is 20% of the mass of the carbonyl substrate.

[0154] The catalyst sources and substrate conversion rates in Examples 13-16 are shown in Table 13 below.

[0155] Table 13

[0156] Example 17

[0157] The difference between this embodiment and embodiment 1 is that in step (1), the reaction condition of shaking at 30°C for 16 hours is changed to shaking at 20°C for 20 hours.

[0158] Example 18

[0159] The difference between this embodiment and Embodiment 1 is that the reaction condition of shaking at 30°C for 16 hours in step (1) is changed to shaking at 40°C for 12 hours.

[0160] Example 19

[0161] The difference between this embodiment and Embodiment 1 is that the reaction condition of shaking at 30°C for 16 hours in step (1) is changed to shaking at 15°C for 10 hours.

[0162] Example 20

[0163] The difference between this embodiment and Embodiment 1 is that the reaction condition of shaking at 30°C for 16 hours in step (1) is changed to shaking at 10°C for 16 hours.

[0164] Example 21

[0165] The difference between this embodiment and Embodiment 1 is that the reaction condition of shaking at 30°C for 16 hours in step (1) is changed to shaking at 50°C for 16 hours.

[0166] The catalyst sources and substrate conversion rates in Examples 17-21 are shown in Table 14 below.

[0167] Table 14

[0168] Example 22

[0169] Furthermore, error-prone PCR mutagenesis was performed on enzymes that exhibited good catalytic activity in the reductive amination reaction of 6,8-dichloro-3,4-dihydro-1H-2-naphthone with significant steric hindrance and L-valine tert-butyl ester. After screening over 5000 mutants, five mutants with higher conversion rates than their respective parent lines were obtained, as shown in the table below. Specifically, SEQ ID NO:13 is a mutant of SEQ ID NO:12, SEQ ID NO:14 is a mutant of SEQ ID NO:7, SEQ ID NO:15 is a mutant of SEQ ID NO:9, SEQ ID NO:16 is a mutant of SEQ ID NO:2, and SEQ ID NO:17 is a mutant of SEQ ID NO:5. The specific sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 are as described in the previous detailed implementation section and will not be repeated here.

[0170] Table 15

[0171] As can be seen from the above description, the embodiments of this application achieve the following technical effects: This application uses imine reductase or reductive amination enzyme or enzyme mutant as a catalyst for the synthesis of chiral amines, which can synthesize chiral amines from non-keto acid ketone compounds and amino donors under mild reaction conditions. It not only has a wide range of substrates, but also is simple and economical to operate, and can directly synthesize a variety of chiral amine compounds, making it suitable for industrial production.

[0172] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for the synthesis of chiral amines, characterized in that, The synthesis method of the chiral amine comprises: performing a reductive amination reaction on a carbonyl substrate and an amine group donor under catalysis of an imine reductase or a reductive amination enzyme to obtain the chiral amine; wherein the carbonyl substrate is a ketone compound, and a structural schematic diagram of the ketone compound is shown in the following formula (I): In formula (I), R1 represents H, a substituted or unsubstituted C6-C10 aryl, a substituted or unsubstituted C6-C10 arylene, or a substituted or unsubstituted C5-C10 heteroarylene; R2 represents a substituted or unsubstituted C1-C5 alkylene, or a 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; The amine donor is an amino acid or an amino acid ester, the structure of which is shown in the following formula (II): In formula (II), 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.

2. The synthesis method according to claim 1, characterized in that, The imine reductase or the reductase is selected from imine reductases or reductases having amino acid sequences as shown in SEQ ID NOs: 1-12, or imine reductases or reductases having more than 70% homology with amino acid sequences shown in SEQ ID NOs: 1-12.

3. The method of synthesis of claim 1, wherein, The imine reductase or the reductase is selected from imine reductases or reductases having the amino acid sequences shown in SEQ ID NOs: 6-12, or imine reductases or reductases having more than 70% homology with the amino acid sequences shown in SEQ ID NOs: 6-12.

4. The method of synthesis of chiral amines according to any one of claims 1 to 3, characterized in that, The reductive amination reaction is carried out at a temperature of 20-40℃ for 12-20 hours.

5. The method of synthesis of chiral amines according to any one of claims 1 to 3, characterized in that, The synthesis method includes: adding the imine reductase or the reductive amination enzyme, the carbonyl substrate, the amine donor, glucose dehydrogenase, glucose, coenzyme I, and coenzyme II to a Tris-HCl solution to carry out the reductive amination reaction, obtaining a chiral amine reaction solution, and purifying the chiral amine reaction solution to obtain the chiral amine.

6. The method of synthesis of chiral amines as claimed in claim 5 wherein, The mass ratio of the imine reductase or the reductase 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 glucose to the carbonyl substrate is (1-5):

1.

7. The method of synthesis of chiral amines as claimed in claim 5 wherein, The molar ratio of 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.

8. The method of synthesis according to any one of claims 1 to 3, wherein, The carbonyl base material is selected from at least one of cyclohexanone, 5-methoxy-2-naphthoone, β-tetrahydronaphthoone, and 6,8-dichloro-3,4-dihydro-1H-2-naphthoone.

9. The method of synthesis according to any one of claims 1 to 3, wherein, The amine donor is selected from at least one of L-methionine, L-leucine, L-isoleucine, L-valine, L-valine, L-alanine, glycine, L-threonine, L-aspartic acid, L-tyrosine, L-phenylalanine, L-valine methyl ester, and L-valine tert-butyl ester.

10. The method of synthesis according to any one of claims 1 to 3, wherein, The carbonyl base is 6,8-dichloro-3,4-dihydro-1H-2-naphthone, the amino donor is L-valine tert-butyl ester, and the imine reductase or the reductoaminerase is selected from imine reductases or reductoaminers having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO:

17. An imine reductase or reductase that has more than 70% homology with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17; Preferably, the imine reductase or the reductase is selected from imine reductases or reductases having amino acid sequences as shown in SEQ ID NOs: 13-17.

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