Process for reducing aromatic imine with neoimine reductase (IRED)

Abstract

The present invention relates to a process for producing a chiral aromatic amine, said process comprising the steps of: a) providing an aromatic imine, and b) contacting said aromatic imine from step a) with an imine reductase, thereby stereoselectively reducing said aromatic imine to a chiral aromatic amine with said imine reductase; and to novel imine reductases capable of catalytic reactions.

Description

[0001] The present invention relates to a novel method for preparing chiral aromatic amines, comprising a) providing an aromatic imine and b) contacting the aromatic imine from step a) with an imine reductase, thereby stereoselectively reducing the aromatic imine to a chiral aromatic amine by the imine reductase.

[0002] The present invention also relates to a novel imine reductase, which can catalyze the stereoselective reduction of aromatic imines to chiral aromatic amines.

[0003] Chiral aromatic amines are structural units for developing novel therapeutic drug candidates. In fact, the stereoselective synthesis of chiral aromatic amines has been an area of ​​development in recent decades, as these compounds recur in a variety of active pharmaceutical intermediates with different therapeutic targets.

[0004] In the art, various chemical synthetic methods for chiral aromatic amines are known, such as stereoselective reductive amination of ketones and amines, in which an imine is formed briefly as an intermediate and then reduced to an amine (Afanasyev et al. 2019).

[0005] Reductive amination of ketone precursors and aromatic amines using reductive aminoases is also known (Aleku et al., 2017; France et al., 2018; Roiban et al., 2017; Scheller et al., 2015).

[0006] However, these enzymes often fail to perform reductive amination reactions, especially when aromatic ketones and / or aromatic amines are involved.

[0007] Therefore, the object of the present invention is to find a method that allows for the preparation of chiral aromatic amines on a preparative scale with high selectivity from suitable precursor compounds.

[0008] The objective of this invention can be achieved by the method described below, which includes:

[0009] a) Provide aromatic imines and

[0010] b) Contact the aromatic imine from step a) with imine reductase, thereby stereoselectively reducing the aromatic imine to a chiral aromatic amine by imine reductase.

[0011] The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the present invention.

[0012] As used herein, the terms "peptide" or "enzyme" refer to macromolecular biocatalysts, typically proteins, that accelerate chemical reactions. Enzymes act on molecules that serve as substrates, and they convert the substrates into products. Like all catalysts, enzymes increase reaction rates by lowering the activation energy of the reaction.

[0013] "Imine reductase / reductoaminoase" refers to enzymes that catalyze the reduction of imines to amines, most commonly secondary amines. The term "imine reductase" encompasses all enzymes capable of imine reduction, while "reductoaminoase" specifically refers to enzymes particularly adept at the direct reductive amination of amines and ketones, as imines are believed to be transiently formed at the enzyme's active site, although imine reductases can also perform reductive amination. Both imine reductases and reductoaminoases catalyze the reduction of imines to amines using a reducing equivalent provided by NADH or NADPH.

[0014] According to the present invention, a protein as defined herein is used as an imine reductase. This means that, under conditions that allow for reduction, the protein comes into contact with a substrate containing the imine to be reduced, and the reduction reaction occurs.

[0015] The term "unsubstituted alkyl" includes straight-chain or branched alkyl groups with 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl, and their isomers. Preferred alkyl groups are those having 1 to 4 carbon atoms.

[0016] The term "substituted alkyl" includes the aforementioned alkyl chain, which may carry one or more substitutions selected from halogen, halogen alkyl, alkyl, hydroxy, aryl, alkoxy, aryloxy or alkoxycarbonyl or aryloxycarbonyl, cyano, nitro.

[0017] The term "alkenyl" includes straight-chain or branched alkenyl groups with 2 to 6 carbon atoms, carrying one or more double bonds in the chain. Typical alkenyl groups are represented by vinyl or allyl.

[0018] The term "substituted alkenyl" includes the aforementioned alkyl or alkenyl chains that may carry one or more substitutions selected from halogen, halogen alkyl, alkyl, hydroxyl, aryl, alkoxy, aryloxy or alkoxycarbonyl or aryloxycarbonyl, cyano, nitro.

[0019] The term "alkoxy" refers to an alkyl group, as defined above, attached to an oxygen atom. A typical example is the methoxy group.

[0020] The term "alkoxycarbonyl" refers to an alkyl group as defined above, attached to a carbonyl group. A typical example is an acetyl group.

[0021] The term "aryl" includes phenyl, naphthyl, anthraceneyl, or phenanthryl, but is preferably phenyl.

[0022] The term "substituted aryl" includes the aryl groups listed above, which may carry one or more substitutions selected from halogen, halogenated alkyl, alkyl, hydroxyl, aryl, alkoxy, aryloxy or alkoxycarbonyl or aryloxycarbonyl, cyano, nitro.

[0023] The term "aryloxy group" refers to an aryl group, as defined above, attached to an oxygen atom. A typical example is the phenoxy group.

[0024] The term "aryloxycarbonyl" refers to an aryl group, as defined above, attached to a carbonyl group. A typical example is the benzoyl group.

[0025] The term "heteroaryl" refers to an aromatic 5- to 6-membered monocyclic or 9- to 10-membered bicyclic ring that may contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and / or sulfur, such as pyridyl, pyridinyl, pyrazolyl, pyrimidinyl, benzimidazolyl, quinolinyl and isoquinolinyl, thiophenyl, furanyl, pyrroleyl, pyrazolyl, isoxazolyl, oxazolyl, thiazolyl, or imidazolyl.

[0026] The term "halogen" includes F, Cl, Br, and I.

[0027] The term "cyano" indicates a CN substituent.

[0028] The term "nitro" represents a NO2 substituent.

[0029] The term "carbon ring" refers to C 3-10 - The cycloalkane moiety, such as cyclohexane, can fused to an aromatic ring system. A typical example is tetrahydronaphthalene, in which cyclohexane is fused to a benzene ring.

[0030] The term "heterocycle" refers to a C-ring in which at least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, or sulfur. 3-10 -Cycloalkyl moiety. Heterocyclic rings can fused into aromatic ring systems.

[0031] The term "chirality" is used to describe molecules that cannot be superimposed on their mirror images. Chiral molecules have non-superimposed mirror images and exist in two distinct forms known as enantiomers.

[0032] The terms "E" or "Z" configuration describe the relative orientation of the two substituents on the double bond. The E configuration indicates that the two substituents are on opposite sides of the double bond, while the Z configuration indicates that the two substituents are on the same side of the double bond.

[0033] As outlined above, the method includes

[0034] a) Provide aromatic imines and

[0035] b) Contact the aromatic imine from step a) with imine reductase, thereby stereoselectively reducing the aromatic imine to a chiral aromatic amine by imine reductase.

[0036] In a preferred embodiment, the method includes stereoselectively reducing an aromatic imine to a chiral primary or secondary aromatic amine using an imine reductase.

[0037] In the context of this invention, "providing aromatic imines" preferably means that the aromatic imines are pre-formed, i.e., have been synthesized in advance and provided as substrates for the reduction reaction. Therefore, they do not appear as intermediates.

[0038] The preparation of aromatic imines can be carried out according to methods known in the art (Carlson et al., 1992, Yasukawa et al., 2022).

[0039] Suitable aromatic imines have formula I

[0040]

[0041] in

[0042] R 1 For substituted or unsubstituted aryl or heteroaryl groups,

[0043] R 2 C is hydrogen, substituted or unsubstituted 1-6 -alkyl or C 2-6 -Alkenyl, substituted or unsubstituted C 1-6 -alkoxycarbonyl or R 1 and R 2 Together they form a carbon ring, and

[0044] R 3 It is hydrogen or substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C 1-6 -Alkyl or substituted or unsubstituted C 1-6 -alkoxycarbonyl,

[0045] And among them

[0046] Substituents are selected from halogens, halogen C 1-6 -alkyl, C 1-6 -alkyl, hydroxyl, aryl, C 1-6 -alkoxy, aryloxy, or C 1-6 -Alkoxycarbonyl or aryloxycarbonyl, cyano or nitro.

[0047] In the preferred embodiment

[0048] R 1 For substituted or unsubstituted aryl or heteroaryl groups,

[0049] R 2 For hydrogen, C 1-6 -alkyl or C 1-6 -alkoxycarbonyl or

[0050] R 1 and R 2 Together they form carbon rings or heterocycles, and

[0051] R 3 It is hydrogen or substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C 1-6 -alkyl or C 2-6 -Alkenyl

[0052] And among them

[0053] Substituents are selected from C 1-6 -alkyl, C 1-6 -alkoxy or aryl.

[0054] In a further embodiment

[0055] R 1 It is a substituted or unsubstituted phenyl, naphthyl, or an aromatic 5- to 6-membered monocyclic or 9- to 10-membered bicyclic ring that may contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and / or sulfur.

[0056] R 2 For hydrogen, C 1-6 -alkyl or C 1-6 -alkoxycarbonyl or

[0057] R 1 and R 2 Together they form C 3-10 -Carbon rings or heterocyclic rings, which can fused to aromatic rings, and

[0058] R 3 It is a hydrogen-containing or substituted or unsubstituted phenyl, naphthyl, or an aromatic 5- to 6-membered monocyclic or 9- to 10-membered bicyclic, substituted or unsubstituted C-type compound that may contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and / or sulfur. 1-6 -alkyl or C 2-6 -Alkenyl

[0059] And among them

[0060] Substituents are selected from C 1-6 -alkyl, C 1-6 -alkoxy or aryl.

[0061] The aromatic imine of formula I can exist in either the E or Z configuration.

[0062] Preferably, the concentration of the aromatic imine is from 2 mM to 1000 mM, more preferably from 20 mM to 500 mM, and most preferably from 50 mM to 300 mM.

[0063] The stereoselective reduction in step b) is typically carried out in the presence of a cofactor regeneration system, which contains cofactors and enzymes capable of reducing such cofactors.

[0064] In a preferred embodiment, the cofactor is NADP. + or NAD + Furthermore, the enzymes capable of reducing cofactors are phosphorous acid dehydrogenase or glucose dehydrogenase.

[0065] Preferably, the cofactor is NADP. + Furthermore, the enzymes capable of reducing cofactors are phosphorous acid dehydrogenase or glucose dehydrogenase.

[0066] Imine reductase can be administered in solution or as a cell-free extract in lyophilized form.

[0067] In a preferred embodiment, the reduction reaction occurs in an aqueous environment having a pH of 5.0 to 10.0, preferably 6.5 to 8.5, at a temperature ranging from 5°C to 50°C, preferably 20°C to 40°C, at a sodium phosphite or glucose concentration of 100 to 1 M, preferably 100 mM to 500 mM, and at a phosphite dehydrogenase or glucose dehydrogenase concentration of 0.1 g / L to 10 g / L, preferably 0.1 g / L to 8 g / L, wherein the concentration of aromatic imine is 2 mM to 1000 mM, preferably 20 mM to 500 mM.

[0068] Typically, solvents selected from polar protic solvents (such as lower fatty alcohols, like methanol) and dipolar aprotic solvents (such as dimethyl sulfoxide, cyrene) are used. TM Co-solvents of nonpolar solvents (such as cyclohexane) or nonpolar solvents.

[0069] Contact / incubation time can range from about 5 minutes to many hours, preferably from about 1 hour to 100 hours, such as from 4 hours to 50 hours.

[0070] However, the incubation time can depend on the assay format, solution volume, substrate and catalyst concentrations, and the amount of aromatic imine to be reduced. Those skilled in the art are familiar with how to apply the optimal incubation time.

[0071] The same applies to the addition of aromatic imines, which can occur at the start of the reaction or at specific doses at different points during the incubation period.

[0072] At the end of the reaction, the chiral aromatic amine is typically separated by increasing the pH of the reaction (e.g., by adding an alkali metal carbonate base), followed by liquid-liquid extraction with a suitable organic solvent such as ethyl acetate. Subsequently, the solvent can be evaporated under reduced pressure, and the chiral aromatic amine can be separated.

[0073] The resulting chiral aromatic amines have formula II

[0074]

[0075] Where R 1 R 2 and R 3 As defined above, and the same preferred options therein also apply.

[0076] The enzyme capable of catalyzing the stereoselective reduction of aromatic imines to chiral aromatic amines may be selected from imine reductases having a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 98% sequence identity with any of SEQ ID NO. 1 to 33.

[0077] SEQ ID 1 (IR00005)

[0078] MHHHHHHKTVAVIGLGQMGTTLARLFIEAGMQVRVWNRTRSKAEPLASRGAIVAATAAAAMADAEAVVICVHDYRATHDILSDVAVKSALKGKLLLQLTTGSPQDARDMAELAARIGAGYLDGALQVAPEQMGQPDTTVLVSGSGEDHALA RELLAVLGGNVVYLGEDVAAAATMDLATLSYVYGASMGFFQGAALAQAEGLDVGVYGGIVEAMSPSFGAFLRHEGNVIDNGDYAVSQSPLSISIDATGRIEQAMRQKGLRSELPSLIARLLRDAEEAGYGNEEFAAVAKILRGAAEPAPVR

[0079] SEQ ID 2 (IR00010)

[0080] MHHHHHHTTTPTVTVLGLGPMGQALSRALLDAGHTVTVWNRTESKAQALRDRGALSAPTPAAAIAASDLALVNVVDHDAVDAILTAAGDAPAGRTVIGLSSDTPDRARRTAKLVGNVGGRYLDGAIMTPIDTIGTRGASILFAGPQALFDEHRGVLDTLGQLTWVGEDHGRAAAFDMALLDLFWTSVGGFGHALMVARANGIEPSELMPHAHGIVGILSPIFTEVAQRVEDDRHSDASASVSSVASSVRHLIAASREAGVDAGLLEAFRGYVDATVAAGHGDDEISRIASEMTTLTRG

[0081] SEQ ID 3 (IR00014)

[0082] MHHHHHHTNNATPVSILGLGLMGQALARAFLKAGHPTTVWNRTPGKADQLMAEGAQVAPTAAEAIDASSLTVICVSDYPAMYELLDASDLAGTTLLNLTSGDSAQARQAARWAEQRGAHYLDGAIMAIPQAIGTDDAVILISGAQADADAHRPTLEALGTLTYLGADHGLASLYDVAGLAMMWSVLNAWLQGTALLRTAGVDAATFAPFAQQMAAGVAGWLPGHAQEIDAGSFATEVASLDTHVRTMDHLIEECEAAGINAELPRLIKSMADRSLAAGHGAASYSVLIEEFAKPA

[0083] SEQ ID 4 (IR00015)

[0084] MHHHHHHMKAPVTVVGLGPMGKAMAETFLKNGHPTTVWNRTASKAAPLVEQGATLAATPDDALAASGLVVISQTDYKAMYDSLDGAEMKGRVLVNLSSGSSPDELRRAEAAGKGAELLTGGVMPPPPGIGQPGAYIMYSGPEALLDRHRETLRVLGDTTYVGADVGLSNLYYQAQLYLFWSTLTAYLHSIAMLQSAGVSAEQFRPFATETVTSLGVDGPMGFLRILAEEADAGHSPGGENSMLMMAVGADHMVEAAEAAGIDTMGPRALRDLFWRTVNAGHGADGLSVIEVVRKGA

[0085] SEQ ID 5 (IR00020)

[0086] MHHHHHHSATTNTTSADGVAGPGGPGGRPPVTVLGLGQMGAAIAGALLAAGHPVTVWNRTPGKAAPLVEQGAVLAGSVAEAVAASPLVLSVVLDYPALYGILDPEPDALKGRALVNLTTGTPEQAGEAEAWAARHGVDYLDGAIMTTPPGVGTREVMFLYSGDRAVFDAHHAALDVLGEPLHLGTEPGLAALYDVNLLGLMWATMAGWLHGTAVVGAEGTRAVDFTEVAIRWLGTVNNFIRRYAAQVDEGVYPGDDATVDVQIAVVEHQLHAAEARGVDNRLPELLKTLMLEANAKGHGQDSFGSVVEVLRKGARR

[0087] SEQ ID 6 (IR00032)

[0088] MHHHHHHRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLVCVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAFSEAEPVLSLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGFGQQEVSALIKVLRARNGAAQ

[0089] SEQ ID 7 (IR00033)

[0090] MHHHHHHRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDINLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPDQIGTSDASIICSGASAAFSDAEPVLRLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPIAKVCAQFSELSPILGGDVAHLGKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGLGQQEVSALIKVLRARNGAAL

[0091] SEQ ID 8 (IR00041)

[0092] MHHHHHHMSSVSIFGLGAMGTALASRFLEEKYKVAVWNRSPEKASSLLGKGATLSHTAVDGINASDLIIICLLDNAAVEATLAGALDHLHGKTIINLTNGTPDQARKLSDRFVSHGARYVHGGIMATPSMIGSPYALVLYSGSPDAFKAAEGDLSVLAKCVFLGEDAGTASLHDLALLSGMYGLFSGFLHATALVRSSTPAVKFMDLLVPWLGAMTEYTKGMAKQIDEGKYTSEGSNLAMQLVGIQNIIDASEAQQVSAEFIRPMKEFMQKAVAAGHGGDDISSLIDFVKST

[0093] SEQ ID 9 (IR00049)

[0094] MHHHHHHMSGDNRAPVTVIGLGMMGAALAGAYLKAGHPTTVWNRSAAKAAPLVEQGARRATDVAEAVAASGIVVVCVFDYTVARSLLAPVRDQLAGKVIVNLTSGLPDEARETAAWAEDAGARYLDGYVMTVPPAVGLPQTLLFYGGDKEIFDAHEETLKVLGGNSVHLGTDPGIAALYDLALLGILWSTLTSALHGFALVGSENVPAAALMPFAESWITHVVLPTVQGAAQQVDAGHYATDISTVELNAMGLPKMIKASEAQGVRADLMVPIKDFLEKRVADGHGADALASLIEVIRDGDR

[0095] SEQ ID 10 (IR00052)

[0096] MHHHHHHMSNTKAAQAPVSVIGLGLMGQALAAAFLKAGHPTTVWNRTAAKADQLVGEGAALAGSTADAIAASPLVVVCVTDYTAVRELLDPLAGALKGKVLVNLTTGTSTQARETAEWAADKEITYLDGAIMAIPPDIATDAAVLLYSGPKAAFDEHEATLRALGAAGTTYLDTDHGLSALYDMSLLGIMWGILNGFLHGAALLGTAEVKATTFAPLANTMINVVTEYVTAYAPQIDEGKYPAGDATMTVHQDALEHLAEESETLGINAEMPRFFKALVDRSVAAGHAESGYAALIEQFRKPAV

[0097] SEQ ID 11 (IR00054)

[0098] MHHHHHHMKSNSQNEKNGSETTNAVGNRKSVTVIGLGPMGQAMADVFLEYGYSVTVWNRTSSKADQLVAKGAIRVSTVNEALAANELVILSLTDYNVMYSILEPVSENLFGKVLVNLSSDTPEKARKAAKWLEDRGARHITGGVQVPPSGIGKSESYTYYSGDRVVFEAHRETLEVLTSSDYRGEDPGLAMLYYQIQMDIFWTAMLSYLHALAIANANGITAEQFLPYASAMMSSLPKFVEFYTPRLDEGEHPGDVDRLAMGLASVEHVVHTTQEAGIDIALPATVLEVFRRGMKTGHASDSFTSLIEIFKNSDIRS

[0099] SEQ ID 12 (IR00060)

[0100] MHHHHHHMSTVSASESPVTVVGLGMGHALAAAFVAKGHPTTVWNRSAGKADDLVAAGATLADSVQAAVEASPLVVVCVSDYDAVHALLDPVGPALAGRTLVNLTTASSSQARDTAEWAAKLDATYLDGAILALPQGIGTDEATLLYAGPKAAFEEHQATLAVLGEAATVYLDEDHGLSALYDMAVLTIMWGVLNSFLHAAALLGTANVKATTFAGMAATAINVTADYVAAYAPQIDAGEYPATDATVNVHVGGMQHLLEESKALGVNAELPRFFLELAGRAVAGGHAEDSYAALIKQFRAPSA

[0101] SEQ ID 13 (IR00063)

[0102] MHHHHHHMKPTLTVIGAGRMGSALIKAFLQSGYTTTVWNRTKAKSEPLAKLGAHLADTVRDAVKRSDIIVVNVLDYDTSDQLLRQDEVTRELRGKLLVQLTSGSPALAREQETWARQHGIDYLDGAIMATPDFIGQAECALLYSGSAAL FEKHRAVLNVLGGATSHVGEDVGHASALDSALLFQMWGTLFGTLQALAISRAEGIPLEKTTAFIKLTEPVTQGAVADVLTRVQQNRLTADAQTLASLEAHNVAFQHLLALCEERNIHRGVADAMYSVIREAVKAGHGKDDFAILTRFLK

[0103] SEQ ID 14 (IR00064)

[0104] MHHHHHHMSTPPHTTAGPAAVTVLGLGRMGSALAAAFLAAGHSTTVWNRTPGKADELAARGARRAGSVAEAVAAAPLVVVCVADDEAVHQLLDPLDGALAGRTLVNLTTGTSAQARANAAWAKERGAAFLDGAIMAVPEDIATGDAVLLYSGPRDAFDAYEEALRVLAPAGTTHLGGDAGLAALHDLALLGIMWGVLNGFLHGAALLGTAGVRAGDFAPLAARMTTVVAGYVTAAAPEVDAGSYPAGDATLTVHQEAMRHLAEESEALGVNAELPRFLQLLAGRAVAEGHAESGYSALVEQFRKA

[0105] SEQ ID 15 (IR00065)

[0106] MHHHHHHMKPHISILGAGRMGSALVKAFLQNEYTTTVWNRTRARCEPLAAAGARIADSVRDAVQTASVVIVNVNDYDTSDALLRQDEVTQELRGKVLVQLTSGSPKLAREQATWARRHGIDYLDGAIMATPDLIGRPDCTLLYAGPKALYDKHQAVLAALGGNTQHVSEDEGHASALDSAILFQLWGSLFSGLQAAAICRAEGIALDALGPHLEAVAAMIQFSMKDLLQRIQKEQYGADPQSPATLDTHNVAFQHLLHLCEERNIHHALPKAMDALIQTARKAGHGQDDFSVLARFLR

[0107] SEQ ID 16 (IR00066)

[0108] MHHHHHHMSVSVLGLGPMGQALTRALLNANHRTTVWNRTAAKADDVVARGAVWADDPASAIAAGDLTLVNVVDQQAVDAVLTAAGDAVAGRVIIGLSAGTPDAARRTASFVTGAGGAYLDGAIMTPTDTIGTASASVLFSGPRELFDDHREVLSALGTLTWLGEDVGRAEAFDVALLDLFWTSVSGFVHALSIAKANGVSAVELLPHANGISEILPAIFTEIAERVESGRHDDASAPVSSVAASLRNLISAARSAGIDAGALETFRDYVDAAVAAGHGDAEISRITPLGIG

[0109] SEQ ID 17 (IR00073)

[0110] MHHHHHHMKNDGVTKGSVALLGLGEMGRVLAERLLDAGYPVTVWNRTPGRDTALVERGARRAETVREAVTAATTVVTCLFDHASVRETLEPVGADLAGRTLVDLTTTTPNEARWLGGWAEERGIEHLDGAIMATPSMIGAPEASLLYSGSAEAFGRHRTLFEVWGSATYDGADHGAASLFDLALLSGMYTMFTGFAHGAAMVTSAGVTAEEFAHRSARLLSAMTGVFPMTAKVIDEGDYTGPGQSLEWTATALDTIARASAEQGVSPGPIEMTRALVLAQIEAGYGNENSDRIYEELRAG

[0111] SEQ ID 18 (IR00075)

[0112] MHHHHHHMAATPTNPSTDSGKTPVTVLGLAMGRALAGAFLKAGHPTVWNRSEHKADELVARGAVRAGSVAEAVAASPLIVVCVVDYEVSHRILEPVGADLAGRVLVNLTSDTPVRSRRAAEWAAGHGVEYLDGAIMVPTPVIGTPEATVLYSGSRRAFDTYEETLKALGGKAPFLGTDHGVAAVYDLAMLSFFYSGMAGLAHAFTLAGEEGVPATDLAPFLDVITGIFPPIAKGMADDLVGGRLDGAGEGNIVMEAAGIAHIVEASRDRGVNTDVLDALKALMDRTIAAGHGESEFVRVTEAMRGAYA

[0113] SEQ ID 19 (IR00100)

[0114] MGSSHHHHHSSGLVPRGSHMPDNPSTKGRMMRNQQAEHTPVTVIGLGLMGQALAGAFLGAGHPTTVWNRTAAAKPARGAKSVAEAVAASPLVVVCVSDYDAVHALLDPLDGTALQGRTLVNLTSGTSAQAREARAAWADGRGADYLDGAILAGPAAIGTADAVVLLSGPRSAFDPHASALGGLGAGTTYLGADHGLASLYDAAGLVMMWSILNGFLQGAALLGTAGVDATTFAPFITQGIGTVADWLPGYARQIDDGAYPADDAAIDTHLATMEHLIHESEFLGVNAELPRFIKALADRAVADGHGGSGYPALIEQFRTHSGK

[0115] SEQ ID 20 (IR00202)

[0116] MGSSHHHHHHSSGLVPRGSMTDTSAKLTLLGLGAMGSALATAWLAADYDITVWNRTASRAEPLRTLGAQVADTAADAVAANDLVVACLLDDASVRSTLDGVDLTGKDLINLTTGTPGSGRELAACATARGARYVDGGIMAVPPMIGVPDSGAYVFYSGSAAAFEAHRDALAVGAGTKFVGEDPGFAALYDVALLSAMTGMFAGVSHAFALVRKENIDPREFAGLVSGWITAMSGYAHGIAEHLASGDYTTGVTSNLAMMVEGNATMLRTAHEQGVSPELLEPFMRLMRQRVDDGHGDEDTTGVIDLLLSRR

[0117] SEQ ID 21 (IR00376)

[0118] MRSEPAAVTVLGLGSMGTALAGALLKCGHATTVWNRSPHKTGPLAERGATVAATPEEAVAASPLVIACVLDYAALHAVLDPVADSLAGKTLVNLTSGSPEQAAEAAAWARSHGAHYLDGAIMTTPPGVGSPEMMFLYSGERTVLDTHRPVLASLGDPLYLGTDPGLASLYDAALLGLMWATMTGWLHGTALVGAEGTPATAFTPVAIRWLSAVTGFLTTYAPQVDAGHYPGDDATVDVQIAAIDHLIHAAAARGVDNALPGLLKAAMERTRAAGHGSSSYASVIEVLRKAADAR

[0119] SEQ ID 22 (IR00393)

[0120] MGSSHHHHHHSSGLVPRGSMGDNRTPVTVIGLGLMGQALAAAFLEAGHTTTVWNRSAGKAEQLVSQGAVQAATPADAVAASELVVVCLSTYDNMHDVIGSLGESLRGKVIVNLTSGSSDQGRETAAWAEKQGVEYLDGAIMITPPGIGTETAVLFYAGTQSVFEKYEPALKLLGGGTTYLGTDHGMPALYDVSLLGLMWGTLNSFLHGVAVVETAGVGAQQFLPWAHMWLEAIKMFTADYAAQIDAGDGKFPANDATLETHLAALKHLVHESEALGIDAELPKYSEALMERVISQGHAKNSYAAVLKAFRKPSE

[0121] SEQ ID 23 (IR00395)

[0122] MGSSHHHHHHSSGLVPRGSHMDNETAPVTVIGLGLMGRALAGAFLRAGHPTTVWNRTASKAEQLVAEGARLAPTVGDALEASSVAIVCLTDYEVVHELLGAGEIKLDGTLLINLTSGDSTQAREAARWAEQRGARYLDGAIMAVPPAIGTAEAMILLSGPQSDFESHKAMLGALGGTTYLGADHGLASLYDVAGLAMMWSILNAWLQGSALVGTADVDAATFTPFAQQLASVVVEWLPGYAEQVDSGSFPAEVSALETDVRAMTHLIEESEAVGVNAEMPRLFKAIADRSIVAGHGGEQYPVLIEEFRKPRDT

[0123] SEQ ID 24 (IR00464)

[0124] MTSEQRASVTVLGTSMGSALARAFLAAGYRTHVWNRSAARTTELVAAGATAHRDITDAVEASPVIIACLSTFAATHASLSPSTGILAGRDIITLNSGTPAQAREFAQWVRCAGARFLGGAIKNVPAAVGDRNTLLYFGGDRVFDAHTGTLRVLGGELDFLGVETDLAALYESAVGATLLPALLGFLEGAAMLASRGLPAHTMVPYSVKWLRMIESLLPVLAEEIDTGNYTLLGSSVELFHNALADDRQLAAESGVDLSWHAPMHDLLRRAVAEGRGDQSITALIELLTARP

[0125] SEQ ID 25 (IR00653)

[0126] MPTGTTVSSAVSSPVTPPLAVTVIGLGAMGSALAGAFLDAGHSVTVWNRTPGKGDALAARGAVVAATAEEAVTASELIVVCLVDYDASEAILTPLAKALSGRTLVNVTADVPDRARTAAAWAAEHGIAYLDGAVMVPTAVVGTPAGLLFYSGDPTTFERYEPVLRALGGRTVHVGDSPDRAAVFDVALLDLFYGAMGAMIHAFALARAHGVPAAEVVPYMTSIVDLLPEALEAMAGDIDARSYPALTAGLGTMAASDHIVHAGRAAGIDSAQMEGIQRMTDRARELGHADSGWAANYEALINPRTRDRS

[0127] SEQ ID 26 (IR00887)

[0128] MEGFPMGTNAHTGTATETAGTLAEQRSVTVIGLGPMGRAMADAYLDRGYEVTVWNRTASRADDLVARGATLAPDVARALTANDLVILSLTDYEAMYAILEPATSALAGRVLVNLTSDTPDKARAAARWAADHGAVQLTGGVTVPPSGIGRPESSTFY SGPREVFDAHRPVLEVVTGKADYRGEDPGLAALYYQMGVMFWTSMLGYWQAIALADANGLAASDILGHAVDTANSLPGFFTFYAGRIDAGAHQGDVDRLAMGLASVEHVLHTNADAGVDTALPAAVVEIFRRGIKAGHGADSFSSLLELMKKGGES

[0129] SEQ ID 27 (IR01134)

[0130] MSNPNAAQAPVTVIGLGLMGQALAAAFIKQGHPTTVWNRTPGKAEQLTADGAILAQSAGKAISASPLVIVCVSDYDGVHEILGPLQGALAGKVLVNLSTGTSAQARETAEWAAQRGARYLDGAIMAIPPVIGTDGAVLLYSGPQDSFEEHAATLRALGTPGTTYLGDHGLSALHDMALLGLMWGILNGFLHGAALLGTAGVKATTFAPLANQMIKEVTEYVTAYAPQVDEGKYPATDATLTVHQDALEHLADESKAVGINSELPRFFKALVDRAAADGRADESYAALIEVFRKEAA

[0131] SEQ ID 28 (IR01136)

[0132] MSTLPASGAPVTVIGLGLMGQALAGAFLAKGHPTTVWNRSAAKADDLVARGAVLADSVQSAVEASPLVVVCVSDYDAVHQLLGPVGGSLAGRTLVNLTTASSTQARETAEWAQKLDAAYLDGAILALPHAIGTEEATLLYAGPQATFEEHRATLEVLGAEATVYLDEDHGLSALYDLSVLSIMWGVLNSFLHGAALLGTANVKASTFAELAVTAINVTAEYASAYAQQIDAGEYPATDATVNVHVGGMTHLVEESETLGVNAELPRFFLELAKRAVAAGDAENSYASLIKQFRKPSA

[0133] SEQ ID 29 (IR01139)

[0134] MSTQQTTVTVLGLGLMGRALAAAFLRAGHPTTVWNRTANKADQLVSEGARQAPTLVDALAASPLTIICLTDYQAVRELLDTHDLDLGATTLINLTSGDSAQARTAAAWAASRGARYLDGAIMAIPSAIGTAEAVILHSGPHADFDSHRPILDALGTVTYLGADHGLASLYDVAGLAMMWSVLNAWLHGTAMLRTAGVDAAAYAPFAQQIAAGVASWLPGYAEQIDRGAFPAQVSALETDVRAMAHLLAESAAVGVNTELPELIKAMADRAIAAGHGAEQYPVLIEEFAKAASAPPR

[0135] SEQ ID 30 (IR01421)

[0136] MNTNPKRFSNGNHPYDAQADGSILGPVTVIGLGAMGSKLARTFVNNGYVTTVWNRTPEKAAELVGLGASGTTDIADAIAASPLLIVCVLDYDAARHILEPMREKLSGKVVVNLTTGTPEEARTMGTWMRTQGAEYLDGGIMAVPSLIATPHAVILYSGSQTAYETYKQVLERLGTSHYLGSDPALAPLNDLALLSGMYGMFGGFIHAVALAGSEGTKAADFTTTLLIPFLQAMIFTMPEMANQIDTGDYSAKDASLAMQAAHDTIGEVSRVQGVSAETFAPIFELMKRRVEEGYGGDDFASVIELIRKRPSS

[0137] SEQ ID 31 (IR01598)

[0138] MGSSHHHHHHSSGLVPRGSMHERPPLSVTVLGLGTMGTALASALLDTGHAVTVWNRTASRATPLITRGAARAGDVGEALAASRVIVTCLLDHASVHDVLDDHASALTGRTLVNVTNTTPEGSAELASWAASHGADFLDGGIMAVPPQIGTPSAFVLYSGSPSALETARPALEAFGDVRYLGSDPSLAALQDTALLSGMYGLFGGILHAFALVRTGGVTARQFAPVLREWLTGMVGWASSAAERIDDGDHARDVASNLAMQATAYEGFLAVARDRGMNPLLLDPLGRLLRRRVAEGFGHEDITGVIDYLTASTNTTGASA

[0139] SEQ ID 32 (IR01855)

[0140] MKPSISVLGTGRMGSALARALLQAGYRTVVWNRTSEKAEPLAALGATVAPTVRQAIDASGIVIVNVSDYAATSTLLRASDVTPGLRGKLIVELTSGTPEGARETSQWTAAHGARYLDGAILATPDFIGTDAGTILLSGALEPFAANED VFRALGGNVQHIGTEPGLANALDSAVLALMWGALFGGLHAIAVCRAEEIDLGELGRQWAATAPVVEGLVADLIKRTSAGRFVSDAETLSSISPHYGAFQHLKELMEARRIDRTVVDGYDAIFRRAIASGHLHDDFAALSQFMGKAEQP

[0141] SEQ ID 33 (IR02159)

[0142] MPNTNGAQAPVTVIGLGLMGRALAAAFLEAGHPTTVWNRTAAKADRLVAEGAVRAGSVGDAIAASPLVVVCVTDYAAVRELLGPSASGSLGGKVVANLTTGTSAQARETAEWAAGLGARYLDGAIMAIPADIATDAAVLLHSGPKEAFE EHEATLRALGAAGTTYLDTDPGLSALYDMSLLGIMWGVLNGFLQGAALLGTAKVRATAFAPLANTMIKVVTEYVTAYAPQIDEGVYPADDATVTVHRDALGHLAEESEKLGVNAELPRFFKALTDRAATDGHADSSYAALIEQFRKPAA

[0143] Preferred is an imine reductase having a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% sequence identity with SEQ ID 01, 02, 03, 04, 06, 10, 12, 14, 15, 17, 18, 20, 21, 23, 27, 28, 31, 32 and 33.

[0144] Even more preferred is an imine reductase having a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% sequence identity with SEQ ID 06, 10, 12, 14, 15, 27 and 32.

[0145] The imine reductase used in this invention is novel and therefore represents a further embodiment of the invention, having a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 98% sequence identity with any of SEQ ID NO. 1 to 33 and being capable of catalyzing the stereoselective reduction of aromatic imines to chiral aromatic amines.

[0146] The preferred options outlined above also apply.

[0147] The imine reductase used in this invention can be obtained by culturing host cells using conventional procedures known to those skilled in the art. That is, the nucleic acid encoding the imine reductase of this invention can be introduced into suitable host cells to produce the corresponding protein by recombination. These host cells can be any type of suitable cell, preferably bacterial cells, such as *Escherichia coli*, which can be cultured. In the first step, the method may include cloning the corresponding gene into a suitable vector, such as a vector according to the second aspect of the invention. Vectors are widely used for gene cloning and can be readily introduced (i.e., transfected) into bacterial cells that have been transiently infiltrated with DNA. After the protein has been expressed in the corresponding host cells, the cells can be harvested and used as starting material for preparing a cell extract containing the protein of interest. The cell extract containing the protein of interest is obtained by lysing the cells. Methods for preparing cell extracts by chemical or mechanical cell lysis are well known to those skilled in the art and include, but are not limited to, hypotonic saline treatment, homogenization, or sonication.

[0148] "Host cell" can be any kind of organism suitable for application to recombinant DNA technology, and includes, but is not limited to, all kinds of bacterial and yeast strains suitable for expressing one or more recombinant proteins. Examples of host cells include, for example, various Bacillus subtilis or Escherichia coli strains. Many Escherichia coli bacterial host cells are known to those skilled in the art, and include, but are not limited to, strains such as DH5-alpha, HB101, MV1190, JM109, JM101, or XL-1 blue, which are available from various suppliers including, for example, Stratagene (California, USA), Promega (Wisconsin, USA), or Qiagen (Hilden, Germany). Particularly suitable host cells are also described in the examples, namely Escherichia coli BL21 (DE3) cells. Bacillus subtilis strains that can be used as host cells include, for example, 1012 wild-type: leuA8 metB5 trpC2 hsdRM1 and 168 Marburg: trpC2 (Trp-), which are commercially available, for example, from MoBiTec (Germany).

[0149] As used herein, the term "nucleic acid" generally refers to any nucleotide molecule encoding the imine reductase of the present invention and may have a variable length. Examples of nucleic acids of the present invention include, but are not limited to, plasmids, vectors, or any kind of DNA and / or RNA fragments that can be isolated by standard molecular biology procedures, including, for example, ion exchange chromatography. The nucleic acids of the present invention can be used for transfection or transduction of specific cells or organisms.

[0150] Nucleic acid molecules can be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for example, cDNA and genomic DNA, obtained, for example, by cloning or by chemical synthesis techniques or by combining thereof. DNA can be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA can be the coding strand (also known as the sense strand), or it can be the non-coding strand (also known as the antisense strand).

[0151] Among other things, nucleic acid molecules as used herein also refer to single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA, or mixtures of single-stranded and double-stranded regions, which may be single-stranded or more typically double-stranded or triple-stranded. Additionally, nucleic acid molecules as used herein refer to triple-stranded regions containing RNA or DNA, or both RNA and DNA.

[0152] In addition, nucleic acids can contain one or more modified bases. These nucleic acids can also contain modifications, such as in the ribose-phosphate backbone, to increase the stability and half-life of these molecules in physiological environments.

[0153] Therefore, DNA or RNA with a backbone modified for stability or other reasons is a "nucleic acid molecule" having the characteristics described herein. Furthermore, to name just two examples, DNA or RNA containing unusual bases (such as inosine) or modified bases (such as triphenylmethylated bases) is a nucleic acid molecule in the context of this invention. It should be understood that DNA and RNA have been modified in a wide variety of ways for many useful purposes known to those skilled in the art. As used herein, the term nucleic acid molecule encompasses chemical, enzymatic, or metabolically modified forms of nucleic acid molecules, as well as the chemical forms of DNA and RNA specific to viruses and cells, including both simple and complex cells.

[0154] Furthermore, the nucleic acid molecule encoding the imine reductase of the present invention can be functionally linked with any desired sequence (such as a regulatory sequence, leader sequence, heterolabel sequence, or heterocoding sequence) using standard techniques (such as standard cloning techniques) to produce a fusion protein.

[0155] Nucleic acids can be initially formed in vitro or in cultured cells and are typically manipulated by endonucleases and / or exonucleases and / or polymerases and / or ligases and / or recombinases or other methods of producing nucleic acids known to those skilled in the art.

[0156] Nucleic acids can be contained in expression vectors, wherein the nucleic acids are operatively linked to promoter sequences that promote the expression of the nucleic acids in host cells.

[0157] In a preferred embodiment, the nucleic acid encodes an imine reductase, wherein the imine reductase consists of or includes an amino acid sequence that is at least 90%, 95%, or 98% identical to the amino acid sequence of any of SEQ ID NO: 1 to 33.

[0158] As used herein, the term "vector" generally refers to any kind of nucleic acid molecule that can be used to express a protein of interest in a cell. In particular, a vector can be any plasmid or vector known to those skilled in the art as suitable for expressing a protein in a specific host cell, including but not limited to mammalian cells, bacterial cells, and yeast cells. A vector can also be a nucleic acid encoding the imine reductase of the present invention and used for subsequent cloning into a corresponding vector to ensure expression. Plasmids and vectors for protein expression are well known in the art and are available from various suppliers, including, for example, Promega35 (Madison, Wisconsin, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, California, USA), or MoBiTec (Germany). Methods of protein expression are well known to those skilled in the art and are described, for example, in Sambrook et al., 2000.

[0159] Vectors may additionally include nucleic acid sequences that allow them to replicate in host cells, such as origins of replication, one or more therapeutic genes and / or selectable marker genes, and other genetic elements known in the art, such as regulatory elements that direct the transcription, translation, and / or secretion of encoded proteins. Vectors can be used to transduce, transform, or infect cells, thereby enabling the cells to express nucleic acids and / or proteins other than those native to the cell. Vectors optionally include materials that facilitate the entry of nucleic acids into the cell, such as viral particles, liposomes, protein coatings, etc. Many types of suitable expression vectors for protein expression are known in the art using standard molecular biology techniques. Such vectors are selected from conventional vector types, including insect, e.g., baculovirus expression, or yeast, fungal, bacterial, or viral expression systems. Other suitable vectors of various types known in the art may also be used for this purpose. Methods for obtaining such vectors are well known (Sambrook et al., 2000).

[0160] As detailed above, the nucleic acid encoding the imine reductase of the present invention is operatively linked to a sequence suitable for driving protein expression in a host cell to ensure protein expression. However, it is covered within this invention that the claimed vector may represent an intermediate product which is subsequently cloned into a suitable vector to ensure protein expression. The vectors of the present invention may further comprise all types of nucleic acid sequences, including but not limited to polyadenylation signals, splice donor and splice acceptor signals, intermediate sequences, transcriptional enhancer sequences, translational enhancer sequences, drug resistance genes, etc. Optionally, the drug resistance gene may be operatively linked to an internal ribosome entry site (IRES), which may be cell cycle specific or cell cycle independent.

[0161] As used herein, the term "operably linked" generally means that gene elements are arranged such that they work synergistically for their intended purpose, such as transcription initiated by a promoter and carried out via a DNA sequence encoding the imine reductase of the present invention. That is, an RNA polymerase transcribes the sequence encoding the imine reductase into mRNA, which is then spliced ​​and translated into a protein. As used in the context of the present invention, the term "promoter sequence" generally refers to any kind of regulatory DNA sequence operably linked to a downstream coding sequence, wherein the promoter is capable of binding to an RNA polymerase and initiating transcription of the encoded open reading frame in the cell, thereby driving the expression of the downstream coding sequence. The promoter sequence of the present invention can be any kind of promoter sequence known to those skilled in the art, including but not limited to constitutive promoters, inducible promoters, cell cycle-specific promoters, and cell type-specific promoters. Furthermore, the present invention also includes a host cell containing the imine reductase of the present invention, a nucleic acid encoding the imine reductase, or a vector containing a nucleic acid encoding the imine reductase.

[0162] Example

[0163] method

[0164] Synthesis of pre-formed imines:

[0165] Imines are synthesized according to one of the following two procedures.

[0166] Program A:

[0167] Synthesize pre-formed imine substrates according to a slightly modified procedure described by Carlson et al. (1992).

[0168] The reaction was carried out in a 100 mL three-necked round-bottom flask equipped with a magnetic stir bar, a 50 mL dropping funnel, and an argon inlet. Dry toluene (10 mL) and the desired amine (9 mmol, 3 equivalents) were added to the flask. TiCl4 (1 M in dichloromethane / toluene, 3 mmol, 1 equivalent) was added via the dropping funnel over a period of 10 minutes while the mixture was cooled in an ice bath. The reaction mixture was then stirred at room temperature for 10 minutes, after which the desired carbonyl group (3 mmol, 1 equivalent) was added. The reaction was stirred overnight at room temperature.

[0169] For post-processing, the formed titanium complex was precipitated by adding diethyl ether (20 mL). After further stirring for 20 minutes, the reaction mixture was filtered through diatomaceous earth and washed with diethyl ether. The solvent was removed under reduced pressure. The product was purified by rapid column chromatography.

[0170] Program B:

[0171] Synthesize pre-formed imine substrates according to the procedure described by Yasukawa et al. (2022).

[0172] The reaction was carried out in a 20 mL glass screw-cap vial equipped with a magnetic stir bar and a molecular sieve (5 Å, 2 mm diameter, 1 g) placed on a stirring hot plate in a glove box. Toluene (5 mL), the desired carbonyl group (3 mmol, 1 equivalent), and the desired amine (3–15 mmol, 1–5 equivalents) were added to the vial, and the reaction was stirred overnight at 120 °C or for 72 h at 60 °C.

[0173] For post-processing, the reaction was cooled to room temperature and removed from the glove box. The mixture was filtered through diatomaceous earth and the solvent was removed under reduced pressure.

[0174] Enzyme preparations:

[0175] For the acquisition of the imine reductase gene and the construction of the expression vector, an imine reductase (IRED) open reading frame was designed and synthesized based on the reported amino acid sequence of imine reductase for expression in *E. coli*, and codon optimization was performed using an algorithm from Twist Bioscience (South San Francisco, USA). Stop codons were added to the ends in all cases. Restriction sites for subsequent cloning in the vector of interest, pET-21(+), were added to the nucleotide sequence; an NdeI restriction sequence was added to the 5′ end, and an XhoI restriction sequence was added to the 3′ end. The vector contained a coding sequence for ampicillin resistance. Expression was controlled by the lac promoter according to the cloning strategy. The resulting plasmid was transformed into *E. coli* BL21(DE3) using standard methods.

[0176] Plasmids from Twist Bioscience were resuspended in sterile water. Inoculation into *E. coli* BL21(DE3) cells was achieved by heating (42°C for 45 s). The pre-culture was incubated overnight at 37°C on Luria Bertani agar plates containing 100 μg / mL ampicillin. Single colonies were selected and incubated overnight at 37°C in liquid LB medium containing 100 μg / mL ampicillin.

[0177] Cells were inoculated into Terrific Broth (TB) medium containing 100 μg / mL ampicillin using liquid LB culture. After incubation at 37°C for 3.5 hours, isopropyl-β-D-thiogalactoside (IPTG) was added to the TB culture at a final concentration of 1 mM to induce IRED expression. Incubation was continued overnight at 20°C. Cells were collected by centrifugation (4°C, 8000 rpm, 20 min), and the supernatant was discarded. Cells were resuspended in potassium phosphate buffer (100 mM, pH 7). They were then sonicated (02:30 min, 2 sec on, 4 sec off, 20% amplitude). The sonicated cells were centrifuged (4°C, 17000 rpm, 20 min), and the lysates were frozen at -20°C and subsequently lyophilized. The lyophilized enzyme preparation was stored at -20°C.

[0178] Reaction settings:

[0179] Small-scale biotransformation using a clear, cell-free extract or lyophilized, cell-free extract of *E. coli* BL21(DE3) overexpressing imine reductase (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted at pH 8.0 in 100 mM bicine buffer. A pre-formed imine was dissolved in methanol at a concentration of 200 mM and then partitioned into the reaction system, resulting in a final concentration of 20 mM. NADP was added... + Sodium phosphite (1 mM), sodium phosphite (200 mM), and phosphite dehydrogenase (4 g / L) or glucose (200 mM) and glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared from water. The reaction system was incubated at 25 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 to 24 hours.

[0180] To determine the conversion rate of the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imine, product amine, ketone, and amine produced from the hydrolysis of imine.

[0181] Preparative-scale biotransformations were carried out using a clarified or lyophilized cell-free extract with IRED overexpression as a biocatalyst in a total volume of 30 mL. Unless otherwise specified, reactions using IRED were conducted at 30 °C in a 100 mL flask equipped with a mechanical stirrer and including pH and temperature sensors. The reactions were carried out at pH 8.0 and kept constant using 1 M NaOH solution and a titration system from Metrohm (Titrando 902, touch-sensitive). The concentrations listed in the following reaction descriptions are relative to the initial volume (30 mL).

[0182] The reaction system was prepared in an aqueous solution containing 100 mM pH 8 bicine buffer, with lyophilized IRED (8-4 g / L) and NADP added. + (1 mM), sodium phosphite (200 mM) and phosphite dehydrogenase (8-4 g / L) or glucose dehydrogenase (200 mM) and glucose dehydrogenase (0.1 g / L). Finally, the pre-formed imine (final concentration in the reaction system 100 mM-200 mM) was dissolved in methanol (10% v / v) and transferred to the reaction system.

[0183] The pH was kept constant by adding 1 M NaOH.

[0184] Samples were taken at different reaction times and analyzed by GC to determine the progress of the reaction. Enantiomer excess was analyzed by GC or HPLC. The GC and HPLC methods used are described below.

[0185] At the end of the reaction, the pH was increased by adding Na₂CO₃, followed by liquid-liquid extraction with an organic solvent (ethyl acetate) to separate the primary or secondary amines. Subsequently, the solvent was evaporated under reduced pressure, and the product amine was separated into a solid or an oil.

[0186] GC methods:

[0187] The sample (500 μL) of the reaction system was alkalized with 2M Na₂CO₃ (200 μL), then extracted with ethyl acetate (500 μL, twice) and the mixture was vigorously shaken. The reaction solution was centrifuged at 14,100 x g for 5 min and the organic layer was transferred to a glass vial for analysis.

[0188] Use GC analysis to determine conversion rates:

[0189] Method 1

[0190] Column: DB-1701 (30 m × 250 μm × 0.25 μm)

[0191] Inlet temperature: 250℃

[0192] Detector temperature: 300℃

[0193] Injection volume: 1 μL

[0194] Flow split ratio: 90:1

[0195] Oven temperature program: 40℃, 2 min; 10℃ / min to 300℃; 300℃, 2 min

[0196] Total running time: 30 min

[0197] Method 2

[0198] Column: RTX5 amine (30 m × 250 μm × 0.25 μm)

[0199] Inlet temperature: 200℃

[0200] Detector temperature: 321℃

[0201] Injection volume: 1 μL

[0202] Flow split ratio: 20:1

[0203] Oven temperature program: 100℃, 0 min; 40℃ / min to 320℃; 320℃, 2 min

[0204] Total running time: 6.5 min

[0205] Method 3 (Chirality)

[0206] Column: BGB-178 (30 m × 250 μm × 0.25 μm)

[0207] Inlet temperature: 200℃

[0208] Detector temperature: 321℃

[0209] Injection volume: 1 μL

[0210] Flow split ratio: 40:1

[0211] Oven temperature program: 85℃, 0 min; 1.3℃ / min to 115℃; 115℃, 0 min

[0212] Total running time: 15 min

[0213] HPLC method:

[0214] The sample (500 µL) of the reaction system was alkalized with 2M Na₂CO₃ (200 µL), then extracted with ethyl acetate (500 µL, twice) and the mixture was vigorously shaken. The reaction solution was centrifuged at 14,100 x g for 5 min. The supernatant was used to determine the enantiomeric excess using HPLC.

[0215] Enantiomeric excess was determined using HPLC analysis.

[0216] Method 1:

[0217] Column: Daicel Chiralcel OD-H (4.6 mm × 250 mm × 5 μm)

[0218] Eluent: 99 / 1 n-heptane / 2-propanol

[0219] Column temperature: 20℃

[0220] Additive: 0.1% DEA

[0221] Flow rate: 0.5 mL / min

[0222] Total running time: 60 min

[0223] DAD: 218 nm

[0224] Method 2:

[0225] Column: Daicel Chiralpak IC column (4.6 mm × 250 mm × 5 μm)

[0226] Eluent: 90 / 10 n-Heptane / 2-Propanol

[0227] Column temperature: 20℃

[0228] Additive: 0.1% DEA

[0229] Flow rate: 0.5 mL / min

[0230] Total running time: 60 min

[0231] DAD: 218 nm

[0232] Method 3:

[0233] Column: Daicel Chiralpak AD-3R column (2.1 mm × 250 mm × 3 μm)

[0234] Eluent: 90 / 10 methanol / water

[0235] Column temperature: 25℃

[0236] Flow rate: 0.7 mL / min

[0237] Total running time: 8 min

[0238] DAD: 245 nm

[0239] Method 4:

[0240] Column: Daicel Chiralpak AD-3R column (2.1 mm × 250 mm × 3 μm)

[0241] Eluent: 65 / 35 20 mM ammonium formate pH 9.0 + 5% acetonitrile / 20 mM ammonium formate + 90% acetonitrile

[0242] Column temperature: 40℃

[0243] Flow rate: 1.5 mL / min

[0244] Total running time: 15 min

[0245] DAD: 218 nm

[0246] Method 5:

[0247] Column: Daicel Chiralpak AD-3R column (2.1 mm × 250 mm × 3 μm)

[0248] Eluent: 60 / 40 20 mM ammonium formate pH 9.0 + 5% acetonitrile / 20 mM ammonium formate + 90% acetonitrile

[0249] Column temperature: 40℃

[0250] Flow rate: 1.2 mL / min

[0251] Total running time: 15 min

[0252] DAD: 218 nm

[0253] Method 6:

[0254] Column: Daicel Chiralpak IG-3 column (4.6 mm × 250 mm × 3 μm)

[0255] Eluent: 70 / 30 20 mM ammonium formate / acetonitrile

[0256] Column temperature: 35℃

[0257] Flow rate: 1.0 mL / min

[0258] Total running time: 12 min

[0259] DAD: 210 nm

[0260] Method 7:

[0261] Column: Daicel Chiralpak IG-3 column (4.6 mm × 250 mm × 3 μm)

[0262] Eluent: 70 / 30 20 mM ammonium formate / acetonitrile

[0263] Column temperature: 30℃

[0264] Flow rate: 1.3 mL / min

[0265] Total running time: 13 min

[0266] DAD: 210 nm

[0267] Method 8:

[0268] Column: Daicel Chiralpak AD-3R column (2.1 mm × 250 mm × 3 μm)

[0269] Eluent: 50 / 50 20 mM ammonium formate pH 9.0 + 5% acetonitrile / 20 mM ammonium formate + 90% acetonitrile

[0270] Column temperature: 40℃

[0271] Flow rate: 1.5 mL / min

[0272] Total running time: 25 min

[0273] DAD: 210 nm

[0274] Method 9:

[0275] Column: Daicel Chiralpak IG-3 column (4.6 mm × 250 mm × 3 μm)

[0276] Eluent: 50 / 50 20 mM ammonium formate / acetonitrile

[0277] Column temperature: 30℃

[0278] Flow rate: 1.3 mL / min

[0279] Total running time: 12 min

[0280] DAD: 210 nm

[0281] Method 10:

[0282] Column: Daicel Chiralpak IG-3 column (4.6 mm × 250 mm × 3 μm)

[0283] Eluent: 55 / 45 20 mM ammonium formate / acetonitrile

[0284] Column temperature: 30℃

[0285] Flow rate: 1.3 mL / min

[0286] Total running time: 18 min

[0287] DAD: 210 nm

[0288] The method used in Example 1:

[0289]

[0290] Comparison examples:

[0291] Reductive amination of selected IRED

[0292] Based on scientific principles (Sharma et al., 2018), the reductive amination of aromatic ketones and primary amines is expected to proceed via an intermediate aromatic imine in the presence of an enzyme with imine reductase activity, which is reduced in the presence of the enzyme to provide a chiral aromatic amine.

[0293] This example illustrates an attempt at direct reductive amination of the aromatic ketone precursor acetophenone and the primary amine 3-phenylpropylamine, and a measurement of the conversion to the desired chiral secondary aromatic amine 3-phenyl-N-(1-phenylethyl)propane-1-amine.

[0294] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) with different imine reductase (IRED) overexpression as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted in bicine buffer at pH 8.0. NADP was added... +Sodium phosphite (1 mM), sodium phosphite (200 mM), phosphorous acid dehydrogenase (4 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared in water. Acetophenone and 3-phenylpropylamine were dissolved in methanol at a concentration of 200 mM and then partitioned into the reaction system. The final substrate concentration was 20 mM. The reaction system was incubated at 25 °C in an Eppendorf thermostatic shaker with vigorous stirring for 24 hours.

[0295] The reaction was quenched by adding 2 M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0296] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0297] Table A: Screening results of IREDs used for the direct reductive amination of acetophenone with 3-phenylpropylamine to 3-phenyl-N-(1-phenylethyl)propane-1-amine

[0298]

[0299]

[0300] Representative reactions from the compounds listed in Example 1 were also tested for direct reductive amination parallel to the imine reduction reaction. For the examples reported below, direct reductive amination did not produce an amine product.

[0301] Example 1:

[0302] Screening for the reduction of different pre-formed imines by IRED.

[0303] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) with different overexpressed imine reductases (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted at pH 8.0 in 100 mM bicine buffer. Pre-formed imines were dissolved in methanol at a concentration of 200 mM and then partitioned into the reaction system, resulting in a final concentration of 20 mM. NADP... +Sodium phosphite (1 mM), sodium phosphite (200 mM), phosphorous acid dehydrogenase (4 g / L), or glucose (200 mM) and GDH (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared from water. The reaction system was incubated at 25 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 to 24 hours.

[0304] In small-scale biotransformation, the reaction was quenched by adding 2M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0305] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0306] The following summarizes the selected best results for imine reduction by IRED. For the determination of enantioselectivity, in the absence of a standard with a known stereochemical attribute, the major enantiomer is indicated by the number 1 or 2, which refers to the elution order of the enantiomers in the chiral method.

[0307] Table 1: Screening results of IRED for the reduction of 1-phenyl-N-(3-phenylpropyl)ethane-1-imine to 3-phenyl-N-(1-phenylethyl)propane-1-amine

[0308]

[0309]

[0310] Table 2: Screening results of IREDs used for the reduction of N-1-diphenylethyleneimine to N-(1-phenylethyl)aniline

[0311]

[0312]

[0313] Table 3: Screening results of IRED for the reduction of N-(4-methoxyphenyl)-1-(naphth-1-yl)ethane-1-imine to 4-methoxy-N-[1-(1-naphthyl)ethyl]aniline

[0314]

[0315]

[0316] Table 4: Screening results of IRED for the reduction of methyl 2-phenyl-2-(phenylimino)acetate to methyl 2-anilino-2-phenyl-acetate

[0317]

[0318]

[0319] Table 5: Screening results of IRED for the reduction of N-(4-methoxyphenyl)-2,2-dimethyl-1-phenylpropane-1-imine to N-(2,2-dimethyl-1-phenyl-propyl)-4-methoxy-aniline

[0320]

[0321]

[0322] Table 6: Screening results of IRED for the reduction of N-(4-methoxyphenyl)tetrahydronaphthalene-1-imine to N-(4-methoxyphenyl)tetrahydronaphthalene-1-amine

[0323]

[0324]

[0325] Table 7: Screening results of IRED used for the reduction of N-methyl-1-phenyl-ethylimine to N-methyl-1-phenyl-ethylamine

[0326]

[0327]

[0328] Table 8: Screening results of IREDs used for the reduction of N-allyl-1-phenyl-ethyleneimine to N-(1-phenylethyl)prop-2-en-1-amine

[0329]

[0330]

[0331] Table 9: Screening results of IRED for the reduction of N-phenyl-1-(3-pyridyl)ethyleneimine to N-[1-(3-pyridyl)ethyl]aniline

[0332]

[0333]

[0334] Table 10: Screening results of IRED for the reduction of 1-phenyl-N-(3-pyridyl)ethyleneimine to N-(1-phenylethyl)pyridin-3-amine

[0335]

[0336]

[0337] Table 11: Screening results of IREDs used for the reduction of N-benzyl-1-phenyl-ethyleneimine to N-benzyl-1-phenyl-ethylamine

[0338]

[0339]

[0340] Table 12: Screening results of IREDs used to reduce diphenylimine to diphenylmethane.

[0341]

[0342]

[0343] Table 13: Screening results of IRED for the reduction of 1-(4-chlorophenyl)-N-phenyl-ethyleneimine to N-[1-(4-chlorophenyl)ethyl]aniline

[0344]

[0345]

[0346] Table 14: Screening results of IRED for the reduction of N-tert-butyl-1-phenyl-ethyleneimine to 2-methyl-N-(1-phenylethyl)propane-2-amine

[0347]

[0348]

[0349] Example 2:

[0350] Optimization of reaction temperature

[0351] For the selected enzymes (IR00005, IR00395, IR01139), further detailed testing of process parameters was conducted to determine the optimal reaction conditions. In particular, the reaction temperature was tested between 20°C and 40°C.

[0352] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) overexpressing imine reductase (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted at pH 8.0 in 100 mM bicine buffer. N-1-diphenylethyleneimine was dissolved in methanol at 200 mM and then partitioned into the reaction system, resulting in a final imine concentration of 20 mM. NADP... + 1 mM glucose, 200 mM glucose and glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in stock solutions prepared from water. The reaction system was incubated at 20 °C, 25 °C, 30 °C, 35 °C and 40 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 h.

[0353] The reaction was quenched by adding 2 M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0354] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0355] The temperature screening results are as follows:

[0356] Table 15: Temperature screening results of selected IREDs used for the reduction of N-1-diphenylethyleneimine to N-(1-phenylethyl)aniline

[0357]

[0358] Example 3:

[0359] Optimization of reaction pH

[0360] For the selected enzymes (IR00005, IR00395, IR01139), further detailed testing of process parameters was conducted to determine optimal reaction conditions. Specifically, the reaction pH was tested between values ​​of 6.5 and 9.0.

[0361] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) overexpressing imine reductase (IRED) as a biocatalyst was carried out in 0.5 mL volumes. Reactions were conducted in 100 mM bicine buffer at pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. N-1-diphenylethyleneimine was dissolved in methanol at 200 mM and then partitioned into the reaction system, resulting in a final imine concentration of 20 mM. NADP... + 1 mM glucose, 200 mM glucose and glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared in water. The reaction mixture was incubated at 30 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 hours.

[0362] The reaction was quenched by adding 2 M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0363] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0364] The pH screening results are as follows:

[0365] Table 16: pH screening results of selected IREDs used for the reduction of N-1-diphenylethyleneimine to N-(1-phenylethyl)aniline

[0366]

[0367] Example 4:

[0368] Cosolvent optimization

[0369] For the selected enzymes (IR00005, IR00395, IR01139), further detailed testing of process parameters was conducted to determine optimal reaction conditions. In particular, the reactions were tested in the presence of several 10% (v / v) co-solvents.

[0370] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) overexpressing imine reductase (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted at pH 8.0 in 100 mM bicine buffer. N-1-diphenylethyleneimine was dissolved at 200 mM in the following solvents (methanol, DMSO, acetonitrile, glycerol, tert-methyl butyl ether, toluene, cyclopentyl methyl ether, isopropyl acetate, isoamyl acetate, cyrene). TM One of the following (and cyclohexane) was then partitioned into the reaction system, with the final concentration of the pre-formed imine being 20 mM. NADP was then added. + 1 mM glucose, 200 mM glucose and glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared in water. The reaction mixture was incubated at 30 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 hours.

[0371] The reaction was quenched by adding 2 M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0372] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0373] The results of the co-solvent screening are as follows:

[0374] Table 17: Cosolvent Screening Results of Selected IREDs for the Reduction of N-1-diphenylethyleneimine to N-(1-phenylethyl)aniline

[0375]

[0376] For the selected enzymes (IR00005, IR00395, IR01139), further detailed testing of process parameters was conducted to determine optimal reaction conditions. In particular, the reactions were tested in the presence of the selected co-solvents at concentrations of 10, 20, and 30% (v / v).

[0377] Small-scale biotransformation using a clear, cell-free extract of *E. coli* BL21(DE3) overexpressing imine reductase (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted at pH 8.0 in 100 mM bicine buffer. N-1-diphenylethyleneimine was dissolved at 200 mM in the following solvents (methanol, DMSO, cyrene). TM The NADP is added to one of the solvents (such as cyclohexane) and then partitioned into the reaction system, with the pre-formed imine having a final concentration of 20 mM. The final concentration of the solvent is adjusted to 10, 20, or 30% (v / v). + 1 mM glucose, 200 mM glucose and glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared in water. The reaction mixture was incubated at 30 °C in an Eppendorf thermostatic shaker with vigorous stirring for 18 hours.

[0378] The reaction was quenched by adding 2 M Na₂CO₃ (200 µL), and the reaction mixture was then extracted with ethyl acetate (500 μL, twice). The organic layer was transferred to a glass vial for analysis.

[0379] To determine the conversion in the reaction, the peaks in the GC chromatogram were compared with peaks from known standards of imines, secondary amines and ketones, and amines obtained from the hydrolysis of imines.

[0380] The results of the co-solvent screening are as follows:

[0381] Table 18: Screening Results of Selected Cosolvent Concentrations for Selected IREDs Used to Reduce N-1-Diphenylethyleneimine to N-(1-Phenylethyl)aniline

[0382]

[0383] Example 5:

[0384] Bioconversion of 2-aniline-2-phenyl-2-phenyl-acetic acid ester to methyl 2-phenyl-2-acetic acid ester on a large scale using IRED and methyl 2-phenyl-2-(phenylimino)acetic acid ester

[0385]

[0386] Preparative-scale biotransformation of methyl 2-phenyl-2-(phenylimino)acetate to methyl 2-phenylamino-2-phenylacetate was carried out using a lyophilized, cell-free extract of IR00060 as a biocatalyst. The reaction was conducted at a scale of 3 mmol (781 mg, 23.9 g / L) at 30 °C with 8 g / L IR00060 in a 30 mL volume.

[0387] Methyl 2-phenyl-2-(phenylimino)acetate (781 mg, 3 mmol, 100 mM) was dissolved in 3 mL of methanol and added to a solution containing lyophilized IR00060 (8 g / L), lyophilized phosphorous acid dehydrogenase (4 g / L), and NADP. + The reaction mixture was prepared in a solution of bicine buffer (100 mM, pH 8.0) containing 1 mM Na2HPO3 (100 mM). The reaction mixture was stirred at 30 °C and 120 rpm for 24 hours.

[0388] Then, saturated Na₂CO₃ solution (15 mL) was added, and the product was extracted with ethyl acetate (2 × 45 mL). The combined organic phases were dried over Na₂SO₄. The product was purified by rapid column chromatography [Biotage® Sfär 25 g; cyclohexane / EtOAc; gradient (EtOAc) = 0% (2 CV), 0–20% (20 CV), 20–25% (2 CV)].

[0389] The purified amine product was obtained with a separation yield of 76%, preferentially forming the second eluting enantiomer (94% ee). The optical rotation was determined to be [α]. 20 D = –98.3° (c = 1.3, CHCl3), which, according to the literature (Zhang et al., 2014), corresponds to the (R)-enantiomer.

[0390] 2-Aniline-2-phenylacetic acid methyl ester: 1 H NMR (300 MHz, CDCl3) δ ppm 7.55 – 7.48 (m, 2H), 7.43 – 7.28 (m, 3H), 7.20 – 7.07 (m, 2H), 6.78 – 6.66 (m, 1H), 6.64 – 6.53(m, 2H), 5.10 (s, 1H), 3.74 (s, 3H).

[0391] Example 6:

[0392] Preparative-scale biotransformation of N-(1-phenylethyl)aniline using IRED and N-1-diphenylimine

[0393]

[0394] Preparative-scale biotransformation of N-1-diphenylethyleneimine to N-(1-phenylethyl)aniline was carried out using a lyophilized, cell-free extract of IR01139 as a biocatalyst. The reaction was conducted at a scale of 2.5 mmol (500 mg, 20 g / L) at 25 °C with 10 g / L IR01139 in a 25 mL volume.

[0395] N-(1-Phenylethyl)aniline (500 mg, 2.5 mmol, 100 mM) was dissolved in 2.5 mL of DMSO and added to a solution containing lyophilized IR01139 (10 g / L), lyophilized glucose dehydrogenase (0.1 g / L), and NADP. + The reaction mixture was prepared in a solution of 1 mM glucose (200 mM) and bicine buffer (100 mM, pH 7.2). The reaction mixture was stirred at 25°C for 18 hours. The pH was kept constant during the reaction using a pH stat.

[0396] After 18 hours, the conversion estimated by GC showed that the product N-(1-phenylethyl)aniline accounted for 95% of the total peak area of ​​the starting material.

[0397] The reaction pH was adjusted to 11 by adding 1 M NaOH, and the product was extracted with ethyl acetate (2 × 25 mL) and filtered through a Dicalite filter. The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product was isolated in 57% yield (290 mg isolated product, 97% purity, enantiomeric excess not determined).

[0398] N-(1-Phenethyl)aniline: 1 H NMR (600 MHz, CDCl3) δ ppm 7.37 – 7.41 (m, 2 H), 7.32 – 7.35 (m, 2 H), 7.22 – 7.26 (m, 1 H), 7.08 – 7.13 (m, 2 H), 6.66 (tt, J=7.3,1.1 Hz, 1 H), 6.51 – 6.56 (m, 2 H), 4.51 (q, J=6.7 Hz, 1 H), 4.04 (br s, 1 H), 1.54 (d, J=6.7 Hz, 3 H)

[0399] Example 7:

[0400] Bioconversion of N-[1-(3-pyridyl)ethyl]aniline to a preparative scale using IRED and N-phenyl-1-(3-pyridyl)ethyleneimine

[0401]

[0402] Preparative-scale biotransformation of N-phenyl-1-(3-pyridyl)ethylimine to N-[1-(3-pyridyl)ethyl]aniline was carried out using a lyophilized, cell-free extract of IR00005 as a biocatalyst. The reaction was conducted at a scale of 2.5 mmol (392.5 mg, 15.7 g / L) at 25 °C with 8 g / L IR00005 in a 25 mL volume.

[0403] N-Phenylacetyl-1-(3-pyridyl)ethyleneimine (392.5 mg, 2.0 mmol, 80 mM) was dissolved in 2.5 mL of methanol and added to a solution containing lyophilized IR00005 (8 g / L), lyophilized glucose dehydrogenase (0.1 g / L), and NADP. + The reaction mixture was prepared in a solution of 1 mM glucose (200 mM) and bicine buffer (100 mM, pH 8.0). The reaction mixture was stirred at 25°C for 24 hours. The pH was kept constant during the reaction using a pH stat.

[0404] After 6 hours, the conversion estimated by GC showed that the product N-[1-(3-pyridyl)ethyl]aniline accounted for 91% of the total peak area of ​​the starting material. The conversion remained constant until the reaction was stopped after 24 hours.

[0405] The reaction pH was adjusted to 11 by adding 1 M NaOH, and the product was extracted with ethyl acetate (3 × 30 mL) and filtered through a Dicalite filter. The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product was isolated in 85% yield (356 mg isolated product, 95% purity, 98% enantiomeric excess).

[0406] N-[1-(3-pyridyl)ethyl]aniline: 1H NMR (600 MHz, CDCl3) δ ppm 8.67 (d, J=2.2 Hz, 1 H), 8.51 (dd, J=4.8, 1.7 Hz, 1 H), 7.69 – 7.72 (m, 1 H), 7.23 – 7.27 (m, 1 H), 7.12 (dd, J=8.6, 7.3 Hz, 2 H), 6.68 – 6.72 (m, 1 H), 6.50 – 6.54 (m, 2 H), 4.52 – 4.61 (m, 1 H), 3.96 – 4.15 (m, 1 H), 1.57 (d, J=6.7 Hz, 3 H)

[0407] Example 8:

[0408] Bioconversion of N-(2,2-dimethyl-1-phenylpropane-1-imine to N-(2,2-dimethyl-1-phenyl-propyl)-4-methoxy-aniline on a preparative scale using IRED and N-(4-methoxyphenyl)-2,2-dimethyl-1-phenylpropane-1-imine

[0409]

[0410] Preparative-scale biotransformation of N-(4-methoxyphenyl)-2,2-dimethyl-1-phenylpropane-1-imine was carried out using the lyophilized, cell-free extract of IR00005 as a biocatalyst. The reaction was conducted at a scale of 2.0 mmol (534.7 mg, 21.4 g / L) at 25 °C with 8 g / L IR00060 in a 25 mL volume.

[0411] N-(2,2-dimethyl-1-phenyl-propyl)-4-methoxy-aniline (392.5 mg, 2.0 mmol, 80 mM) was dissolved in 2.5 mL of methanol and added to a solution containing lyophilized IR00060 (8 g / L), lyophilized glucose dehydrogenase (0.1 g / L), and NADP. + The reaction was carried out in a solution of bicine buffer (100 mM, pH 8.0) containing 1 mM glucose and 200 mM glucose. The reaction was stirred at 25°C for 24 hours.

[0412] After 24 hours, the conversion estimated by GC showed that the product N-(2,2-dimethyl-1-phenyl-propyl)-4-methoxy-aniline accounted for 92% of the total peak area of ​​the starting material.

[0413] The reaction pH was adjusted to 11 by adding 1 M NaOH, and the product was extracted with ethyl acetate (3 × 50 mL) and filtered through a Dicalite filter. The combined organic phases were dried over Na₂SO₄ and the solvent was removed under reduced pressure. The product was isolated in 82% yield (460 mg isolated product, 96% purity, enantiomeric excess not determined).

[0414] N-(2,2-Dimethyl-1-phenyl-propyl)-4-methoxy-aniline: 1 H NMR (600 MHz, CDCl3) δ ppm7.27 – 7.35 (m, 3 H), 7.18 – 7.24 (m, 1 H), 6.61 – 6.69 (m, 2 H), 6.40 – 6.48 (m, 2 H), 4.01 (br s, 1 H), 3.94 – 3.98 (m, 1 H), 3.64 – 3.70 (m, 3 H), 0.96 –1.04 (m, 9 H)

[0415] References

[0416] Afanasyev, OI, Kuchuk, E., Usanov, DL and Chusov, D., 2019. Reductive amination in the synthesis of pharmaceuticals. Chemical reviews, 119(23), pp.11857-11911.

[0417] Aleku, GA, France, SP, Man, H., Mangas-Sanchez, J., Montgomery, SL, Sharma, M., Leipold, F., Hussain, S., Grogan, G. and Turner, NJ, 2017. A reductive aminase from Aspergillus oryzae. Nature Chemistry, 9(10), pp.961-969.

[0418] Carlson, R., Larsson, U. and Hansson, L., 1992. Efficient synthesisof imines by a modified titanium tetrachloride procedure. Acta ChemicaScandinavica, 46, pp.1211-1211.

[0419] France, S.P., Howard, R.M., Steflik, J., Weise, N.J., Mangas‐Sanchez,J., Montgomery, S.L., Crook, R., Kumar, R. and Turner, N.J., 2018.Identification of novel bacterial members of the imine reductase enzymefamily that perform reductive amination. ChemCatChem, 10(3), pp.510-514.

[0420] Roiban, G.D., Kern, M., Liu, Z., Hyslop, J., Tey, P.L., Levine, M.S.,Jordan, L.S., Brown, K.K., Hadi, T., Ihnken, L.A.F. and Brown, M.J., 2017.Efficient biocatalytic reductive aminations by extending the imine reductasetoolbox. ChemCatChem, 9(24), pp.4475-4479.

[0421] Sambrook, J.F. and Russell, D.W., 2000. Molecular cloning: Alaboratory manual, third 39 edition. Cold Spring Harbor Laboratory Press.

[0422] Sharma, M., Mangas-Sanchez, J., France, S.P., Aleku, G.A.,Montgomery, S.L., Ramsden, J.I., Turner, N.J. and Grogan, G., 2018. Amechanism for reductive amination catalyzed by fungal reductive aminases. ACSCatalysis, 8(12), pp.11534-11541.

[0423] Scheller, P.N., Lenz, M., Hammer, S.C., Hauer, B. and Nestl, B.M.,2015. Imine reductase‐catalyzed intermolecular reductive amination ofaldehydes and ketones. ChemCatChem, 7(20), pp.3239-3242.

[0424] Yasukawa, T., Nakajima, H., Masuda, R., Yamashita, Y. and Kobayashi,S., 2022. Effect of Activation Methods of Molecular Sieves on KetimineSynthesis. The Journal of Organic Chemistry, 87(21), pp.13750-13756.

[0425] Zhang, X., Staples, R.J., Rheingold, A.L. and Wulff, W.D., 2014.Catalytic asymmetric α-iminol rearrangement: new chiral platforms. Journal ofthe American Chemical Society, 136(40), pp.13971-13974.

Claims

1. A method for producing a chiral aromatic amine, comprising the steps of: a) providing an aromatic imine, and b) contacting the aromatic imine from step a) with an imine reductase, thereby stereoselectively reducing the aromatic imine to a chiral aromatic amine with the imine reductase.

2. The method according to claim 1, wherein the chiral aromatic amine is a chiral primary or secondary aromatic amine.

3. The method according to claim 1 or 2, wherein the aromatic imine has the formula I and the chiral aromatic amine has the formula II wherein R 1 substituted or unsubstituted aryl or heteroaryl, R 2 R is hydrogen, substituted or unsubstituted C 1-6 -alkyl or substituted or unsubstituted C 1-6 -alkoxycarbonyl, or R 1 and R 2 together form a carbocyclic or heterocyclic ring, and R 3 is hydrogen or substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C 1-6 -alkyl or substituted or unsubstituted C 2-6 -alkenyl, and wherein substituents are selected from halogen, halogen C 1-6 - alkyl, C 1-6 - alkyl, hydroxy, aryl, C 1-6 - alkoxy, aryloxy or C 1-6 - alkoxycarbonyl or aryloxycarbonyl, cyano or nitro.

4. The method according to claim 3, wherein R 1 substituted or unsubstituted aryl or heteroaryl, R 2 R is hydrogen, C 1-6 -alkyl or C 1-6 -alkoxycarbonyl, or R 1 and R 2 together form a carbocyclic or heterocyclic ring, and R 3 is hydrogen or substituted or unsubstituted aryl or heteroaryl, substituted or unsubstituted C 1-6 -alkyl or C 2-6 -alkenyl, and wherein substituents are selected from C 1-6 -alkyl, C 1-6 -alkoxy or aryl.

5. The method according to any one of claims 1 to 4, wherein the concentration of the aromatic imine is 2 mM to 1000 mM.

6. The method according to any one of claims 1 to 5, wherein the stereoselective reduction in step b) is performed in the presence of a cofactor and a cofactor regeneration system comprising an enzyme capable of reducing such cofactor.

7. The method of claim 6, wherein the co-factor is NADP + or NAD + and the enzyme capable of reducing the co-factor is phosphite dehydrogenase or glucose dehydrogenase.

8. The method according to any one of claims 1 to 7, wherein the stereoselective reduction in step b) is performed in an aqueous environment having a pH of 5.0 to 10.0 at a temperature of 5 °C to 50 °C.

9. The method according to any one of claims 1 to 8, wherein the stereoselective reduction in step b) is performed at a sodium phosphite or glucose concentration of 100 mM to 1 M and / or at a phosphite dehydrogenase or glucose dehydrogenase concentration of 0.1 g / L to 10 g / L.

10. The method according to any one of claims 1 to 9, wherein the aromatic imine and the imine reductase contacted in step b) are incubated for 1 h to 100 h for the stereoselective reduction.

11. The method according to any one of claims 1 to 10, wherein the imine reductase has a sequence having at least 80% sequence identity to any one of SEQ ID NO. 1 to 33.

12. An imine reductase having a sequence having at least 90% sequence identity to any one of SEQ ID NO. 1 to 33.

13. Use of an imine reductase in a method for producing a chiral aromatic amine of formula II according to any one of claims 1 to 11. 。

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