Process for the reduction of aromatic imines with novel imine reductases (IREDS)

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

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480047661.1 (22) Application Date 2024.07.17 (30) Priority Data 23185969.5 2023.07.18 EP (85) PCT International Application Entering National Phase Date 2026.01.16 (86) PCT International Application Application Data PCT / EP2024 / 070314 2024.07.17 (87) PCT International Application Publication Data WO2025 / 017089 EN 2025.01.23 (71) Applicant: Hofmeister Roche Ltd. Address: Basel, Switzerland (72) Inventors: S. Berger, S. Bisani, F. Gösselin, C. Grin, H. Eidin, I. Mazzoli, K. Püntner, J. Schlitveiser, S. Frable (74) Patent Agency: China Patent Agency (Hong Kong) Ltd. 72001 Patent Attorneys: Li Tang, Zhang Hua (51) Int.Cl. C12N 9 / 06 (2006.01) C12P 13 / 00 (2006.01) C12P 17 / 12 (2006.01) C12P 41 / 00 (2006.01) C12P 13 / 04 (2006.01) (54) Invention Title: For use with new imine reductase (IRED) Method for Reducing Aromatic Imines (57) Abstract This invention relates to a method for producing chiral aromatic amines, the method 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; and relates to novel imine reductases capable of catalyzing the reaction. Claims 2 pages Description 31 pages Serial Listing (Electronic Publication) CN 121532499 A 2026.02.13 CN 1 21 53 24 99 A 1. A method for producing chiral aromatic amines, 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 of claim 1, wherein the chiral aromatic amine is a chiral primary aromatic amine or a secondary aromatic amine. 3. The method of claim 1 or 2, wherein the aromatic imine has formula I and the chiral aromatic amine has formula II, wherein R1 is a substituted or unsubstituted aryl or heteroaryl group, R2 is hydrogen, a substituted or unsubstituted C1-6-alkyl group, or a substituted or unsubstituted C1-6-alkoxycarbonyl group, or R1 and R2R1 and R2 are hydrogen, or a substituted or unsubstituted aryl or heteroaryl group, a substituted or unsubstituted C1-6-alkyl group, or a substituted or unsubstituted C2-6-alkenyl group, wherein the substituent is selected from halogen, halogenated C1-6-alkyl, C1-6-alkyl, hydroxyl, aryl, C1-6-alkoxy, aryloxy, or C1-6-alkoxycarbonyl or aryloxycarbonyl, cyano, or nitro. 4. The method according to claim 3, wherein R1 is a substituted or unsubstituted aryl or heteroaryl group, R2 is hydrogen, C1-6-alkyl, or C1-6-alkoxycarbonyl, or R1 and R2 together form a carbocyclic or heterocyclic ring, and R3 is hydrogen, or a substituted or unsubstituted aryl or heteroaryl group, a substituted or unsubstituted C1-6-alkyl, or C2-6-alkenyl group, wherein the substituent is selected from C1-6-alkyl, C1-6-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 carried out in the presence of a cofactor regeneration system comprising a cofactor and an enzyme capable of reducing such cofactor. 7. The method according to claim 6, wherein the cofactor is NADP+ or NAD+, and the enzyme capable of reducing the cofactor is phosphorous acid dehydrogenase or glucose dehydrogenase. 8. The method according to any one of claims 1 to 7, wherein the stereoselective reduction in step b) is carried out 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 carried out at a concentration of 100 mM to 1 M sodium phosphite or glucose and / or at a concentration of 0.1 g / L to 10 g / L phosphite dehydrogenase or glucose dehydrogenase. 10. The method according to any one of claims 1 to 9, wherein the aromatic imine and the imine reductase in contact in step b) are incubated for 1 h to 100 h to perform 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 with any of SEQ ID NO. 1 to 33. 12. An imine reductase having a sequence having at least 90% sequence identity with any of SEQ ID NO. 1 to 33. 13. An imine reductase according to claims 1 to 11.Use in any one of the methods for producing chiral aromatic amines of formula II. Claims 2 / 2 Page 3 CN 121532499 A Method for reducing aromatic imines with a novel imine reductase (IRED)

[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 with the imine reductase.

[0002] The present invention also relates to a novel imine reductase capable of catalyzing the stereoselective reduction of aromatic imines to chiral aromatic amines.

[0003] Chiral aromatic amines are structural units for developing new therapeutic drug candidates. In fact, the stereoselective synthesis of chiral aromatic amines has been an area of ​​development in recent decades because these compounds are recurring in various 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, wherein an imine is formed briefly as an intermediate and then reduced to an amine (Afanasyev et al., 2019).

[0005] Reductive amination reactions from ketone precursors and aromatic amines using reductive aminoases are also known (Aleku et al., 2017, France et al., 2018, Roban et al., 2017, Scheller et al., 2015).

[0006] However, these enzymes generally fail to carry out reductive amination reactions, particularly when aromatic ketones and / or aromatic amines are involved.

[0007] Therefore, an 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] It has been found that the object of the present invention can be achieved by a method summarized below, comprising:

[0009] a) providing an aromatic imine and

[0010] 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.

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

[0012] The terms “peptide” or “enzyme” as used herein are macromolecular biocatalysts, typically proteins, that accelerate chemical reactions. Enzymes can act on a substrate molecule, and the enzyme converts the substrate into a product. Like all catalysts, enzymes increase the reaction rate by lowering the activation energy of the reaction.

[0013] “Imine reductase / reductoaminoase” is an enzyme capable of catalyzing the reduction of imines to amino groups, most commonly to secondary amines. The term “imine reductase” includes all enzymes capable of performing imine reduction, while reductoaminoase only covers those particularly capable of...The term "imine reductase" is used to describe enzymes that are adept at the direct reductive amination of amines and ketones, because it is believed that imines are transiently formed at the active site of the enzyme, although imine reductases are also capable of reductive amination. Imine reductases and reductive aminoases catalyze the reduction of imines to amines using the reducing equivalent provided by NADH or NADPH.

[0014] According to the invention, proteins as defined herein are used as imine reductases. This means that, under conditions that allow for a reduction reaction, the protein contacts 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 having 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, hydroxyl, aryl, alkoxy, aryloxy or alkoxycarbonyl or aryloxycarbonyl, cyano, nitro.

[0017] The term “alkenyl” includes a straight-chain or branched alkenyl group with 2 to 6 carbon atoms, which carries 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 chain, which 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” represents an alkyl group as defined above, attached to an oxygen atom. A typical example is methoxy.

[0020] The term “alkoxycarbonyl” represents 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, and nitro.

[0023] The term “aryloxy” represents an aryl group as defined above, attached to an oxygen atom. A typical example is a phenoxy group.

[0024] The term “aryloxycarbonyl” represents an aryl group as defined above, attached to a carbonyl group. A typical example is a 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, pyrimidine, etc.

[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 "carbocyclic" refers to a C3-10-cycloalkane moiety, such as cyclohexane, which can fused to an aromatic ring system. A typical example is tetrahydronaphthalene, in which cyclohexane is fused to a benzene ring.

[0030] The term "heterocyclic" refers to a C3-10-cycloalkane moiety in which at least one carbon atom is replaced by a heteroatom selected from nitrogen, oxygen, or sulfur. Heterocyclic moieties can fused to aromatic ring systems.

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

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

[0033] As outlined above, the method comprises

[0034] a) providing an aromatic imine and

[0035] 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.

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

[0037] In the context of this invention, "providing an aromatic imine" preferably means that the aromatic imine is pre-formed, i.e., has been synthesized in advance and provided as a substrate 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. (Specification 2 / 31 pages, CN 121532499 A)

[0040]

[0041] wherein

[0042] R1 is a substituted or unsubstituted aryl or heteroaryl,

[0043] R2 is hydrogen, a substituted or unsubstituted C1-6-alkyl or C2-6-olefin, a substituted or unsubstituted C1-6-alkoxycarbonyl, or R1 and R2 together form a carbocyclic ring, and

[0044] R3 is hydrogen or a substituted or unsubstituted aryl or heteroaryl, a substituted or unsubstituted C1-6-alkyl, or a substituted or unsubstituted C1-6-alkoxycarbonyl,

[0045] and wherein

[0046] the substituent is selected from halogen, halogenated C1-6-alkyl, C1-6-alkyl, hydroxyl, aryl, C1-6-alkoxy, aryloxy, or...C1-6-alkoxycarbonyl or aryloxycarbonyl, cyano or nitro.

[0047] In a preferred embodiment,

[0048] R1 is a substituted or unsubstituted aryl or heteroaryl,

[0049] R2 is hydrogen, C1-6-alkyl or C1-6-alkoxycarbonyl or

[0050] R1 and R2 together form a carbocyclic or heterocyclic ring, and

[0051] R3 is hydrogen or a substituted or unsubstituted aryl or heteroaryl, a substituted or unsubstituted C1-6-alkyl or C2-6-alkenyl,

[0052] and wherein

[0053] the substituent is selected from C1-6-alkyl, C1-6-alkoxy or aryl.

[0054] In a further embodiment,

[0055] R1 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] R2 is hydrogen, C1-6-alkyl, or C1-6-alkoxycarbonyl, or

[0057] R1 and R2 together form a C3-10-carbon ring or heterocycle that may be fused to an aromatic ring; and

[0058] R3 is hydrogen, or a substituted or unsubstituted phenyl, naphthyl, or an aromatic 5- to 6-membered monocyclic or 9- to 10-membered bicyclic, substituted or unsubstituted C1-6-alkyl, or C2-6-alkenyl ring that may contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and / or sulfur;

[0059] and wherein

[0060] The substituents are selected from C1-6-alkyl, C1-6-alkoxy, or aryl.

[0061] The aromatic imine of Formula I may be present in 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 generally carried out in the presence of a cofactor regeneration system comprising a cofactor and an enzyme capable of reducing such cofactor. Specification 3 / 31 pages 6 CN 121532499 A

[0064] In a preferred embodiment, the cofactor is NADP+ or NAD+, and the enzyme capable of reducing the cofactor is phosphorous acid dehydrogenase or glucose dehydrogenase.

[0065] Preferably, the cofactor is NADP+ and the enzyme capable of reducing the cofactor is phosphorous acid dehydrogenase or glucose dehydrogenase.

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

[0067] In a preferred embodiment, the reduction reaction is carried out in an aqueous environment having a pH of 5.0 to 10.0, preferably 6.5 to 8.5, at a temperature of 5°C to 50°C, preferably 20°C to 50°C.The incubation occurs at a temperature within the range of 40°C, at a concentration of sodium phosphite or glucose of 100 to 1 M, preferably 100 mM to 500 mM, and at a concentration of phosphite dehydrogenase or glucose dehydrogenase 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, a co-solvent selected from polar protic solvents (such as lower fatty alcohols, such as methanol), dipolar aprotic solvents (such as dimethyl sulfoxide, cyrene™), or nonpolar solvents (such as cyclohexane) is applied.

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

[0070] However, the incubation time can depend on the assay form, solution volume, substrate and catalyst concentrations, amount of aromatic imine to be reduced, etc. 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 dosages 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 amine has formula II

[0074]

[0075] wherein R1, R2, and R3 are as defined above and wherein the same preferred embodiments 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%, and even more preferably at least 98% sequence identity with any of SEQ ID NO. 1 to 33.

[0077] SEQ ID 1 (IR00005)

[0078] MHHHHHHKTVAVIGLGQMGTTLARLFIEAGMQVRVWNRTRSKAEPLASRGAIVAATAAAAMADAEAVVI CVHDYRATHDILSDVAVKSALKGKLLLQLTTGSPQDARDMAELAARIGAGYLDGALQVAPEQMGQPDTTVLVSGSGE DHALARELLAVLGGNVVYLGEDVAAAATMDLATLSYVYGASMGFFQGAALAQAEGLDVGVYGGIVEAMSPSFGAFLRHEGNVIDNGDYAVSQSPLSISIDATGRIEQAMRQKGLRSELPSLIARLLRDAEEAGYGNEEFAAVAKILRGAAEPAP VR DESCRIPTION Page 4 / 31 7 CN 121532499 A

[0079] SEQ ID 2 (IR00010)

[0080] MHHHHHHTTTPTVTVLGLGPMGQALSRALLDAGHTVTVWNRTESKAQALRDRGALSAPTPAAAIAASDL ALVNVVDHDAVDAILTAAGDAPAGRTVIGLSSDTPDRARRTAKLVGNVGGRYLDGAIMTPIDTIGTRGASILFAGPQ ALFDEHRGVLDTLGQLTWVGEDHGRAAAFDMALLDLFWTSVGGFGHALMVARANGIEPSELMPHAHGIVGILSPIFT EVAQRVEDDRHSDASASVSSVASSVRHLIAASREAGVDAGLLEAFRGYVDATVAAGHGDDEISRIASEMTTLTRG

[0081] SEQ ID 3 (IR00014)

[0082] MHHHHHHTNNATPVSILGLGLMGQALARAFLKAGHPTTVWNRTPGKADQLMAEGAQVAPTAAEAIDASS LTVICVSDYPAMYELLDASDLAGTTLLNLTSGDSAQARQAARWAEQRGAHYLDGAIMAIPQAIGTDDAVILISGAQA DADAHRPTLEALGTLTYLGADHGLASLYDVAGLAMMWSVLNAWLQGTALLRTAGVDAATFAPFAQQMAAGVAGWLPG HAQEIDAGSFATEVASLDTHVRTMDHLIEECEAAGINAELPRLIKSMADRSLAAGHGAASYSVLIEEFAKPA

[0083] SEQ ID 4 (IR00015)

[0084] MHHHHHHMKAPVTVVGLGPMGKAMAETFLKNGHPTTVWNRTASKAAPLVEQGATLAATPDDALAASGLV VISQTDYKAMYDSLDGAEMKGRVLVNLSSGSPDELRRAAEWAAGKGAELLTGGVMVPPPGIGQPGAYIMYSGPEALLDRHRETLRVLGDTTYVGADVGLSNLYYQAQLYLFWSTLTAYLHSIAMLQSAGVSAEQFRPFATETVTSLGVDGPMGF LRILAEEADAGHSPGGENSMLMMAVGADHMVEAAEAAGIDTMGPRALRDLFWRTVNAGHGADGLGSVIEVVRKGA

[0085] SEQ ID 5 (IR00020)

[0086] MHHHHHHSATTNTTSADGVAGPGGPGGRPPVTVLGLGQMGAAIAGALLAAGHPVTVWNRTPGKAAPLVE QGAVLAGSVAEAVAASPLVLSVLDYPALYGILDPEPDALKGRALVNLTTGTPEQAGEAEAEWAARHGVDYLDGAIMT TPPGVGTREVMFLYSGDRAVFDAHHAALDVLGEPLHLGTEPGLAALYDVNLLGLMWATMAGWLHGTAVVGAEGTRAV DFTEVAIRWLGTVNNFIRRYAAQVDEGVYPGDDATVDVQIAVVEHQLHAAEARGVDNRLPELLKTLMLEANAKGHGQ DSFGSVVEVLRKGARR

[0087] SEQ ID 6 (IR00032)

[0088] MHHHHHHRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDITLV CVDNYAVSQLLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPAQIGTSDASIICSGASAAF SEAEPVLSLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPVAKVCAQFSELSPILGGDVAHL GKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQFGGQQEVSALIKVLRARNGAAQ

[0089] SEQ ID 7 (IR00033)

[0090] MHHHHHHRHLSVIGLGAMGSALATTLLKAGHPVTVWNRSAAKAAPLQALGATLAPSVGAAIAASDINLVCVDNYAVSQQLLDEASDAVAGKLLVQLSTGSPQGARALESWSHARGARYLDGAILCFPDQIGTSDASIICSGASAAF SDAEPVLRLLAPTLDHVAEAVGAAAAQDCAVAAYFAGGLLGALHGALICEAEGLPIAKVCAQFSELSPILGGDVAHL GKTLASGDFDHPYASLKTWSAAISRLAGHATDAGIDSRFPRFAADLFEEGVAQGLGQQEVSALIKVLRARNGAAL

[0091] SEQ ID NO: 8 (IR00041)

[0092] MHHHHHHMSSVSIFGLGAMGTALASRFLEEKYKVAVWNRSPEKASSLLGKGATLSHTAVDGINASDLII ICLLDNAAVEATLAGALDHLHGKTIINLTNGTPDQARKLSDRFVSHGARYVHGGIMATPSMIGSPYALVLYSGSPDA FKAAEGDLSVLAKCVFLGEDAGTASLHDLALLSGMYGLFSGFLHATALVRSSTPAVKFMDLLVPWLGAMTEYTKGMA KQIDEGKYTSEGSNLAMQLVGIQNIIDASEAQQVSAEFIRPMKEFMQKAVAAGHGGDDISSLIDFVKST

[0093] SEQ ID NO: 9 (IR00049)

[0094] MHHHHHHMSGDNRAPVTVIGLGMMGAALAGAYLKAGHPTTVWNRSAAKAAPLVEQGARRATDVAEAVAA SGIVVVCVFDYTVARSLLAPVRDQLAGKVIVNLTSGLPDEARETAAWAEDAGARYLDGYVMTVPPAVGLPQTLLFYG DESCRIPTION Page 5 / 31 8 CN 121532499 A GDKEIFDAHEETLKVLGGNSVHLGTDPGIAALYDLALLGILWSTLTSALHGFALVGSENVPAAALMPFAESWITHVV LPTVQGAAQQVDAGHYATDISTVELNAMGLPKMIKASEAQGVRADLMVPIKDFLEKRVADGHGADALASLIEVIRDG DR

[0095] SEQ ID NO: 10 (IR00052)

[0096] MHHHHHHMSNTKAAQAPVSVIGLGLMGQALAAAFLKAGHPTTVWNRTAAKADQLVGEGAALAGSTADAI AASPLVVVCVTDYTAVRELLDPLAGALKGKVLVNLTTGTSTQARETAEWAADKEITYLDGAIMAIPPDIATDAAVLL YSGPKAAFDEHEATLRALGAAGTTYLDTDHGLSALYDMSLLGIMWGILNGFLHGAALLGTAEVKATTFAPLANTMIN VVTEYVTAYAPQIDEGKYPAGDATMTVHQDALEHLAEESETLGINAEMPRFFKALVDRSVAAGHAESGYAALIEQFR KPAV

[0097] SEQ ID 11 (IR00054)

[0098] MHHHHHHHMKSNSQNEKNGSETTNAVGNRKSVTVIGLGPMGQAMADVFLEYGYSVTVWNRTSSKADQLVA KGAIRVSTVNEALAANELVILSLTDYNVMYSILEPVSENLFGKVLVNLSDTPEKARKAAKWLEDRGARHITGGVQV PPSGIGKSESYTYYSGDRVVFEAHRETLEVLTSSDYRGEDPGLAMLYYQIQMDIFWTAMLSYLHALAIANANGITAE QFLPYASAMMSSLPKFVEFYTPRLDEGEHPGDVDRLAMGLASVEHVVHTTQEAGIDIALPATVLEVFRRGMKTGHAS DSFTSLIEIFKNSDIRS

[0099] SEQ ID 12 (IR00060)

[0100] MHHHHHHMSTVSASESPVTVVGLGMGHALAAAFVAKGHPTTVWNRSAGKADDLVAAGATLADSVQAAV EASPLVVVCVSDYDAVHALLDPVGPALAGRTLVNLTTASSSQARDTAEWAAKLDATYLDGAILALPQGIGTDEATLL YAGPKAAFEEHQATLAVLGEAATVYLDEDHGLSALYDMAVLTIMWGVLNSFLHAAALLGTANVKATTFAGMAATAINVTADYVAAYAPQIDAGEYPATDATVNVHVGGMQHLLEESKALGVNAELPRFFLELAGRAVAGGHAEDSYAALIKQFR APSA

[0101] SEQ ID 13 (IR00063)

[0102] MHHHHHHMKPTLTVIGAGRMGSALIKAFLQSGYTTTVWNRTKAKSEPLAKLGAHLADTVRDAVKRSDII VVNVLDYDTSDQLLRQDEVTRELRGKLLVQLTSGSPALAREQETWARQHGIDYLDGAIMATPDFIGQAECALLYSGS AALFEKHRAVLNVLGGATSHVGEDVGHASALDSALLFQMWGTLFGTLQALAISRAEGIPLEKTTAFIKLTEPVTQGA VADVLTRVQQNRLTADAQTLASLEAHNVAFQHLLALCEERNIHRGVADAMYSVIREAVKAGHGKDDFAILTRFLK

[0103] SEQ ID 14 (IR00064)

[0104] MHHHHHHMSTPPHTTAGPAAVTVLGLGRMGSALAAAFLAAGHSTTVWNRTPGKADELAARGARRAGSVA EAVAAAPLVVVCVADDEAVHQLLDPLDGALAGRTLVNLTTGTSAQARANAAWAKERGAAFLDGAIMAVPEDIATGDA VLLYSGPRDAFDAYEEALRVLAPAGTTHLGGDAGLAALHDLALLGIMWGVLNGFLHGAALLGTAGVRAGDFAPLAAR MTTVVAGYVTAAAPEVDAGSYPAGDATLTVHQEAMRHLAEESEALGVNAELPRFLQLLAGRAVAEGHAESGYSALVE QFRKA

[0105] SEQ ID 15 (IR00065)

[0106] MHHHHHHMKPHISILGAGRMGSALVKAFLQNEYTTTVWNRTRARCEPLAAAGARIADSVRDAVQTASVV IVNVNDYDTSDALLRQDEVTQELRGKVLVQLTSGSPKLAREQATWARRHGIDYLDGAIMATPDLIGRPDCTLLYAGPKALYDKHQAVLAALGGNTQHVSEDEGHASALDSAILFQLWGSLFSGLQAAAICRAEGIALDALGPHLEAVAAMIQFS MKDLLQRIQKEQYGADPQSPATLDTHNVAFQHLLHLCEERNIHHALPKAMDALIQTARKAGHGQDDFSVLARFLR

[0107] SEQ ID NO: 16 (IR00066)

[0108] MHHHHHHMSVSVLGLGPMGQALTRALLNANHRTTVWNRTAAKADDVVARGAVWADDPASAIAAGDLTLV DESCRIPTION Page 6 / 31 9 CN 121532499 A NVVDQQAVDAVLTAAGDAVAGRVIIGLSAGTPDAARRTASFVTGAGGAYLDGAIMTPTDTIGTASASVLFSGPRELF DDHREVLSALGTLTWLGEDVGRAEAFDVALLDLFWTSVSGFVHALSIAKANGVSAVELLPHANGISEILPAIFTEIA ERVESGRHDDASAPVSSVAASLRNLISAARSAGIDAGALETFRDYVDAAVAAGHGDAEISRITPLGIG

[0109] SEQ ID NO: 17 (IR00073)

[0110] MHHHHHHMKNDGVTKGSVALLGLGEMGRVLAERLLDAGYPVTVWNRTPGRDTALVERGARRAETVREAV TAATTVVTCLFDHASVRETLEPVGADLAGRTLVDLTTTTPNEARWLGGWAEERGIEHLDGAIMATPSMIGAPEASLL YSGSAEAFGRHRTLFEVWGSATYDGADHGAASLFDLALLSGMYTMFTGFAHGAAMVTSAGVTAEEFAHRSARLLSAM TGVFPMTAKVIDEGDYTGPGQSLEWTATALDTIARASAEQGVSPGPIEMTRALVLAQIEAGYGNENSDRIYEELRAG

[0111] SEQ ID NO: 18 (IR00075)

[0112] MHHHHHHMAATPTNPSTDSGKTPVTVLGLGAMGRALAGAFLKAGHPTTVWNRSEHKADELVARGAVRAGSVAEAVAASPLIVVCVVDYEVSHRILEPVGADLAGRVLVNLTSDTPVRSRRAAEWAAGHGVEYLDGAIMVPTPVIGT PEATVLYSGSRRAFDTYEETLKALGGKAPFLGTDHGVAAVYDLAMLSFFYSGMAGLAHAFTLAGEEGVPATDLAPFL DVITGIFPPIAKGMADDLVGGRLDGAGEGNIVMEAAGIAHIVEASRDRGVNTDVLDALKALMDRTIAAGHGESEFVR VTEAMRGAYA

[0113] SEQ ID 19 (IR00100)

[0114] MGSSHHHHHHSSGLVPRGSHMPDNPSTKGRMMRNQQAEHTPVTVIGLGLMGQALAGAFLGAGHPTTVWN RTAAKAEPLVARGAKSAGSVAEAVAASPLVVVCVSDYDAVHALLDPLDGTALQGRTLVNLTSGTSAQARERAAWADG RGADYLDGAILAGPAAIGTADAVVLLSGPRSAFDPHASALGGLGAGTTYLGADHGLASLYDAAGLVMMWSILNGFLQ GAALLGTAGVDATTFAPFITQGIGTVADWLPGYARQIDDGAYPADDAAIDTHLATMEHLIHESEFLGVNAELPRFIK ALADRAVADGHGGSGYPALIEQFRTHSGK

[0115] SEQ ID 20 (IR00202)

[0116] MGSSHHHHHHSSGLVPRGSMTDTSAKLTLLGLGAMGSALATAWLAADYDITVWNRTASRAEPLRTLGAQ VADTAADAVAANDLVVACLLDDASVRSTLDGVDLTGKDLINLTTGTPGSGRELAACATARGARYVDGGIMAVPPMIG VPDSGAYVFYSGSAAAFEAHRDALAVGAGTKFVGEDPGFAALYDVALLSAMTGMFAGVSHAFALVRKENIDPREFAG LVSGWITAMSGYAHGIAEHLASGDYTTGVTSNLAMMVEGNATMLRTAHEQGVSPELLEPFMRLMRQRVDDGHGDEDT TGVIDLLLSRR

[0117] SEQ ID 21 (IR00376)

[0118] MRSEPAAVTVLGLGSMGTALAGALLKCGHATTVWNRSPHKTGPLAERGATVAATPEEAVAASPLVIACV LDYAALHAVLDPVADSLAGKTLVNLTSGSPEQAAEAAAWARSHGAHYLDGAIMTTPPGVGSPEMMFLYSGERTVLDT HRPVLASLGDPLYLGTDPGLASLYDAALLGLMWATMTGWLHGTALVGAEGTPATAFTPVAIRWLSAVTGFLTTYAPQ VDAGHYPGDDATVDVQIAAIDHLIHAAAARGVDNALPGLLKAAMERTRAAGHGSSSYASVIEVLRKAADAR

[0119] SEQ ID 22 (IR00393)

[0120] MGSSHHHHHHSSGLVPRGSMGDNRTPVTVIGLGLMGQALAAAFLEAGHTTTVWNRSAGKAEQLVSQGAV QAATPADAVAASELVVVCLSTYDNMHDVIGSLGESLRGKVIVNLTSGSSDQGRETAAWAEKQGVEYLDGAIMITPPG IGTETAVLFYAGTQSVFEKYEPALKLLGGGTTYLGTDHGMPALYDVSLLGLMWGTLNSFLHGVAVVETAGVGAQQFL PWAHMWLEAIKMFTADYAAQIDAGDGKFPANDATLETHLAALKHLVHESEALGIDAELPKYSEALMERVISQGHAKN SYAAVLKAFRKPSE

[0121] SEQ ID 23 (IR00395)

[0122] MGSSHHHHHHSSGLVPRGSHMDNETAPVTVIGLGLMGRALAGAFLRAGHPTTVWNRTASKAEQLVAEGA DESCRIPTION Page 7 / 31 10 CN 121532499 A RLAPTVGDALEASSVAIVCLTDYEVVHELLGAGEIKLDGTLLINLTSGDSTQAREAARWAEQRGARYLDGAIMAVPP AIGTAEAMILLSGPQSDFESHKAMLGALGGTTYLGADHGLASLYDVAGLAMMWSILNAWLQGSALVGTADVDAATFTPFAQQLASVVVEWLPGYAEQVDSGSFPAEVSALETDVRAMTHLIEESEAVGVNAEMPRLFKAIADRSIVAGHGGEQY PVLIEEFRKPRDT

[0123] SEQ ID 24 (IR00464)

[0124] MTSEQRASVTVLGTSMGSALARAFLAAGYRTHVWNRSAARTTELVAAGATAHRDITDAVEASPVIIAC LSTFAATHASLSPSTGILAGRDIITLNSGTPAQAREFAQWVRCAGARFLGGAIKNVPAAVGDRNTLLYFGGDRVFD AHTGTLRVLGGELDFLGVETDLAALYESAVGATLLPALLGFLEGAAMLASRGLPAHTMVPYSVKWLRMIESLLPVLA EEIDTGNYTLLGSSVELFHNALADDRQLAAESGVDLSWHAPMHDLLRRAVAEGRGDQSITALIELLTARP

[0125] SEQ ID 25 (IR00653)

[0126] MPTGTTVSSAVSSPVTPPLAVTVIGLGAMGSALAGAFLDAGHSVTVWNRTPGKGDALAARGAVVAATAE EAVTASELIVVCLVDYDASEAILTPLAKALSGRTLVNVTADVPDRARTAAAWAAEHGIAYLDGAVMVPTAVVGTPAG LLFYSGDPTTFERYEPVLRALGGRTVHGDSPDRAAVFDVALLDLFYGAMGAMIHAFALARAHGVPAAEVVPYMTSI VDLLPEALEAMAGDIDARSYPALTAGLGTMAASDVDHIVHAGRAAGIDSAQMEGIQRMTDRARELGHADSGWAANYEA LINPRTRDRS

[0127] SEQ ID 26 (IR00887)

[0128] MEGFPMGTNAHTGTATETAGTLAEQRSVTVIGLGPMGRAMADAYLDRGYEVTVWNRTSRADDLVARGA TLAPDVARALTANDLVILSLTDYEAMYAILEPATSALAGRVLVNLTSDTPDKARAAARWAADHGAVQLTGGVTVPPSGIGRPESSTFYSGPREVFDAHRPVLEVVTGKADYRGEDPGLAALYYQMGMVMFWTSMLGYWQAIALADANGLAASDI LGHAVDTANSLPGFFTFYAGRIDAGAHQGDVDRLAMGLASVEHVLHTNADAGVDTALPAAVVEIFRRGIKAGHGADS FSSLLELMKKGGES

[0129] SEQ ID 27 (IR01134)

[0130] MSNPNAAQAPVTVIGLGLMGQALAAAFIKQGHPTTVWNRTPGKAEQLTADGAILAQSAGKAISASPLVI VCVSDYDGVHEILGPLQGALAGKVLVNLSTGTSAQARETAEWAAQRGARYLDGAIMAIPPVIGTDGAVLLYSGPQDS FEEHAATLRALGTPGTTYLGGDHGLSALDMALLGLMWGILNGFLHGAALLGTAGVKATTFAPLANQMIKEVTEYVT AYAPQVDEGKYPATDATLTVHQDALEHLADESKAVGINSELPRFFKALVDRAAADGRADESYAALIEVFRKEAA

[0131] SEQ ID 28 (IR01136)

[0132] MSTLPASGAPVTVIGLGLMGQALAGAFLAKGHPTTVWNRSAAKADDLVARGAVLADSVQSAVEASPLVV VCVSDYDAVHQLLGPVGGSLAGRTLVNLTTASSTQARETAEWAQKLDAAYLDGAILALPHAIGTEEATLLYYAGPQAT FEEHRATLEVLGAEATVYLDEDHGLSALYDLSVLSIMWGVLNSFLHGAALLGTANVKASTFAELAVTAINVTAEYAS AYAQQIDAGEYPATDATVNVHVGGMTHLVEESETLGVNAELPRFFLELAKRAVAAGDAENSYASLIKQFRKPSA

[0133] SEQ ID 29 (IR01139)

[0134] MSTQQTTVTVLGLGLMGRALAAAFLRAGHPTTVWNRTANKADQLVSEGARQAPTLVDALAASPLTIICLTDYQAVRELLDTHDLDLGATTLINLTSGDSAQARTAAAWAASRGARYLDGAIMAIPSAIGTAEAVILHSGPHADFDS HRPILDALGTVTYLGADHGLASLYDVAGLAMMWSVLNAWLHGTAMLRTAGVDAAAYAPFAQQIAAGVASWLPGYAEQ IDRGAFPAQVSALETDVRAMAHLLAESAAVGVNTELPELIKAMADRAIAAGHGAEQYPVLIEEFAKAASAPPR

[0135] SEQ ID NO: 30 (IR01421)

[0136] MNTNPKRFSNGNHPYDAQADGSILGPVTVIGLGAMGSKLARTFVNNGYVTTVWNRTPEKAAELVGLGAS GTTDIADAIAASPLLIVCVLDYDAARHILEPMREKLSGKVVVNLTTGTPEEARTMGTWMRTQGAEYLDGGIMAVPSL DESCRIPTION Page 8 / 31 11 CN 121532499 A IATPHAVILYSGSQTAYETYKQVLERLGTSHYLGSDPALAPLNDLALLSGMYGMFGGFIHAVALAGSEGTKAADFTT TLLIPFLQAMIFTMPEMANQIDTGDYSAKDASLAMQAAHDTIGEVSRVQGVSAETFAPIFELMKRRVEEGYGGDDFA SVIELIRKRPSS

[0137] SEQ ID NO: 31 (IR01598)

[0138] MGSSHHHHHHSSGLVPRGSMHERPPLSVTVLGLGTMGTALASALLDTGHAVTVWNRTASRATPLITRGA ARAGDVGEALAASRVIVTCLLDHASVHDVLDDHASALTGRTLVNVTNTTPEGSAELASWAASHGADFLDGGIMAVPP QIGTPSAFVLYSGSPSALETARPALEAFGDVRYLGSDPSLAALQDTALLSGMYGLFGGILHAFALVRTGGVTARQFA PVLREWLTGMVGWASSAAERIDDGDHARDVASNLAMQATAYEGFLAVARDRGMNPLLLDPLGRLLRRRVAEGFGHED ITGVIDYLTASTNTTGASA

[0139] SEQ ID NO: 32 (IR01855)

[0140] EDVFRALGGNVQHIGTEPGLANALDSAVLALMWGALFGGLHAIAVCRAEEIDLGELGRQWAATAPVVEGLVADLIKR TSAGRFVSDAETLSSISPHYGAFQHLKELMEARRIDRTVVDGYDAIFRRAIASGHLHDDFAALSQFMGKAEQP

[0141] SEQ ID 33 (IR02159)

[0142] MPNTNGAQAPVTVIGLGLMGRALAAAFLEAGHPTTVWNRTAAKADRLVAEGAVRAGSVGDAIAASPLVV VCVTDYAAVRELLGPSAGSLGGKVVANLTTGTSAQARETAEWAAGLGARYLDGAIMAIPADIATDAAVLLHSGPKEA FEEHEATLRALGAAGTTYLDTDPGLSALYDMSLLGIMWGVLNGFLQGAALLGTAKVRATAFAPLANTMIKVVTEYVT AYAPQIDEGVYPADDATVTVHRDALGHLAEESEKLGVNAELPRFFKALTDRAATDGHADSSYAALIEQFRKPAA

[0143] Preferably, it 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 preferably, 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 NO. 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 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.The sequence is identical and capable of catalyzing the stereoselective reduction of aromatic imines to chiral aromatic amines.

[0146] The preferred embodiments 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 kind of suitable cells, preferably bacterial cells, such as Escherichia coli, which can be cultured in a culture. 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 collected 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. The specification, page 9 / 31, CN 121532499 A

[0148] , states that "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, namely Escherichia coli BL21 (DE3) cells, are also described in the examples. 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 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, which 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 combination thereof. DNA can be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA can be a coding strand (also known as the sense strand), or it can be a 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, hybrid molecules comprising DNA and RNA, which may be single-stranded or more typically double-stranded or triple-stranded, or mixtures of single-stranded and double-stranded regions. Additionally, nucleic acid molecules as used herein refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA.

[0152] Furthermore, nucleic acids may contain one or more modified bases. Such nucleic acids may also contain modifications, for example, in the ribose-phosphate backbone, to increase the stability and half-life of such molecules in physiological environments.

[0153] Thus, DNA or RNA having 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. The term nucleic acid molecule, as used herein, encompasses chemical, enzymatic, or metabolically modified forms of nucleic acid molecules, as well as chemical forms of DNA and RNA specific to viruses and cells, particularly including simple and complex cells.

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

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

[0156] The nucleic acid can be contained in an expression vector in which the nucleic acid is operatively linked to a promoter sequence capable of promoting the expression of the nucleic acid in a host cell.

[0157] In a preferred embodiment, the nucleic acid encodes an imine reductase, wherein the imine reductase comprises or includes the following: 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 (page 10 / 31, CN 121532499 A).

[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 particular 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, Promega 35 (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 cells. Vectors optionally include materials that facilitate the entry of nucleic acids into the cells, 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, such as baculovirus expression, or yeast, fungal, bacterial, or viral expression systems. Other suitable vectors of many 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 the present 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, splicing sequences, etc.Donor and splice acceptor signals, intermediate sequences, transcription enhancer sequences, translation enhancer sequences, drug resistance genes, etc. Optionally, drug resistance genes may be operatively linked to internal ribosome entry sites (IRES), which may be cell cycle specific or cell cycle independent.

[0161] As used herein, the term “operatively 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 operatively linked to a downstream coding sequence, wherein the promoter is capable of binding to 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 sequences of the present invention may be any kind of promoter sequences 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] Examples

[0163] Methods

[0164] Synthesis of pre-formed imines:

[0165] Imines were synthesized according to one of the following two procedures. Specification 11 / 31 pages 14 CN 121532499 A

[0166] Procedure A:

[0167] A pre-formed imine substrate was synthesized according to a slightly modified procedure described in 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 a dropping funnel over a period of 10 minutes while cooling in an ice bath. The reaction mixture was then stirred at room temperature for 10 minutes, followed by the addition of the desired carbonyl group (3 mmol, 1 equivalent). The reaction was stirred at room temperature overnight.

[0169] For post-treatment, 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] Procedure B:

[0171] According to Yasukawa et al. 2022The pre-formed imine substrate was synthesized using the procedure described in

[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 equivalent) were added to the vial and the reaction was stirred overnight at 120 °C or stirred at 60 °C for 72 h.

[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 preparation:

[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 Escherichia coli (E. coli), and codon optimization was performed using an algorithm from Twist Bioscience (South San Francisco, USA). In all cases, a stop codon was added to the end. 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] The plasmid from Twist Bioscience was resuspended in sterile water. Inoculation of *E. coli* BL21(DE3) cells was achieved by heat treatment (42°C for 45 s). The preculture was incubated overnight at 37°C on Luria Bertani agar plates containing 100 μg / mL ampicillin. Single microbial colonies were selected and incubated overnight at 37°C in liquid LB medium containing 100 μg / mL ampicillin.

[0177] Terrific Broth (TB) medium containing 100 μg / mL ampicillin was inoculated 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, amplitude...).20%. The sonicated cells were centrifuged (4°C, 17000 rpm, 20 min), and the lysates were frozen at -20°C and then lyophilized. The lyophilized enzyme preparation was stored at -20°C.

[0178] Reaction setup:

[0179] Small-scale biotransformation using a clear cell-free extract of Escherichia coli BL21(DE3) with overexpression of imine reductase (IRED) or a lyophilized cell-free extract as a biocatalyst was carried out in a volume of 0.5 mL. The reaction was carried out in 100 mM bicine buffer at pH 8.0. The pre-formed imine was dissolved in methanol at a concentration of 200 mM and then partitioned into the reaction system, with a final concentration of 20 mM for the pre-formed imine. Dissolve NADP+ (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) in a stock solution prepared from water. Incubate the reaction system at 25°C in an Eppendorf thermostatic shaker with vigorous stirring for 18 to 24 hours.

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

[0181] Preparative-scale biotransformation was carried out using a clear cell-free extract or lyophilized cell-free extract with IRED overexpression as a biocatalyst in a total volume of 30 mL. Unless otherwise stated, reactions using IRED were carried out at 30°C in 100 mL flasks equipped with mechanical stirrers and including pH and temperature sensors. The reaction was carried out at pH 8.0 and kept constant using 1 M NaOH solution and a titration system from Metrohm (Titrando 902, touch). The concentrations listed in the following reaction description 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, wherein lyophilized IRED (8–4 g / L), NADP+ (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) were added. Finally, the pre-formed imine (final concentration 100 mM–200 mM in the reaction system) was dissolved in methanol (10% v / v) and transferred to the reaction system.

[0183] By adding1 M NaOH was used to maintain a constant pH.

[0184] Samples were taken at different reaction times and analyzed by GC to determine the progress of the reaction. The enantiomeric 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 of the reaction was increased by adding Na2CO3, followed by liquid-liquid extraction with an organic solvent (i.e., 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 method:

[0187] A 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 shaken vigorously. 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] Conversion rate was determined using GC analysis:

[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] Split ratio: 90:1

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

[0196] Total run time: 30 min

[0197] Method 2

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

[0199] Inlet temperature: 200℃ (Instructions for Use, Page 13 / 31, 16 CN 121532499 A)

[0200] Detector temperature: 321℃

[0201] Injection volume: 1 μL

[0202] Split ratio: 20:1

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

[0204] Total run time: 6.5 min

[0205] Method 3 (Chiral)

[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] Split ratio: 40:1

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

[0212] Total run 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] Enantiomer excess rate 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 run 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 run 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 (Instructions for Use 14 / 31 pages 17 CN 121532499 A)

[0237] Total run 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 run 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 run 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 run 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 run 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 run time: 25 min

[0273] DAD: 210 nm

[0274] Method 9:

[0275] Column: Daicel Chiralpak IG-3 column (4.6 mm × 250 mm × 3 μm) Instruction manual 15 / 31 pages 18 CN 121532499 A

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

[0277] Column temperature: 30℃

[0278] Flow rate: 1.3 mL / min

[0279] Total run time: 12 min

[0280] DAD: 210 nm

[0281] Method 10:

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

[0283] Eluent: 55 / 45 20 mMAmmonium formate / acetonitrile

[0284] Column temperature: 30°C

[0285] Flow rate: 1.3 mL / min

[0286] Total run time: 18 min

[0287] DAD: 210 nm

[0288] Method used in Example 1:

[0289]

[0290] Comparative example:

[0291] Reductive amination of selected IREDs

[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 an 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 overexpressed imine reductases (IRED) as a biocatalyst was carried out in 0.5 mL volumes. The reaction was conducted in bicine buffer at pH 8.0. NADP+ (1 mM), sodium phosphite (200 mM), phosphite dehydrogenase (4 g / L), and IRED (4 g / L) were dissolved in stock solutions prepared from 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 h.

[0295] The reaction was quenched by adding 2 M Na2CO3 (200 µL) and 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 IRED for the direct reductive amination of acetophenone with 3-phenylpropylamine to 3-phenyl-N-(1-phenylethyl)propane-1-amine

[0298]

[0299]

[0300] Tests were also performed on representative reactions from the compounds listed in Example 1 regarding 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] By IREDScreening for the reduction of different pre-formed imines was performed.

[0303] Small-scale biotransformation was carried out in 0.5 mL volumes using a clear, cell-free extract of *E. coli* BL21(DE3) with different overexpressed imine reductases (IRED) as a biocatalyst. The reaction was carried out at pH 8.0 in 100 mM bicine buffer. The 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 for the pre-formed imines. Dissolve NADP+ (1 mM), sodium phosphite (200 mM), phosphite dehydrogenase (4 g / L), or glucose (200 mM) and GDH (0.1 g / L), and IRED (4 g / L) in a stock solution prepared from water. Incubate the reaction system 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 system 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 (page 17 / 31 of CN 121532499 A).

[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 IRED for the reduction of N-1-diphenylethylimine to N-(1-phenylethyl)aniline

[0311] Specification 18 / 31 pages 21 CN 121532499 A

[0312]

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

[0314]

[0315]

[0316] Table 4: Instructions for the reduction of methyl 2-phenyl-2-(phenylimino)acetate to 2-anilino-2-phenyl-acetic acidPage 22, 19 / 31 CN 121532499 A Screening results of IRED for methyl esters

[0317]

[0318]

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

[0320] Specification Page 23, 20 / 31 CN 121532499 A

[0321]

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

[0323]

[0324]

[0325] Table 7: Screening results of IRED for reducing N-methyl-1-phenyl-ethyleneimine to N-methyl-1-phenyl-ethylamine

[0326] Specification 21 / 31 Page 24 CN 121532499 A

[0327]

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

[0329]

[0330] Specification 22 / 31 Page 25 CN 121532499 A

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

[0332]

[0333]

[0334] Table 10: Screening results of IREDs for reducing 1-phenyl-N-(3-pyridyl)ethyleneimine to N-(1-phenylethyl)pyridine-3-amine

[0335]

[0336]

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

[0338] Specification 23 / 31 Page 26 CN 121532499 A

[0339]

[0340] Table 12: Screening results of IREDs for reducing diphenylimine to diphenylmethylamine

[0341]

[0342]

[0343] Table 13: Screening results of IREDs for reducing 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] Specification 24 / 31 pages 27 CN 121532499A

[0348]

[0349] Example 2:

[0350] Optimization of reaction temperature

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

[0352] Small-scale biotransformation was carried out in 0.5 mL volumes using a clear, cell-free extract of Escherichia coli BL21(DE3) with overexpression of imine reductase (IRED) as a biocatalyst. The reaction was carried out at pH 8.0 in 100 mM bicine buffer. N-1-diphenylethylimine was dissolved in methanol at a concentration of 200 mM and then partitioned into the reaction system, with the pre-formed imine having a final concentration of 20 mM. NADP+ (1 mM), glucose (200 mM), 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 for 18 hours at 20°C, 25°C, 30°C, 35°C, and 40°C in an Eppendorf thermostatic shaker with vigorous stirring.

[0353] The reaction was quenched by adding 2 M Na2CO3 (200 µL), and the reaction system was 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 the selected IRED for the reduction of N-1-diphenylethylimine to N-(1-phenylethyl)aniline

[0357] Specification 25 / 31 pages 28 CN 121532499 A

[0358] Example 3:

[0359] Optimization of reaction pH

[0360] For the selected enzymes (IR00005, IR00395, IR01139), the process parameters were further tested in detail to determine the optimal reaction conditions. In particular, the reaction pH was tested between values ​​of 6.5 and 9.0.

[0361] Small-scale biotransformation was carried out in a volume of 0.5 mL using a clear, cell-free extract of Escherichia coli BL21(DE3) with overexpression of imine reductase (IRED) as a biocatalyst. The reaction was carried out in 100 mM bicine buffer at pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. N-1-diphenylethyleneimine was added at 200 mM...The concentration was dissolved in methanol and then partitioned into the reaction system, with the final concentration of the pre-formed imine being 20 mM. NADP+ (1 mM), glucose (200 mM), glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved in a stock solution prepared in water. The reaction system 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 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 imine, secondary amines and ketones, and amines obtained from the hydrolysis of imine.

[0364] The pH screening results are as follows:

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

[0366]

[0367] Example 4:

[0368] Cosolvent optimization

[0369] For the selected enzymes (IR00005, IR00395, IR01139), the process parameters were further tested in detail to determine the optimal reaction conditions. In particular, the reaction was tested in the presence of several 10% (v / v) concentration cosolvents.

[0370] Small-scale biotransformation using a clear cell-free extract of Escherichia coli BL21(DE3) with overexpression of imine reductase (IRED) as a biocatalyst was carried out in a volume of 0.5 mL. The reaction was carried out at pH 8.0 in 100 mM bicine buffer. N-1-Diphenylethyleneimine was dissolved at a concentration of 200 mM in one of the following solvents (methanol, DMSO, acetonitrile, glycerol, tert-methyl butyl ether, toluene, cyclopentyl methyl ether, isopropyl acetate, isoamyl acetate, cyrene™, and cyclohexane) and then partitioned into the reaction system, with the final concentration of the pre-formed imine being 20 mM. NADP+ (1 mM), glucose (200 mM), 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 30°C in an Eppendorf thermostatic shaker with vigorous stirring for 18 hours.

[0371] The reaction was quenched by adding 2 M (200 µL) of Na2CO3, and then followed by ethyl acetate (500 μL, twice).The reaction system was extracted. 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 cosolvent screening results are as follows:

[0374] Table 17: Cosolvent screening results of selected IREDs for the reduction of N-1-diphenylethylimine to N-(1-phenylethyl)aniline

[0375]

[0376] For the selected enzymes (IR00005, IR00395, IR01139), the process parameters were further tested in detail to determine the optimal reaction conditions. In particular, the reaction was tested in the presence of selected cosolvents 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 one of the following solvents (methanol, DMSO, cyrene™, and 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 was adjusted to 10, 20, or 30% (v / v). NADP+ (1 mM), glucose (200 mM), glucose dehydrogenase (0.1 g / L), and IRED (4 g / L) were dissolved from stock solutions prepared in water. The reaction system 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 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 cosolvent screening results are as follows:

[0381] Table 18: Screening results of selected cosolvent concentrations of selected IREDs for the reduction of N-1-diphenylethylimine to N-(1-phenylethyl)aniline. (Page 27 / 31, 30 CN 121532499 A)

[0382]

[0383] Example 5:

[0384] Preparative-scale bioconversion of 2-anilino-2-phenyl-acetic acid methyl ester to 2-anilino-2-phenyl-acetic acid methyl ester using IRED and 2-phenyl-2-(phenylimino)acetic acid methyl ester

[0385]

[0386] Preparative-scale biotransformation of methyl 2-phenyl-2-(phenylimino)acetate to methyl 2-aniline-2-phenyl-acetate 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), NADP+ (1 mM), and Na2HPO3 (100 mM) in bicine buffer (100 mM, pH 8.0). The reaction mixture was stirred at 120 rpm for 24 hours at 30 °C.

[0388] A saturated Na2CO3 solution (15 mL) was then 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 in 76% separation yield, preferentially forming the second eluting enantiomer (94% ee). The optical rotation was determined to be [α]20D = –98.3° (c = 1.3, CHCl3), which corresponds to the (R)-enantiomer according to the literature (Zhang et al., 2014).

[0390] Methyl 2-aniline-2-phenylacetic acid: 1H 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). Instruction manual, pages 28 / 31, CN 121532499 A

[0391] Example 6:

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

[0393]

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

[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), NADP+ (1 mM), and glucose (200 mM) in bicine buffer (100 mM, pH 7.2). The reaction system was stirred at 25 °C for 18 h. 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 Dicalite. The combined organic phases were dried with Na2SO4 and the solvent was removed under reduced pressure. The isolated product yield was 57% (290 mg isolated product, purity 97%, enantiomeric excess not determined).

[0398] N-(1-phenylethyl)aniline: ¹H 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] Using IRED Preparative-scale bioconversion of N-phenyl-1-(3-pyridyl)ethyleneimine to N-[1-(3-pyridyl)ethyl]aniline

[0401]

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

[0403] N-phenyl-1-(3-pyridyl)ethylimine (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), NADP+ (1 mM), and glucose (200 mM) in bicine buffer (100 mM, pH 8.0). The reaction system 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. Instructions for Use, pages 29 / 31, CN 121532499 A

[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 Dicalite. The combined organic phases were dried with Na2SO4 and the solvent was removed under reduced pressure. The product was isolated in 85% yield (356 mg isolated product, purity 95%, enantiomeric excess 98%).

[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 bioconversion of N-(4-methoxyphenyl)-2,2-dimethyl-1-phenylpropane-1-imine was carried out using lyophilized cell-free extract of IR00005 as a biocatalyst. The reaction was carried out at a scale of 2.0 mmol (534.7 mg,The reaction was carried out at 25 °C with 21.4 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), NADP+ (1 mM), and glucose (200 mM) in bicine buffer (100 mM, pH 8.0). 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 Dicalite. The combined organic phases were dried with Na2SO4 and the solvent was removed under reduced pressure. The product was isolated in 82% yield (460 mg isolated product, purity 96%, enantiomeric excess not determined).

[0414] N-(2,2-dimethyl-1-phenyl-propyl)-4-methoxy-aniline: ¹H NMR (600 MHz, CDCl₃) δ ppm 7.27–7.35 (m, 3H), 7.18–7.24 (m, 1H), 6.61–6.69 (m, 2H), 6.40–6.48 (m, 2H), 4.01 (br s, 1H), 3.94–3.98 (m, 1H), 3.64–3.70 (m, 3H), 0.96–1.04 (m, 9H)

[0415] References

[0416] Afanasyev, O.I., Kuchuk, E., Usanov, D.L. 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, S.L., Sharma, M., Leipold , F ., Hussain , S ., Grogan , G . and Turner , N .J ., 2017. A reductive aminase from Aspergillus oryzae. Nature Chemistry, 9(10) , pp.961‑969. Description Page 30 of 31 33 CN 121532499 A

[0418] Carlson, R ., Larsson, U. and Hansson, L., 1992. Efficient synthesis of imines by a modified titanium tetrachloride procedure . Acta Chemica Scandinavica, 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 enzyme family 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 reductase toolbox. ChemCatChem, 9(24) , pp.4475‑4479.

[0421] Sambrook , J .F . and Russell , D .W .,2000 . Molecular cloning: A laboratory 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 . A mechanism for reductive amination catalyzed by fungal reductive aminases. ACS Catalysis, 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 of aldehydes 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 Ketimine Synthesis. 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 of the American Chemical Society,136(40), pp.13971-13974. Instruction manual, 31 / 31 pages, 34 CN 121532499 A

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. 。