WO2025003017A2 - Procédé de préparation de dérivés d'aldéhydes gamma, delta-insaturés - Google Patents

Procédé de préparation de dérivés d'aldéhydes gamma, delta-insaturés Download PDF

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WO2025003017A2
WO2025003017A2 PCT/EP2024/067536 EP2024067536W WO2025003017A2 WO 2025003017 A2 WO2025003017 A2 WO 2025003017A2 EP 2024067536 W EP2024067536 W EP 2024067536W WO 2025003017 A2 WO2025003017 A2 WO 2025003017A2
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group
firmenich
compound
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WO2025003017A3 (fr
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Kerstin Steiner
Hervé MOSIMANN
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Firmenich SA
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Firmenich SA
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Priority to CN202480042986.0A priority patent/CN121399271A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/48—Preparation of compounds having groups
    • C07C41/50—Preparation of compounds having groups by reactions producing groups
    • C07C41/54—Preparation of compounds having groups by reactions producing groups by addition of compounds to unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/48—Preparation of compounds having groups
    • C07C41/50—Preparation of compounds having groups by reactions producing groups
    • C07C41/56—Preparation of compounds having groups by reactions producing groups by condensation of aldehydes, paraformaldehyde, or ketones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/30—Compounds having groups
    • C07C43/303—Compounds having groups having acetal carbon atoms bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/30—Compounds having groups
    • C07C43/315—Compounds having groups containing oxygen atoms singly bound to carbon atoms not being acetal carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/51—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by pyrolysis, rearrangement or decomposition
    • C07C45/511—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by pyrolysis, rearrangement or decomposition involving transformation of singly bound oxygen functional groups to >C = O groups
    • C07C45/515—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by pyrolysis, rearrangement or decomposition involving transformation of singly bound oxygen functional groups to >C = O groups the singly bound functional group being an acetalised, ketalised hemi-acetalised, or hemi-ketalised hydroxyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
    • C12Y106/99001—NADPH dehydrogenase (1.6.99.1)

Definitions

  • the present invention relates to the field of organic synthesis and more specifically it concerns a process for preparing compound of formula (II) starting from compound of formula (I) and a process for preparing compound of formula (I) starting from compound of formula (III) via valuable new chemical intermediates such as compound of formula (IV) and the compound of formula (V).
  • the compound (IV) and the compound of formula (V) are also part of the invention. Background of the invention In the perfumery industry, there is a constant need to provide compounds imparting novel organoleptic notes.
  • the rearrangement step provides the desired intermediate or product with moderate to low yield. Being products of industrial interest, there is always a need for new processes showing an improved yield and productivity. In the meantime, today there is a need to foster sustainable processes, for examples using enzymatic transformation.
  • the present invention allows to solve the above problems by using an oxidoreductase in the process to prepare compound of formula (II).
  • the process herein disclosed represents a novel route through novel intermediates, never disclosed before, while improving yield and environmental impact.
  • the invention s conditions and the compounds of formula (IV) and (V) which are an object of the present invention, have never been reported in the prior art.
  • the invention relates to a novel process allowing the preparation of compound of formula (II) with a high yield and high selectivity starting from compound of formula (III) via compound of formula (IV), (V) and (I).
  • the invention process represents a new efficient route toward compound of formula (II).
  • the first object of the present invention is a process for the reduction by hydrogenation of conjugated dienal of formula in the form of any and wherein each R 1 , R 2 3 and R , a atom, a C1-3 alkoxy group, a C1-6 alkyl group or a C2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3-8 cycloalkyl or C5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; into a deconjugated enal of formula in the form of any one and wherein R 1 to R 6 have the same meaning as defined in formula (I); said process being carried out in the presence of an oxidoreductase.
  • a second object of the present invention is a process for the preparation of a compound of formula Firmenich SA 3 in the form of any one of its or a mixture thereof, and wherein each R 1 , R 2 and R 3 , independently from each other, represent a hydrogen atom, a C1-3 alkoxy group, a C1-6 alkyl group or a C2-6 alkenyl group, each optionally substituted by a hydroxy or C 1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3-8 cycloalkyl or C5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; comprising the steps of a) converting a compound of formula in the form of thereof, and wherein R 1 , R 2 , R 3 , R 4 and R 5 have the same meaning as defined in formula (I); into an acetal of formula in the form of 1 thereof, and wherein
  • a third object of the present invention is a compound of formula in the form of any one 1 2 and wherein R , R and R 3 , independently from each other, represent a hydrogen atom, a C 1-3 alkoxy group, a C 1-6 alkyl group or a C2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C 3-8 cycloalkyl or C 5-8 cycloalkenyl group; R 4 and R 5 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C 1-4 alkyl group or R a and R b , when taken together, represent a C2-5 alkanediyl group.
  • a further object of the present invention is a compound of formula in the form of and and wherein R 1 , R 2 and R 3 , independently from each other, represent a hydrogen atom, a C1-3 alkoxy group, a C 1-6 alkyl group or a C 2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3- Firmenich SA 5 8 cycloalkyl or C5-8 cycloalkenyl R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C1-4 alkyl group or R a and R b , when taken together, represent a C 2-5 alkanediyl group; R c represents a C 1-4 alkyl group.
  • a further object of the present invention is a compound of formula (I) and/or formula (II) and an oxidoreductase.
  • a further object of the present invention is a reaction medium comprising an oxidoreductase enzyme according to the invention and a compound of formula (I) and/or formula (II).
  • a further object of the present invention is a compound of formula (II) obtained or obtainable by the process of the invention.
  • perfuming ingredients of formula (II) can be obtained from a new class of precursors (or chemical intermediates), as defined herein below in formula (IV) and (V), and that said new intermediates allow the corresponding perfuming ingredients to be obtained with overall higher yield, compared to the methods known from the prior art and using more sustainable conditions.
  • the first object of the invention is a process for the reduction by hydrogenation of conjugated dienal of formula in the form of any and wherein each R 1 , R 2 3 and R , a atom, a C1-3 alkoxy group, a C 1-6 alkyl group or a C 2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3-8 cycloalkyl or C5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; into a deconjugated enal of formula Firmenich SA 6 in the form of any one and wherein R 1 to R 6 have the same meaning as defined in formula (I); said process being carried out in the presence of an oxidoreductase.
  • any one of its stereoisomers or a mixture thereof can be a pure enantiomer or a mixture of enantiomers.
  • the compounds of formula (I) and (II) may possess at least one stereocenter which can have two different stereochemistries (e.g. R or S).
  • the compounds of formula (I) and (II) may even be in the form of a pure enantiomer or in the form of a mixture of enantiomers.
  • the compounds of formula (I) and (II) may even be in the form of a pure diastereoisomer or in the form of a mixture of diastereoisomers when compounds of formula (I) and (II) possess more than one stereocenter.
  • the compounds of formula (I) and (II) can be in a racemic form or scalemic form. Therefore, the compounds of formula (I) and (II) can be one stereoisomer or in the form of a composition of matter comprising, or consisting of, various stereoisomers.
  • by the wavy bond in compound of formula (II), or the similar it is meant the normal meaning understood by a person skilled in the art, i.e.
  • the double bond may have a cis configuration corresponding to the Z isomer, a trans configuration corresponding to the E isomer or a mixture thereof.
  • the compound of formula (II) may be in the form of its E or Z isomer or of a mixture thereof, e.g. the invention process leads to a composition of matter consisting of one or more compounds of formula (II), having the same chemical structure but differing by the configuration of the double bond.
  • compound (II) can be in the form of a mixture consisting of isomers E and Z and wherein said isomer E represents at least 25% of the total mixture, at least 35%, at least 50%, or even at least 75% (i.e a mixture E/Z comprised between 75/25 and 100/0), or even at least 88%, or even at least 95%.
  • isomer E represents at least 25% of the total mixture, at least 35%, at least 50%, or even at least 75% (i.e a mixture E/Z comprised between 75/25 and 100/0), or even at least 88%, or even at least 95%.
  • Firmenich SA 7 The terms “alkyl” and “alkenyl” are as comprising branched and linear alkyl and alkenyl groups.
  • alkenyl and “cycloalkenyl” are understood as comprising 1, 2 or 3 olefinic double bonds, preferably 1 or 2 olefinic double bonds.
  • cycloalkyl and “cycloalkenyl” are understood as comprising a monocyclic or fused, spiro and/or bridged bicyclic or tricyclic cycloalkyl and cycloalkenyl, groups, preferably monocyclic cycloalkyl and cycloalkenyl groups.
  • R 1 and R 2 are taken together and form a C 3-8 cycloalkyl or C 5-8 cycloalkenyl group”, it is meant that the carbon atoms to which both groups are bonded are included into the C5-8 cycloalkyl or C5-8 cycloalkenyl group.
  • R 4 may be a hydrogen atom or a methyl group. Even more particularly, R 4 may be a hydrogen atom.
  • R 5 may be a methyl or an ethyl group. Even more particularly, R 5 may be a methyl group.
  • R 6 may be a hydrogen atom or a methyl group. Even more particularly, R 6 may be a hydrogen atom.
  • R 3 may be, independently from each other, a hydrogen atom, a methoxy group, an ethoxy group, a C1-4 alkyl group or a C2-4 alkenyl group, each optionally substituted by a hydroxy, methoxy or ethoxy group.
  • R 3 may be, independently from each other, a hydrogen atom, a C1-3 alkyl group or a C 2-3 alkenyl group, each optionally substituted by a hydroxy or methoxy group.
  • R 3 may be, independently from each other, a hydrogen atom or a C1-3 alkyl group.
  • R 3 may be, independently from each other, a hydrogen atom or a methyl or ethyl group.
  • R 3 may be a hydrogen atom.
  • the compound of formula (I) is of formula in the form of any and wherein each R 1 and R 2 have the same as and said compound of formula (II) is of formula Firmenich SA 8 in the form of any and wherein each R 1 2 and R have the same as
  • R 1 may be, independently from each other, a hydrogen atom, a methoxy group, an ethoxy group, a C 1-4 alkyl group or a C 2-4 alkenyl group, each optionally substituted by a hydroxy, methoxy or ethoxy group.
  • R 1 may be, independently from each other, a hydrogen atom, a C 1-3 alkyl group or a C2-3 alkenyl group, each optionally substituted by a hydroxy or methoxy group.
  • R 1 may be, independently from each other, a hydrogen atom or a C 1-3 alkyl group.
  • R 1 may be, independently from each other, a hydrogen atom or a methyl or ethyl group. Even more particularly, R 1 may be a methyl group.
  • R 2 may be, independently from each other, a hydrogen atom, a methoxy group, an ethoxy group, a C 1-4 alkyl group or a C 2-4 alkenyl group, each optionally substituted by a hydroxy, methoxy or ethoxy group.
  • R 2 may be, independently from each other, a hydrogen atom, a C 1-3 alkyl group or a C2-3 alkenyl group, each optionally substituted by a hydroxy or methoxy group.
  • R 2 may be, independently from each other, a hydrogen atom or a C 1-3 alkyl group.
  • R 2 may be, independently from each other, a hydrogen atom or a methyl or ethyl group. Even more particularly, R 2 may be a hydrogen atom.
  • Non limiting examples of compound of formula (II) may include (E)-4-methyl-5- (p-tolyl)pent-4-enal, (4E)-2,4-dimethyl-5-(4-methylphenyl)-4-pentenal, (4E)-4-methyl-5- (3-methylphenyl)-4-pentenal, (E)-5-(4-ethylphenyl)-4-methylpent-4-enal, (E)-5-(4- isopropylphenyl)-4-methylpent-4-enal, (E)-5-(4-methoxyphenyl)-4-methylpent-4-enal, (E)-5-(2,3-dihydro-1H-inden-5-yl)-4-methylpent-4-enal, (E)-5-(1,1-dimethyl-2,3-
  • Non limiting examples of compound of formula (I) may include (2E,4E)-4-Methyl- 5-(4-methylphenyl)-2,4-pentanedienal, (2E,4E)-2,4-dimethyl-5-(p-tolyl)penta-2,4-dienal, (2E,4E)-4-methyl-5-(m-tolyl)penta-2,4-dienal, (2E,4E)-5-(4-ethylphenyl)-4-methylpenta- 2,4-dienal, (2E,4E)-5-(4-isopropylphenyl)-4-methylpenta-2,4-dienal, (2E,4E)-5-(4- methoxyphenyl)-4-methylpenta-2,4-dienal, (2E,4E)-5-(2,3-dihydro-1H-inden-5-yl)-4- methylpenta-2,4-dienal, (2E,4E
  • polypeptide means an amino acid sequence of consecutively polymerized amino acid residues, for instance, at least 15 residues, at least 30 residues, at least 50 residues.
  • a polypeptide comprises an amino acid sequence that is an enzyme, or a fragment, or a variant thereof.
  • protein refers to an amino acid sequence of any length wherein amino acids are linked by covalent peptide bonds, and includes oligopeptide, peptide, polypeptide and full length protein whether naturally occurring or synthetic.
  • cinnamon The term “isolated” polypeptide to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods.
  • nucleic acid sequence refers to a sequence of nucleotides.
  • a nucleic acid sequence may be a single-stranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and non-coding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and/or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes.
  • nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U).
  • nucleotide sequence should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.
  • nucleic acid sequences are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms.
  • “Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press.
  • the term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an mRNA in a cell, operably linked to suitable regulatory regions, e.g., a promoter.
  • a gene may thus comprise several operably linked sequences, Firmenich SA 11 such as a promoter, a 5’ leader sequence e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and/or a 3’non- translated sequence comprising, e.g., transcription termination sites.
  • “Expression of a gene” encompasses “heterologous expression” and “over- expression” and involves transcription of the gene and translation of the mRNA into a protein.
  • Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and/or enzyme activity in transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background.
  • “Expression vector” as used herein means a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology for delivery of foreign or exogenous DNA into a host cell.
  • the expression vector typically includes sequences required for proper transcription of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for an RNA, e.g., an antisense RNA, siRNA and the like.
  • An “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system.
  • the expression vector includes the nucleic acid of an embodiment herein operably linked to at least one regulatory sequence, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker.
  • Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleic acid of an embodiment herein.
  • the term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template.
  • the term “host cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence.
  • the host cell is particularly a bacterial cell, a fungal cell or a plant cell.
  • the host cell may contain a recombinant gene which has been integrated into the nuclear or organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally.
  • Methods for introducing nucleic acid sequences into cells are well known in the art. For example, Firmenich SA 12 where the cell is a prokaryotic cell (e.g.
  • Such methods include among others heat shock of chemically prepared competent cells (chemical transformation) and electroporation of electrocompetent cells. Both techniques are well-known and no further explanation is needed.
  • the cell is a eukaryotic cell such as a fungal cell
  • the most widely methods used for transformation are for example ATMT, PEG-mediated protoplast transformation, and electroporation.
  • oxidoreductase known in the art, specifically ene reductase, catalyzing selective reduction of conjugated dienals ( ⁇ , ⁇ , ⁇ , ⁇ -di-unsaturated aldehyde).
  • R 4 , R 5 and/or R 6 of formula (I) are a methyl or an ethyl group
  • additional steric hindrances in the active site of ene reductases are present.
  • no compound of formula (I) with a methyl or an ethyl group at R 4 , R 5 and/or R 6 has been converted with an enzyme to reduce the ⁇ , ⁇ -double bond.
  • the process for making a compound of formula (II) comprises reducing a precursor compound of formula (I) with an oxidoreductase enzyme.
  • Oxidoreductase is an enzyme catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor.
  • Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, oxidoreductases have much utility in the development of biotech methods of synthesis of desirable compounds.
  • There are several different classes of oxidoreductases which are primarily defined according to their substrate and/or mode of action. For example, ene reductases, ketoreductases, peroxidases, hydroxylases and oxygenases, and reductases.
  • the oxidoreductase is an ene reductase (also termed ERED herein).
  • OYEs can be found in bacteria, fungi and plants and are divided in several subfamilies depending on their sequence homology and structural features. Examples of OYEs are represented by SEQ ID NOs: 11 to 44.
  • the catalytic site of OYEs harbors a flavin mononucleotide (FMN) cofactor, which donates a hydride to the Cb atom of the substrate.
  • FMN flavin mononucleotide
  • the enzyme used in the method of the invention requires an FMN cofactor
  • said cofactor may be introduced to the reaction.
  • the growth media provides sufficient quantities of the FMN cofactor so that it is not necessary to further supplement the reaction with this compound.
  • SEQ ID NOs: 1 to 7 are double bond reductase-like enzymes (DBR, cd08295) and SEQ ID NOs: 8 to 10 belong to the prostaglandin dehydrogenase subfamily (PGDH, cd05288).
  • an embodiment of the invention is wherein the ERED is a MDR.
  • the ene reductase (ERED) enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity of any of SEQ ID NOs: 1 to 44.
  • ERED reactions typically require a cofactor.
  • cofactor refers to a non-protein compound that operates in combination with an ERED enzyme.
  • Cofactors suitable for use with the ERED enzymes in the processes of the invention described herein include, but are not limited to, NADP + (nicotinamide adenine dinucleotide phosphate), NADPH (the reduced form of NADP + , NAD + (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD + ).
  • NADP + nicotinamide adenine dinucleotide phosphate
  • NADPH the reduced form of NADP +
  • NAD + nicotinamide adenine dinucleotide
  • NADH the reduced form of NAD +
  • the reduced form of the cofactor is added to the reaction mixture.
  • An embodiment of the process of the invention is wherein the ERED reduction is performed in the presence of a cofactor; preferably the cofactor is NAD(P)H or NAD(P) + .
  • the reduced NAD(P)H form can be optionally regenerated from the oxidized NAD(P) + form using a cofactor regeneration system.
  • a cofactor regenerating system can push the equilibrium of the process of the invention towards the generation of the desired product.
  • the process of the invention can be more optimized and efficient in terms of reagents used, therefore more time and cost effective than without the use of a cofactor regenerating system.
  • an embodiment of the process of the invention is wherein the ERED reduction is performed in the presence of a cofactor regeneration system.
  • cofactor regeneration system refers to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NAD(P) + to NAD(P)H).
  • oxidized form of the cofactor e.g., NAD(P) + to NAD(P)H.
  • Firmenich SA 15 Cofactors oxidized by the ERED- of the substrate are regenerated in reduced form by the cofactor regeneration system.
  • Cofactor regeneration systems comprise a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor.
  • the cofactor regeneration system may further comprise a catalyst, for example a cofactor regeneration enzyme, that catalyzes the reduction of the oxidized form of the cofactor by the reductant.
  • Cofactor regeneration systems to regenerate NADH or NADPH from NAD + or NADP + , respectively, are known in the art and may be used in the processes described herein.
  • the cofactor regeneration system can be in vivo or in vitro. While not wishing to be bound to any specific embodiments, examples of in vivo cofactor regeneration systems include where the cofactor regeneration enzyme that catalyzes the reduction of the oxidized form of the cofactor by the ERED, is synthesized in a cell which also synthesizes the ERED enzyme. Hence there is a cofactor regeneration within a single cell. In such embodiments the cell is genetically modified to express both the ERED enzyme and the cofactor regeneration enzyme. Examples of polypeptide sequences encoding the ERED enzyme provided herein.
  • the cofactor regeneration enzyme is an alcohol dehydrogenase (ADH), a formate dehydrogenase (FDH), a glucose dehydrogenase (GDH), a phosphite dehydrogenase, or a 6-phosphate glucose dehydrogenase, and examples of such enzymes and their polypeptide sequences are well known in the art. Wild type organisms such as baker’s yeast have been traditionally used for the reduction of alkenes using e.g. glucose as the co-substrate.
  • the cofactor regeneration system is an in vitro system. In such embodiments, the ERED enzyme and the cofactor regeneration enzyme are synthesized in two separate cells or in a single cell.
  • a further aspect of the invention comprises a reaction medium comprising a oxidoreductase enzyme as defined herein and a compound of formula (I) and/or formula (II).
  • the reaction medium may further comprise a cofactor regeneration system as described herein.
  • the oxidoreductase enzyme my be provided in the form of a recombinant cell comprising the oxidoreductase enzyme, or crude or cell free lysates, or purified recombinant enzymes.
  • the cofactor regeneration system comprises an alcohol dehydrogenase, a formate dehydrogenase (FDH), or a glucose dehydrogenase (GDH) system.
  • the cofactor regeneration system comprises an ADH
  • the cofactor regeneration system may further comprise an alcohol as a substrate for the regeneration system.
  • the cofactor regeneration system comprises an GDH
  • the cofactor regeneration system may further comprise a glucose as a substrate for the regeneration system.
  • the cofactor regeneration system comprises an FDH
  • the cofactor regeneration system may further comprise a formate as a substrate for the regeneration system.
  • the cofactor regenerating system may comprise a formate dehydrogenase.
  • formate dehydrogenase and “FDH” are used interchangeably herein to refer to an NAD + or NADP + -dependent enzyme that catalyzes the conversion of formate and NAD + or NADP + to carbon dioxide and NADH or NADPH, respectively.
  • Formate dehydrogenases that may be suitable for use as cofactor regenerating systems in the ERED-catalyzed reduction reactions described herein include both naturally occurring formate dehydrogenases, as well as non-naturally occurring formate dehydrogenases.
  • the cofactor regeneration system is a formate dehydrogenase (FDH), for example LbFDH (the amino acid sequence for which is provided in SEQ ID NO: 133, the nucleotide sequences for which are provided in SEQ ID NOs: 135 and 137) and MvFDH-var (the amino acid sequence for which is provided in SEQ ID NO 134, the nucleotide sequences for which are provided in SEQ ID NOs: 136 and 138).
  • FDH formate dehydrogenase
  • LbFDH the amino acid sequence for which is provided in SEQ ID NO: 133, the nucleotide sequences for which are provided in SEQ ID NOs: 135 and 137
  • MvFDH-var the amino acid sequence for which is provided in SEQ ID NO 134, the nucleotide sequences for which are provided in SEQ ID NOs: 136 and 138.
  • EREDs oxidoreductases
  • a further aspect of the invention is the use of a polypeptide having oxidoreductase activity comprising an amino acid sequence having least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity or more to any of SEQ ID NOs: 1 to 44 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 44 in the process of the invention for preparing a compound of formula (II).
  • a further aspect of the invention provides an isolated polypeptide having Firmenich SA 17 oxidoreductase activity comprising an sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of SEQ ID NOs: 1 to 4 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 4. This is the first time a polypeptide having such an amino acid sequence has been shown to have oxidoreductase activity.
  • nucleic acid molecule encoding a polypeptide having oxidoreductase activity comprising a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 45 to 132, or the reverse complement thereof, or comprising nucleotide sequence of any of SEQ ID NOs: 45 to 132, or the reverse complement thereof, in the process of the invention for preparing a compound of formula (II).
  • a further aspect of the invention provides an isolated nucleic acid molecule encoding a polypeptide having oxidoreductase activity comprising a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 45 to 48 or 89 to 92 or the reverse complement thereof, or comprising nucleotide sequence of any of SEQ ID NOs: 45 to 48 or 89 to 92, or the reverse complement thereof.
  • This is the first time a nucleic acid sequence has been shown to encode a polypeptide having oxidoreductase activity.
  • a vector comprising the nucleic acid molecules described herein.
  • the vector is an expression vector.
  • the vector is a prokaryotic vector, viral vector or a eukaryotic vector.
  • a non-human host organism or a host cell comprising (1) a nucleic acid molecule described above, or (2) an expression vector comprising said nucleic acid molecule.
  • the non-human organism or host cell is a prokaryotic or eukaryotic cell.
  • the host cell is a bacterial cell, a plant cell, a fungal cell or a yeast.
  • the bacterial cell is Escherichia coli and the yeast cell is Saccharomyces cerevisiae.
  • a nucleotide sequence obtained by modifying any of SEQ ID NOs: 45 to 132 or the reverse complement thereof which encompasses any sequence that has been obtained by modifying the sequence of any of SEQ ID NOs: 45 to 132, or of the reverse complement thereof using any method known in the art, for example, by introducing any type of mutations such as deletion, insertion and/or substitution mutations.
  • nucleic acids comprising a sequence obtained by mutation of any of SEQ ID NOs: 45 Firmenich SA 18 to 132 or the reverse complement thereof encompassed by an embodiment herein, provided that the sequences they comprise share at least the defined sequence identity of any of SEQ ID NOs: 45 to 132 or the reverse complement thereof and provided that they encode a polypeptide having oxidoreductase activity, as defined in any of the above embodiments.
  • Mutations may be any kind of mutations of these nucleic acids, for example, point mutations, deletion mutations, insertion mutations and/or frame shift mutations of one or more nucleotides of the DNA sequence of any of SEQ ID NOs: 45 to 132.
  • the nucleic acid of an embodiment herein may be truncated provided that it encodes a polypeptide as described herein.
  • a variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system.
  • bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by particular codons. Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, multiple nucleic acid sequences can code for the same protein or polypeptide, all these DNA sequences being encompassed by an embodiment herein.
  • the nucleic acid sequences encoding the oxidoreductase may be optimized for increased expression in the host cell.
  • nucleotides of an embodiment herein may be synthesized using codons particular to a host for improved expression.
  • Provided herein are also cDNA, genomic DNA and RNA sequences. Any nucleic acid sequence encoding the oxidoreductase or variants thereof is also referred herein as an oxidoreductase encoding sequence.
  • a fragment of a polynucleotide of any of SEQ ID NOs: 45 to 132 refers to contiguous nucleotides that is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and/or at least 60 bp in length of the polynucleotide of an embodiment herein.
  • the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein.
  • the fragment of the polynucleotides herein may be used as a PCR primer, and/or as a probe, or for anti-sense gene silencing or RNAi.
  • genes including the polynucleotides of an embodiment herein, can be cloned on basis of the available nucleotide sequence information, such as found in the attached sequence listing, by methods known in the art. These include e.g. the design of DNA primers representing the flanking sequences of such gene of which one is generated in sense orientations and which initiates synthesis of the sense strand and the other is created in reverse complementary fashion and generates the antisense strand. Thermostable DNA polymerases such as those used in polymerase chain reaction are commonly used to carry out such experiments. Alternatively, DNA sequences representing genes can be chemically synthesized and subsequently introduced in DNA vector molecules that can be multiplied by e.g.
  • PCR primers and/or probes for detecting nucleic acid sequences encoding an oxidoreductase are provided.
  • the skilled artisan will be aware of methods to synthesize degenerate or specific PCR primer pairs to amplify a nucleic acid sequence encoding the oxidoreductase or fragments thereof, based on any of SEQ ID NOs: 45 to 132.
  • a detection kit for nucleic acid sequences encoding the oxidoreductase may include primers and/or probes specific for nucleic acid sequences encoding the oxidoreductase, and an associated protocol to use the primers and/or probes to detect nucleic acid sequences encoding the oxidoreductase in a sample.
  • Such detection kits may be used to determine whether a plant, organism or cell has been modified, i.e., transformed with a sequence encoding the oxidoreductase.
  • the sequence of interest is operably linked to a selectable or screenable marker gene and expression of the reporter gene is tested in transient expression assays with protoplasts or in stably transformed plants.
  • DNA sequences capable of driving expression are built as modules. Accordingly, expression levels from shorter DNA fragments may be different than the one from the longest fragment and may be different from each other.
  • nucleic acid sequence coding the oxidoreductase proteins provided herein, i.e., nucleotide sequences that hybridize under stringent conditions to the nucleic acid sequence of any of SEQ ID NOs: 45 to 132.
  • the percentage of sequence identity is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100.
  • the optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web.
  • the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity.
  • NCBI National Center for Biotechnology Information
  • NCBI National Center for Biotechnology Information
  • provided herein is also an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.
  • Polypeptides are also meant to include variants and truncated polypeptides provided that they have oxidoreductase activity.
  • the at least one polypeptide having a oxidoreductase activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein-described embodiments comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 44, obtained by genetic engineering, provided that said variant has oxidoreductase activity and has the required percentage of identity to any of SEQ ID NOs: 1 to 44 as described herein.
  • the at least one polypeptide having oxidoreductase activity used in any of the herein-described embodiments or encoded by the Firmenich SA 21 nucleic acid used in any of the herein- embodiments is a variant of any of SEQ ID NOs: 1 to 44 that can be found naturally in other organisms provided that it has oxidoreductase activity.
  • the polypeptide includes a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides provided that they have oxidoreductase activity and that they share at least the defined percentage of identity with the corresponding fragment of any of SEQ ID NOs: 1 to 44.
  • variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of an embodiment herein.
  • Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of an embodiment herein, as described thereafter, are also encompassed by an embodiment herein.
  • Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of an embodiment herein.
  • a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system.
  • additional peptide sequences may be signal peptides, for example.
  • Another aspect encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides. Polypeptides resulting from a fusion with another functional protein can also be advantageously used in the methods of an embodiment herein.
  • a variant may also differ from the polypeptide of an embodiment herein by attachment of modifying groups which are covalently or non-covalently linked to the polypeptide backbone.
  • the variant also includes a polypeptide which differs from the polypeptide provided herein by introduced N-linked or O-linked glycosylation sites, and/or an addition of cysteine residues.
  • the skilled artisan will recognize how to modify an amino acid sequence and preserve biological activity.
  • DNA sequence polymorphisms may exist within a given population, which may lead to changes in the amino acid sequence of the Firmenich SA 22 polypeptides disclosed herein.
  • Such polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents.
  • nucleic acid encoding the polypeptide or variants thereof of an embodiment herein is a useful tool to modify non-human host organisms or cells and to modify non-human host organisms or cells intended to be used in the methods described herein.
  • An embodiment provided herein provides amino acid sequences of oxidoreductase proteins including orthologs and paralogs as well as methods for identifying and isolating orthologs and paralogs of the oxidoreductase in other organisms.
  • the oxidoreductase polypeptide can be obtained by extraction from any organism expressing it, using standard protein or enzyme extraction technologies. If the host organism is a unicellular organism or cell releasing the polypeptide of an embodiment herein into the culture medium, the polypeptide may simply be collected from the culture medium, for example by centrifugation, optionally followed by washing steps and re- suspension in suitable buffer solutions.
  • the polypeptide may be obtained by disruption or lysis of the cells and optionally further extraction of the polypeptide from the cell lysate.
  • the at least one polypeptide having oxidoreductase can be used in the processes of the invention.
  • the functionality or activity of any oxidoreductase protein, variant or fragment may be determined using various methods. For example, transient or stable overexpression in plant, bacterial or yeast cells can be used to test whether the protein has activity. Oxidoreductase activity may be assessed in an assay described in the examples herein, indicating functionality.
  • a variant or derivative of an oxidoreductase polypeptide of an embodiment herein retains an ability to have oxidoreductase activity.
  • Amino acid sequence variants of the oxidoreductase provided herein may have additional desirable biological functions including, e.g., altered substrate utilization, reaction kinetics, product distribution or other alterations.
  • at least one vector comprising the nucleic acid molecules Firmenich SA 23 described herein.
  • nucleic acid sequences of an embodiment herein encoding oxidoreductase proteins can be inserted in expression vectors and/or be contained in chimeric genes inserted in expression vectors, to produce oxidoreductase proteins in a host cell or non- human host organism.
  • the vectors for inserting transgenes into the genome of host cells are well known in the art and include plasmids, viruses, cosmids and artificial chromosomes.
  • Binary or co-integration vectors into which a chimeric gene is inserted can also be used for transforming host cells.
  • An embodiment provided herein provides recombinant expression vectors comprising a nucleic acid sequence of an oxidoreductase gene, or a chimeric gene comprising a nucleic acid sequence of an oxidoreductase gene, operably linked to associated nucleic acid sequences such as, for instance, promoter sequences.
  • a chimeric gene comprising a nucleic acid sequence of any of SEQ ID NO: 45 to 132 or a variant thereof may be operably linked to a promoter sequence suitable for expression in plant cells, bacterial cells or fungal cells, optionally linked to a 3’ non-translated nucleic acid sequence.
  • the promoter sequence may already be present in a vector so that the nucleic acid sequence which is to be transcribed is inserted into the vector downstream of the promoter sequence.
  • Vectors can be engineered to have an origin of replication, a multiple cloning site, and a selectable marker.
  • an expression vector comprising a nucleic acid as described herein can be used as a tool for transforming non-human host organisms or host cells suitable to carry out the method of an embodiment herein in vivo.
  • the expression vectors provided herein may be used in the methods for preparing a genetically transformed non-human host organism and/or host cell, in non-human host organisms and/or host cells harboring the nucleic acids of an embodiment herein and in the methods for making polypeptides having oxidoreductase activity, as described herein.
  • Recombinant non-human host organisms and host cells transformed to harbor at least one nucleic acid of an embodiment herein so that it heterologously expresses or over- expresses at least one polypeptide of an embodiment herein are also very useful tools to Firmenich SA 24 carry out the method of an embodiment Such non-human host organisms and host cells are therefore provided herein.
  • a further aspect of the invention provides a recombinant cell comprising a compound of formula (I) and/or formula (II) and an oxidoreductase.
  • a host cell or non-human host organism comprising at least one of the nucleic acid molecules described herein or comprising at least one vector comprising at least one of the nucleic acid molecules.
  • a nucleic acid according to any of the above-described embodiments can be used to transform the non-human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides.
  • the non-human host organism or host cell is a prokaryotic cell.
  • the non-human host organism or host cell is a bacterial cell.
  • the non-human host organism or host cell is Escherichia coli. In one embodiment, the non-human host organism or host cell is a eukaryotic cell. In another embodiment, the non-human host organism or host cell is a yeast cell. In a further embodiment, the non-human host organism or cell is Saccharomyces cerevisiae. In one embodiment the non-human host organism or host cell expresses a polypeptide, provided that the organism or cell is transformed to harbor a nucleic acid encoding said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the host organism or cell. Suitable methods to transform a non-human host organism or a host cell have been previously described and are also provided herein.
  • the host organism or host cell is cultivated under conditions conducive to the production of a compound of formula (II).
  • conditions conducive to the production of a compound of formula (II) may comprise addition of suitable cofactors to the culture medium of the host.
  • a culture medium may be selected, so as to maximize compound of formula (II) synthesis. Examples of optimal culture conditions are described in a more detailed manner in the examples.
  • Non-human host organisms suitable to carry out the method of an embodiment herein in vivo may be any non-human multicellular or unicellular organisms.
  • the non-human host organism used to carry out an embodiment herein in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used.
  • the non-human host organism to carry out the method of an embodiment herein in vivo is a microorganism. Any microorganism can be used, for example, the microorganism can be a bacteria or yeast, such as Escherichia coli or Saccharomyces cerevisiae. Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of an embodiment herein in vivo.
  • Suitable eukaryotic cells may be any non-human cell, such as plant or fungal cells. Further provided here is a method comprising transforming a host cell or a non- human host organism with a nucleic acid encoding a polypeptide having oxidoreductase activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 45 to 132, preferably 45 to 48 or 89 to 92, or comprising the nucleic acid sequence of any of SEQ ID NOs: 45 to 132, preferably 45 to 48 or 89 to 92.
  • a method provided herein comprises cultivating a non-human host organism or a host cell transformed to express a polypeptide wherein the polypeptide comprises a sequence of amino acids that has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1 to 44, preferably 1 to 4, under conditions that allow for the production of the polypeptide.
  • aldehydes can be reduced by endogenous ketoreductases present in host strains such as E. coli and yeasts, causing unwanted reduction of substrate or product of the process of the invention to the respective alcohol during in vivo and in vitro production processes.
  • ketoreductases can be downregulated, engineered or removed so as to improve the efficiency of the process of the invention.
  • ketoreductases include: ADH6 and ADH7 in yeast, yahK, dkgA. dkgB, yeaE, yjgB, yqhD, ybbO, yghZ, adhE, entA, gldA, fucO, eutG, adhP, yghAyiaY, ydjL, ydjJ, ybdR, yphC or betA.
  • Multiple ketoreductases can be targeted at the same time.
  • an embodiment of the process of the invention is wherein the host cells has reduced expression (and even no expression) of one or more endogenous ketoreductases, preferably the ketoreductase is selected from: ADH6 and ADH7 in yeast, yahK, dkgA. dkgB, yeaE, yjgB, yqhD, ybbO, yghZ, adhE, entA, gldA, fucO, eutG, adhP, yghAyiaY, ydjL, ydjJ, ybdR, yphC or betA.
  • the ketoreductase is selected from: ADH6 and ADH7 in yeast, yahK, dkgA. dkgB, yeaE, yjgB, yqhD, ybbO, yghZ, adhE, entA, gldA, fucO, eu
  • the invention further relates to methods for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a Firmenich SA 26 polypeptide-producing microorganism is optionally the expression of the polypeptides is induced by applying at least one inducer inducing gene expression and the expressed polypeptides are isolated from the culture.
  • the polypeptides can also be produced in this way on an industrial scale, if desired.
  • the microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method or in the fed-batch method or repeated fed-batch method. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioreatechnik 1. Consumable Biobacteritechnik [Bioprocess technology 1.
  • the culture medium to be used must suitably meet the requirements of the respective strains. Descriptions of culture media for various microorganisms are given in the manual “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C., USA, 1981).
  • These media usable according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugars can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
  • oils and fats for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example palmitic acid, stearic acid or linoleic acid, alcohols, for example glycerol, methanol or ethanol and organic acids, for example acetic acid or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials that contain these compounds.
  • nitrogen sources comprise ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources, such as corn-steep liquor, soya flour, soya protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used alone or as a mixture.
  • Inorganic salt compounds that can be present in the media comprise the chloride, Firmenich SA 27 phosphorus or sulfate salts of calcium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Inorganic sulfur-containing compounds for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source.
  • Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • Chelating agents can be added to the medium, in order to keep the metal ions in solution.
  • Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid or aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA).
  • the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine, or cofactors such as, flavin mononucleotide and flavin-adenine dinucleotide.
  • Growth factors and salts often originate from the components of complex media, such as yeast extract, molasses, corn-steep liquor and the like.
  • suitable precursors can be added to the culture medium.
  • the exact composition of the compounds in the medium is strongly dependent on the respective experiment and is decided for each specific case individually. Information on media optimization can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0199635773).
  • Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like. All components of the medium are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components can either be sterilized together, or separately if necessary.
  • All components of the medium can be present at the start of culture or can be added either continuously or batchwise.
  • the culture temperature is normally between 15°C and 45°C, preferably 25°C to 40°C and can be varied or kept constant during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric Firmenich SA 28 acid or sulfuric acid.
  • Antifoaming agents example fatty acid polyglycol esters, can be used for controlling foaming.
  • suitable selective substances for example antibiotics, can be added to the medium.
  • oxygen or oxygen-containing gas mixtures for example ambient air
  • the culture is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours.
  • the fermentation broth is then processed further.
  • the biomass can be removed from the fermentation broth completely or partially by separation techniques, for example centrifugation, filtration, decanting or a combination of these methods or can be left in it completely. If the polypeptides are not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolation of proteins.
  • the cells can optionally be disrupted with high-frequency ultrasound, high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the aforementioned methods.
  • the polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography, affinity chromatography and hydrophobic chromatography, and with other usual techniques such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described, for example in Cooper, T.
  • anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be used as packing in a chromatography column, Firmenich SA 29 or can be used on a microtiter plate or on other carrier.
  • a solid carrier for example a polymer matrix, which can for example be used as packing in a chromatography column, Firmenich SA 29 or can be used on a microtiter plate or on other carrier.
  • the enzymes or polypeptides according to the invention or for use in the processes of the invention can be used free or immobilized in the method described herein.
  • An immobilized enzyme is an enzyme that is fixed to an inert carrier.
  • Suitable carrier materials include for example clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion and cation exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene.
  • the carrier materials are usually employed in a finely divided, particulate form, porous forms being preferred.
  • the particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (particle-size distribution curve).
  • Carrier materials are e.g. Ca-alginate, and carrageenan.
  • Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and other immobilization techniques are described for example in J. Lalonde and A. Margolin “Immobilization of Enzymes” in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out methods according to the invention are also given for example in Rehm et al.
  • the at least one oxidoreductase enzyme which is present during a process of the invention or an individual step of a multistep-method as defined herein, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form.
  • the at least one enzyme may be present in solution or as an enzyme immobilized on a carrier or encapsulated. One or several enzymes may simultaneously be present in soluble and/or immobilized form. Also included in the scope of the present invention is the use of various commercially available laboratory kits.
  • kits can include reagents, including ERED enzymes which can be used in the process of the invention. Also included in the scope of the invention is wherein the ERED enzyme is purchased from suppliers of laboratory reagents and/or enzymes. Such kits and enzyme suppliers are well known the skilled person. Firmenich SA 30 The processes according to the can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few milliliters to dozens of liters of reaction volume) to an industrial scale (several liters to thousands of cubic meters of reaction volume).
  • a chemical reactor can be used.
  • the chemical reactor usually allows controlling the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium.
  • the process will be a fermentation.
  • the biocatalytic production will take place in a bioreactor (fermenter), where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled.
  • Cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods.
  • detergents examples include digitonin, n- dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3- [(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate), Nonidet® P40 (Ethylphenolpoly(ethyleneglycolether), and the like.
  • living cells biomass of non-living cells containing the required biocatalyst(s) may be applied for the biotransformation reactions of the invention as well. If the at least one enzyme is immobilized, it can be attached to an inert carrier as described above.
  • the conversion reaction can be carried out batch wise, fed-batch, semi-batch wise or continuously.
  • Reactants and optionally nutrients
  • the reaction of the invention may be Firmenich SA 31 performed in an aqueous, aqueous-organic non-aqueous reaction medium.
  • An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 11, like 6 to 10.
  • an organic solvent miscible, partly miscible or immiscible with water may be applied.
  • Non-limiting examples of suitable organic solvents might be selected from aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, chlorinated hydrocarbons, aromatic hydrocarbons like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, esters, such as ethylacetate, isopropylmyristate, ethers, like diethylether, methyl-tert.-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutylether, tetrahydrofuran or 2-methyltetrahydrofuran, ketones and alcohols.
  • aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane,
  • Additional mediums include DMF, DMSO, deep eutectic solvent or ionic liquids. Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols like glycerol.
  • the ratio between aqueous and organic phase in a biphasic reaction system with water immiscible organic solvents may be 20:1 to 1:10, preferably 10:1 to 1:1, more preferably 8:2 to 8:1.8.
  • the non-aqueous medium may be substantially free of water, i.e. may contain less that about 1 wt.-% or 0.5 wt.-% of water. Biocatalytic methods may also be performed in an organic non-aqueous medium.
  • a suitable organic solvents might be selected from aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, chlorinated hydrocarbons, aromatic hydrocarbons like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, esters, such as ethylacetate, isopropylmyristate, ethers, like diethylether, methyl-tert.-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutylether, tetrahydrofuran or 2-methyltetrahydrofuran, ketones and alcohols.
  • aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentan
  • Additional mediums include DMF, DMSO, deep eutectic solvent or ionic liquids.
  • concentration of the reactants/substrates may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied.
  • the initial substrate concentration may be in the 0.001 to 1 M, preferably 0.01 to 0.5 M, preferably 0.05 to 0.2 M, preferably 0.1 to 0.2 M,
  • Cofactors, such as NADP + , NAD + , NADH, NADPH, or FMN and FAD may be Firmenich SA 32 added to the reaction medium. The of such cofactors may depend on the specific enzyme applied.
  • the concentration of cofactors such as NADP + , NAD + , NADH, NADPH may be 0.1 to 1.5 mM, preferably, 0.2 to 1 mM, and the concentration of cofactors such as FMN and FAD may be 0.5 to 50 ⁇ M, preferably 1 to 5 ⁇ M.
  • the reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70°C, as for example 20 to 50 or 25 to 40°C.
  • reaction temperatures are about 25°C, 28°C, 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C and about 60°C.
  • the process may proceed until equilibrium between the substrate and the product(s) is achieved but may be stopped earlier.
  • Usual process times are in the range from 10 minutes to 48 hours, in particular 5 hours to 40 hours, as for example in the range from 10 hour to 30 hours, most preferably approximately 24 hours. These parameters are non-limiting examples of suitable process conditions.
  • the methodology of the present invention can further include a step of recovering an end or intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form.
  • the term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture or reaction media.
  • Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.
  • a conventional resin e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.
  • a conventional adsorbent e.g.
  • a further aspect of the invention provides a cell culture media comprising the recombinant cell comprising an oxidoreductase, preferably an ERED, and a compound of formula (I) and/or formula (II).
  • the composition of such media has been disclosed above in relation to the performance of Firmenich SA 33 the process of the invention.
  • Identity and purity of the isolated product may be determined by known techniques, like High Performance Liquid Chromatography (HPLC), gas chromatography (GC), Spectroscopy (like IR, UV, NMR), Coloring methods, TLC, NIRS, enzymatic or microbial assays.
  • HPLC High Performance Liquid Chromatography
  • GC gas chromatography
  • Spectroscopy like IR, UV, NMR
  • Coloring methods TLC, NIRS, enzymatic or microbial assays.
  • Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S.521-540, S.540-547, S.
  • Another object of the present invention is a process for the preparation of a compound of formula in the form of thereof, and wherein each R 1 , R 2 and R 3 , independently from each other, represent a hydrogen atom, a C 1-3 alkoxy group, a C 1-6 alkyl group or a C 2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C 3-8 cycloalkyl or C 5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an eth
  • R a and R b independently from each other, may be a C1-3 alkyl group.
  • R a and R b independently from each other, may be a methyl or ethyl group.
  • R a and R b independently from each other, may be a methyl group.
  • R c may be a C 1-3 alkyl group.
  • R c may be a methyl or ethyl group.
  • R c is an ethyl group.
  • the conversion of the compound of formula (III) into acetal of formula (IV) may be carried out under normal conditions known by the person skilled in the art, i.e. in the presence of an acid such as Bronsted acid or a Lewis acid compatible with alcohols, such as Lanthanide triflates, and a reagent selected from the group consisting of C1-4 trialkyl orthoformate, C1-4 alcohol, C2-5 diol and a mixture thereof.
  • an acid such as Bronsted acid or a Lewis acid compatible with alcohols, such as Lanthanide triflates
  • a reagent selected from the group consisting of C1-4 trialkyl orthoformate, C1-4 alcohol, C2-5 diol and a mixture thereof is well known and has been largely reported in the prior art. So, the person skilled in the art will be able to set up the best conditions in order to convert compound of formula (III) into compound of formula (IV).
  • the step a) may be performed under the conditions reported in Green Chemistry, 2013, 15(10), 2740-2746; Synthesis, 2009, (23), 4082-4086; Synlett, 2002, (2), 319-321; Tetrahedron Letters, 2004, 45(26), 5135- 5138; Current Organocatalysis, 2018, 5(3), 196-200 or Tetrahedron Letters, 2004, 45(44), 8141-8144.
  • the acid used in step a) may have a pKa below 3.
  • Bronsted acid may be selected from the group consisting of para toluenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, methanedisulfonic acid, methanetrisulfonic acid, 2,4 dinitrobenzenesulfonic acid.
  • the Bronsted acid may be para-toluenesulfonic acid.
  • Lewis acid compatible with alcohols may be selected from the group consisting of metal triflates such as Al(OTf)3, Lanthanide triflates such as Sc(OTf) 3 , Bi(OTf) 3 , metal tetrafluoroborates such as Zn(BF 4 ) 2 , and zinc halides such as ZnCl2, ZnBr2.
  • metal triflates such as Al(OTf)3, Lanthanide triflates such as Sc(OTf) 3 , Bi(OTf) 3 , metal tetrafluoroborates such as Zn(BF 4 ) 2 , and zinc halides such as ZnCl2, ZnBr2.
  • C1-4 trialkyl orthoformate, C1- 4 alcohol or C 2-5 diol may be selected from the group consisting of methanol, ethanol, ethylene glycol, propylene glycol, trimethyl orthoformate, triethyl orthoformate.
  • the C 1-4 trialkyl orthoformate, C 1-4 alcohol or C 2-5 diol can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • concentration values those ranging from about 1 to about 2 equivalents, relative to the amount of the of substrate, preferably from 1 to about 1.5 equivalents, relative to the amount of the of substrate.
  • C1-4 alcohol concentration values those ranging from about 2 to about 15 equivalents, relative to the amount of the of substrate, preferably from 3 to about 5 equivalents, relative to the amount of the of substrate
  • the optimum concentration of the C 1-4 trialkyl orthoformate, C 1-4 alcohol or C 2-5 diol will Firmenich SA 36 depend, as the person skilled in the art on the nature of the latter, on the nature of the substrate, on the reaction temperature as well as on the desired time of reaction.
  • the acid of step a) can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • the optimum concentration of the acid of step a) will depend, as the person skilled in the art knows, on the nature of the latter, on the nature of the substrate, on the nature of the C 1-4 trialkyl orthoformate, C 1-4 alcohol or C 2-5 diol, on the reaction temperature as well as on the desired time of reaction.
  • the invention’s process to form compound of formula (IV) is carried out at a temperature comprised between 20°C and 55°C.
  • the temperature is in the range between 20°C and 30°C.
  • a person skilled in the art is also able to select the preferred temperature as a function of the melting and boiling point of the starting and final products as well as the desired time of reaction or conversion.
  • the acetal formation can be carried out in the presence or absence of a solvent. When a solvent is required or used for practical reasons, then any solvent current in such reaction type can be used for the purposes of the invention.
  • Non-limiting examples include C6-12 aromatic solvents such as toluene, 1,3-diisopropylbenzene, cumene or pseudocumene, or mixtures thereof, alcoholic solvent such as methanol, ethanol, or mixtures thereof, hydrocarbon solvents such as cyclohexane or heptane, ethyl acetate or ethereal solvents such as methyl tetrahydrofuran, tetrahydrofuran or mixtures thereof.
  • the choice of the solvent is function of the nature of the substrate and/or catalyst and the person skilled in the art is well able to select the solvent most suitable in each case to optimize the reaction.
  • Specific and non-limiting examples of acid used in step b) may be selected from the group consisting of Boron trifluoride complexes, such as BF3.OEt2, BF3.OBu2, BF3.(AcOH)2 or BF3.MeCN, anhydrous zinc chloride, para toluene sulfonic acid.
  • the acid use is step b) is a Lewis acid.
  • the compound of formula R 6 - OR c can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • concentrations those ranging from about 1 to about 5 equivalents, relative to the amount of the substrate, preferably from 1.0 to about 1.2 equivalents, relative to the amount of the of substrate.
  • the acid used in step b) can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • the invention s process to form compound of formula (V) is carried out at a temperature comprised between 10°C and 100°C. In particular, the temperature is in the range between 5°C and 25°C.
  • step b) of the invention processes can be carried out in the presence or absence of a solvent.
  • a solvent is required or used for practical reasons, then any solvent current in such reaction type can be used for the purposes of the invention.
  • Non-limiting examples include C6-12 aromatic solvents such as toluene, 1,3-diisopropylbenzene, cumene or pseudocumene, or mixtures thereof, ethyl acetate or ethereal solvents such as methyl tetrahydrofuran, tetrahydrofuran or mixtures thereof or chlorinated solvents such dichloromethane, dichloroethane or a mixture thereof.
  • the choice of the solvent is function of the nature of the substrate and/or catalyst and the person skilled in the art is well able to select the solvent most suitable in each case to optimize the reaction. Particularly, the step b) may be carried out in absence of solvent.
  • steps a) and b) of the invention’s process may be done in one pot with an acid such as boron trifluoride acetic Firmenich SA 38 acid complex, para toluene sulfonic acid, sulfonic acid.
  • the acid used in step c) may be selected from the group consisting of carboxyl acid such as formic, acetic acid, aqueous acetic acid or propionic acid, inorganic acid such aqueous sulfuric acid, sulfuric acid, aqueous hydrochloric acid.
  • the acid used in step c) may be acetic acid.
  • the acid used in step c) can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • acid concentration values those ranging from about 1 to about 10 equivalents, relative to the amount of the of substrate, preferably from 3 to about 8 equivalents, relative to the amount of the of substrate
  • the optimum concentration of the acid used in step c) will depend, as the person skilled in the art knows, on the nature of the latter, on the nature of the substrate, on the reaction temperature as well as on the desired time of reaction.
  • the step c) may be carried out in a presence of a base in addition to the acid.
  • Non-limiting example of base selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, sodium formate, potassium formate, sodium propionate and potassium propionate.
  • the base further added in step c) may be sodium acetate.
  • the base used in step c) can be added into the reaction medium of the invention’s process in a large range of concentrations.
  • the invention s process to form compound of formula (I) is carried out at a temperature comprised between 25°C and 150°C. In particular, the temperature is in the range between 90°C and 120°C.
  • step c) can be carried out in the presence or absence of a solvent.
  • any solvent current in such reaction type can Geneva SA 39 be used for the purposes of the invention.
  • C6-12 aromatic solvents such as toluene, 1,3-diisopropylbenzene, cumene or pseudocumene, or mixtures thereof
  • alcoholic solvent such as methanol, ethanol, or mixtures thereof
  • hydrocarbon solvents such as but not limited to, cyclohexane or heptane, ethyl acetate or ethereal solvents such as methyl tetrahydrofuran, tetrahydrofuran, 1,4-dioxane or mixtures thereof.
  • the choice of the solvent is function of the nature of the substrate and/or catalyst and the person skilled in the art is well able to select the solvent most suitable in each case to optimize the reaction.
  • the invention’s process may be carried out in one pot; i.e. step a) to c) may be performed without isolation step of any intermediate.
  • the invention’s process for the preparation of compound of formula (I) may be carried out under batch or continuous conditions.
  • the compound of formula (IV) and (V) are, generally, novel compounds and present a number of advantages as explained above and shown in the Examples.
  • another object of the present invention is a compound of formula in the form of any one and wh 1 2 erein R , R and R 3 , independently from each other, represent a hydrogen atom, a C1-3 alkoxy group, a C1-6 alkyl group or a C2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3-8 cycloalkyl or C5-8 cycloalkenyl group; R 4 and R 5 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C1-4 alkyl group or R a and R b , when taken together, represent a C 2-5 alkanediyl group.
  • Another object of the present invention is a compound of formula Firmenich SA 40 in the form of and wherei 1 2 n R , R and R 3 , independently from each other, represent a hydrogen atom, a C1-3 alkoxy group, a C 1-6 alkyl group or a C 2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3-8 cycloalkyl or C 5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C 1-4 alkyl group or R a and R b , when taken together, represent a C2-5 alkanediyl group; R c represents a C1-4 alkyl group.
  • Another object of the present invention is the use of a compound of formula in the form of any 1 2 and wherein R , R and R 3 , independently from each other, represent a hydrogen atom, a C 1-3 alkoxy group, a C1-6 alkyl group or a C2-6 alkenyl group, each optionally substituted by a hydroxy or C 1-3 alkoxy group; or or R 1 and R 2 , are taken together and form a C 3-8 cycloalkyl or C 5- 8 cycloalkenyl group; R 4 and R 5 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C 1-4 alkyl group or R a and R b , when taken together, represent a C2-5 alkanediyl group; in the process for preparing compounds of formula (I) and (II).
  • Another object of the present invention is the use of a compound of formula Firmenich SA 41 in the form of and and wherein R 1 , R 2 and R 3 , independently from each other, represent a hydrogen atom, a C1-3 alkoxy group, a C 1-6 alkyl group or a C 2-6 alkenyl group, each optionally substituted by a hydroxy or C1-3 alkoxy group; or R 1 and R 2 , are taken together and form a C3- 8 cycloalkyl or C 5-8 cycloalkenyl group; R 4 , R 5 and R 6 , independently from each other, are a hydrogen atom, a methyl or an ethyl group; R a and R b , independently from each other, represent a C 1-4 alkyl group or R a and R b , when taken together, represent a C2-5 alkanediyl group; R c represents a C1-4 alkyl group; in the process for preparing compounds of formula (I) and (II
  • Step 2 Preparation of 1-[(1E)-3,3-Dimethoxy-2-methyl-1-propenyl]-4- methylbenzene (Compound of formula (IV)
  • Methyl orthoformate (1948.0 Firmenich SA 43 g, 18.36 mol.) was added over 2 hours. 15 after the end of the addition, sodium methylate (30% solution in methanol, 33.4 g, 0.185 mol.) was added and the mixture was concentrated by distillation.
  • Step 3 Preparation of 1-[(1E)-5-Ethoxy-3,5-dimethoxy-2-methyl-1-pentenyl]-4- methylbenzene (Compound of formula (V)) Boron trifluoride acetate (2.86 g, 0.0153 mol.) was added to 1-[(1E)-3,3-Dimethoxy-2- methyl-1-propenyl]-4-methylbenzene (1584.0 g, 7.62 mol.) cooled to 15°C. Vinyl ethyl ether (577.0 g, 8.00 mol.) was added in 3 hours. 30 minutes after the end of the addition, sodium methylate (30% solution in methanol, 13.7 g, 0.076 mol.) was added.
  • Step 4 Preparation of (2E,4E)-4-Methyl-5-(4-methylphenyl)-2,4- pentanedienal (Compound of formula (I)) Sodium hydroxide (30% aqueous solution, 277 g, 2.08 mol.) was added in 10 minutes to acetic acid (686.0 g, 11.4 mol., 5.5 eq.).
  • LB or TB medium containing kanamycin 50 ⁇ g/mL was inoculated with the E. coli strains harboring the constructs and incubated at 37 °C and 200 rpm overnight.
  • the precultures were used to inoculate the main cultures in LB or TB medium containing kanamycin (50 ⁇ g/mL) at OD 0.1, which was incubated at 37°C until the optical density at 600 nm (OD600) reached approximately 0.8.
  • Expression of the genes was induced by 0.1-0.2 mM IPTG (isopropyl-D-thiogalactopyranoside) and the cultures were shaken at 20-25°C for 20 h.
  • the cells were harvested by centrifugation, and then resuspended in 50 mM potassium phosphate buffer (KPi, pH 7 to pH 8) to an OD of 50, aliquoted and stored at -20°C. Protein production was confirmed by SDS-PAGE.
  • Example 3 Screening of ene reductases for the reduction of (2E,4E)-4-methyl-5-(4-methylphenyl)-2,4- pentanedienal to (4E)-4-methyl-5-(4-methylphenyl)-pent-4-enal
  • the screening ERED library was performed in deep well plates using the following procedure: whole cells corresponding to 1 mL cultures containing EREDs, were lysed using 150 ⁇ L of a lysis buffer containing lysozyme, Triton X-100 and DNAse, and used directly in an assay at 0.8 mL scale adding 2 g/L (2E,4E)-4-methyl-5-(4- methylphenyl)-2,4-pentanedienal, 10 U/mL GDH, 50 mM glucose, 0.2 mM NADP + and NAD + , with optionally 10% EtOH or 10% cyclohexane in 100 mM phosphate buffer, pH 7.5, 25°C, 1000
  • ketoreductases which are present in E. coli. This has been observed and described many times before in literature.
  • a well-known approach to reduce the formation of alcohol is the down-regulation or knock-out of ketoreductases as mentioned in the description section above.
  • the reaction was performed at 1 mL scale using the following procedure: whole cells containing EREDs at OD10, whole cells containing FDH at OD5, 20 mg/mL (2E,4E)-4-methyl-5-(4- methylphenyl)-2,4-pentanedienal, 1 M sodium formate, 1 mM NADP + , optionally 2 ⁇ M FMN, 10% toluol in 200 mM phosphate buffer, pH 7.5, 25°C, 1000 rpm. Reactions were done in duplicate. The reactions were extracted with EtOAc and analyzed by GC-FID as above. Table 3.

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Abstract

La présente invention concerne le domaine de la synthèse organique et concerne plus particulièrement un procédé de préparation d'un composé de formule (II) à partir d'un composé de formule (I) et un procédé de préparation d'un composé de formule (I) par l'intermédiaire de nouveaux intermédiaires chimiques précieux tels que le composé de formule (IV) et le composé de formule (V). Le composé (IV) et le composé de formule (V) font également partie de l'invention.
PCT/EP2024/067536 2023-06-27 2024-06-21 Procédé de préparation de dérivés d'aldéhydes gamma, delta-insaturés Ceased WO2025003017A2 (fr)

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