EP3645500A1 - Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective pour la préparation énantiosélective de composés alcool-amines - Google Patents

Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective pour la préparation énantiosélective de composés alcool-amines

Info

Publication number
EP3645500A1
EP3645500A1 EP18735275.2A EP18735275A EP3645500A1 EP 3645500 A1 EP3645500 A1 EP 3645500A1 EP 18735275 A EP18735275 A EP 18735275A EP 3645500 A1 EP3645500 A1 EP 3645500A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
hydroxy
fusion protein
compounds
oxidation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18735275.2A
Other languages
German (de)
English (en)
Inventor
Tarek SHANATI
Marion Ansorge-Schumacher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Dresden
Original Assignee
Technische Universitaet Dresden
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technische Universitaet Dresden filed Critical Technische Universitaet Dresden
Publication of EP3645500A1 publication Critical patent/EP3645500A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C213/02—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton by reactions involving the formation of amino groups from compounds containing hydroxy groups or etherified or esterified hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C209/00—Preparation of compounds containing amino groups bound to a carbon skeleton
    • C07C209/82—Purification; Separation; Stabilisation; Use of additives
    • C07C209/86—Separation
    • C07C209/88—Separation of optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C221/00—Preparation of compounds containing amino groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
    • C07C29/132—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group
    • C07C29/136—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group of >C=O containing groups, e.g. —COOH
    • C07C29/143—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring by reduction of an oxygen containing functional group of >C=O containing groups, e.g. —COOH of ketones
    • 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
    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/64—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by introduction of functional groups containing oxygen only in singly bound form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/29—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by introduction of oxygen-containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/32—Oxygen atoms
    • C07D209/38—Oxygen atoms in positions 2 and 3, e.g. isatin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/40—Radicals substituted by oxygen atoms
    • C07D307/42—Singly bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/14—Radicals substituted by singly bound hetero atoms other than halogen
    • C07D333/16—Radicals substituted by singly bound hetero atoms other than halogen by oxygen atoms
    • 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.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the invention relates to enzymes and processes for the stereoselective reduction of carbonyl compounds for isomerization between ephedrine and pseudoephedrine and for the stereoselective reductive amination of chiral alpha-hydroxy ketones to the amine and the corresponding counterreactions. Also included are the corresponding fusion proteins, nucleotide sequences and vector, cell and organism. Furthermore, the invention relates to the corresponding methods.
  • Alcohol dehydrogenases are oxidoreductases (enzyme class 1), which catalyze the hydride transfer depending on the co-factor nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). Alcohol dehydrogenases may be (R) - or (S) - specific according to the hydride transfer on the Si side or the re side of the prochiral carbonyl group.
  • Alcohol dehydrogenases are described for use in the synthesis of chiral arylaliphatic compounds.
  • One of these chiral arylaliphatic compounds is phenylacetylcarbinol (PAC), which serves as an intermediate in the pharmaceutical production of ephedrine and pseudoephedrine.
  • PAC phenylacetylcarbinol
  • Toukoniitty et al. describe the chemical synthesis of (R) -PAC via a platinum complex catalyst with an enantiomeric excess of 65% and a yield of 90% for both enantiomers (Toukoniitty et al., 2004).
  • Torres et al. disclose the synthesis of PAC with a rhodium catalyst (Rh / MCM-41), wherein Enantiomeric excess of up to 70% ee and a yield of 80% is achieved (Torres et al., 2014).
  • CN 101486998 A discloses a specific Morganella morganii carbonyl reductase (CMCC (B) NO 49208), its nucleic acid sequence and protein sequence.
  • the carbonyl reductase asymmetrically reduces the substrate 2-amino-1-phenyl-propanone to (D) - pseudoephedrine ((-) - (1 S, 2S) -2-methylamino-1-phenylpropan-1-ol) with addition of NAD + as a cofactor with a yield of 89%.
  • the enantiomeric purity of the product is not disclosed.
  • asymmetric ⁇ -hydroxycarbonyl compounds are prepared using pyruvate decarboxylases or their mutants.
  • WO 90/04639 A1 discloses a process for the preparation of (R) -phenylacetylcarbinol (PAC) from benzaldehyde and pyruvate, an immobilized cell mass of non-viable cells of mutant yeast strains which are resistant to aldehyde inhibition during PAC formation, preferably Saccharomyces cerevisiae P- 2180-1A-8pa, for use in the method and a method of making the immobilized cell mass.
  • the yeast strains form a pyruvate decarboxylase. By the method, yields of up to 73% are achieved.
  • WO 2001044486 A1 describes a process for the preparation of carbinols, in particular (R) -phenylacetylcarbinol (PAC), by means of yeast strains, in particular Saccharomyces cerevisiae, with a yield of up to 84% with an enantiomeric purity of 90% ee or an enantiomeric purity of up to 94% ee with a yield of 51%.
  • PAC -phenylacetylcarbinol
  • WO 99/09195 A1 discloses a process for preparing enantiomerically pure phenylacetylcarbinols, in particular (R) -phenylacetylcarbinol, from acetaldehyde and benzaldehydes in the presence of pyruvate-decarboxylase from Zymomonas, preferably Zymomonas mobilis, preference being given to reacting unsubstituted benzaldehyde.
  • the disadvantage is the formation of by-products and a low reaction rate in the event of deviations from the optimal acetaldehyde concentration of 20 to 50 mM.
  • DE 10142574 A1 describes the use of a two-phase system with at least one aqueous phase and preferably one organic phase, particularly preferably octanol.
  • a two-phase system with at least one aqueous phase and preferably one organic phase, particularly preferably octanol.
  • WO 2016/041535 A1 discloses a mutated lyase from Aceobacter pasteurianus and a method for the asymmetric synthesis of (S) -phenylacetylcarbinol (S-PAC) using the mutated lyase, wherein yields of up to 73% after 48 h and enantiomeric purity of up to 98% ee can be achieved.
  • DE3408850C2 describes the isomerization of similar compounds (with free amine or acetylated amino function) by acid treatment via an oxazolinium intermediate.
  • the disadvantage of the process under very harsh conditions very high temperatures, concentrated H2SO4 or HCl, temperatures close to acetic acid reflux, 50% NaOH). Enantiomeric purities are not published.
  • stereoselective in this case the diastereoselective
  • reductive amination of such chiral hydroxy ketones is a reaction of industrial interest.
  • the object of the present invention is to provide enzymes and processes for the stereoselective reduction of various carbonyl compounds to chiral Hydroxy compound and for the oxidation of these chiral hydroxy compounds (back) to the carbonyl compound and finally also to the isomerization between ephedrine and pseudoephedrine (combination of the latter reduction and oxidation). It is highly desirable to prepare both the chiral (R) and (S) compounds.
  • the object is achieved by an enzyme having an amino acid sequence with at least 85% identity to one of the sequences SEQ ID NO. 1 (EDH) or 2 (PseDH), where the enzyme is present as a monomer, dimer, polymer or immobilized.
  • EDH amino acid sequence with at least 85% identity to one of the sequences SEQ ID NO. 1 (EDH) or 2 (PseDH), where the enzyme is present as a monomer, dimer, polymer or immobilized.
  • enzyme stands for the enzyme with at least 85% identity to SEQ ID NO. 1
  • enzyme (PseDH) for the enzyme with at least 85% identity to SEQ ID NO. Second
  • Identity means the number of matching amino acids relative to the total number of amino acids.
  • the crosslinking of the proteins or the covalent compound and the compound via interactions to solid phases such as solid surfaces, polymers, polymer beads, glass beads or resin in, for example, particulate or particulate form.
  • the invention likewise provides a fusion protein comprising at least one amino acid sequence which has at least 85% identity to the sequence SEQ ID NO. 1 (EDH) or 2 (PseDH), wherein the fusion protein is present as a monomer, dimer, polymer or immobilized.
  • this also includes that the fusion protein according to the invention has both of the abovementioned amino acid sequences.
  • the fusion protein further comprises a label, a peptide linker, and / or another enzyme.
  • a marker or affinity tag is understood to mean a short peptide sequence which serves to detect and / or purify the fusion protein.
  • the marker is selected from a polyhistidine (His) tag, a streptavidin (Strep) tag, a maltose-binding protein (MBP) tag, an S-tag, or a glutathione S-transferase (GST). Day.
  • a peptide linker is understood to mean a short peptide sequence for linking components of the fusion protein while retaining their activity.
  • the peptide linker has 5 to 20 amino acids.
  • the additional enzyme is selected from NAD + or NADP + dependent enzymes.
  • NAD + - or NADP + -dependent enzymes can regenerate spent NAD (P) H.
  • NAD + - or NADP + -dependent enzymes are derived from alcohol dehydrogenases (ADH), formate dehydrogenases (FDH), glucose dehydrogenases (GDH), glucose-6-phosphate dehydrogenases (G6PDH), glutamate dehydrogenases (GluDH), NADH oxidases (NOX), NAD + - dependent hydrogenases or phosphite dehydrogenases (PtDH) selected.
  • the further enzyme is selected from formate dehydrogenases, for example from Candida boidinii (SEQ ID NO. 9).
  • the enzymes with an amino acid sequence SEQ ID NO. 1 or 2 can be isolated from Arthrobacter Speeles TS-15 (German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, 38124 Braunschweig, International Patent Application Number: DSM 32400, hereinafter referred to as DSM 32400).
  • the enzyme is a dehydrogenase, in particular a short-chain dehydrogenase (short-chain dehydrogenase / reductase superfamily, SDR) (Kavanagh et al., 2008).
  • a dehydrogenase in particular a short-chain dehydrogenase (short-chain dehydrogenase / reductase superfamily, SDR) (Kavanagh et al., 2008).
  • the enzyme having an amino acid sequence with at least 85% identity to SEQ ID NO. 1 is a prelog-specific ephedrine dehydrogenase (EDH).
  • EH prelog-specific ephedrine dehydrogenase
  • a prelog-specific ephedrine dehydrogenase (EDH) in the context of the invention is an enzyme which catalyzes the oxidation of (R) -hydroxy groups, in particular the oxidation of ephedrine according to formula (I): (1R, 2S) -2-methylamino-1-phenylpropane -1-ol - (S) -2-methylamino-1-phenylpropane-1-one
  • the enzymes of the present invention catalyze the reduction of the carbonyl compound, which is advantageously stereoselective.
  • the enzyme having an amino acid sequence with at least 85% identity to SEQ ID NO. 2 an anti-prelog specific pseudoephedrine dehydrogenase (PseDH).
  • An anti-prelog specific pseudoephedrine dehydrogenase (PseDH) in the sense of the invention is an enzyme which catalyzes the oxidation of (S) -hydroxy groups, in particular the oxidation of pseudoephedrine according to formula (II):
  • Diastereoselectivity and enantioselectivity includes. Diastereoselectivity is used when one of the possible diastereomeric products of one reaction is preferentially formed. Thus, in the case of enantioselectivity, the possible products are enantiomers and one of the possible enantiomers is preferentially formed.
  • the invention enables the catalytic oxidation of, in particular, ephedrine and pseudoephedrine to the product (S) -2-methylamino-1-phenylpropan-1-one.
  • the oxidation of the hydroxy group (of ephedrine or pseudoephedrine) by means of one of the enzymes according to the invention is combined with the stereoselective reduction of the resulting carbonyl group with the other enzymes according to the invention (SEQ ID NO. 1 or 2).
  • SEQ ID NO. 1 or 2 the other enzymes according to the invention
  • isomerized between ephedrine and pseudoephedrine proceeds via the intermediate in Figure 4 (Formula IV).
  • the enzyme has an amino acid sequence with at least 90% identity to one of the sequences SEQ ID NO. 1 or 2, particularly preferably at least 95% identity to one of the sequences SEQ ID NO. 1 or 2.
  • the enzyme has an amino acid sequence with at least 90% identity, preferably 95% identity in the active center of the enzyme having the sequence SEQ ID NO. 1 or 2 on.
  • An active center is understood to be the region of an enzyme which is responsible for the catalytic action by binding and reaction of the substrate.
  • the active site of ephedrine dehydrogenase (EDH) having the sequence SEQ ID NO. 1 comprises 54 amino acids, in particular amino acids 142 to 160 (SEQ ID NO: 3), amino acids 186 to 214 (SEQ ID NO: 4) and amino acids 242 to 247 (SEQ ID NO: 5).
  • the active site of pseudoephedrine dehydrogenase (PseDH) having the sequence SEQ ID NO. Figure 2 comprises 71 amino acids, particularly amino acids 135 to 156 (SEQ ID NO: 6), amino acids 182 to 220 (SEQ ID NO: 7), and amino acids 246 to 255 (SEQ ID NO: 8).
  • the enzyme of the invention has a length of 240 amino acids to 300 amino acids.
  • the enzyme according to the invention comprises one of the sequences SEQ ID NO. 1 or 2.
  • the enzyme comprises the sequence SEQ ID NO. 1 has a length of 249 amino acids to 280 amino acids. In a further embodiment, the enzyme comprising the sequence SEQ ID NO. 2 has a length of 274 amino acids to 300 amino acids.
  • the enzyme consists of one of the sequences SEQ ID NO. 1 or 2.
  • an enzyme obtained by isolation from the same organism Arthrobacter species TS-15 (DSM 32400), having an amino acid sequence with at least 85% identity to the sequence SEQ ID NO. 13 (AlAmDH),
  • this enzyme having an amino acid sequence with at least 85% identity to SEQ ID NO. 13 an anti-prelog specific alanine amine dehydrogenase (AlAmDH).
  • enzyme stands for the last-mentioned enzyme with at least 85% identity to SEQ ID NO. 13. In a preferred embodiment, the identity is 90%, more preferably 95%.
  • the latter enzyme catalyzes the stereoselective reductive amination of chiral alpha-hydroxy ketones to the 1-hydroxy-2-amino product and the opposite oxidation reaction of 1-hydroxy-2-amino compounds to the alpha-hydroxy ketone.
  • oxidation takes place in the presence of the EDH, PseDH or AlAmDH-encoding enzyme of the invention or fusion protein and cofactor NAD +; on the other hand, in the presence of NADH instead of NAD + , a reduction takes place.
  • the present enzyme regulates which functional group can be reacted by lowering the activation energy of the reaction (and acting as a catalyst).
  • NADH and NAD + are present simultaneously before or during the reaction, the equilibrium between reaction and backreaction is controlled by the present regenerant.
  • an enzyme is additionally used for the regeneration of the cofactor.
  • FDH with formic acid salts as a regenerating agent regenerate NAD +, whereas NADH is regenerated by, for example, NADH oxidase with O2.
  • the enzyme for regenerating the cofactor is preferably selected from alcohol dehydrogenases (ADH), formate dehydrogenases (FDH), glucose dehydrogenases (GDH), glucose-6-phosphate dehydrogenases (G6PDH), glutamate dehydrogenases (GluDH), NAD + -dependent hydrogenases or phosphite dehydrogenases (PtDH). It is preferably selected from formate dehydrogenases or NADH oxidases, particularly preferably from Candida boidinii (SEQ ID NO. 9).
  • the enzymes according to the invention advantageously have a high stereoselectivity in the reduction of carbonyl compounds, preferably in the reduction of carbonyl compounds of the formula (III), particularly preferably in the case of dicarbonyl compounds.
  • stereoselectivity is meant the preferred formation of one of several possible stereoisomers.
  • the enzymes according to the invention have a low substrate specificity.
  • substrate specificity is understood to mean the ability of enzymes to bind and convert only one particular substrate or group of substrates in their active site.
  • fusion protein according to the invention with a further enzyme selected from NAD + - or NADP + -dependent enzymes in a process for the stereoselective reduction of carbonyl compounds no co-factor is needed.
  • Another aspect of the invention relates to a nucleotide sequence comprising a sequence coding for an inventive enzyme or a fusion protein.
  • the nucleotide sequence comprises one of the sequences SEQ ID NO. 10 or SEQ ID NO. 1 1.
  • the nucleotide sequences SEQ ID NO. 10 or SEQ ID NO. 1 1, encoding the enzymes of the invention can be isolated from genomic material of Arthrobacter species TS-15 (DSM 32400).
  • a further aspect of the invention relates to a vector comprising at least one nucleotide sequence according to the invention.
  • a vector is understood to mean a delivery vehicle for the transfer of a nucleotide sequence into a cell.
  • the vector is recombinant.
  • Recombinant means an artificial molecule which has been assembled by means of genetic engineering methods.
  • the vector comprises one of the nucleotide sequences SEQ ID NO. 10 or SEQ ID NO. 1 1.
  • the vector is selected from a plasmid or cosmid.
  • a plasmid is understood to mean a small, annular, extrachromosomally present, double-stranded DNA molecule.
  • a cosmid is understood as meaning a plasmid which contains so-called cos sites (cohesive sites).
  • the vector further comprises a nucleotide sequence encoding an NAD + or NADP + -dependent enzyme.
  • NAD + - or NADP + -dependent enzymes can regenerate spent NAD (P) H.
  • NAD + - or NADP + -dependent enzymes are from alcohol dehydrogenases (ADH), formate dehydrogenases (FDH), glucose dehydrogenases (GDH), glucose-6-phosphate dehydrogenases (G6PDH), glutamate dehydrogenases (GluDH), NAD + -dependent hydrogenases or phosphite dehydrogenases ( PtDH) selected.
  • the vector comprises a nucleotide sequence encoding formate dehydrogenases, more preferably from Candida boidinii (SEQ ID NO: 12).
  • the invention furthermore relates to a cell or organism containing at least one nucleotide sequence according to the invention. Also included in one embodiment is that this nucleotide sequence is included as part of a vector in the cell or organism of the invention. In a preferred embodiment, the cell or organism is not human.
  • the nucleotide sequence of the invention is integrated into the genome of the cell or organism.
  • the cell or organism is from E. coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, Pichia pastoris or from the genus Arthrobacter, Rhodococcus, preferably Arthrobacter aurescens; Bacillus, preferably Bacillus subtilis; Streptomyces or Pseudomonas, preferably Pseudomonas sp., Selected.
  • the cell or organism is Arthrobacter aurescens, preferably Arthrobacter species TS-15 (DSM 32400).
  • One aspect is also a fusion protein comprising an amino acid sequence that is at least
  • the enzyme is present as a monomer, dimer, polymer or immobilized.
  • One aspect is also the nucleotide sequence comprising a sequence coding for the last-mentioned inventive enzyme or the last-mentioned inventive fusion protein.
  • One aspect is also vector, cell or organism containing this latter nucleotide sequence.
  • the invention likewise provides a process for the stereoselective reduction of carbonyl compounds or for the oxidation of hydroxy compounds, comprising the steps of a) providing an enzyme according to the invention (EDH or PseDH) or a corresponding fusion protein,
  • the provision of enzymes in the methods of the invention involves, in addition to providing in isolated form, also providing a cell or organism comprising a nucleotide sequence encoding the corresponding enzyme of the invention. Under certain conditions, the cell or organism produces and deposits this enzyme free.
  • the cell or organism in the provision of the cell or the organism in the method according to the invention eliminates the protein purification of the enzyme. Further advantageously, the enzyme has a higher stability in the cell or organism.
  • alpha-aryl ketones are stereoselectively reduced to the corresponding 1-aryl-1-hydroxy compounds with the inventive method.
  • the carbonyl compound is selected from symmetrical or unsymmetrical, substituted or unsubstituted ketones, ⁇ - or ⁇ -diketones, ⁇ -ketoesters, ⁇ -aminoketones, ⁇ -hydroxyketones or ⁇ -haloketones.
  • the carbonyl compound comprises the following substrates:
  • Substituted according to the invention means the replacement of any number of hydrogen atoms to halogen, preferably Cl, F or I, against hydroxy, hydroxyalkyl, amino, aminoalkyl, carbonyl, carbamoyl, carboxylic acid chloride, acetyl, cyano, alkyl, alkenyl or alkenyl.
  • halogen preferably Cl, F or I
  • Alkyl, alkenyl or alkynyl in the context of the invention preferably denotes substituted or unsubstituted C1-C4 chains.
  • the at least one carbonyl compound is selected from dicarbonyl compounds, preferably from ⁇ - or ⁇ -diketones.
  • Dicarbonyl compounds are advantageously reacted selectively with the process according to the invention or with the enzyme according to the invention to give hydroxycarbonyl compounds.
  • the stereoselective reduction of the process according to the invention, in the case of a diketone starting compound, wherein one of the two carbonyl carbon atoms is substituted with an aromatic proceeds regioselectively, i. the carbonyl function is selectively reacted on the aromatic-substituted carbonyl carbon.
  • the ⁇ - or ⁇ -diketones are cyclic ⁇ - or ⁇ -diketones.
  • the at least one carbonyl compound is a carbonyl compound of the formula (III)
  • X is selected from unsubstituted or substituted aryl, preferably phenyl radicals; or heterocyclic radicals, preferably furanyl radicals, thiophene radicals or pyridine radicals; wherein Y is selected from unsubstituted or substituted alkyl, aryl, heterocyclic or substituted carbonyl radicals.
  • the carbonyl compound of formula (III) wherein X is selected from substituted phenyl radicals is selected from phenyl-a-carbonyl compounds having at least one alkyl group, one hydroxy group, one alkoxy group, preferably one methoxy group; and / or a halogen on the phenyl ring.
  • the carbonyl group of a dicarbonyl compound having a phenyl radical which is in opposition to the phenyl radical is advantageously regioselectively reduced by the process according to the invention or with the enzyme according to the invention.
  • the at least one carbonyl compound is a phenyl a-diketone.
  • the reaction steps according to the invention are carried out in at least one solvent.
  • a solvent is understood as meaning an inorganic or organic liquid which brings solid or liquid substances to solution by physical means.
  • the prerequisite for suitability as a solvent is that neither the solvent nor the solute chemically changes during the dissolution process.
  • the at least one solvent is selected from an aqueous solution or a two-phase system comprising an aqueous solution and at least one organic solvent.
  • the at least one organic solvent is particularly preferably made of long-chain alkanes, preferably hexane; long-chain alkanols, preferably 1-hexanol, 1-octanol or 1-decanol; or ethers, preferably cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE) or 2-methyltetrahydrofuran (2-MTHF); selected.
  • long-chain alkanes preferably hexane
  • long-chain alkanols preferably 1-hexanol, 1-octanol or 1-decanol
  • ethers preferably cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE) or 2-methyltetrahydrofuran (2-MTHF); selected.
  • the process according to the invention is carried out for the stereoselective reduction of carbonyl compounds or for the oxidation of hydroxy compounds in a two-branched system, with the specification for steps a) to c):
  • the solution of the sparingly soluble carbonyl compounds in the at least one organic solvent and the enzymatic activity of the enzyme is retained in the aqueous solution.
  • the provision of an enzyme or a fusion protein according to the invention is carried out in step a) and the at least one solvent is selected from an organic solvent, the organic solvent being selected from alcohols, preferably from alkanols, more preferably 1-decanol ,
  • step c) is carried out at a temperature between 0 ° C and 80 ° C, preferably at a temperature between 5 ° C and 50 ° C, more preferably at 20 ° C to 35 ° C.
  • At least one cofactor is added in step c).
  • Co-factors are understood to mean molecules and molecular groups which are necessary for the enzymatic activity of enzymes.
  • the cofactor is selected from NADH, NADPH and NAD + , preferably the cofactor is NADH or NAD + .
  • the cofactor is reacted in step c).
  • the co-factor is regenerated in step c). In one embodiment of the method according to the invention, the co-factor is regenerated by addition of an NAD + or NADP + -dependent enzyme.
  • NAD + - or NADP + -dependent enzymes are derived from alcohol dehydrogenases (ADH), formate dehydrogenases (FDH), glucose dehydrogenases (GDH), glucose-6-phosphate dehydrogenases (G6PDH), glutamate dehydrogenases (GluDH), NAD + -dependent hydrogenases or phosphite dehydrogenases ( PtDH) selected.
  • the NAD + or NADP + -dependent enzyme is selected from formate dehydrogenases, more preferably from Candida boidinii (SEQ ID NO. 9).
  • formate dehydrogenases more preferably from Candida boidinii (SEQ ID NO. 9).
  • no cofactor is required for the process according to the invention by addition of an NAD + or NADP + -dependent enzyme.
  • the co-factor is regenerated by addition of a formate dehydrogenase, more preferably from Candida boidinii.
  • a formate dehydrogenase more preferably from Candida boidinii.
  • the addition of formate (methanoic acid), preferably potassium formate takes place in step c); in a concentration of 0.2 mol / l to 2 mol / l, preferably 0.5 mol / l to 1 mol / l.
  • conversion rates of at least 5 mg per hour in 1 ml of solvent, preferably at least 10 mg per hour in 1 ml of solvent, more preferably even 15, are achieved. Furthermore, yields of up to 99% and stereoselectivities of up to 99% ee are advantageously achieved.
  • the invention also provides a process for the isomerization between ephedrine and pseudoephedrine, comprising the steps:
  • a cofactor such as NADH or NAD + is present during the contacting in step c) and d).
  • the cofactor in step c) differs from that in step d).
  • steps c) and d) at least one purification or isolation step for purifying or isolating the intermediate compound (S) -2-methylamino-1-phenylpropan-1-one is carried out.
  • the invention also provides a process for the stereoselective reductive amination of chiral alpha-hydroxy ketones to the 1-hydroxy-2-amino product or to the opposite oxidation of 1-hydroxy-2-amino compounds to the alpha-hydroxy ketone, comprising the steps
  • AlAmDH an enzyme according to the invention
  • AlAmDH a fusion protein according to the invention
  • Alanine-amine dehydrogenase also requires a cofactor such as NADH for reductive amination.
  • the opposite reaction, ie the reverse reaction, of the oxidation of 1-hydroxy-2-amino compounds to the alpha-hydroxy ketone also requires a cofactor, for example NAD + .
  • methylamine is additionally used for the reductive amination.
  • the reduction / oxidation process of the present invention is combined with the present invention's reductive amination of a hydroxyketone (previously obtained from the dicarbonyl compound) to a hydroxy-amino compound.
  • a hydroxyketone previously obtained from the dicarbonyl compound
  • a hydroxy-amino compound Preferably, an alpha-dicarbonyl compound used, so that according to the invention stereoselective reduction to the chiral alpha-hydroxy ketones and subsequent reductive amination of a 1-hydroxy-2-amino compound is formed.
  • the reductive amination proceeds preferably diastereoselectively.
  • 1-phenyl-1,2-propanedione is selectively converted to ephedrine or pseudoephedrine by this combination (see FIG. 4).
  • the reaction sequence via the R-alcohol (R-PAC) to the 1R, 2S-ephedrine can be catalyzed by means of EDH and AlAmDH.
  • the reaction sequence can be catalyzed by means of PseDH and AlAmDH via the S-alcohol (S-PAC) to the 1S, 2S-pseudoephedrine.
  • a chiral aminoalcohol can be prepared from the corresponding diketone in a cascade reaction enantioselectively with more than 98% ee and without by-products.
  • the chiral alpha-hydroxy ketone (starting material) is an alpha-aryl-alpha-hydroxy ketone, in particular (R) -PAC or (S) -PAC.
  • the invention also provides the use of at least one enzyme according to the invention (EDH or PseDH), at least one fusion protein according to the invention (EDH or PseDH) or at least one vector, cell or organism according to the invention (EDH or PseDH) in the processes according to the invention, for: - stereoselective reduction of carbonyl compounds or oxidation of hydroxy compounds, or
  • the invention also provides the use of an enzyme according to the invention (AlAmDH), a fusion protein (AlAmDH) according to the invention or a vector, cell or organism (AlAmDH) according to the invention in the method according to the invention, for:
  • the invention also provides a kit comprising:
  • At least one of the enzymes according to the invention (EDH, PseDH and AlAmDH),
  • EDH EH, PseDH and AlAmDH
  • the cofactor is a molecule that is essential for the function of the enzyme or fusion protein of the invention.
  • it is NAD + or NADH.
  • the kit further contains an enzyme for regenerating the cofactor, which is as defined above.
  • the sequencing technique MiSeq Sequencer (lllumina, 2x250 bp paired-end sequencing, v3 chemistry, 2.9 million reads) is used and the assembly of the reads is done via CLC Genomics software.
  • the software "mauve” is used and the resulting order is checked manually.
  • the expression of the native peptide sequences of EDH and PseDH is carried out by cloning the nucleotide sequences SEQ ID NO. 10 or SEQ ID NO. 1 1 without marker into a pETDuet vector. Furthermore, the nucleotide sequence of formate dehydrogenase (FDH) from Candida boidinii is in each case cloned into the second rear multiple cloning site.
  • FDH formate dehydrogenase
  • EDH and PseDH are expressed in the form of a fusion protein with formate dehydrogenase (FDH) from Candida boidinii in the recombinant strain E. coli SHuffleT7.
  • FDH formate dehydrogenase
  • a polyhistidine tag is inserted at the N-terminus as well as at the C-terminus.
  • the N-terminal marker consists of 6 histidine residues followed by a linker peptide "SSGHIDDDDKH" and FDH
  • the C-terminal marker consists of 6 histidine residues between EDH and PseDH and FDH, respectively, and EDH and PseDH showed enzymatic activity as fusion proteins.
  • EDH fusion protein was generated in which the N-terminus with the additional amino acid sequence "MNGRI” begins and a rigid short peptide linker "AAAKEAAAKAA” was inserted. This EDH fusion protein showed 30% higher enzymatic activity than the wild-type but was less soluble.
  • the two generated E. co // 'strains shuffle T7 with the vectors pETDuet_PseDH_FDH or pETDuet_EDH_FDH be used for two-phase whole cell biotransformation or two-phase cell-free biotransformation.
  • lyophilized cells or 10 mg for two-phase cell-free biotransformation are incubated for 30 min in 100 mM Kpi buffer (pH 7.5) and mixed until a homogeneous suspension is obtained. Subsequently, potassium formate is added at a final concentration of 0.1M to 1M. To a volume of the aqueous phase of 1 ml, 1 ml of organic solvent (cyclopentyl methyl ether (CPME) or hexanol) is added to the substrate.
  • organic solvent cyclopentyl methyl ether (CPME) or hexanol
  • the substrate concentration varies according to the enzyme activity and the solubility of the respective substrate from 20 mM to 600 mM, in particular 371, 5 mM acetylbenzoyl, 500 mM methyl benzyl formate, 50 mM 1-phenylglyoxal, 40 mM isatin, 500 mM 1-phenyl-1, 3- butanedione, 600 mM 1-phenyl-1-propanone, 145 mM desylbromide, 163 mM 4- Chlorobenzil, 20 mM 2,2'-furil, 380 mM benzil, 143 mM 2,2'-dichlorobenzil, 324 mM 4,4'-difluorobenzil and 335 mM 4,4'-dimethylbenzil.
  • Detection of the products was by 1 H NMR measurement at 400 MHz and deuterated chloroform (CDC) was used as the solvent).
  • the chiral separation of the substrates is carried out in the Knauer Smartline HPLC system with a CHIRALPAK IA column (250 x 4.6 mm particle size 5 ⁇ ) and by means of the eluent n-hexane: 2-isopropanol 9: 1 with a flow rate of 0.8 ml / min and a temperature of 25 ° C.
  • the analysis is performed at a wavelength of 256 nm.
  • the chirality of the products is determined by means of the rotation values measured by means of a polarimeter (Perkin Elmer) and compared with literature values (Giovannini et al., 2016).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des enzymes pour une utilisation dans un procédé de réduction stéréosélective de composés carbonyle ou d'oxydation de composés hydroxylés, dans un procédé d'isomérisation entre l'éphédrine et la pseudoéphédrine, et dans un procédé d'amination réductive stéréosélective d'alpha-hydroxycétones chirales en amine, et la contre-réaction. L'invention concerne également leur séquence d'acides aminés, des protéines de fusion comprenant l'enzyme, ainsi qu'un vecteur, des cellules ou un organisme contenant une séquence nucléotidique codant pour l'enzyme. En outre, les procédés et l'utilisation correspondants sont englobés dans ce procédé.
EP18735275.2A 2017-06-28 2018-06-28 Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective pour la préparation énantiosélective de composés alcool-amines Withdrawn EP3645500A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102017210944.0A DE102017210944B4 (de) 2017-06-28 2017-06-28 Alkoholdehydrogenasen und Verfahren zur stereoselektiven Reduktion von Carbonylverbindungen
PCT/EP2018/067407 WO2019002459A1 (fr) 2017-06-28 2018-06-28 Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective – pour la préparation énantiosélective de composés alcool-amines

Publications (1)

Publication Number Publication Date
EP3645500A1 true EP3645500A1 (fr) 2020-05-06

Family

ID=62784158

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18735275.2A Withdrawn EP3645500A1 (fr) 2017-06-28 2018-06-28 Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective pour la préparation énantiosélective de composés alcool-amines

Country Status (3)

Country Link
EP (1) EP3645500A1 (fr)
DE (1) DE102017210944B4 (fr)
WO (1) WO2019002459A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109824579B (zh) * 2019-03-11 2020-08-11 浙江工业大学 一种(s)-苯基(吡啶-2-基)甲醇衍生物的制备方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3408850A1 (de) 1983-03-12 1984-09-13 Knoll Ag, 6700 Ludwigshafen Verfahren zur herstellung von threo-1-phenyl-2-amino-propanolen-(1)
WO1990004639A1 (fr) 1988-10-21 1990-05-03 Synergen, Inc. Procede permettant de produire l-phenyl acetyl carbinol (pac), une masse cellulaire immobilisee pour cette fin et un procede de preparation de ladite masse cellulaire
DE19736104A1 (de) 1997-08-20 1999-02-25 Basf Ag Verfahren zur Herstellung von enantiomerenreinen Phenylacetylcarbinolen aus Acetaldchyd und Benzaldehyd in Gegenwart von Pyruvatdecarboxylase aus Zymomonas
AUPQ462599A0 (en) 1999-12-13 2000-01-13 Polychip Pharmaceuticals Pty Ltd Yeast-based process for production of l-pac
DE10142574A1 (de) 2001-09-01 2003-03-20 Basf Ag Verfahren zur Herstellung von R-Phenylacetylcarbinol durch ein enzymatisches verfahren in einem Zweiphasensystem
DE102004022686A1 (de) * 2004-05-05 2005-11-24 Basf Ag Verfahren zur Herstellung optisch aktiver Alkohole
CA2735769C (fr) * 2008-09-17 2018-10-09 Basf Se Procede de preparation d'alcools optiquement actifs
CN101486998A (zh) 2008-12-29 2009-07-22 江南大学 摩尔根氏变形杆菌的(d)-专一性羰基还原酶的基因序列和氨基酸组成
DE102013009631A1 (de) * 2013-06-10 2014-12-11 Forschungszentrum Jülich GmbH Verfahren zur Herstellung von Cathin
DE102014013642A1 (de) 2014-09-16 2016-03-17 Forschungszentrum Jülich GmbH Lyase sowie Verfahren zur asymmetrischen Synthese von (S)-Phenylacetylcarbinol

Also Published As

Publication number Publication date
WO2019002459A1 (fr) 2019-01-03
DE102017210944B4 (de) 2019-05-23
DE102017210944A9 (de) 2019-02-28
DE102017210944A1 (de) 2019-01-03

Similar Documents

Publication Publication Date Title
JP2014524245A (ja) 二級アルコールの酸化及びアミン化
JPH09503658A (ja) 酵素触媒作用アシル化による１級及び２級アミンのラセミ分割
EP1974045B1 (fr) Procede de fabrication enzymatique de citronellal
Díaz-Rodríguez et al. Why leave a job half done? Recent progress in enzymatic deracemizations
WO2013156454A1 (fr) Alanine déshydrogénase nadp dépendante
WO2009033957A1 (fr) Procédé de fabrication d'amines enrichies en énantiomères
EP2171074B1 (fr) Procédé de production d'alcools optiquement actifs en utilisant d'une déshydrogénase dérivée de Azoarcus sp. EbN1
WO2008058951A1 (fr) Procédé de réduction enzymatique de dérivés d'alcène
DE69920568T2 (de) Verfahren zur Herstellung von optisch aktivem 4-Halo-3-hydroxybuttersäureester
CN107586793B (zh) 制备具有多个手性中心的醇化合物的方法
EP3645500A1 (fr) Enzymes et procédé pour la réduction stéréosélective de composés carbonyle, l'oxydation, ainsi que l'amination réductive stéréosélective pour la préparation énantiosélective de composés alcool-amines
Dold et al. Transaminases and their applications
DE60038281T2 (de) Verfahren zur herstellung von optisch aktiven pyridinethanol-derivaten
EP0828841A2 (fr) Procede de preparation d'acyloines, decarboxylases de pyruvate appropriees et leur preparation, et sequence d'adn du gene pdc les codant
WO2009074524A2 (fr) Procédé de réduction enzymatique d'alpha- et de bêta-déhydroaminoacides
JP3703928B2 (ja) 光学活性ｎ−ベンジル−３−ピロリジノールの製造方法
JP2005533497A (ja) ２相のアルコールデヒドロゲナーゼをベースとする共役酵素反応系
CN107406861B (zh) 手性-1,1-二氟-2-丙醇的工业的制造方法
DE102013211075B9 (de) Biotechnologisches Verfahren zur Herstellung von substituierten oder unsubstituierten Phenylessigsäuren und Ketonen mit Enzymen des mikrobiellen Styrolabbaus
JP2000175693A (ja) （ｒ）−２−ヒドロキシ−１−フェノキシプロパン誘導体の製造方法
JP2021502798A (ja) 高効率酵素法による（ｒ）−３−キヌクリジノールの製造方法
Aguillón et al. Biocatalysis toward the synthesis of chiral amines
JP2004065049A (ja) Ｄ−マンデル酸脱水素酵素をコードするポリヌクレオチド、及びその用途
JP2005533496A (ja) カンジダボイジニイから誘導されたギ酸デヒドロゲナーゼを用いた共役酵素反応系
WO2020165417A1 (fr) Variants de l-lysine epsilon-déshydrogénase et leurs utilisations

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20191213

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20220221

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20220906