Classifications

• • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
• • 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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • 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
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
• • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
• • 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
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the invention relates to a process for preparing enantiomerically pure alcohols of the general formula Ia or Ib
• R 1, R 2 , R 3 , R 4 , R 5 and R 6 each represent hydrogen, halogen, a C 1 -C 6 -alkyl or C 1 -C 6 -alkoxy group, with the proviso that at least one of the radicals Ri , R 2 , R 3 , R 4 , R 5 and R 6 is different from the other five, and the proviso that at least one of Ri, R 2 , R 3 , R 4 , R 5 and R 6 is a halogen ,
• the invention relates to a process for the preparation of enantiomerically pure alcohols of the general formula HIa or IHb
• R 7 , R 8 and R 9 represent a Ci-C 6 alkyl group.
• Enantiomerically pure alcohols of the general formulas Ia or Ib and IHa or IHb are valuable chiral building blocks for the synthesis of a variety of chiral compounds, which are of interest for the preparation of pharmaceutically active substances.
• many of these enantiomerically pure alcohols are chemically not or only very expensive representable and are therefore not available in larger quantities.
• the invention therefore has as its object to provide a process which enables the economical preparation of enantiomerically pure alcohols of general formula Ia or Ib and IHa or IHb in high yield and high enantiomeric purity.
• This object is achieved in relation to the alcohols of the general formula Ia or Ib according to the invention characterized in that a ketone of the general formula II
• Ri, R 2, R 3, R 4, R 5 and R 6 are each hydrogen, halogen, a Ci-C 6 alkyl or Ci-C 6 - represent alkoxy group, with the proviso that at least one of the radicals Ri, R 2 , R 3 , R 4 , R 5 and R 6 are different from the other five, and with the proviso that at least one of Ri, R 2 , R 3 , R 4 , R 5 and R 6 is a halogen, in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as cofactor is enzymatically reduced.
• R 9 wherein R 7 , R 8 and R 9 represent a Ci-C 6 alkyl group, is enzymatically reduced in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as a cofactor.
• NADH is understood to mean reduced jSficotinarmd-adenine dmucleotide and by the term “NAD” nicotinamide adenine dinucleotide.
• NADPH reduced nicotinamide adenine dinucleotide phosphate and by the term “NADP” nicotinamide adenine dinucleotide phosphate.
• ketones of the general formula II or IV which are used according to the invention as starting material are generally available in a simple and inexpensive manner.
• the dehydrogenase used for the enzymatic reduction is obtained according to a preferred embodiment of microbial starting material. Which configuration of the products is formed predominantly or exclusively depends on the type of dehydrogenase / oxidoreductase as well as the type of cofactor.
• a secondary alcohol dehydrogenase from Lactobacilli of the genus Lactobacilliales, in particular Lactobacillus kefir, Lactobacillus brevis or Lactobacillus minor, or from Pseudomonas used ,
• Such R-specific secondary alcohol dehydrogenases are described, for example, in US Pat. No. 5,200,335, DE 196 10 984 A1, DE 101 19 274 or US Pat. No. 5,385,833.
• S-specific dehydrogenase is preferably a secondary alcohol dehydrogenase from the genus Pichia or Candida, in particular Candida boidinii ADH, Candida parapsilosis or Pichia capsulata.
• S-specific dehydrogenases are described, for example, in US Pat. No. 5,523,223 or DE 103 27 454.
• the enzyme does not have to be used in pure form. Likewise, it is also possible to use enzyme-containing microorganisms or more or less purified lysates thereof. If the reaction is to be carried out continuously, immobilized enzymes can also be used.
• the immobilization can be carried out, for example, by including the enzymes - in particular in polymeric networks or in semipermeable membranes - or by binding to a support, for example by absorption or by ionic or covalent bonds.
• the dehydrogenases are used in free form.
• the enzymatic reduction itself proceeds under mild conditions, so that the alcohols produced do not continue to react.
• the inventive method have a long service life, an enantiomeric purity of more than 95% of the prepared chiral alcohols of formulas Ia and Ib and purple or Erb and a high yield based on the amount of keto compounds of formula II or IV.
• the oxidoreductases can either be completely purified or partially purified in the inventive process, used in the form of cell lysates or in the form of whole cells.
• the cells used may be native or permeabilized.
• Cloned and overexpressed oxidoreductases are preferably used.
• the volume activity of the oxidoreductase used is 10 U / ml to 5000 U / ml, preferably 100 U / ml to 1000 U / ml.
• the enzyme unit 1 U corresponds to the amount of enzyme required to react 1 ⁇ mol of the keto compound of the formula II or IV per minute.
• a preferred embodiment of the invention is further characterized in that the NAD or NADP formed during the reduction is continuously reduced with a cosubstrate to NADH or NADPH.
• Preferred cosubstrates are primary and secondary alcohols, such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.
• cosubstrates are converted by means of an oxidoreductase and NAD or NADP to the corresponding aldehydes or ketones and NADH or NADPH. This leads to the regeneration of NADH or NADPH.
• the proportion of the cosubstrate for the regeneration is from 5 to 95% by volume, based on the total volume.
• an alcohol dehydrogenase may additionally be added.
• Suitable NADH-dependent alcohol hydro genases are obtainable, for example, from baker's yeast, from Candida boidinii, Candida parapsilosis or Pichia capsulata.
• Suitable NADPH-dependent alcohol dehydrogenases are also found in Lactobacillus brevis (DE 196 10 984 A1), Lactobacillus minor (DE 101 19 274), Pseudomonas (US 5,385,833) or in Thermoanaerobium brockii.
• Suitable cosubstrates for these alcohol dehydrogenases are the abovementioned secondary alcohols, such as ethanol, 2-propanol (isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.
• cofactor regeneration may also be performed, for example, with NAD- or NADP-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol., Bioeng., 1999, 64, 187-193, Pilot-scale production and isolation of recombinant NAD and NADP specific Formats dehydrogenase) are performed.
• Suitable cosubstrates of formate dehydrogenase are, for example, salts of formic acid, such as ammonium formate, sodium formate or calcium formate.
• the processes according to the invention are carried out without such an additional dehydrogenase, i. a substrate-coupled coenzyme regeneration takes place.
• the aqueous portion of the reaction mixture in which the enzymatic reduction takes place preferably contains a buffer, for example potassium phosphate, tris / HCl or triethanolamine buffer, having a pH of from 5 to 10, preferably a pH of from 6 to 9.
• the buffer may additionally contain ions for stabilizing or activating the enzymes, for example zinc ions or magnesium ions.
• the temperature during the execution of the inventive method expediently from about 10 ° C to 7O 0 C, preferably from 20 ° C to 40 ° C.
• the enzymatic reaction is carried out in the presence of an organic solvent which is immiscible or only slightly miscible with water.
• This solvent is, for example, a symmetrical or unsymmetrical di (C 1 -C 6 ) alkyl ether, a straight-chain or branched alkane or cycloalkane or a water-insoluble secondary alcohol which simultaneously represents the cosubstrate.
• the preferred organic solvents are, for example, diethyl ether, tert-butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methyl-2-pentanol or cyclohexane.
• the reaction mixture consists of the use of water-insoluble solvents or cosubstrates of an aqueous and an organic phase.
• the substrate is distributed according to its solubility between organic and aqueous phase.
• the organic phase generally has a content of from 5 to 95%, preferably from 20 to 90%, based on the total reaction volume.
• the two liquid phases are preferably mechanically mixed so that a large surface is created between them.
• the NAD or NADP formed during the enzymatic reduction can be reduced again to NADH or NADPH with a cosubstrate as described.
• the concentration of the cofactor NADH or NADPH in the aqueous phase is generally 0.001 mM to ImM, in particular 0.01 mM to 0.1 mM.
• a stabilizer of the oxidoreductase / dehydrogenase is also possible.
• Suitable stabilizers are, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethyl sulfoxide (DMSO).
• the process according to the invention is carried out, for example, in a closed reaction vessel made of glass or metal.
• the components are transferred individually into the reaction vessel and stirred under an atmosphere of, for example, nitrogen or air.
• the reaction time is from 1 hour to 48 hours, especially from 2
• the reaction mixture is worked up.
• the aqueous phase is separated, the organic phase is filtered. If appropriate, the aqueous phase can be extracted once more and, like the organic phase, worked up further. Thereafter, if necessary, the solvent is evaporated from the filtered organic phase.
• POL-S2 measured at a layer thickness of 1 dm.

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• 2004
  • 2004-10-27 AT AT0180804A patent/AT501928B1/de not_active IP Right Cessation
• 2005
  • 2005-10-26 EP EP05800828A patent/EP1805313A1/de not_active Withdrawn
  • 2005-10-26 CA CA002585411A patent/CA2585411A1/en not_active Abandoned
  • 2005-10-26 CN CN2005800447796A patent/CN101120094B/zh not_active Expired - Fee Related
  • 2005-10-26 WO PCT/EP2005/011459 patent/WO2006045598A1/de active Application Filing
  • 2005-10-26 US US11/718,118 patent/US20090148917A1/en not_active Abandoned
  • 2005-10-26 KR KR1020077011888A patent/KR20070085458A/ko not_active Application Discontinuation
  • 2005-10-26 JP JP2007538326A patent/JP2008517612A/ja active Pending

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