WO2008074506A1 - Résolution optique d'un mélange d'énantiomères de butynol ou de buténol - Google Patents

Résolution optique d'un mélange d'énantiomères de butynol ou de buténol Download PDF

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Publication number
WO2008074506A1
WO2008074506A1 PCT/EP2007/011267 EP2007011267W WO2008074506A1 WO 2008074506 A1 WO2008074506 A1 WO 2008074506A1 EP 2007011267 W EP2007011267 W EP 2007011267W WO 2008074506 A1 WO2008074506 A1 WO 2008074506A1
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WIPO (PCT)
Prior art keywords
cofactor
butyn
compound
process according
buten
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PCT/EP2007/011267
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English (en)
Inventor
Stefaan Marie André WILDEMAN DE
Wolfgang Skranc
Henricus Martinus Maria Gerardus Straatman
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Dsm Ip Assets B.V.
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Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2008074506A1 publication Critical patent/WO2008074506A1/fr

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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
    • C12P7/02—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
    • 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
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • 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 the preparation of a desired enantiomer of a compound selected from the group of 3-butyn-2-ol and 3-buten-2-ol, from a (racemic) mixture of enantiomers of the compound by selective oxidation of the non-desired enantiomer and isolation of the desired enantiomer.
  • optical resolution of said compounds from a (racemic) mixture poses serious problems to the man skilled in the art. Due to minimal sterical difference between both methyl and ethynyl ethenyl respectively groups within said compounds neither the use of an enzyme such as lipase nor the use of classical chemical methods for optical resolution of these alkynol compounds do provide preferred processes in terms of selectivity for and yield of the desired enantiomer. Derivatization at the ⁇ -position of the triple or double bonds of the C 4 -compounds forming derivatives such as trimethylsilyl-alkynols, partially can solve this problem, but this adds chemical steps to the synthesis route.
  • the present invention provides a solution to these problems avoiding derivatization of the compound or other additional steps while maintaining a high stereoselectivity due to the recognition of only one of both enantiomers by certain dehydrogenase enzymes.
  • the resolution of 3-butyn-2-ol has never been shown to proceed well in literature, the present invention surprisingly provides a solution for this problem.
  • the desired enantiomerically enriched compound selected from the group of 3-butyn-2-ol and 3-buten-2-ol can be prepared from a (racemic) aqueous mixture of the enantiomers by selectively oxidating the non-desired enantiomer of the compound in the presence of an alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, and isolating the desired enantiomerically enriched compound.
  • the present invention relates to optical resolution of a racemic mixture containing an alkynol compound of the general formula [1]
  • the invention results in the separation of the desired enantiomeric form of 3-butyn-2-ol, or 3-buten-2-ol, respectively, from 3-butyn-2-one or 3-buten-2-one, respectively, and the target enantiomer of 3-butyn-2-ol or 3-buten-2-ol may subsequently be isolated and purified to the desired purity.
  • a cofactor suitable for use in the process of the present invention includes any oxidized cofactor, for instance an oxidized nicotinamide cofactor, preferably nicotinamide adenine dinucleotide (NAD + ) or nicotinamide adenine dinucleotide phosphate (NADP + ).
  • an oxidized cofactor for instance an oxidized nicotinamide cofactor, preferably nicotinamide adenine dinucleotide (NAD + ) or nicotinamide adenine dinucleotide phosphate (NADP + ).
  • NAD + nicotinamide adenine dinucleotide
  • NADP + nicotinamide adenine dinucleotide phosphate
  • the concentration of cofactor used in the process of the present invention is not critical.
  • 0.01 mol/l and 10 mmol/l are used, more preferably between 0.1 mmol/l and 1 mmol/l, in particular between 0.2 mmol/l and 0.5 mmol/l.
  • alcohol dehydrogenase is defined as an enzyme capable of catalyzing the oxidation of an alcohol to the corresponding ketone or corresponding aldehyde, preferably, also capable of catalyzing the reduction of a ketone or an aldehyde to the corresponding alcohol.
  • Alcohol dehydrogenases suitable for the invention include: alcohol dehydrogenase from EC class: 1.1 , preferably from EC class 1.1.1. Alcohol dehydrogenases are abundant and may for instance be isolated from living organisms, preferably microorganisms, such as yeasts, bacteria and fungi. Examples of alcohol dehydrogenases include lactate dehydrogenases.
  • An alcohol dehydrogenase may for example be selected for the process of the invention by screening several enzymes or host cells expressing genes encoding alcohol dehydrogenases.
  • Suitable ADH enzymes can for example be selected from the group of ADH enzymes represented by the sequences SEQ ID NO. 2 and SEQ ID NO. 4.
  • the reduced form of the cofactor will be formed.
  • the reduced cofactor is being oxidized to the oxidized form of the cofactor by a cofactor regeneration enzyme during the enzymatic resolution reaction. More preferably this oxidation of reduced cofactor, for example
  • NAD(P)H into oxidized co-factor, for example NAD(P)+, is effected by a cofactor regeneration enzyme, for example a NAD(P)H oxidase.
  • a cofactor regeneration enzyme for example a NAD(P)H oxidase.
  • Suitable NAD(P)H oxidases are for example derived from Bacillus sp., Lactobacillus sp.and Streptococcus sp.
  • this oxidation of reduced cofactor is catalyzed by a cofactor regeneration enzyme in the presence of a ketone forming a second phase with water.
  • Enzymes may be used as cell free extracts or as part of a whole cell catalytic system co-expressing both required enzymes. In a preferred embodiment, a whole cell catalytic system co-expressing both required enzymes is used.
  • concentration of the compound used in the process of the present invention is not critical. Preferably, the compound is used in a concentration of at least 1 mmol/l, more preferably at least 10mmol/l, in particular at least 0.1 mol/l, more in particular at least 1 mol/l.
  • the compound is used in a concentration of not more than 5 mol/l, more preferably not more than 4 mol/l.
  • the amount of enzyme (alcohol dehydrogenase and/or NAD(P)H oxidase) used is in principle not critical.
  • Unit (U) 1 micromole substrate converted per minute at 37°C at pH 7.0 and at 1 bar pressure.
  • U 1 micromole substrate converted per minute at 37°C at pH 7.0 and at 1 bar pressure.
  • the alcohol dehydrogenase and the cofactor regeneration enzyme may be used in any form.
  • the alcohol dehydrogenase and the cofactor regeneration enzyme may be used - for example in the form of a dispersion, emulsion, a solution or in immobilized form - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess alcohol dehydrogenase and/or cofactor regeneration enzyme activity, or in a lysate of cells with such activity.
  • both alcohol dehydrogenase and cofactor regeneration enzyme are coexpressed in one host allowing whole cell application shuttling the cofactor within the cell and avoiding difficult work-up procedures.
  • the process of the present invention may be performed in a batch process. Alternatively, the process of the present invention is performed in a
  • the oxidized compound is (semi)continuously removed from the reactor.
  • the enantiomerically enriched compound is removed from the reaction mixture, for instance by distillation. It is known to the person skilled in the art which conditions are suited for distilling the enantiomerically enriched compound.
  • solvents may be chosen from a wide range of solvents .
  • a ketone different from 3-butyn-2-one may function both as a solvent and as a cofactor oxidizing agent, for example: formaldehyde, acetaldehyde, acetone, 2-butanone, 4-methyl-2-pentanone, 2- pentanone, 3-pentanone, 2-hexanone, 3-hexanone, cyclo-hexanone, methyl iso-butyl ketone, 2-heptanone, 2-octanone.
  • Water may also be chosen as the sole solvent, which is advantageous from a practical and environmental point of view.
  • solvents for example combinations of solvents with water and a solvent as mentioned above.
  • the reaction is carried out in a two phase system. More preferably, the process is carried out in a system comprising water and a ketone having a boiling point exceeding 135 0 C at atmospheric pressure, for example 2-octanone.
  • the water concentration is preferably at least 1 vol%, more preferably at least 5 vol%, even more preferably at least 10 vol%, in particular at least 20 vol%, more in particular at least 30 vol%.
  • Using a two-phase system has as an advantage that it allows higher compound concentrations to be used, exceeding 1wt% of compound, mor prefereably exeding 2wt% of compound, relative to the total weight of the reaction mixture, while still achieving conversions exeeding 50% and enantiomeric excess exceeding 60%, preferably 70%, more preferably 90%.
  • the ee of the process according to the invention exceeds 95%.
  • the choice of the reaction conditions of the process of the invention depends on the choice of the enzyme system used for the optical resolution.
  • the temperature of the process is chosen between 0 and 90 0 C, in particular between 10 and 70 0 C, more in particular between 20 and 50 0 C; usually the pH of the process is chosen between 5 and 12, more preferably between 6 and 11.
  • the pH is preferally chosen between 8-10, more preferably between 8.50-9.50.
  • the used oxygen concentration is as high as possible in view of solubility and safety.
  • the temperature and the chosen solvent more or less oxygen will dissolve in the reaction mixture.
  • Oxygen transfer to the reaction mixture can be enhanced using methods known to the person skilled in the art, for example by oxygen transfer systems used in large-scale fermentors, for example by the use of nozzles.
  • Isolation of the enantiomerically enriched compound may be performed by methods known to the person skilled in the art. For example, it may be isolated by evaporation of the organic phases, such as the organic solvents that may be present and the reduced form of the ketone. Such conditions are known to the person skilled in the art.
  • the process according to the invention is a process wherein 3-butyn-2-one or 3-buten-2-one, respectively, is wholly or partially removed from the reaction mixture, or wholly or partially neutralized.
  • Neutralization of 3-butyn-2-one and 3-buten-2-one may for example be carried out by adding NaHSO 3 .
  • Enantiomerically enriched compound according to the present invention may be used as a building block in e.g. pharmaceutical and agro products.
  • Synthetic E. coli codon-optimized gene constructs of (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (SEQ ID No. 1) and NADH oxidase (NOX) of Streptococcus mutans (SEQ ID No. 5) were prepared for co- expression in a single E. coli host cell. Cloning was done with Gateway technology (Invitrogen) towards pBAD-DEST expression vectors. The E. coli host cells were TOP10 cells; competent cells were used in transformation experiments (purchased at Invitrogen). The expression clones were transformed via the standard heat-shock transformation protocol of Invitrogen. Medium used in fermentation experiments was Luria Bertani broth, applying 100 mg/l carbenicillin as antibiotic and 0.02 wt%
  • L-arabinose as inducer. Induction of cells occurred at OD 62 o 0.6 under fermentation conditions. Cell densities of co-expressing E. coli cells reached 30 gcww/l- Cells were harvested after centrifugation.
  • Racemic 3-butyn-2-ol and NAD + were purchased at Sigma-Aldrich. LB broth was purchased at Difco (BD).
  • Second phase is 1 liter of 2-octanone.
  • E. coli codon-optimized gene constructs of (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (SEQ ID No. 1 ) were prepared for co-expression in a single E. coli host cell. Cloning was done with Gateway technology (Invitrogen) towards pBAD-DEST expression vectors. The E. coli host cells were TOP10 cells; competent cells were used in transformation experiments (purchased at Invitrogen). The expression clones were transformed via the standard heat-shock transformation protocol of Invitrogen.
  • Racemic 3-butyn-2-ol and NAD + were purchased at Sigma-Aldrich. LB broth was purchased at Difco (BD).
  • Biooxidation reaction Ingredients needed: 1 liter of water containing racemic 3-butyn-2-ol

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

Abstract

La présente invention concerne un énantiomère souhaité d'un composé choisi parmi le groupe constitué de 3-butyn-2-ol et 3-butèn-2-ol, qui peut être préparé à partir d'un mélange racémique contenant l'énantiomère souhaité dudit composé par oxydation sélective de l'énantiomère non souhaité et par isolement de l'énantiomère souhaité. Une telle oxydation sélective peut de préférence être menée à l'aide d'un système enzymatique approprié. Un tel système enzymatique peut contenir un alcool déshydrogénase approprié, un cofacteur et une enzyme de régénération de cofacteur.
PCT/EP2007/011267 2006-12-21 2007-12-20 Résolution optique d'un mélange d'énantiomères de butynol ou de buténol WO2008074506A1 (fr)

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EP06026565 2006-12-21
EP06026565.9 2006-12-21

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009153325A1 (fr) * 2008-06-19 2009-12-23 Dsm Ip Assets B.V. Résolution optique d'un mélange d'énantiomères de butynol ou de buténol
WO2011157717A1 (fr) * 2010-06-14 2011-12-22 Dsm Fine Chemicals Austria Nfg. Gmbh & Co Kg Nouveaux polypeptides ayant une activité nad(p)h oxydase et leur utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5385833A (en) * 1992-02-26 1995-01-31 The Scripps Research Institute Pseudomonas sp. ATCC No. 49794 alcohol dehydrogenase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5385833A (en) * 1992-02-26 1995-01-31 The Scripps Research Institute Pseudomonas sp. ATCC No. 49794 alcohol dehydrogenase

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE UniProt [online] 5 July 2004 (2004-07-05), KURODA, M. ET AL: "Alcohol dehydrogenase from Staphylococcus aureus", XP002436502, retrieved from EBI accession no. ADH_STAAN Database accession no. Q7A742 *
EDEGGER ET AL: "Biocatalytic oxidation of sec-alcohols via hydrogen transfer", JOURNAL OF MOLECULAR CATALYSIS. A, CHEMICAL, ELSEVIER, AMSTERDAM, NL, vol. 251, no. 1-2, 17 May 2006 (2006-05-17), pages 66 - 70, XP005403839, ISSN: 1381-1169 *
KROUTIL W ET AL: "Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols", CURRENT OPINION IN CHEMICAL BIOLOGY, CURRENT BIOLOGY LTD, LONDON, GB, vol. 8, no. 2, 2004, pages 120 - 126, XP002416332, ISSN: 1367-5931 *
STAMPFER W ET AL: "Biocatalytic oxidative kinetic resolution of sec-alcohols: stereocontrol through substrate-modification", TETRAHEDRON: ASYMMETRY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 14, no. 2, 17 January 2003 (2003-01-17), pages 275 - 280, XP004404190, ISSN: 0957-4166 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009153325A1 (fr) * 2008-06-19 2009-12-23 Dsm Ip Assets B.V. Résolution optique d'un mélange d'énantiomères de butynol ou de buténol
WO2011157717A1 (fr) * 2010-06-14 2011-12-22 Dsm Fine Chemicals Austria Nfg. Gmbh & Co Kg Nouveaux polypeptides ayant une activité nad(p)h oxydase et leur utilisation

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