DD287052A5 - METHOD FOR THE BIOCATALYTIC MANUFACTURE OF STEREOISOMER PURE SEKUNDAERIC ALCOHOLS - Google Patents

Abstract

The invention relates to a process for the preparation of stereoisomerically pure secondary alcohols of the general formula I from mixtures of isomers, preferably racemates, taking advantage of the high stereoselectivity of fatty alcohol oxidases from alkane-utilizing yeasts. Field of application is biocatalysis {stereoisomerically pure alcohols; racemates; stereoselectivity; Fettalkoholoxidasen; alkane-utilizing yeasts}

Description

and optionally the unreacted stereoisomer further purified and isolated.

2. The method according to claim 1, characterized in that the enzyme from Candida maltosa, Candida tropicalis, Pichia guilliermondii, Trichosporon adeninovorans or Yarrowia lipolytica or the respective microorganism is used as the fatty alcohol oxidase.

3. The method according to claim 1-2, characterized in that as the fatty alcohol producing organism a yeast of the genera Candida, Debaryomyces, Pichia, Rhodotorula, Torulopsis, Trichosporon or Yarrowia is used.

4. The method according to claim 1-3, characterized in that the yeast is a wild strain or a mutant obtained by genetic processing.

Field of application of the invention

The invention relates to a process for the preparation of stereoisomerically pure secondary alcohols having 4-28 C atom of the general formula I (see Appendix) from the isomeric mixtures of the respective alcohols.

The field of application of the invention is biocatalysis. The stereoisomerically pure secondary alcohols can be used for the synthesis of natural products, for. B. fragrances and pheromones and as terminal ligands of smectic liquid crystals.

Characteristic of the known state of the art

The chemical synthesis of secondary alcohols z. As by catalytic hydrogenation of ketones, catalytic hydration of olefins or Grignard reactions with aldehydes always leads to racemates. Their chromatographic separation into the isomers as diastereomeric derivatives, eg. B. Camphansäure ester (Mosandl, A., Z. Lebensm. Unters. Forsch. 185,379, 1987) is possible, but expensive. For some time, attempts have been made to solve this problem by exploiting the more or less pronounced stereoselectivity of enzymatic reactions. So far, NAD (P) -dependent dehydrogenases or stereoselective esterase reactions have been used, the latter also for the separation of chlalic hydroxyalkanoic acids (eg Scileinati, A. et al., Tetrahedron Lett., 29, 497, 1988. Feichter et al. , Tetrahedron, Lett., 30, 1989). The enzymatic reactions used so far all have at least one of the disadvantages mentioned below, often also several; 1. low, z. T. very low specific activity, 2. incomplete substrate turnover, 3. only moderate "ereo selectivity (in some cases << 90%), 4. usable only for a limited chain length range, 5. necessity of cofactor regeneration, all these disadvantages can be avoided by using fatty alcohol oxidases.

Object of the invention

The object of the invention is to provide a process for obtaining stereoisomerically pure secondary alcohols of the general formula I (see Appendix) from chemically prepared racemic forms of these compounds.

Explanation of the essence of the invention The invention is based on the object, a biocatalytic process for the preparation of stereoisomerically pure secondary To develop alcohols of general formula I (see Appendix 1) according to the objective. According to the invention the object is achieved in that for the very high stereoselectivity of fatty alcohol oxidases in the Oxidation of medium and long-chain secondary alcohols is used. The inventive method is characterized in that a mixture of isomers of the alcohols of the general formula I.

(see Appendix) is brought into contact with a fatty alcohol oxidase or with organisms that produce fatty alcohol oxidases, wherein if necessary catalase is added. In this case, preference is given to a stereoisomer according to the reaction equation

sec. Alcohol + O ₁ - * ketone + H ₂ O ₂ oxidized.

The unreacted stereoisomer is isolated, optionally after further purification. Preferred fatty alcohol oxidases are enzymes from Candida mattosa, Candida tropicalis, Pichia guilliermondii, Trichosporon adeninovorans or Yarrowia lipolyt ^! about to use. The method can be equally with the respective Perform microorganism. As a fatty alcohol oxidase producing organism continue to be yeasts of the genera Candida, Debariomyces, Pichia, Rhodotorula, Torulopsis, Trichosporon or Yarrowia. The yeasts used may be wild or genetically engineered mutants. For carrying out the method according to the invention, the following additional statements are made: The reaction takes place in aqueous systems with a preferred pH of 8.8. The substrate is miscible in water

organic solvents, e.g. Dissolved acetone, methanol or dimethyl sulfoxide and added in this form the reaction mixture.

In general, only a maximum of 50% are implemented at high speed, the remaining 50% not at all or only with

very low speed. This reaction is due to the fact that only one enantiomer of the secondary

Alcohol is a good enzyme substrate for fatty alcohol oxidases, the second enantiomer from steriscl for reasons not or only slowly

can be oxidized. The unreacted enantiomer is isolated from the reaction mixture by known methods.

Depending on the origin of the enzyme, either the R or the S form is oxidized to the ketone and becomes corresponding

then recovered the S or R enantiomer.

For this method, both a membrane fraction, after mechanical cell disruption of n-alkanes as Carbon-grown yeast is obtained by differential centrifugation (fraction between 6000 and

100000 x g), as well as the purified enzyme suitable.

The subcellular fraction has the advantage that it simultaneously contains the enzyme catalase, whereby the second product of the Reaction, the hydrogen peroxide, is destroyed immediately. The enzyme fatty alcohol oxidase from the yeast Candida maltosa allows z. Example, the recovery of the S-enantiomer, since the R-form

is oxidized. The pH optimum of this reaction is 8.8, at a temperature of 3O ⁰ C and dodecane-2-ol as a substrate conversion rate of 30nmol / min χ mg protein is reached. By using fatty alcohol oxidases, which work with O ₂ as an electron acceptor, the complex cofactor regeneration, which is necessary in the use of dehydrogenases, avoided.

The invention will be explained in more detail by an embodiment. embodiments example 1 Recovery of stereoisomerically pure dodecan-2-ol To 100 ml of buffered (10 mM Tris-HCl pH 8.8) aqueous solution, the 270mg protein of a membrane fraction from on n-alkanes

cultured cells of Candida maltosa (microsome fraction after mechanical cell disruption according to MAUERSBERGER et al.

Basic Microbiol. 1987), 2.5 ml of a 5OmM dodecan-2-ol solution in acetone (corresponds to 23mg as the racemate of

stereoisomeric forms R and S) was added and incubated with intensive stirring in a vessel at 25 ⁰ C. The demolition of

Reaction occurs after about 35 minutes by addition of 50 ml of 8% H ₂ SO ₄ , if in a parallel batch in a closed

3 ml measuring cell equipped with an Oj electrode, no more O ₂ consumption is observed.

The reaction mixture is treated with 750 ml of isopropanol-hexane-1N sulfuric acid (40: 10: 1 by volume, according to P.P. DOLE, J. Clin. Invest. 35, 150, 195) or other suitable solvent mixtures. The recovered organic phase contains the reaction product dodecan-2-one and the unreacted S-form of the dodecane-2-one

ol, since the fatty alcohol oxidase from C. maltosa stereoselectively oxidizes the R-form of the secondary alcohol. The determination of

Isomeric purity, upon conversion to the isopropyl carbamate, was gas chromatographed on a chiral valine amide phase

and gave an e.e. value of> 95%. The stereoisomeric S-dodecan-2-ol can be separated from the reaction product by preparative thin-layer or gas chromatography or Hochdruckflüssigkeitschromatograohie.

Comparable results are obtained in the incubation of secondary alcohols of other chain length. For the

longer-chain substrates, the incubation time or the amount of enzyme used is increased.

Annex formula I

R ₁ -CHOH-R _{2 f}

Ri = CH ₃ -, CH ₃ - -CH ₂ - or CH ₂ = CH- and

R ₂ = an alkyl group having 1-25 C atoms, in which 1 or more non-adjacent CH ₂ groups may be substituted by -O- and / or -CH = CH- and / or C = O and / or the CH ₃ group and / or one or more CH ₂ groups by F ", Cl", Br ^- , J ", CN", NO ₂ ", an aromatic or heterocyclic ring may be modified or the terminal has a carboxyl radical.

Claims (1)

1. A process for the biocatalytic recovery of stereoisomerically pure secondary alcohols of general formula I (see Appendix), characterized in that from a mixture of isomers of the alcohols of general formula I with a fatty alcohol or with organisms that produce fatty alcohol oxidases, optionally with the addition of catalase, preferably a stereoisomer according to the reaction equation sec. Alcohol + O ₂ -> ketone + H ₂ O ₂ oxidized