DE102008064575A1 - Preparing 1-hydroxy ketone compounds, useful e.g. in agrochemicals, comprises contacting dihydroxy compound with microorganism that is capable for stereoselective oxidation, in a reaction mixture, and isolating the product from the mixture - Google Patents

Abstract

Preparing 1-hydroxy ketone compounds (I) by stereoselective oxidation of secondary hydroxy group of a dihydroxy compound (II), which contains a primary and a secondary hydroxy group, comprises contacting the dihydroxy compound with a microorganism or its part, which is capable for stereoselective oxidation, in a reaction mixture, and isolating the produced 1-hydroxy ketone compounds from the reaction mixture, where the microorganism is Rhodococcus erythropolis. Preparing 1-hydroxy ketone compounds of formula (HO-CH 2-(C(R2)(R3)) n-C(=O)-CH 2-R1) (I) by stereoselective oxidation of secondary hydroxy group of dihydroxy compound of formula (HO-CH 2-(C(R2)(R3)) n-CH(OH)-CH 2-R1) (II), which contains a primary and a secondary hydroxy group, comprises contacting the dihydroxy compound with a microorganism or its part, which is capable for stereoselective oxidation, in a reaction mixture, and isolating the produced 1-hydroxy ketone compounds from the reaction mixture, where the microorganism is Rhodococcus erythropolis. R1-R3 : H, halo, nitro, cyano, amino, -SH, 1-6C-alkyl, 2-6C-alkenyl, 2-6C-alkynyl, 6-12C-aryl and/or 1-6C-alkoxy, where 1-6C-alkyl, 2-6C-alkenyl, 2-6C-alkynyl and/or 6-12C-aryl are unsubstituted or one or more times substituted with OH, halo, nitro and/or cyano, and the carbon skeleton of 1-6C-alkyl is one or more times substituted by N, O, S and/or C=O groups and/or can contain one or more double bonds; and n : 1-20, preferably 1-10.

Description

The The present invention relates to a process for the preparation of 1-Hydroxyketone Compounds by Stereoselective Oxidation of secondary hydroxy group of a primary and a secondary hydroxyl-containing dihydroxy compound, comprising contacting the dihydroxy compound with a Microorganism or parts of the microorganism.

The Preparation of enantiomerically pure compounds for use in medicine or food industry plays one for the industry great importance, as often only one of the two possible Enantiomers shows the desired biological activity. In addition, by such manufacturing methods, for example the cost of cleaning the final products and disposal lowered by-products.

ketones are valuable compounds used as raw materials in the synthesis play an important role in organic compounds. A significant one Use as starting material for the representation of chiral, enantiomerically pure compounds such as optically active alcohols with pharmacological activity, pheromones or aroma active compounds in the food industry, as well as agrochemicals.

Particularly in the pharmaceutical sector, there is an increasing demand for chiral compounds and thus for chiral synthesis technologies, since racemic compounds, ie mixtures of enantiomers, will scarcely be used as pharmaceuticals in the future. For this purpose it is necessary to provide appropriate precursor compounds. For the differentiation of the isomeric species from each other, see eg the IUPAC rules section E ( Pure Appl. Chem. 45, 11-30, 1976 ).

at the stereoselective oxidation of alcohol compounds are known chemical oxidation processes often unsuitable, especially when they have more functional groups.

A chemical, selective oxidation involving the secondary hydroxy group of 1,4-decanediol and 1,5-decanediol to the hydroxyketones 1-hydroxy-4-decanone and 1-hydroxy-5-decanone by means of IBX / n-Bu ₄ NBr / CH ₂ Cl ₂ -H ₂ O is oxidized, for example Kuhakarn et al. 2005, Tetrahedron 61: 8995-9000 known. However, the oxidation is predominantly stereoselective. In addition to the secondary hydroxy group, the primary hydroxy group is also oxidized. The yields are 70 percent GC (1-hydroxy-4-decanone) and 74 percent GC (1-hydroxy-5-decanone).

adversely in this synthesis is in addition to the effort of synthesis in particular also the cleaning or separation effort to separate the desired 1-hydroxy ketone compounds and the final care of the synthesis residues.

Increasingly, therefore, biocatalytic processes are used for the stereoselective oxidation of alcohol compounds to ketones. Subsequent asymmetric reduction of ketones as precursors to optically active alcohols is one of the key strategies for chiral biosynthesis. An overview of industrially used biotransformations is available Breuer et al. (2004) Angew. Chem. 116: 806-843 ,

So describes Romano et al. Microorganisms, such as acetogenic bacteria of the genera Gluconobacter and Acetobacter, which oxidize, for example, 1,4-nonanediol at the primary hydroxyl group under the conditions described, whereby 4-hydroxynonanoic acid and gamma-lactones are formed in the course of the overall reaction ( Romano et al. 2002, J. Mol. Catal. B: enzyme. 17: 235-240 ).

Furthermore, it is known that the microorganism Rhodococcus erythropolis DSM 44534, under the specified conditions (growth medium with ethanol, yeast extract, 1,2-propanol) also the primary hydroxy group of 1,4-nonanediol ( Moreno-Horn et al. 2007, J. Mol. Catal. B: enzyme. 49: 24-27 ) is oxidized. The selective oxidation of the secondary hydroxy group of these dialcohols, however, was not observed.

task Therefore, it is the object of the present invention to provide a process for oxidation the secondary hydroxy group is a primary one and a secondary hydroxy group-containing dihydroxy compound to provide, wherein the oxidation is to proceed stereoselectively, whereby the process, in particular with regard to the yield of desired reaction product and the correspondingly low Costs for cleaning and use of starting materials especially is cheap.

The The object is achieved by the method according to claim 1. Advantageous embodiments are in the dependent claims specified.

The inventive method thus relates to a Process for the preparation of 1-hydroxyketone compounds stereoselective oxidation of the secondary hydroxy group one is a primary and one secondary hydroxy groups containing dihydroxy compound comprising contacting the dihydroxy compound with a microorganism or parts of the Microorganism capable of stereoselective oxidation are, in a reaction mixture, wherein the microorganism Rhodococcus erythropolis, and the isolation of the produced 1-hydroxyketone compounds from the reaction mixture.

Under stereoselective oxidation in the context of the present invention a preferred oxidation of the starting substrates, the dihydroxy compounds, understood in the reaction mixture in which the 1-hydroxy ketone compounds arise in the reaction mixture after the reaction, which opposite other possible hydroxy ketone compounds in excess, preferably in excess of more than 20 percent, preferably more than 50 percent, more preferably more than 90 percent exist. The determination and separation of isomers takes place according to the methods customary to the person skilled in the art, for example liquid chromatography.

ist,
welche durch stereoselektive Oxidation der sekundären Hydroxygruppe der Dihydroxy-Verbindung der allgemeinen Formel (II)

hergestellt wird, wobei n = 1–20 ist, bevorzugt n = 1–10 und
R¹, R², R³ gleich oder verschieden und einfach oder mehrfach unabhängig voneinander ausgewählt sind aus der Gruppe, umfassend Wasserstoff, Halogen, Nitro, Cyano, Amino, -SH, -C₁-C₆-Alkyl, -C₂-C₆-Alkenyl, -C₂-C₆-Alkinyl, -C₆-C₁₂-Aryl, -C₁-C₆-Alkoxy, wobei -C₁-C₆-Alkyl, -C₂-C₆-Alkenyl, -C₂-C₆-Alkinyl und/oder -C₆-C₁₂-Aryl unsubstituiert sind oder einfach oder mehrfach unabhängig voneinander mit Hydroxy, Halogen, Nitro und/oder Cyano substituiert sind, wobei das Kohlenstoffgerüst des -C₁-C₆-Alkyls einfach oder mehrfach unabhängig voneinander Stickstoff-, Sauerstoff-, Schwefelatome und/oder C=O-Gruppen und/oder eine oder mehrere Doppelbindungen enthalten kann,
sowie die Solvate, Hydrate, Stereoisomere, Diastereomere, Enantiomere und Salze der 1-Hydroxyketon-Verbindungen.Preferred is the method in which the 1-hydroxyketone compound is a compound of the general formula (I)

is
which by stereoselective oxidation of the secondary hydroxy group of the dihydroxy compound of the general formula (II)

where n = 1-20, preferably n = 1-10 and
R ¹ , R ² , R ^{3 are the} same or different and are singly or multiply independently selected from the group comprising hydrogen, halogen, nitro, cyano, amino, -SH, -C ₁ -C ₆ -alkyl, -C ₂ - C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₆ -C ₁₂ aryl, C ₁ -C ₆ alkoxy, where -C ₁ -C ₆ alkyl, -C ₂ -C ₆ alkenyl , -C ₂ -C ₆ -alkynyl and / or -C ₆ -C ₁₂ -aryl are unsubstituted or monosubstituted or polysubstituted independently of one another by hydroxy, halogen, nitro and / or cyano, wherein the carbon skeleton of the -C ₁ -C _6- alkyls may contain, once or several times independently of one another, nitrogen, oxygen, sulfur atoms and / or C =O groups and / or one or more double bonds,
and the solvates, hydrates, stereoisomers, diastereomers, enantiomers and salts of the 1-hydroxyketone compounds.

Under Alkyl is in each case a straight-chain or branched alkyl radical, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec. Butyl, tert-butyl, pentyl, isopentyl and hexyl to understand.

Under Alkoxy is in each case a straight-chain or branched alkoxy radical, such as methyloxy, ethyloxy, propyloxy, isopropyloxy, Butyloxy, isobutyloxy, sec. Butyloxy, pentyloxy, isopentyloxy and Hexyloxy to understand.

Under Alkenyl is in each case to be understood as straight-chain or branched, where For example, the following radicals are meant: vinyl, propen-1-yl, Propen-2-yl, but-1-en-1-yl, but-1-en-2-yl, but-2-en-1-yl, but-2-en-2-yl, 2-methyl prop-2-en-1-yl, 2-methyl-prop-1-en-1-yl, but-1-en-3-yl, but-3-en-1-yl, allyl.

Alkynyl is in each case to be understood as meaning a straight-chain or branched alkynyl radical which contains two to six, preferably two to four, C atoms. For example, the following radicals are suitable: ethynyl, Propyn-1-yl, propyn-3-yl, but-1-yn-1-yl, but-1-yn-4-yl, but-2-yn-1-yl, but-1-yn-3 yl.

Under Aryl is in each case an aryl radical having 6-12 carbon atoms to understand. Aryl can be mono- or bicyclic, for example naphthyl, biphenyl and especially phenyl.

Under Halogen is to be understood in each case fluorine, chlorine, bromine or iodine.

Particularly preferred in the process according to the invention are the dihydroxy compounds and correspondingly the 1-hydroxy ketone compounds which are selected from the group in which R ¹ , R ² , R ^{3 are} identical or different and are monosubstituted or polysubstituted independently of one another from the group are hydrogen, halogen, nitro, cyano, amino, -SH, -C ₁ -C ₄ -alkyl. Finally, particularly preferred herein are the 1-hydroxyketone compounds and correspondingly the dihydroxy compounds which are selected from the group in which R ¹ , R ^{2 is} hydrogen. It is further preferred if in addition R ^{3 is} hydrogen.

When Starting substrates for the inventive Especially preferred are the dihydroxy compounds, which are selected from the group comprising 1,3-butanediol, 1,4-pentanediol, 1,4-hexanediol, 1,5-hexanediol, 1,4-heptanediol, 1,4-octanediol, 1,5-octanediol, 1,4-nonanediol, 1,5-nonanediol, 1,4-decanediol, 1,5-decanediol, 1,4-undecanediol, 1,5-undecanediol, 1,4-dodecanediol and 1,5-dodecanediol.

to Implementation of the method according to the invention is the wild type DSM 44534 of the microorganism Rhodococcus erythropolis (Deposit: German Collection of Microorganisms (DSZM), Inhoffenstr. 7B, 38124 Braunschweig), because it is less susceptible against genetic changes. Next to it is the use of wild types the significant advantage that costs are reduced since the use of GMOs (genetically modified organisms) with sometimes considerable requirements and restrictions which are regulated in the Genetic Engineering Act. By the renunciation genetically engineered recombinant microorganisms The procedure also significantly increases occupational safety.

Next living cells, such as growing and / or dormant cells of the microorganism or dead cells, the method can also be used with the microorganism in the form of ground cells, immobilized cells or lyophilized Cells are performed. Similarly, cell-free, d. H. Essentially separated from cell walls extracts, eg. B. cell-free extracts or Zentrifugatüberstände the ground or lyophilized cells for the reaction used. The extracts contain cell proteins which are released by the Centrifugation are obtained. The proportion of proteins in the extract can by conventional methods, eg. B. after Bradford determined become.

Next the stereoselective oxidation may be added to the process in an upstream step, the cultivation of the microorganism.

To can be solid, semi-solid or liquid nutrient media are used for growing the microorganism, with preference used aqueous-liquid nutrient media become.

The Cultivation takes place under aerobic or semi-anaerobic conditions and may, according to the usual methods, z. B. in shake flasks or in Submerskulturen performed become. Preferably, the cultivation is carried out in the aerobic Submersverfahren in ventilated fermenters. It is possible the Cultivation continuously or discontinuously. Preferably, work is carried out batchwise.

In a preferred embodiment, the nutrient medium one or more 2,3-alkanediol compounds for the growth of the microorganism added. Is the 2,3-alkanediol compounds the nutrient medium added, it may during or after completion of the growth phase of the microorganism are added. This can be done once Add or by successive addition. It has become demonstrated that the addition of these compounds the formation of the excess increased at formed 1-hydroxy ketone compounds. Preferably the 2,3-alkanediol compound is 2,3-butanediol.

In a further preferred embodiment of the process, one or more 2,3-alkanediol compounds are preferably added to the reaction mixture at the beginning of the reaction. The 2,3-alkanediol compounds can be used racemically or enantiomerically pure. Preferably, the 2,3-alkanediol compound is 2,3-butanediol and / or 2,3-pentanediol. 2,3-butanediol and / or 2,3-Pentanediol can thereby, as (R, R) -configured enantiomer, (S, S) -configured enantiomer, meso-configured diastereomer, or as any mixture of the enantiomers, especially as a racemate, or as any mixture of the corresponding Diastereomers are present.

In another embodiment, both the Nutrient medium and the reaction mixture, the above 2,3-alkanediol compounds are added.

For the oxidation of one primary and one secondary Hydroxy group-containing dihydroxy compounds are cultivated microorganism according to the invention in the corresponding Make these dihydroxy compounds either directly or previously centrifuged and resuspended in suitable buffers Cells added or in the case of using parts of the microorganism These compounds are with the parts of the microorganism in Reaction solutions brought into contact. The can Shake the jars on shakers become.

In a particular embodiment of the invention Procedure, the living microorganism is pretreated by the permeability of the cell membrane for the compounds increased compared to the intact system. Prefers is a method in which the microorganism, for example, by Freezing and / or treatment with an organic solvent, such as Example, toluene, is pretreated.

Prefers the process is carried out at a temperature of 18 ° C to 50 ° C, particularly preferably at 25 ° C to 40 ° C, and a pH between pH 4 and pH 9, more preferably between pH 6,8 and 7,4.

to Isolation and purification of 1-hydroxyketone compounds can conventional methods are used, e.g. B. by extraction the entire reaction mixture or parts of the reaction mixture with an organic extraction phase and subsequent Column chromatography, distillation and / or crystallization.

following some examples are presented to illustrate the invention, without reducing the invention to these examples.

Cultivation of the microorganism

A 2 L shake flask containing 500 ml of nutrient medium (4.5 g / L Na ₂ HPO, 2.2 g / L KH ₂ PO 4 × 2H ₂ O, 1.0 g / L ammonium sulfate, 0.02 g / L yeast extract, 1 g / L 2,3-butanediol, pH 7.2 +/- 0.2) was inoculated after autoclaving with 20 ml Rhodococcus erythropolis DSM 44534 inoculum culture. The inoculum was bred as a shake culture in a medium of the same composition.

The inoculated culture was 48 h under aerobic conditions at 28 ° C on a shaker, z. B. in a shaker, cultivated. After reaching a CD _{620 nm} of 1.0, the cells were separated by centrifugation at about 6000 × g for 10 min at 4 ° C from the nutrient medium. The cells were washed in K / Na phosphate buffer (100 mM, pH 7.2) and then 1 g of cell mass was resuspended in 2 ml of buffer. The cell suspension was either used directly for the reaction or stored at -20 ° C.

Stereoselective oxidation

example 1

Oxidation of dihydroxy compounds as Starting substrate with living cells

In To a 10 ml glass jar was added 4 ml of K / Na phosphate buffer (100 mM, pH 7.2) with 1 mM 1,4-nonanediol presented. The biotransformation was added by adding 1 ml of cell suspension (about 4 mg dry weight per ml) started. After 100 min reaction time at 28 ° C. in the shaker, the reaction mixture was treated with 1 ml of dichloromethane stopped, extracted and the product mixture analyzed by GC-MS. The starting substrate was 15 percent in the reaction mixture after the reaction available. There was a conversion of 81 percent 1-hydroxy-4-nonanone determined in GC-MS. Other hydroxyketones were not detected.

The procedure of Example 1 was repeated with the following starting substrates according to Table 1: Table 1 Dihydroxy compound product byproduct 1,3-butanediol 4-hydroxy-2-butanone None 1,4-pentanediol 5-hydroxy-2-pentanone None 1,4-nonanediol 1-hydroxy-4-nonanone None 1,4-decanediol 1-hydroxy-4-dekanon None 1,5-nonanediol 1-hydroxy-5-nonanone None 1,5-decanediol 1-hydroxy-5-dekanon None

Example 2

Oxidation of dihydroxy compounds as Starting substrate with a cell-free crude extract containing a protein fraction.

For the oxidation was previously 20 ml of one at -20 ° C Frozen cell suspension thawed, mixed with glass beads and 5 × 2 min by means of a ball mill digested. The cell-free crude extract was then purified by centrifugation at 6000 x g for 10 min; 4 ° C as supernatant won.

The oxidation was carried out in a 1 ml reaction mixture in a 1 ml measuring cell. The activity was determined by photometric measurement. 0.9 ml of 100 mM K / Na buffer, pH 7.2 with 1.5 mM of a dihydroxy compound and 3 mM NAD ⁺ as co-factor were introduced. The increase in NADH was monitored after addition of 100 μl of the crude cell-free extract at 340 nm.

100 percent spec. Activity corresponds to 0.2 U or an oxidation of 0.2 .mu.mol dihydroxy compound at the secondary hydroxy group per mg of crude extract per min at 25 ° C. Table 2 Dihydroxy compound Relative activity in percent 1,3-butanediol 6 1,4-pentanediol 18 1,4-nonanediol 16 1,5-nonanediol 23 1,5-decanediol 17

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• - Pure Appl. Chem. 45, 11-30, 1976 [0004]
• - Kuhakarn et al. 2005, Tetrahedron 61: 8995-9000 [0006]
• - Breuer et al. (2004) Angew. Chem. 116: 806-843 [0008]
• - Romano et al. [0009]
• - Romano et al. 2002, J. Mol. Catal. B: enzyme. 17: 235-240 [0009]
• - Moreno-Horn et al. 2007, J. Mol. Catal. B: enzyme. 49: 24-27 [0010]

Claims (11)

Process for the preparation of a 1-hydroxyketone compound by stereoselective oxidation of the secondary hydroxy group one is a primary and one secondary hydroxy groups containing dihydroxy compound comprising contacting the dihydroxy compound with a microorganism or parts of the Microorganism capable of stereoselective oxidation are, in a reaction mixture, wherein the microorganism Rhodococcus erythropolis, and the isolation of the produced 1-hydroxyketone compound from the reaction mixture. A method according to claim 1, characterized in that the 1-hydroxy ketone compound is a compound of general formula (I)

which by stereoselective oxidation of the secondary hydroxy group of the dihydroxy compound of the general formula (II)

wherein R ¹ , R ² , R ^{3 are the} same or different and are singly or multiply independently selected from the group comprising hydrogen, halogen, nitro, cyano, amino, -SH, -C ₁ -C ₆ -alkyl, -C ₂ -C ₆ -alkenyl, -C ₂ -C ₆ -alkynyl, -C ₆ -C ₁₂ -aryl, -C ₁ -C ₆ -alkoxy, where -C ₁ -C ₆ -alkyl, -C ₂ - C ₆ -alkenyl, -C ₂ -C ₆ -alkynyl and / or -C ₆ -C ₁₂ -aryl are unsubstituted or monosubstituted or polysubstituted independently of one another by hydroxyl, halogen, nitro and / or cyano, where the carbon skeleton of the - C ₁ -C ₆ -alkyl may contain, singly or multiply independently of one another, nitrogen, oxygen, sulfur and / or C =O groups and / or one or more double bonds, n = 1-20, preferably n = 1-10, and the solvates, hydrates, stereoisomers, diastereomers, enantiomers and salts thereof. A method according to claim 2, characterized in that the 1-hydroxy ketone compound and correspondingly the dihydroxy compound are selected from the group in which R ¹ , R ² , R ^{3 are the} same or different and are singly or multiply independently selected from A group comprising hydrogen, halogen, nitro, cyano, amino, -SH, -C ₁ -C ₄ alkyl. Method according to one of claims 2 or 3, characterized in that the 1-hydroxy ketone compound and correspondingly the dihydroxy compound are selected from the group in which R ¹ , R ^{2 is} hydrogen. Method according to one of the preceding claims, characterized in that the dihydroxy compound is selected from the group selected from 1,3-butanediol, 1,4-pentanediol, 1,4-hexanediol, 1,5-hexanediol, 1,4-heptanediol, 1,4-octanediol, 1,5-octanediol, 1,4-nonanediol, 1,5-nonanediol, 1,4-decanediol, 1,5-decanediol, 1,4-undecanediol, 1,5-undecanediol, 1,4-dodecanediol and 1,5-dodecanediol. Method according to one of the preceding claims, characterized in that the microorganism Rhodococcus erythropolis is the wild-type DSM 44534. Method according to one of the preceding claims, characterized in that the microorganism in the form of growing Cells, resting cells, ground cells, immobilized cells, lyophilized cells or cell-free extracts of these cells becomes. Method according to one of the preceding claims, characterized in that the microorganism is in contact before bring with at least one 2,3-alkanediol compound in contact is brought. Method according to claim 8, characterized in that the 2,3-alkanediol compound is 2,3-butanediol and / or 2,3-pentanediol is. Method according to one of the preceding claims, characterized in that the reaction mixture at least one Contains 2,3-alkanediol compound. Method according to claim 10, characterized in that that the reaction mixture 2,3-butanediol and / or 2,3-pentanediol contains.