WO2009056614A1

WO2009056614A1 - Vegetable biocatalysts for the production of optically-active hydroxy compounds

Info

Publication number: WO2009056614A1
Application number: PCT/EP2008/064759
Authority: WO
Inventors: Ralf Zuhse; Thomas Grimm
Original assignee: Chiracon Gmbh
Priority date: 2007-10-30
Filing date: 2008-10-30
Publication date: 2009-05-07
Also published as: DE102007052112A1

Abstract

The present invention relates to novel biocatalysts made of plants of the genera Pastinaca, Petroselinum, Anethum, or Levisticum, which are suitable for the production of optically-active hydroxy compounds made of prochiral ketones, and to the use thereof.

Description

Plant biocatalysts for the preparation of optically active hydroxy compounds

The present invention relates to novel biocatalysts from plants of the genera Pastinaca, Petroselinum, Anethum or Levisticum for the preparation of optically active hydroxy compounds. The biocatalysts can be used as vegetative parts of the plants or as isolated enzymes. The optically active hydroxy compounds are prepared from the corresponding prochiral ketones.

The enantioselective synthesis of chiral compounds has gained in importance in recent years. Optically active hydroxy compounds are important chiral precursors for developments in pharmacy, catalyst production and agrochemistry. [R. N. PATEL: "Biocatalytic Synthesis of Chiral Pharmaceutical Intermediates", Food Technol. Biotechnol., 42 (4) 305-325 (2004)].

The preparation of optically active hydroxy compounds takes place in various chemical and biocatalytic processes (Faber et al., "Biotransformations in Organic Chemistry", 5th ed., 2004, Springer Verlag, Berlin, New York.) Chemical syntheses often have low yields or low optical purities Enantiomeric excesses are usually only possible by using expensive catalysts or under extreme reaction conditions. In biocatalytic processes, living biological material such as microorganisms, plant or animal cell cultures for whole-cell biotransformation or enzymes obtained from the biological material are used for enzymatic biotransformation. These systems can also be used for the reduction of prochiral ketones to optically active hydroxy compounds. The large number of publications and patents stands in contrast to the previously implemented industrial processes (Wandrey et al., "Industrial Biotransformations", Wiley-VCH Verlag GmbH Weinheim, 2000) .The reason for this is the often high process costs, caused by cofactors and enzymes and the limited substrate spectrum of the biocatalysts used.

Most publications on the use of biocatalysts for the production of optically active hydroxy compounds describe the use of microorganisms or microbially produced enzymes. The rapid growth, the high achievable cell densities and the controllability of the biotransformation processes are advantages of the use of microorganisms. However, microorganisms have to be grown under sterile conditions, the enzymes need to be isolated and the biotransformations carried out in closed reactors.

The use of biocatalysts from plant sources allows an easy-to-use biotransformation without high expenditure on equipment. For this purpose, many studies have been carried out in recent years (Nakajiama et al., "Biotransformations using plant cultured cells", J. Molecular Catalysis B: Enz. 23 (2003) 145-170).) Thus, by Marioni et. (J. Molecular Catalysis B: Enz. 11 (2000) 55-58) Studies on the Preparative Synthesis of Reductio

CHA-23533 version.doc performed with Daucus carota roots. By Mironowicz et al. (Tetrahedron Asymmetry 13 (2002) 2299-2302) investigations were carried out on the enantioselective reduction of ketones by means of carrots, celery and horseradish.

Yadav et. al. in Tetrahedron: Asymmetry 12 (2001) 3381-3385 and J. Org. Chem. 2002, 67, 3900-3903 describes the use of Daucus carota roots for the reduction of ketones, the method has filed in the US to the US patent , The application was published as US 2004/0082043 A1.

The ongoing worldwide investigations underline the work at the University of Sao Paulo, BRA of Leandroh et. al. (J. Molecular Cat. B: Enz. 38 (2006) 84-90) for the bioreduction of aromatic ketones with various plant species. However, many of the plant species used have only a small conversion, if the enantioselectivity is in part good.

The object of the invention was therefore to find novel enantiose- lektive biocatalysts that are cheap available and can be used easily and are suitable for the production of optically active hydroxy compounds from the corresponding prochiral ketones.

According to the invention, the object has been achieved by biocatalysts of plants of the genera Pastinaca, Petroselinum, Anethum or Levisticum, which have been produced by comminuting these plants or parts thereof and optionally further processing steps.

Although the mentioned plants belong to the carrot family like the carrots mentioned in the introduction. The umbels' family includes a wide range of

CHA-23533 version.doc most plant species, which vary greatly by different growth and fruit shape. These plant species were studied alongside different plants from other families, with few species yielding usable results. The surprising suitability of the said plants was thus revealed only after lengthy investigations.

The plant biocatalyst is used in vegetative form as a plant or plant part, as a crude extract of the homogenized plant parts or as an isolated enzyme of the plant.

The biocatalysts according to the invention are preferably prepared from plants of the genus Pastinaca or Petroselinum.

It is preferred that the biocatalysts be prepared from vegetative plants or plant parts. In particular, the biocatalysts according to the invention are prepared from stems and / or roots of these plants.

It is particularly preferred to produce them from plant roots of plants of the genus Pastinaca.

In a preferred embodiment, the biocatalysts according to the invention are characterized in that they have been prepared by isolation of the enzymes contained in the plants. It is preferred that they have been prepared by enzyme crude extraction.

The biocatalysts according to the invention are suitable for the preparation of optically active hydroxy compounds from the corresponding prochiral ketones.

The invention therefore also relates to the use of biocatalysts from plants of the genera Pastinaca, Petra

CHA-23533 version.doc roselinum, anethum or levisticum, which have been prepared by comminuting these plants or parts thereof and optionally further processing steps, for the preparation of optically active hydroxy compounds from prochiral ketones.

In a preferred embodiment, plants of the genus Pastinaca or Petroselinum are used. The use according to the invention is characterized in particular by the use of vegetative plants or plant parts. Stems and / or roots of these plants are preferably used. It is particularly preferred to use plant roots of plants of the genus Pastinaca.

In a preferred embodiment, enzymes isolated from the plants are used. It is particularly preferred to use enzyme crude extracts.

Aromatic and aliphatic organic compounds of the general formula (I) are used as prochiral ketones

(D, wherein the substituents R ¹ and R ^{2 are} selected from the

Group of

alkyl,

alkenyl,

alkynyl,

cycloalkyl,

CHA-23533 version.doc Cycloalkenyl radicals, aryl radicals, aralkyl radicals, cycloalkylalkyl radicals, aryloxyalkyl radicals and heteroaryl radicals, where existing carbon chains are rectilinear or branched and wherein the cyclic and aromatic systems are monovalent rings or multi-membered annelated rings and optionally at least one further substituent on the substituents R ¹ , R ^{2 is} present and wherein R ¹ and R ^{2 are the} same or different.

Preferably, the substituents R ¹ and R ^{2 are} selected from the group of

C 1 -C ₄ -alkyl radical,

C ₃ -C ₄ alkenyl radical,

C ₃ -C ₄ -alkyl radical,

C ₃ -C ₄ -cycloalkyl radical,

C ₃ -C ₄ cycloalkylene,

CeH ₅ aryl radical (phenyl radical),

C ₆ H ₅ -aryl-C 1 -C ₅ -alkyl radical,

C ₃ -C ₄ -cycloalkyl-C 1 -C ₅ -alkyl radical,

C ₆ H ₅ -aryl-O-C 1 -C ₅ -alkyl radical, or

C ₄ -C 10 heteroaryl radical which contains at least one nitrogen,

Having sulfur or oxygen atom.

It is particularly preferred that the substituents R ¹ and

R ^{2 are} selected from the group of

C 1 -C ₈ -alkyl radical,

C ₃ -C ₅ alkenyl radical,

C ₃ -C ₅ -alkynyl radical,

CHA-23533 version.doc C3-C6-cycloalkyl,

C3-C6 cycloalkenyl group,

C ₆ H ₅ -aryl-C ₁ -C ₃ -alkyl radical,

C ₃ -C ₆ -cycloalkyl-C ₁ -C ₃ -alkyl radical,

C ₆ H ₅ -aryl-O-C ₁ -C ₃ -alkyl radical or

C ₄ -Cio heteroaryl radicals from the group of the 5-ring systems, the 6-ring systems or the multi-membered ring systems which contain at least one

Have nitrogen, sulfur or oxygen atom.

Examples of suitable 5-ring systems include pyrrole, fluorine, thiophene and imidazole. In the context of the 6-ring systems, for example, pyrazine, pyridine, pyrimidine and the pyrrimidine bases can be mentioned. Preferred multi-membered ring systems are, for example, indole, purine or the purine bases.

The substituents R ¹ and R ² preferably have at least one further substituent selected from the group of the halogens, the nitrogen-containing substituents, the phosphorus-containing substituents, the oxygen-containing substituents or the sulfur-containing substituents, these being identical or different in the case of several further substituents.

Preferably, the at least one further substituent is selected from the group of fluorine, chlorine, bromine, iodine,

Amino group or nitro group,

Phosphate group,

Hydroxy group, C 1 -C 8 -alkyloxy group, in particular methoxy group or ethoxy group, carboxyl group, carboxyl-C 1 -C ₅ -

CHA-23533 version.doc Alkyl ester group, or SO 3 ^~ group and So ₄ ^{2 ~} group.

The following is a general scheme for the course of a ketone reduction by enzymatic processes.

Cofactor M 2 cofactor

: Recycling System>

With the biocatalysts according to the invention, the corresponding alcohols are prepared from the ketones.

Surprisingly, it has been found that the species Pasinaca sativa (parsnip), Petroselinum crispum (parsley root), anethum graveolens (dill) and Levisticum officinalis (lovage) from the umbelliferae produce the S-form of the alcohols in high selectivity ,

When comparing the individual plant parts, different results were obtained for roots and stems. Surprisingly, Pastinaca sativa (parsnip) and Petroselinum crispum (parsley root) have shown a much better conversion and enantioselectivity with fresh plant roots. In contrast, in Anethum graveolens (Dill) and Levisticum officinale (Lovage) the shoots or stems proved to be good for the implementation.

CHA-23533 version.doc Crucial is the difference in the available product and thus the corresponding focus of use: the tuber of parsnip or root parsley or the herb of lovage or dill. Lovage or dill roots are hardly available and very small, parsnip or root parsley is also difficult to obtain. In the case of lovage, thick roots that are used for biotransformations are only formed after several years of growth in free-range culture. As a possible source for example Gartenliebstöckel comes into question, which is not a commercially exploitable resource.

When using the method according to the invention, for example with Pastinaca sativa (parsnip) enantiomeric excesses (ee) of more than 60%, preferably more than 65%, most preferably more than 95% can be achieved.

In the case of roots of Petroselinum crispum (parsley root), ee values are greater than 50%, preferably greater than 80%, most preferably greater than 90%.

For the stems of Anethum graveolens (dill), ee values of more than 80%, preferably more than 90%, most preferably more than 95%, can be achieved.

In this sense, the use of stems of Levisticum officinale (lovage, young plant) allows ee values of more than 80%, preferably more than 90%.

In lovage the availability of fresh plants is important for the implementation. With young stems, conversions were achieved with an ee of 97-98%. Thick roots, which can be used for biotransformations, as mentioned above, formed by lovage only after several years of growth in open-field culture. This sausage

CHA-23533 version.doc They are also well suited for realizations, but they are not suitable for preparative use because they are not available on the market. Theoretically, lovage could work with both, ie the stems of young plants or the roots of perennial plants.

With the process according to the invention, for example, complete conversion of acetophenone to (S) -I-phenylethanol can be carried out by cutting plant parts in water with an acetophenone concentration of 1 g / l in high enantiomeric purity.

The plants can be grown and harvested under the usual conditions. Commercially available varieties can be used.

The plants are cleaned for the process and cut evenly. For isolation of the enzyme as crude extract or purest enzyme, the plants are homogenized and the enzyme-containing crude extract obtained. By further steps, the enzyme can be stabilized and isolated.

Cut plants should be used in unit sizes of 0.1 to 5 cm, preferably 0.2 to 2 cm. As the amount of 10 to 1000 g / l, preferably 100 to 500 g / l should be used.

To carry out the process, cofactors such as NAD or NADP and for the regeneration of cofactors cosubstrates such as 2-propanol may be added.

The reaction can be carried out in water without buffer.

However, the reaction may also be carried out in water at a pH of 2-10 using buffers from the group of

CHA-23533 version.doc Acetate buffer, phosphate buffer, Tris buffer or phosphate citrate buffer. Preferably, the reaction is then carried out in water at a pH of 6-8 using buffers from the group of acetate buffer, phosphate buffer, Tris buffer or phosphate citrate buffer. More preferably, the reaction is in water at pH 7 using buffers from the group of acetate buffer, phosphate buffer, Tris buffer or phosphate citrate buffer.

The buffer concentration is generally 0.001 to 5.0 M. Preferably, a buffer concentration of 0.01 to 0.3 M is used. Most preferred is the use of a buffer concentration of 0.1M.

The reaction is generally carried out at 5 ° C to 40 ° C. Preferably, the reaction is at 15 ° C to 30 ° C. Most preferably, the reaction is carried out at 25 ° C.

The reaction is carried out in a period of between 5 and 100 hours. Preferably, the reaction takes place in a period of between 24 and 72 hours.

The concentrations of prochiral ketones are between 0.1 and 10% (w / v), preferably between 0.2 and 5.0% (w / v).

As reactors simple mixable stirred tank and shake flasks can be used. Alternatively, the use of fixed bed reactors is possible. Important is a uniform contact of the biocatalyst with the reaction medium.

CHA-23533 version.doc The invention will be explained in more detail by way of examples.

EXAMPLES

Example 1: Production of vegetable biocatalysts from plants of Pastinaca sativa and Petroselinum crispum

The roots of the plants Pastinaca sativa (parsnip) and Petroselinum crispum (parsley root) are washed and peeled. Subsequently, the roots are cut into cubes with 1 cm edge length. The cut cubes are washed with sterile water.

After this treatment, the vegetable biocatalyst is ready for use in the process for the catalytic production of optically active hydroxy compounds.

Example 2: Production of vegetable biocatalysts from plants of Anethum graveolens and Levisticum officinal

The stems and leaves of the plants Anethum graveolens (dill) and Levisticum officinale (lovage) are washed. Subsequently, the stems and leaves are cut in 1 cm in length. The cut pieces are washed with sterile water.

CHA-23533 version.doc After this treatment, the vegetable biocatalyst is ready for use in the process for the catalytic production of optically active hydroxy compounds.

Example 3 Preparation of (S) -1-phenylethanol from acetophenone by reduction with plant biocatalysts from plants of Petroselinum crispum

Roots of Petroselinum crispum (parsley root) were freshly prepared as described in Example 1. After washing the plant material with sterile water, the batch was prepared in a shake flask as follows:

Preparation: 30 g of plant material from Petroselinum crispum (parsley root) 100 ml of sterile water

100 μl of acetophenone

The batch in the shake flask was provided with a gas-permeable plug and incubated on a shaker at 25 ° C and 100 U / min.

After each 24 h and 48 h, one sample each was taken, starting material and product were extracted and analyzed by gas chromatography. The analyzes were carried out on a chiral separating column, whereby both the contents of

CHA-23533 version.doc not yet reacted starting material and the enantiomers formed (S) -1-phenylethanol and (R) -1-phenylethanol could be determined.

Conditions for analysis by gas chromatography (GC):

A column of the type ß-DEX 225 with a length of 30 m and an internal diameter of 0.25 mm (Supelco) was used. Helium was used as the carrier gas. The settings at the GC were: injector temperature: 230 ° C; FID: 230 ° C; Injection volume lμl; Column temperature 120 ° C.

Fig. 1 shows the reaction of acetophenone to the enantiomers of (S) -1-phenylethanol and (R) -1-phenylethanol.

As can be seen in Figure 1, the acetophenone was very selectively converted in 48h to 94% to 1-phenylethanol with an enantiomeric ratio of 99% S to 1% R.

Example 4 Preparation of (S) -1-phenylethanol from acetophenone by reduction with plant biocatalysts from plants of Pastinaca sativa (parsnips)

As described in Example 1, roots of Pastinaca sativa (parsnip) were washed, peeled and cut. The batch was prepared in a shake flask as follows:

CHA-23533 version.doc Approach: 30 g of plant material from Pastinaca sativa

(Parsnips) 100 ml of sterile water

100 μl of acetophenone

On a shaker at 25 ° C and 100 U / min, the mixture was incubated for 48 h. After 24 h and 48 h, one sample each was taken, starting material and product were extracted and analyzed by gas chromatography as in Example 2. The course of the implementation is shown in Figure 2.

The acetophenone was selectively reacted with 91% to 1-phenylethanol (R / S, 1.3: 98.7).

Example 5: Comparison of the activity and enantioselectivity of root and stem of the plant Levisticum officinale (lovage)

As described in Example 1 (root) and 2 (stems), the entire plants Levisticum officinale (lovage) were washed and cut. The batch was prepared in a root and stem shake flask as follows:

CHA-23533 version.doc Approach: 30 g plant material of Levisticum officinale (Lovage)

100 ml of sterile water

100 μl of acetophenone

On a shaker at 25 ° C and 100 U / min, the mixture was incubated for 48 h. After 24 h and 48 h, a sample starting material and product was in each case taken, extracted and analy Siert ^¬ by gas chromatography as in Example 2. FIG. The results of the reaction after 48 h are shown in Tabel ^¬ le. 1

Table 1: Reaction of acetophenone to (R / S) -l-phenylethanol with Levisticum officinale (stems / root)

Plant part Acetophenone (R / S) -l- ee.

phenylethanol

[% GC%] [%]

[Share GC%]

Stems 27, 9 72,1 94, 6 (S)

Young plant (pot)

Root garden 4,9 95,1 97, 1 (S) lovage

(several years)

The results of Table 1 show a good or very gu ^¬ te reaction (72, 1% and 95.1%) of acetophenone to 1-phenylethanol with very good enantioselectivity (ee. Of

CHA-23533 version.doc 94.6 and 97.1 (S)) for the stems of young plants and the roots of garden lovage, respectively.

Example 6: Reaction of various substrates with the plants Pastinaca sativa (parsnip), Petroselinum crispum (parsley root), Anethum graveolens (dill) and Levisticum officinale (lovage)

As described in Example 1 (root) and 2 (stem), the whole plants Pastinaca sativa (parsnip), Petroselinum crispum (parsley root), Anethum graveolens (dill) and Levisticum officinale (lovage) were washed and cut. The batch was prepared in a root and stem shake flask as follows:

Approach: 30 g plant material

100 ml of sterile water

100 μl of substance

On a shaker at 25 ° C and 100 U / min, the mixture was incubated for 48 h. After 24 h and 48 h, one sample each was taken, starting material and product were extracted and analyzed by gas chromatography as in Example 2. The results of the reaction after 24/48 h are shown in Table 2.

CHA-23533 version.doc Table 2: Reaction of various substances with plant biocatalysts as root (W) and as stalks (S) after 24/48 h

Plant substance implementation ee. Starting material / product [share GC [%]%]

Pastinaca sativa acetophenone / phenylethanol 90.0 (48h) 98.8 (S) (root)

Pastinaca sativa 2-octanone / 2-octanol 97.8 (24h) 88.6 (S) (root)

Pastinaca sativa Ethylacetoacetate / 3-98.2 (24h) 96.7 (S) (Root) Hydoxybutyric Acid

Pastinaca sativa 4-hydroxy-2-butanone / l, 3-3.9 (24h) 64.1 (S) (root) butanediol

Petroselinum crispum acetophenone / phenylethanol 94.3 (48h) 98.7 (S) (root)

Petroselinum crispum 2-octanone / 2-octanol 99.9 (24h) 88.9 (S) (root)

Petroselinum crispum Ethyl acetoacetate / 3-92.5 (24h) 91.7 (S) (root) Hydoxybutyric Acid

Petroselinum crispum 4-hydroxy-2-butanone / 1,3,3,3 (24h) 52,2 (S) (root) butanediol

Anethum graveolens acetophenone / phenylethanol 86.4 (48h) 98.0 (S) (stem)

Anethum graveolens 2-octanone / 2-octanol 91.4 (24h) 93.4 (S) (stem)

Anethum graveolens Ethyl acetoacetate / 3-95.9 (24h) 93.3 S) (stem) Hydoxybutyric acid

Anethum graveolens 4-hydroxy-2-butanone / l, 3-6.6 (24h) 10 (S) (stem) butanediol

Levisticum officina- acetophenone / phenylethanol 95.1 (48h) 97.1 (S) Ie (root, perennial)

Levisticum officina-2-octanone / 2-octanol 92.5 (24h) 88.7 (S) Ie (stem, young plant)

Levisticum officina- ethylacetoacetate / 3- 89.3 (24h) 86.7 (S) Ie (root, several yearsHydoxybutyric acid plant)

CHA-23533 version.doc Example 7: Preparation of enzyme crude extract from plants of Petroselinum crispum

As described in Example 1, 1 kg of roots of Petroselinum crispum (parsley root) were washed, peeled and cut. The plant pieces were homogenized in a crusher and the juice separated from the fiber components. By subsequent centrifugation (3000 g, 20 min, 4 ° C) cell debris were separated. The 0.5 liter crude extract obtained in this way was filtered through a 0.45 μm membrane filter and can be used as crude extract for reactions.

CHA-23533 version.doc Description of the pictures

Fig. 1: Reaction of acetophenone to (R / S) -l-

Phenylethanol with Petroselinum crispum (roots)

Fig. 2: Reaction of acetophenone to (R / S) -l-

Phenylethanol with Pastinaca sativa (roots)

CHA-23533 version.doc

Claims

1. Biocatalysts of plants of the genera Pastinaca, Petroselinum, Anethum or Levisticum, characterized in that they have been prepared by comminuting these plants or parts thereof and optionally further processing steps.

2. Biocatalysts according to claim 1, characterized in that they have been produced from plants of the genus Pastinaca or Petroselinum.

3. Biocatalysts according to claim 1 or 2, characterized in that they have been produced from vegetative plants or parts of plants.

4. Biocatalysts according to one of claims 1 to 3, characterized in that they have been prepared from stems and / or roots of these plants.

5. Biocatalysts according to one of claims 1 to 4, characterized in that they have been prepared from plant roots of plants of the genus Pastinaca.

6. Biocatalysts according to one of claims 1 to 5, characterized in that they have been prepared by isolation of the enzymes contained in the plants.

7. Biocatalysts according to one of claims 1 to 6, characterized in that they have been prepared by enzymatic crude extraction.

8. Use of biocatalysts from plants of

Genera Pastinaca, Petroselinum, Anethum or Levisticum, which have been prepared by comminuting these plants or parts thereof and optionally further processing steps, for the preparation of optically active hydroxy compounds from prochiral ketones.

9. Use of biocatalysts according to claim 8, characterized in that plants of the genus Pastinaca or Petroselinum are used.

10. Use of biocatalysts according to claim 8 or 9, characterized in that vegetative plants or plant parts are used.

11. Use of biocatalysts according to one of claims 8 to 10, characterized in that stems and / or roots of these plants are used.

12. Use of biocatalysts according to one of claims 8 to 11, characterized in that plant roots of plants of the genus Pastinaca are used.

13. Use of biocatalysts according to one of claims 8 to 12, characterized in that enzymes isolated from the plants are used.

14. Use of biocatalysts according to any one of claims 8 to 13, characterized in that enzyme crude extracts are used.

CHA-23533 version.doc

15. Use of biocatalysts according to any one of claims 8 to 14, characterized in that chiral ketones of the general formula (I):

(I:

are used, wherein the substituents R ¹ and R ^{2 are} selected from the group of alkyl radicals, alkenyl radicals, alkynyl radicals, cycloalkyl radicals, cycloalkenyl radicals, aryl radicals, aralkyl radicals, Cycloalkylalkylreste, Aryloxyalkylreste and heteroaryl radicals, wherein existing carbon chains are straight or branched and wherein it cyclic and aromatic systems to single-membered rings or multi-membered annelated rings and optionally at least one further substituent on the substituent R ¹ , R ^{2 is} present and wherein R ¹ and R ^{2 are the} same or different.

16. Use of biocatalysts according to one of claims 8 to 15, characterized in that the substituents R ¹ and R ^{2 are} selected from the

CHA-23533 version.doc Group of Ci-Ci ₄ alkyl, C ₃ -C ₄ alkenyl, C ₃ -C ₄ alkynyl, C ₃ -C ₄ cycloalkyl, C3-Ci ₄ cycloalkenyl, C ₆ H ₅ -aryl (phenyl), C ₆ H ₅ -aryl-C ₁ -C ₅ -alkyl, C ₃ -C ₄ -cycloalkyl-C ₁ -C ₅ -alkyl, C ₆ H ₅ -aryl-O-C 1 -C ₅ -alkyl, or

C ₄ -Cio heteroaryl radical which has at least one nitrogen, sulfur or oxygen atom.

17. Use of biocatalysts according to one of claims 8 to 16, characterized in that the substituents R ¹ and R ^{2 are} selected from the group of C ₁ -C ₈ -alkyl radical, C ₃ -C ₅ -alkenyl radical, C ₃ -C ₅ Alkynyl radical, C ₃ -C ₆ -cycloalkyl radical, C ₃ -C ₆ -cycloalkenyl radical, C ₆ H ₅ -aryl-C ₁ -C ₃ -alkyl radical, C ₃ -C ₆ -cycloalkyl-C ₁ -C ₃ -alkyl radical, C ₆ H ₅ -aryl-O-C ₁ -C ₃ -alkyl radical or C ₄ -Cio-heteroaryl radicals from the group of the 5-ring systems, the 6-ring systems or the multi-membered ring systems which have at least one nitrogen, sulfur or oxygen atom.

CHA-23533 version.doc

18. Use of biocatalysts according to one of claims 8 to 17, characterized in that the

Substituents R ¹ and R ² at least one other

Substituents from the group of halogens, the nitrogen-containing substituents, the phosphorus-containing substituents, the oxygen-containing substituents or the sulfur-containing substituents, wherein in the case of several other substituents, these are the same or different.

19. Use of biocatalysts according to any one of claims 8 to 18, characterized in that the at least one further substituent is selected from the group of fluorine, chlorine, bromine, iodine,

Amino group or nitro group,

Phosphate group,

Hydroxy group, C 1 -C ₅ -alkyloxy group, in particular methoxy group or ethoxy group, carboxyl group, carboxyl-C 1 -C ₅ -alkyl ester group, or

Sθ3 ^~ group and So ₄ ^{2 ~} group.

20. Use of biocatalysts according to any one of claims 8 to 19, characterized in that the reaction is carried out in water without buffer.

21. Use of biocatalysts according to any one of claims 8 to 20, characterized in that the reaction in water at a pH of 2-10 by using buffers from the group of acetate buffer, phosphate buffer, Tris buffer or phosphate citrate Buffer is performed.

CHA-23533 version.doc

22. Use of biocatalysts according to any one of claims 8 to 21, characterized in that the reaction in water at a pH of 6-8 by using buffers from the group of acetate buffer, phosphate buffer, Tris buffer or phosphate citrate Buffer is performed.

23. Use of biocatalysts according to any one of claims 8 to 22, characterized in that the reaction is carried out in water at a pH of 7 by using buffers from the group of Acetatpuf- fer, phosphate buffer, Tris buffer or phosphate citrate buffer becomes.

24. Use of biocatalysts according to one of claims 8 to 23, characterized in that the buffer concentration is 0.001 to 5.0 M.

25. Use of biocatalysts according to any one of claims 8 to 24, characterized in that the buffer concentration is 0.01 to 0.3 M.

26. Use of biocatalysts according to one of claims 8 to 25, characterized in that the buffer concentration is 0.1 M.

27. Use of biocatalysts according to any one of claims 8 to 26, characterized in that the reaction at 5 ° C to 40 ° C is performed.

28. Use of biocatalysts according to one of claims 8 to 27, characterized in that the reaction is carried out at 15 ° C to 30 ° C.

CHA-23533 version.doc

29. Use of biocatalysts according to any one of claims 7 to 28, characterized in that the reaction is carried out at 25 ° C.

30. Use of biocatalysts according to one of claims 8 to 29, characterized in that the reaction is carried out in a period of between 5 and 100 hours.

31. Use of biocatalysts according to any one of claims 8 to 30, characterized in that the reaction is carried out in a period of between 24 and 72 hours.

32. Use of biocatalysts according to one of claims 8 to 31, characterized in that the concentrations of prochiral ketones are between 0.1 and 10% (w / v).

33. Use of biocatalysts according to one of claims 8 to 32, characterized in that the concentrations of prochiral ketones are between 0.2 and 5.0% (w / v).

34. Use of biocatalysts according to any one of claims 8 to 33, characterized in that the concentrations of the prochiral ketones 0.5 to 2.0% (w / v) amount.

35. Use of biocatalysts according to any one of claims 8 to 34, characterized in that are used as reactors shake flasks, simple stirred tanks or fixed bed reactors.

CHA-23533 version.doc