WO2009006662A2

WO2009006662A2 - Process for the oxidative cleavage of vinylaromatics using peroxidases or laccases

Info

Publication number: WO2009006662A2
Application number: PCT/AT2008/000250
Authority: WO
Inventors: Wolfgang Kroutil; Miguel Lara
Original assignee: Universität Graz
Priority date: 2007-07-10
Filing date: 2008-07-09
Publication date: 2009-01-15
Also published as: WO2009006662A4; WO2009006662A3

Abstract

The invention relates to a process for the oxidative cleavage of vinylaromatics of the formula (1), characterized in that compounds of the formula (1) are oxidized in the presence of molecular oxygen using at least one enzyme selected from among peroxidases and laccases as catalysts according to the reaction scheme below to form aldehydes or ketones of the formulae (2) and (3); where n is an integer from 0 to 5; the radicals R1 are selected from among saturated or unsaturated hydrocarbon groups which have from 1 to 10 carbon atoms and in which carbon atoms may be replaced by heteroatoms and may be further substituted, amino, C1-6-alkylamino and C1-6-dialkylamino groups, halogens, hydroxy and cyano, where two of the substituents R1 may be joined to form a ring; R2 and R3 are each, independently of one another, hydrogen or one of the options for R1, where R2 and/or R3 may be joined to an R1 to form a ring and R2 or R3 may each be a chemical bond.

Description

Process for the oxidative cleavage of vinylaromatics

The invention relates to processes for the oxidative cleavage of ethylenic double bonds conjugated with aromatic rings using enzyme catalysts.

Mild and selective oxidation methods as well as new ecological and economical chemical processes are in demand because of the economic conditions and the higher environmental consciousness like never before. The oxidative cleavage of alkenes

0 to the corresponding aldehydes or ketones is a commonly used synthetic method in organic chemistry, (i) to introduce oxygen functionalities into molecules, (ii) to divide complex molecules into smaller building blocks, and (iii) to remove protecting groups. Among the currently available methods for the chemical oxidative cleavage of alkenes, reductive ozonolysis is considered the

5 "cleanest" viewed. However, in practice this method has some disadvantages, such as the need to use special equipment (ozonizer), cryogenic technology (usually -78 ° C) and the additional need for stoichiometric amounts of reducing agents (e.g., dimethyl sulfide, zinc, hydrogen, phosphines, etc.) for reductive work-up. In addition, special safety precautions

0 to meet in order to prevent serious accidents, such as explosions.

Other methods using metal oxides as oxidants require (at least) stoichiometric amounts of salt or peroxides. However, these variants show moderate to low chemo-, regio- and stereoselectivity.

5 In many cases, the over-oxidation of the intermediately obtained aldehydes to the corresponding acids is difficult to suppress secondary reaction. For example, the use of OsO ₄ and NaIO ₄ ^[1] , OsO ₄ and Oxon ^® (2 KHSO ₅ + KHSO ₄ + K ₂ SO ₄ ), ^[2] RuCI ₃ in combination with NaIO ₄ or Oxon ^{® t3 ]} , and of ruthenium nanoparticles with NaIO ₄ ^[4] .

^■ 0

Thus, an oxidation process for alkenes would be desirable, which avoids the above disadvantages and in particular a non-toxic, easily available bares oxidizing agent, such as oxygen, can be used.

However, the only known chemical-catalytic method, which uses molecular oxygen as the oxidant, again requires a Co (II) compound as a catalyst, is only moderately selective and, moreover, limited to isoeugenol derivatives ^[5] .

One possible alternative seemed to be biocatalysis. However, enzymatic alkene cleavages are described using only a mixture of lipoxygenases and hydroperoxide lyases for a few, very specific substrates ^[6] .

Furthermore, they are described as undesirable side reactions, ie, with oxidation products in analytical amounts, for peroxidase-catalyzed processes. ^{[7] "} ^[13] In all these reactions, of course, no molecular oxygen is used as the oxidant.

Although enzymatic alkene cleavage with oxygen under enzyme catalysis has also been attempted, but only with certain mono- and dioxygenases as enzymes and with yields in analytical amounts ^[14H17] . In addition, oxygenases have very high substrate specificity ^{[18], [29]} so that only a very limited selection of substrates is possible.

Against this background, the inventors of the present application and their coworkers had already found in previous research that certain arylalkenes can be oxidized to the corresponding aldehydes or ketones by using molecular oxygen as the oxidant, by dissolving cells or cell extracts of a particular fungus, viz of Trametes hirsuta (Pale Tramete), which catalyze the oxidation ^{[30], [31]} . It was thus a biocatalyzed reaction, probably by enzymatic catalysis, but it could not be determined which enzyme (s) were responsible for it. Continuing this research, it has now surprisingly been found by the inventors that certain peroxidases and laccases, and in no case only those of fungal origin, are capable, under specific conditions, of catalyzing the oxidative cleavage of special ethylenic double bonds by oxygen to aldehydes and ketones. This result was surprising because oxygen is normally not (or, in the case of laccases, at least not a preferred) substrate for such enzymes and, on the other hand, the resulting oxidation products are those commonly produced in ozonolysis reactions. For example, peroxidases can actually - like the

0 name indicates - only process peroxide bonds, and furthermore haloperoxidases give only halogenated, e.g. chlorinated or brominated oxidation products.

DISCLOSURE OF THE INVENTION

The present invention is to provide a process for the oxidative cleavage of aromatic ring-conjugated ethylenic double bonds, i. optionally substituted vinylaromatics of the following formula (1), which is characterized in that one or more compounds of For¬

1) in the presence of molecular oxygen with at least one enzyme selected from peroxidases and laccases as catalyst according to the general scheme below, to form aldehydes or ketones of formulas (2) and (3), respectively:

in which n is an integer from 0 to 5, such that the aromatic ring in ortho, meta and / or para position to the vinyl group is substituted by 0 to 5 substituents R ^1. may be identical or different and are selected from: a) saturated or unsaturated hydrocarbon groups having 1 to 10 carbon atoms, in which one or more carbon atoms are optionally replaced by a heteroatom selected from oxygen, nitrogen and sulfur, and the groups optionally _-6 alkyl with one or more substituents selected from Ci, Ci _-6 alkylene, Ci_6-alkoxy, amino, Ci _-6 alkylamino, and Ci- ₆ - dialkylamino groups, halogens, hydroxy, oxo, and cyano b) amino, d _-6- alkylamino and Ci _-6- dialkylamino groups, and c) halogens, hydroxy and cyano, wherein any two of the substituents R ¹ may be connected to an alicyclic or aromatic ring, and wherein the substituents R ² and R ^{3 are} each independently hydrogen or one of the options described under a), b) and c), wherein R ² and / or R ³ each having a substituent en R ¹ may be connected to an alicyclic ring, wherein R ² and R ³ may represent a chemical bond between the carbon atom of the vinyl group to which they are bonded, and the substituent R ¹ in this case, respectively.

In this way, an oxidation method for the above-mentioned compounds is provided, with which the previously defined goal can be achieved. That is, aryl alkenes can be prepared by using oxygen, an omnipresent, innocuous oxidizing agent, and by specific natural enzymes that are readily and inexpensively available by biological or biotechnological means, to the desired aldehydes and ketones, e.g. Vanillin, to be oxidized. Thus, neither expensive nor toxic (heavy metals) catalysts nor complicated and also costly equipment (ozonizer, cryogenic cooling systems) are required, and there are no consuming waste products to be disposed of.

In preferred embodiments, the at least one enzyme is selected from fungal peroxidases and laccases, haloperoxidases, lignin peroxidases, horseradish peroxidase and bovine milk peroxidase, more preferably fungal peroxidases from Coprinus cinereus (scrubby Tintling), from laccases from Coriolus.

- A - color (butterfly stramete), agaricus bisporus (breeding mushroom) and candida rugosa (a yeast species), laccase of Rhus vernicifera (Japanese lacquer tree), chloroperoxidase of Caldariomyces fυmago (a filamentous fungus) and bromoperoxidases, eg of Streptomyces aureofaciens (a bacterium) or corallina officinalis (coral moss), in particular from horseradish peroxidase, from peroxidases of Coprinus cinereus and from laccases of Coriolus versicolor and Agaricus bisporus. Generally, as preferred peroxidases, those from EC class 1.11.1.x can be given. With the above enzymes, very good results can be achieved under respectively optimized conditions, as explained below.

The process is preferably carried out in a buffer in order to be able to keep the reaction conditions, in particular the pH, stable during the oxidation. Preferably, the reaction is carried out in bis-tris buffer, acetate buffer, formate buffer or phosphate buffer. The pH of the reaction mixture is preferably adjusted to 2 to 7, more preferably to 2 to 4, since in these areas the respective enzymes have their maximum activity.

In further preferred embodiments, the process according to the invention is carried out under O ₂ overpressure so as to increase the yields. However, it is not simply shifted the reaction equilibrium, which can be seen from the fact that some enzymes after reaching a maximum activity at higher pressures turn lower yields. The oxidation is preferably carried out under an O ₂ overpressure of 1 to 6 bar, preferably 2 to 3 bar. Even higher values result in little or no additional improvement, often even lower turnovers, and would significantly increase the expenditure on equipment. In the above pressure ranges, for example, a conventional Parr apparatus can be used easily.

In further preferred embodiments, the process is carried out under the action of light, ie under irradiation, since in this way the yields, in particular when laccases are used as enzymes, can be increased many times over. In additional preferred embodiments, the process is carried out in the presence of an organic solvent or solvent mixture, which is preferably selected from C 1-4 -alkanols, dimethylsulfoxide, toluene, acetone, dioxane, tetrahydrofuran, dimethylformamide and mixtures thereof, and preferably 5 in a proportion of 1 to 20, more preferably 5 to 15, Vol .-% of the reaction mixture is contained.

In a further aspect, the invention thus relates to the use of fungal peroxidases and laccases, fungal halogen peroxidases, bacterial halogenated

0 oxidases, lignin peroxidases, horseradish peroxidase or bovine milk peroxidase for catalysing the oxidative cleavage of aromatic ring-conjugated ethylenic double bonds of optionally substituted vinylaromatic with molecular oxygen, the same enzymes are preferred as before for the method of the first aspect of the invention has been described.

5

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1 to 3 show the change in conversion in the process according to the invention with the addition of various organic solvents. ! 0

The invention will now be described in more detail by way of specific examples which are given by way of illustration only and not by way of limitation.

EXAMPLES

.5

After the reactivity of different enzymes as catalysts of the oxidation of aryl alkenes with molecular oxygen had been determined in preliminary experiments, the pH optimum of the individual enzymes for such reactions was first determined in a model reaction with trans-anethole as the vinyl aromatic of the formula (1). 0 For adjusting pH values of 2 to 7, known buffer systems were used. Examples 1 to 16

Oxidation of trans-anethole at different pH

trans-anethole p-anisaldehyde acetaldehyde

The respective enzymes (in each case 3 mg of the consistently solid preparations) were filled into the wells of "Riplate LV" 5 ml Deep Well Plate (HJ-Bioanalytik GmbH). Thereafter, 900 μl of the respective buffer and 6 μl (0.04 mmol) of trans-anethole were added. The plates were then placed in an upright position in an O ₂ pressure reactor.

0 The reactor was purged with pure molecular oxygen and the pressure was adjusted to 2 bar oxygen. After 24 h at 170 U / min and 25 ⁰ C, the reaction mixtures were transferred to 2 ml test tubes, and the wells were washed with EtOAc (600 ul). These 600 .mu.f were added to the respective Eprouvetten so that also a first extraction of the aqueous reaction mixtures through-

5 feed. After a second extraction with pure EtOAc (600 μl), the combined organic phases were dried over Na ₂ SO ₄ and analyzed by GC for the conversion to p-anisaldehyde (4-methoxybenzaldehyde).

The pH adjusting buffers were as follows: pH 0 - trimethylammonium formate / formic acid, 20 mM pH 3 - trimethylammonium formate / formic acid, 20 mM pH 4 - sodium acetate / acetic acid, 50 mM pH 5 - sodium acetate / acetic acid, 50 mM pH 6 - Bis Tris buffer, 50mM »5 pH 7 - Bis Tris buffer, 50mM

The enzymes used in the respective examples and the conversions of trans-anethole to p-anisaldehyde achieved at different pH values are given in Table 1 below. JO

nb: not determined

The surprising catalytic activity of the tested peroxidases, haloperoxidases and laccases in the above oxidation reaction is clearly evident from this table. Although peroxidases are clearly more reactive in comparison to haloperoxidases and laccases, the conversions of some other enzymes, in particular of Agaricus bisporus laccase, are quite sufficient for a preparative performance of the process according to the invention without having to carry out any of the optimizations described below.

Next, the influence of oxygen pressure on the activity of the enzymes was tested as biocatalysts. Examples 17 to 31

Oxidation of trans-anethole at different oxygen pressure

The reaction and GC measurement were essentially as in Examples 1 to 16, except that the pressure for each enzyme tested was varied between 2 and 6 bar. Due to the high expenditure on equipment, no higher pressures were investigated. The results of the tests are shown in Table 2 below.

Table 2

*) Liquid preparation of which 20 μl (instead of 3 mg) was used.

It is striking that there is no general preference for higher or lower pressures. The expectation that higher oxygen pressures would shift the reaction equilibrium to the product side has thus not been fulfilled. Rather, each enzyme seems to have not only an optimal pH, but also an optimal pressure range. In further experiments, the various enzymes with different substrates, ie arylalkenes of the formula (1), were tested under otherwise substantially identical conditions in order to be able to derive substrate specificities. The exception was only the execution of the reactions at the previously determined pH optimum of the respective enzyme

Examples 32 to 36

Enzyme comparison by the oxidation of trans-anethole

trans-anethole p-anisaldehyde acetaldehyde

The reactions, work-up and GC measurements were carried out as described for Examples 1 to 16 (2 bar oxygen) and with each of the pH optimum corresponding buffer. The enzymes used, buffers, pH values and the conversions of trans-anethole to p-anisaldehyde are given in Table 3 below.

It was once again clear that trans-anethole can be oxidized to p-anisaldehyde in sometimes very good yield by enzyme catalysis, and that peroxidases are clearly superior to laccases, although the latter can also be used for preparative purposes. Examples 37 to 40

Enzyme comparison by the oxidation of 4-aminostyrene

4-aminostyrene 4-amine ibenzalde formaldehyde

Analogously to Examples 32 to 36, instead of trans-anethol, 4-aminostyrene was oxidized with various enzymes and in different buffers to 4-aminobenzaldehyde. In addition, in the work-up, the pH of the aqueous phase was adjusted to 10 to prevent salt formation of the amino groups. The results of the experiments as well as the GC measurements are given in Table 4.

Table 4

It emerges that under the experimental conditions of the four enzymes tested, only laccase from Coriolus versicolor catalyzed the oxidation of 4-aminostyrene with good conversion. This fact, as well as the fact that this enzyme has more than doubled its performance in the case of trans-anethole, clearly demonstrates substrate specificity of the enzymes. Example 41

Oxidation of 4-methoxystyrene

4-methoxystyrene p-anisaldehyde formaldehyde

Analogously to Example 32, instead of trans-anethol, 4-methoxystyrene was oxidized with the peroxidase from Coprinus cinereus, batch 1 to p-anisaldehyde. The results of the two experiments are shown in Table 5.

Table 5

When enzyme buffer pH conversion to play anisaldehyde (%)

32 Coprinus cinereus peroxidase, lot 1 Me ₃ N / HCOOH 2 61

41 Coprinus cinereus peroxidase, lot 1 Me ₃ N / HCOOH 2 3

This example again demonstrates the substrate specificity of the enzymes. The peroxidase tested can obviously catalyze the oxidation of the double-substituted double bond of trans-anethole very well, but the one-sided unsubstituted of methoxy- styrene hardly. The substituents on the vinyl group thus exert a significant influence on the catalytic reaction.

Examples 42 and 43

Oxidation of 2-bromostyrene

2-bromostyrene 2-bromobenzene «formaldehyde

Analogously to Examples 32 and 33, instead of trans-anethol, 2-bromostyrene was oxidized with the peroxidase from Coprinus cinereus, batch 1 and the horseradish peroxidase, batch 1, in this case to 2-bromobenzaldehyde. The results of the four experiments are shown in Table 6.

This result proves once again that the substituents on the aromatic also exert a significant influence on the catalytic reaction.

Example 44

Oxidation of ω.co-dimethylstyrene (2-methyl-1-phenyl-1-propene)

ω, ω-dimethylstyrene benzaldehyde acetone

Analogously to Example 33, instead of trans-anethole, ω, ω-dimethylstyrene was oxidized with radish peroxidase, batch 1, in this case to benzaldehyde. The results of both experiments and those of Example 43 are shown in Table 7 for comparison.

Again, the substrate specificity is proven. The presence of two methyl groups on the ω-carbon of styrene rather than just one and the lack of aromatic substitution have a significant effect on catalysis.

Examples 45 to 47

Oxidation of indene

In the 2- (formylmethyl) benzaldehyde

Analogously to Examples 32, 33 and 36, instead of trans-anethole indene, it was oxidized with Coprinus cinereus peroxidase batch 1, horseradish peroxidase batch 1 and the laccase of Coriolus versicolor, in this case 2- (formylmethyl) - benzaldehyde. The results of the six experiments are shown in Table 8.

While the two peroxidases show significantly lower catalytic activity with indene than substrate, the (less active) laccase shows little difference to trans-anethole. In any case, however, it has been proven that ethylenic double bonds contained in cyclic structures can also be oxidized by the process according to the invention. Comparative Examples 1 and 2

Oxidation of the nonconjugated double bond of 5-o-tolyl-2-pentene

enzyme

5-o-tolyl-2-pentene 3-o-tolylpropionaldehyde acetaldehyde

Analogously to Examples 32 and 33, instead of trans-anethol, 5-o-tolyl-2-pentene was oxidized with the peroxidase from Coprinus cinereus, batch 1 and horseradish peroxidase, batch 1, in this case to 3-o- Tolylpropionaldehyd. The results of the two experiments are shown in Table 9.

Table 9

These results show that aryl alkenes with non-conjugated double bonds are not oxidizable by the process according to the invention. The oxidation products can only be detected in analytical quantities (<1%).

Examples 48 to 51

Oxidation of trans-anethole under different lighting conditions

Reactions and GC measurements were made for each enzyme tested in duplicate essentially as in Examples 32-36 in the buffers indicated there for the particular enzyme. However, in a first series of experiments, a lamp (PAR 38 EC Spot, Osram Concentra, 120 W, 230 V, 448) was placed at a distance of 50 cm above the reactor in order to illuminate the reaction mixtures during the oxidation, while in a second Experimental series of the reactor for darkening was covered with an aluminum foil which was perforated to allow oxygen exchange with the environment. The results of the four best of all enzymes tested are given in Table 10 below.

Table 10

The results show a significant increase in the catalytic activity of all four enzymes under light irradiation. This effect is particularly pronounced in the two laccases, since their effectiveness has been increased to eight to nine times. Thus, even with the tendency for less active laccases very good sales for preparative purposes can be achieved.

Examples 52 to 85

Oxidation of trans-anethole in the presence of various organic solvents

Analogously to the process described for Examples 1 to 16, trans-anethol was oxidized by means of Coprinus cinereus peroxidase, batch 1 or horseradish peroxidase, batch 1 as catalyst. The thus obtained conversions to p-anisaldehyde of 44% and 58%, respectively, served as blank for subsequent repetitions of the procedures except that 17 μl of an organic solvent was added to each of the 900 μl of aqueous trimethylammonium formate / formic acid buffer (20 mM).

The results are given in Tables 11 and 12 below and are plotted in Figures 1 and 2, with the horizontal lines indicating the values of the tests without solvent ("blank").

Table 11

Table 12

DMSO: dimethyl sulfoxide; DMF: dimethylformamide; Tween 80: polyoxyethylene (20) sorbitan monooleate; THF: tetrahydrofuran

All results prove that the presence of an organic solvent is possible in principle without completely inhibiting the oxidation reaction. As can be seen from Table 11 and Fig. 1, peroxidase of Coprinus cinereus is more sensitive to the addition of solvent, since the sales through the solvent

I O - with the exception of DMSO - consistently lower.

In contrast, the addition of solvents in horseradish peroxidase as a catalyst in most cases leads to an increase in sales. Only cyclohexanol and Tween 80 decrease, and 2-propanol gives the same value as the blank. Without wishing to be bound by any particular theory, it is believed that the solvent here serves as a solubilizer for the compound of formula (1) to be oxidized, although only 1.8% by volume of solvent was added to the aqueous buffer. To check whether horseradish peroxidase also larger amounts of solvent positive effect on sales, another series of tests with increasing amounts of DMSO, the only solvent that had been shown in peroxidase of Coprinus cinereus positive effect performed.

Examples 86 to 96

Oxidation of trans-anethole in the presence of increasing amounts of DMSO

Analogously to Examples 69 to 85 above, trans-anethole was oxidized with horseradish peroxidase as the catalyst, replacing the 900 μl of aqueous buffer with increasing percentages of DMSO. Table 13 shows the conversion achieved for each DMSO content. The data are also shown graphically in FIG.

Table 13

It can be seen that horseradish peroxidase at a level of 40% DMSO in the Medium is about as active as without organic solvent. At higher concentrations the activity decreases rapidly, and from 60% DMSO substantially no enzymatic effect is detectable. With 20 to 30% DMSO in the medium, the conversion was increased by about 20%. The best results, ie an approximately 40% increase in sales, were achieved with 5 to 15% solvent.

To check whether this effect is also observable in other reactions, the following series of tests were carried out for the preparation of vanillin (4-hydroxy-3-methoxybenzaldehyde), one of the possible, valuable reaction products of the method according to the invention.

Examples 97-104

Oxidation of isoeuqenol in the presence of DMSO

Isoeugenol vanillin acetaldehyde

Analogously to Examples 52 to 85, instead of trans-anethol, isoeugenol (2-methoxy-4-propen-1-ylphenol) was oxidized with peroxidase from Copήnus cinereus or radish peroxidase as catalyst. The aqueous buffer was replaced by 10 to 20 vol .-% DMSO. The results are shown in Table 14 below.

Table 14

It was found that DMSO in Coprinus c / nereus peroxidase caused an approximately 15-20% increase in all concentrations tested, while horseradish peroxidase barely benefited (i.e., maximum 5%) in this reaction from the presence of DMSO.

Examples 105 to 114

Oxidation of coniferyl alcohol in the presence of DMSO

Coniferyl alcohol Vanillin Glycolaldehyde

Analogously to Examples 97 to 104, instead of isoeugenol, coniferyl alcohol (4-hydroxy-3-methoxycinnamyl alcohol) was oxidized with the two enzymes, which provides an alternative route to vanillin using the process according to the invention and additionally gives glycolaldehyde (hydroxyacetaldehyde) as by-product is a useful reagent in protein chemistry. The aqueous buffer was replaced again, in this case with 5 to 20% DMSO. Table 15 keeps the results achieved.

Table 15

Here are several circumstances clearly visible. On the one hand, coniferyl alcohol was quantitatively oxidized to vanillin with both enzymes as catalyst, as long as no organic solvent was present. On the other hand horseradish peroxidase 5% DMSO was still well tolerated (6% deterioration) and only then suffered significant activity losses (40 to 70%), Coprinus c / nereus peroxidase dropped to 50% of its activity even at 5% DMSO. At 15% DMSO around 90% of the activity had already been lost. These results once again demonstrate the high specificity of the enzyme catalysts in the process of the present invention.

Thus, the effectiveness of the enzymes was clearly demonstrated in the process of the invention. The person skilled in the art can easily determine the optimal conditions with regard to pH, pressure, light irradiation and organic solvents for individual, specific enzymes in specific oxidation reactions by means of optimization series by means of the above teachings. The invention therefore adds a significant contribution to the field of biocatalysis for the production of organic compounds. Materials and sources of trans-anethol: Sigma-Aldrich, Cat. 11, 787-0, Lot .: S16146-283 4-aminostyrene: Lancaster, 11845, 216-185-8, batch FA008716 2-bromostyrene: Sigma-Aldrich, 132683

5 2-Methyl-1-phenyl-1-propene: Sigma-Aldrich, 282510, 13028 BE Inden: Merck-Schuchard, S17014 843, 8.20701.0005 Isoeugenol: Sigma-Aldrich, 117206 Conferyl Alcohol: Sigma-Aldrich, 223735

Peroxidase from Coprinus cinereus, Lot 1: NovoNordisk A / S, Peroxidase SP 502, Batch PPX 3829

Peroxidase from Coprinus cinereus, Lot 2: Novozymes, 51004, OON00008, (produced in Asp. Oryzae) Peroxidase from Coprinus cinereus, lot 3: Biesterfeld Chemiehandel GmbH & Co. KG,

5 "Baylase"

Horseradish peroxidase, lot 1: Sigma-Aldrich, P2088 horseradish peroxidase, lot 2: Sigma-Aldrich, P8250, 031 K74711 horseradish peroxidase, lot 3: Sigma-Aldrich, P6140, 051 K7490 horseradish peroxidase, lot 4: Roche , POD10108090001, Lot .: 93350720

Horseradish Peroxidase, Lot 5: Sigma-Aldrich, P8125, 031K7465

Horseradish Peroxidase, Lot 6: Roche, POD10814407001, Lot .: 93396221 Lignin Peroxidase: Fluka, Lot. & Fillingcode 1239384, 32506 171, 42603 Bovine Milk Peroxidase: Sigma-Aldrich, Lactoperoxidase from bovine milk, L-2005, Lot. 16H38311

! 5 Chloroperoxidase from Caldaromyces fumago: Biochemika, 25810

Bromperoxidase from Corallina officinalis: Sigma-Aldrich, B2170, 123K3783

Laccase of Rhus vernicifera: Sigma-Aldrich, L-2157, Lot .: 67H0281

Laccase of Coriolus versicolor. Fluka, 38429, Lot. & Filling code 414571/1, 43001

Laccase from Candida rugosa: Jülich Fine Chemicals Life Science Technology, Laccase CV,

IO Ord.No. 14:10

Laccase of Agaricus bisporus: Fluka, 40452, Lot. & Filling code 443928/1 42703431 Laccase DeniLite Il Base: Novozymes, OM30402613, Chemical Abtracts Service (CAS) Registry No .: 80498-15-3 references

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Claims

1. A process for the oxidative cleavage of optionally substituted vinyl aromatics of the following formula (1), characterized in that one or more compounds of formula (1) in the presence of molecular oxygen with at least one enzyme selected from peroxidases and laccases as catalyst according to the following general reaction scheme to aldehydes or ketones of the formulas (2) and (3) is oxidized or are:

3

(1) (2) (3) wherein n is an integer of 0 to 5 such that the aromatic ring may be substituted with 0 to 5 substituents R ¹ in ortho, meta and / or para position to the vinyl group, which may be the same or different and are selected from: a) saturated or unsaturated hydrocarbon groups having from 1 to 10 carbon atoms, in which one or more carbon atoms are optionally replaced by a heteroatom selected from oxygen, nitrogen and sulfur, and optionally with one or more carbon atoms more substituents selected from C _1-6 alkyl groups, Ci-e-alkylene, Ci-β-alkoxy, amino, C _1-6 alkylamino and Ci- ₆ dialkylamino, halogen, hydroxy, oxo, and cyano are further substituted, b) amino, alkylamino CI_ ₆ and Ci- ₆ -dialkylamino groups, and c) halo, hydroxy and cyano, wherein any two of the substituents R ¹ may be connected to form an alicyclic or aromatic ring, and wherein the substituent R ² and R ^{3 are} each, independently of one another, hydrogen or one of the options described under a), b) and c), where R ² and / or R ^{3 may} each be joined to a substituent R ¹ to form an alicyclic ring, wherein In this case, R ² and R ³ each represent a chemical bond between the carbon atom of the vinyl group to which they are attached are, and the substituent R ^{1 can} stand.

2. The method according to claim 1, characterized in that the at least one enzyme from fungal peroxidases and laccases, haloperoxidases, lignin peroxidases, horseradish peroxidase and bovine milk peroxidase is selected.

3. The method according to claim 2, characterized in that the at least one enzyme from fungal peroxidases of Coprinus cinereus, from laccases of Coriolus versicolor, Agaricus bisporus and Candida rugosa, laccase of Rhus vernicifera, chloroperoxidase of Caldariomyces fumago and bromoperoxidases, e.g. of Streptomyces aureofaciens or Corallina officinalis.

4. Process according to claim 2 or 3, characterized in that the at least one enzyme is selected from horseradish peroxidase, from peroxidases of Coprinus cereus and from laccases of Coήolus versicolor and Agaricus bisporus.

5. The method according to any one of the preceding claims, characterized in that the oxidation is carried out in a buffer.

6. The method according to claim 5, characterized in that the buffer is selected from Bis-Tris buffer, acetate buffer, formate buffer and phosphate buffer.

7. The method according to any one of the preceding claims, characterized in that the pH of the reaction to 2 to 7, preferably to 2 to 4, is set.

8. The method according to any one of the preceding claims, characterized in that the oxidation is carried out under O ₂ overpressure.

9. The method according to claim 8, characterized in that the oxidation under an O ₂ overpressure of 1 to 6 bar, preferably 2 to 3 bar performed becomes.

10. The method according to any one of the preceding claims, characterized in that the oxidation is carried out under the action of light.

11. The method according to any one of the preceding claims, characterized in that the oxidation is carried out in the presence of an organic solvent or solvent mixture.

12. The method according to claim 11, characterized in that the solvent or solvent mixture in a proportion of 1 to 20, preferably 5 to 15, Vol .-% of the reaction mixture is used.

13. The method according to claim 11 or 12, characterized in that the organic solvent of C _1-4 alkanols, dimethyl sulfoxide, toluene, acetone, dioxane,

Tetrahydrofuran, dimethylformamide and mixtures thereof is selected.

14. The method according to any one of the preceding claims, characterized in that is prepared as aldehyde of the formula (2) vanillin.

15. Use of fungal peroxidases and laccases, fungal halogen peroxidases, bacterial haloperoxidases, lignin peroxidases, horseradish peroxides or bovine milk peroxidase for catalysing the oxidative cleavage of aromatic ring-conjugated ethylenic double bonds of optionally substituted vinylaromatics with molecular oxygen.

16. Use according to claim 15, characterized in that at least one of fungal Peroxidasen of Cophnus cinereus, laccases of Coriolus versicolor, Agaricus bisporus or Candida rugosa, laccases of Rhus vemicifera, chloroperoxidase of Caldariomyces fumago and Bromperoxidasen, for example of Streptomyces aureofaciens or Corallina officinalis, selected enzyme is used as a catalyst.

17. Use according to claim 15 or 16, characterized in that at least one of horseradish peroxidase, peroxidases of Coprinus cinereus or laccases of Coriolus versicolor or Agaήcus bisporus is used as a catalyst.