WO2006024502A2 - Method for producing polymethyl-branched omega-1, omega-2 or omega-3 alcohols or ketones, using the bacillus megaterium hydroxylase system - Google Patents

Abstract

The invention relates to a method for regioselective oxidation of a compound having a multiple methyl hydrocarbon chain branched by an enzyme system.

Description

 Process for regio-selective oxidation

The present invention relates to a process for the regioselective oxidation of a compound having a poly-methyl branched hydrocarbon chain by an enzyme system.

Selective functionalization of multi-methyl branched hydrocarbon chain compounds plays an important role in providing building blocks for the chemical synthesis of complex compounds, e.g. Natural products and macrolide antibiotics. An example of such a macrolide antibiotic is borrelidin.

borrelidin

Borrelidin is a broad-spectrum antibiotic from Streptomyces rochei with anti-bacterial, anti-viral and anti-fungicidal properties. It is used, inter alia, in agriculture in pest control (animal pests and weeds) and as a germination stimulator. It also works against antibiotic-resistant plasmodia (malaria pathogens). In addition, recent results show that borrelidin is a potent angiogenesis inhibitor and thus a potential candidate for the development of an innovative cancer therapeutics. Therefore, great efforts are currently being made to produce borrelidin in various ways. In addition to the fermentative production using the isolated from Streptomyces biosynthetic gene cluster is trying to produce borrelidin synthetically. The chemical synthesis is therefore mainly interesting because modifications that may be necessary for improved efficacy can be more easily introduced in such a process. However, the synthesis methods hitherto known are disproportionately difficult and there is a demand to simplify the synthesis.

Known chemical reactions for the oxidation of long-chain compounds such as fatty acids or alcohols only produce mixtures of substances that can not be separated synthetically. Furthermore, it is known that long-chain, non-branched alcohols, fatty acids, amides or methyl esters can be oxidized by microbial processes. However, the previously known microbial processes only provide mixtures of oxidation products that are not separable. For example, it has been described that an enzyme system from Bacillus megaterium can be used for hydroxylation of long-chain, unbranched fatty acids, amides, methyl esters and alcohols (see Y. Miura and AJ Fulco, J. Biol. Chem. 249 (1974) 1880-1888, Y. Miura and AJ Fulco, Biochim, Biophys, Acta 388 (1975), pp. 305-317, HJ Schafer and E. Cramer, Fat Sci., Technol., 90: 351 (1988) Sawamura et al., Biochim Biophys Acta 1168 (1993), pp. 30-36). The use of the known enzyme system, a special hydroxylase, in the oxidation of long-chain fatty acids, amides, methyl esters or alcohols leads to mixtures of oxidation products, which in the (ω-1) -, (ω-2) - or (ω- 3) - are hydroxylated position. However, these product mixtures resulting from the reaction of straight-chain substrates are not separable. Examples of methyl-branched hydrocarbon chain compounds are 2, 4, 6, 8-tetramethyl decanoic acid, 2,4,6,8-tetramethyl undecanoic acid and 2,4,6-trimethyloctanoic acid which are in the form of R, R, R, R or R , R, R isomers from the rumen glands, eg of domestic geese, by the in the document DE 196 27 899 A1 or M. Morr et al. , Liebigs Ann. Chem. 1992, p 433 can be obtained. It is also known that the hydrocarbon chain of multi-methyl branched hydrocarbon chain compounds can be extended by chain extension reactions. For example, (4R, 6R, 8R) -4,6,8-trimethyldecanoic acid can be prepared by chain extension from (2R, 4R, 6R) -2,4,6-trimethyloctanoic acid (see J. Fortkamp, Dissertation, Technische Universität Braunschweig 1994).

In view of the above-described problems, it is an object of the present invention to provide a process which makes it possible to separate essentially a single oxidation product.

The object of the present invention is achieved by a process for the regioselective oxidation of a compound having a methyl-branched hydrocarbon chain by an enzyme system, wherein essentially a single oxidation product is isolated. The multi-methyl branched hydrocarbon chain compound preferably used in the process of the present invention is a compound of the general formula (1):

(D wherein R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0, 1 or 2, and z is 0, 1, 2, 3, 4 or 5. Also preferred in the process of the present invention is the use of a compound of general formula (2):

(2) wherein R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0, 1 or 2, and z is 0, 1, 2, 3, 4 or 5.

Surprisingly, it has been found that compounds with multiple methyl branched hydrocarbon chain, and in particular compounds of general formula (1) or (2), using an enzyme system can be oxidized substantially to a single oxidation product. Accordingly, essentially this single oxidation product can be isolated. Here, "essentially" means a purity which is sufficient for subsequent syntheses, without additional purification steps are necessary. The substantially only oxidation product can thus be advantageously used as a synthesis component in subsequent synthesis steps, in particular because a technically difficult to carry out separation of other oxidation products with very similar to almost identical properties deleted.

More preferably in the process of the present invention, the compound having a methyl-branched hydrocarbon chain used is a compound selected from 4,6-dimethyloctan-2-ol, 2,4,6-trimethyloctanol, 2,4,6-trimethyloctanoic acid, 2,4,6-Trimethyloctansäuremethylester, 2, 4, 6-trimethylnonanol, 2,4,6-trimethylnonanoic acid, 2,4,6-trimethylnonanoic acid methyl ester, 2,4,6,8-tetramethyldecanol, 2,4,6,8-tetramethyldecanoic acid, 2,4,6, 8th-

Tetramethyldecansäuremethylester, 2,4,6, 8-Tetramethylundecanol, 2,4, 6, 8-Tetratnethylundecansäure and 2, 4, 6, 8-Tetramethylundecans acid methyl ester. Most preferably, in the process of the present invention, the multi methyl branched hydrocarbon chain compound used is a compound selected from 4,6-dimethyloctan-2-ol, 2,4,6-trimethyloctanol, 2,4,6-trimethyloctanoic acid, 2 , 4,6- Trimethyloctansäuremethylester, 2,4,6, 8-tetramethyldecanol, 2, 4, 6, 8-Tetramethyldecansäuremethylester and 2,4,6,8- Tetramethylundecansäuremethylester. In this case, the compound having a methyl-branched hydrocarbon chain is preferably used as the racemate. In another embodiment, the multi-methyl branched hydrocarbon chain compound can be exactly one enantiomer. The compound having a methyl-branched hydrocarbon chain is particularly preferably selected from (2R, 4R, 6R) -4,6-dimethyloctan-2-ol, (2S, 4R, 6R) -4,6-dimethyloctan-2-ol, (2R, 4R, 6R) -2,4,6-trimethyloctanol, (2R, 4R, 6R) -2,4,6-trimethyloctanoic acid, (2R, 4R, 6R) -2,4,6-trimethyloctanoic acid methyl ester, (2R, 4R, 6R, 8R) -2,4,6, 8-tetramethyl decanol, (2R, 4R, 6R, 8R) -2,4,6, 8-tetramethyl decanoic acid methyl ester and (2R, 4R, 6R, 8R) -2,4 , 6, 8-Tetramethylundecansäuremethylester.

Preferably, the enzyme system is present in the process of the present invention in the form of living cells. In another embodiment, the method of the present invention may also be carried out with an enzyme system which is in the form of an enzyme system isolated from a microorganism, more preferably in the form of an isolated soluble enzyme system. Preferred is an enzyme system from Bacillus used megaterium. An enzyme system from Bacillus megaterium strain ATCC 14945 is particularly preferably used. Likewise preferred is an enzyme system from Bacillus megaterium, in particular from Bacillus megaterium strain ATCC 14945, used, which is heterologously expressed in the form of a suitable vector in another organism. Thus, the method according to the invention can advantageously be accelerated since the slow growth of the organism as a speed-limiting effect is eliminated.

More preferably, in the process of the present invention, exactly one oxidation product is isolated. Preferably, the isolated oxidation product is a ketone or an alcohol. Preferably, the compound obtained as an oxidation product may be a compound oxidized in the position (ω-1). Alternatively, the compound obtained as an oxidation product may be a compound oxidized in the position (ω-2). Alternatively, the compound obtained as an oxidation product may be a compound oxidized in position (co-3).

Preferably, the process of the present invention is conducted by (i) performing oxidation of a multi-methyl branched hydrocarbon chain compound using an enzyme system, (ii) isolating and analyzing all oxidation products, and (iii) using unsatisfactory oxidation another enzyme system is performed, wherein steps (i) to (iii) are run through until essentially a desired oxidation product is isolated. Another aspect of the present invention is an oxidation product which is isolated according to the method of the present invention.

In the present invention, a compound having a methyl-branched hydrocarbon chain means an organic compound which consists essentially of a long-chain hydrocarbon chain with a plurality of substituents in the form of methyl groups. A long-chain hydrocarbon chain in the context of the invention is an unbranched hydrocarbon chain having 6 to 24 carbon atoms in the chain, preferably 8 to 20 carbon atoms, more preferably 8 to 16 carbon atoms, and most preferably 8 to 12 carbon atoms. The methyl-substituted carbon atoms are preferably separated from each other by unsubstituted carbon atoms, especially in the form of methylene groups. The compound having a methyl-branched hydrocarbon chain is preferably completely saturated. Alternatively, the compound having a methyl branched hydrocarbon chain can be partly unsaturated by one, two or more carbon-carbon double bonds being present in the carbon chain. In this case, the double bonds are preferably separated from each other by at least one single bond. The connection can be in any absolute configuration. The compound having a methyl-branched hydrocarbon chain is preferably a compound which carries a functional group at one end of the hydrocarbon chain, particularly preferably at the terminal hydrocarbon atom. Preferably, the compound carries a functional group which is characteristic of an alcohol, an alkyl ether, an aldehyde, a ketone or a carboxylic acid. A carboxylic acid may be present in free form or in the form of salts, such as an alkali metal salt or ammonium salt, or in the form of derivatives, such as carboxylic acid amide or carboxylic acid. alkyl esters. Preference is given to a carboxylic acid in free form or in the form of a carboxylic acid amide or carboxylic acid alkyl ester. A carboxylic acid amide can also carry suitable substituents on the nitrogen atom in addition to hydrogen atoms, with alkyl groups being preferred. For alkyl ether, carboxylic acid alkyl ester and Carbonsäurealkylamide of the invention, alkyl groups are preferred alkyl groups having 1 to 4 carbon atoms, particularly preferably methyl groups or ethyl ^groups'. The compound is particularly preferably an alcohol, a free carboxylic acid, a carboxylic acid amide or a carboxylic acid alkyl ester. The compound is particularly preferably a compound of the general formulas (1) or (2)

(2) wherein each R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0, 1 or 2 , and z is 0, 1, 2, 3, 4 or 5.

The multi-methyl branched hydrocarbon chain compound includes compounds which may exist in various absolute configurations. Thus, each of the methyl-substituted carbon atoms can be one independently of the other absolute configuration R or S have. Accordingly, for compounds with y = 0, compounds having the following absolute configurations are included: (R, R, R), (R, R, S), (R, S, R), (S, R, R), (R , S, S), (S, R, S), (S ₁ S, R) and (S, S, S); and for compounds with y = 1: (R ₁ R ₁ R ₁ R), (R, R, R, S), (R, R, S, R), (R, S, R, R), (S , R, R, R), (R, R, S, S), (R, S, S, R), (R, S, R, S), (S, R, S, R), (S , S, R, R), (R, S, S, S), (S, R, S, S), (S, S, R, S), (S, S, S, R) and (S , S, S, S). The compound may be employed as a racemic mixture of one or more pairs of enantiomers (racemate) or in the form of one or more stereoisomers which are not enantiomeric to one another. If the compound is not used as a racemate, it is preferably used in the form of exactly one stereoisomer (enantiomer).

In the present invention, an enzyme system means a system of one or more enzymes which is suitable for the oxidative biotransformation of the compound having a poly-methyl-branched hydrocarbon chain. The enzyme system may be in the form of living cells of a microorganism. Alternatively, it may be one or more enzymes isolated from cells of a microorganism, preferably one or more soluble enzymes. The enzymes may be stable under the reaction conditions or be stabilized by suitable means. Preferably, an enzyme system from Bacillus megaterium is used, and more preferably one from Bacillus megaterium strain ATCC 14945. More preferably, the enzyme system is in the form of living cells of B. megaterium.

The process of the invention is carried out using an oxidizing agent, preferably using oxygen as the oxidizing agent. Oxygen in the form of atmospheric oxygen is particularly preferably used. It is preferred isolated in the process of the invention oxidized by an enzyme system with oxygen oxidation product, more preferably exactly one oxidation product is isolated. Particular preference is given to isolating an oxidation product which is oxidized by the process of the invention in accordance with the following reaction equation from a compound having a methyl-branched hydrocarbon chain, for example of the formula (1):

wherein R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0, 1 or 2, and z is 0 , 1, 2, 3, 4 or 5.

Particularly preferred is a (ω-1) -KetoVerbindung isolated as an oxidation product. example

1. Biotransfoπnation of tetramethyldecanol with Bacillus megaterium in a 15 liter reactor

A 15 liter reactor with a culture volume of 10 liters is charged with 100 g peptone (Marcor), 200 g malt extract, 30 g yeast extract, 1 g behenyl alcohol and 8.5 liters deionized water, heat sterilized and after cooling with 1 liter of a 2 day old culture of Bacillus megaterium ATCC 14945. At the same time, 0.5 liter of a 20% glucose solution which has been sterilized by filtration is added. The fermentation is carried out at 30 ° C and aeration rate of 1 liter per minute. The speed is initially set to 90 rpm, but upshifted from a pO ₂ value of 30% to avoid oxygen limitation. The pH is initially controlled by pumping of 50% acetic acid or 5 N KOH between 7.0 and 7.5, intercepted after 8 days at 7.8.

After 1, 2, 3 and 4 days, 2 ml of tetramethyldecanol are added. After 1, 2, 3, 4, 5 and 9 days each 20% glucose is added, normally 150 ml, after 5 days 250 ml. After 6 days 500 ml of 10X concentrated culture medium is added. The fermenter broth is harvested after 32 days.

2. Processing of the fermentation broth after the biotransformation of (2R, 4R, 6R, 8R) -2,4,6,8-tetramethyl decanol with Bacillus megaterium After removal of the Bacillus on a glass frit with diatomaceous earth and washing with water about 10 1 of the dark brown culture broth with 3 1 ethyl acetate are extracted. The ethyl acetate phase is concentrated by evaporation on a rotary evaporator with sodium sulfate. 4.5 g of crude product are purified after column chromatography over silica gel and elution with chloroform and chloroform / methanol mixtures (9: 1, 8: 2). The detection of the biotransformation is carried out with an ammonium molybdate / cerium (IV) sulphate / 10-15% sulfuric acid or Sprüh¬ Tauchreagenz on TLC silica gel, and heating to about 15O ⁰ C. Blue color indicates the individual fractions.

Rechromatography of the 2 g fraction over silica gel and elution with petroleum ether (40/60 ° C) / ethyl acetate 9: 1/8: 2 provides 1.7 g of 10-hydroxy- (3R, 5R, 7R, 9R) -3.5 , 7,9-tetramethyldecan-2-one (ketoalcohol, Table 1), in addition to 0.17 g of 9- (R / S) -hydroxy- (2R, 4R, 6R, 8R) -2,4,6,8-tetramethyldecanol (Table 1) . The purity of the isolated compounds was determined after trimethylsilination by GC / MS, the structure by means of ¹ H and ¹³ C NMR spectra (see Tables 1 and 2).

Rotation of the ketoalcohol: +7.23 (c = 2.24, chloroform)

Table 1

NMR data of 10-hydroxy-3, 5, 7, 9-tetramethyldecan-2-one in CDCl ₃

Footnotes: a) exchangeable.

Table 2

NMR data of 1, 9-dihydroxy-2,4,8,6-tetramethyldecane in CDCl ₃

Footnotes: a) Abbreviations for multiplicity are: q = quartet, t = triplet, d = doublet, m = multiplet; b) Only 78% of the major isomer is present. The NMR data of the minor isomer are indicated only if they differ from those of the major isomer.

Table 3

Comparative NMR data of 1, 9-Dihydroxy-2,4, 6, 8-tetramethyldecane (Compound A ^''; Table 1) and 1, 8-dihydroxy-2, 4, 6, 8-tetramethyldecane (Compound B) in _CDCl3

C-9 71,4/70,6 d 34,9 t

C-9 71.4 / 70.6 d 34.9 t

C-IO 18, 6/20, 6 q 8.4 q

Me-2 17.9 q 17.7 q

Me-4 21.3 q 21.1 q

Me-6 21.3 q 23.0 q

Me-8 14, 6/15, 1 q 27.1 q

Footnotes: a) Abbreviations for multiplicity are: q = quartet, t = triplet, d = doublet, s = singlet; b) There are 93% of an isomer identical to the minor isomer prepared by the synthesis of these compounds by reduction of the keto starting compound (see Table 2, above). The NMR data of the minor isomer are given only if they differ from those of the major isomer; c) This compound also occurs in trace amounts in the above synthesis product (Table 2). The HMBC spectrum of the synthesis product allows a direct assignment of C-5 to C-10, Me-6 and Me-8. The remaining associations are from a comparison with compound A; d) It was assumed that this signal was superimposed on that of compound A in the mixture.

Claims

claims A process for the regioselective oxidation of a multi-methyl branched hydrocarbon chain compound by an enzyme system, wherein substantially a single oxidation product is isolated. 2. The method according to claim 1, characterized in that the compound with multiple methyl-branched hydrocarbon chain is a compound of general formula (1):

wherein R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0 or 1, and z is 0, 1 , 2, 3, 4 or 5. 3. The method according to claim 1, characterized in that the compound with multiple methyl-branched hydrocarbon chain is a compound of general formula (2):

wherein R is a group of the formula -CH ₂ -OX, -OX, -COOX or -CONX ₂ , X is independently a hydrogen atom or a methyl group, x is 0 or 1, y is 0 or 1, and z is 0, 1 , 2, 3, 4 or 5. 4. The method according to any one of claims 1, 2 or 3, characterized in that the compound with multiple methyl-branched hydrocarbon chain is selected from 4, 6-Dimethyloctan-2-ol, 2,4, 6-Trimethyloctanol, 2,4,6- Trimethyloctanoic acid, 2,4,6-trimethyloctanoic acid methyl ester, 2,4,6-trimethylnonanol, 2,4,6-trimethylnonanoic acid, 2,4,6-trimethylnonanoic acid methyl ester, 2,4,6,8-tetramethyldecanol, 2,4,6, 8-tetramethyldecanoic acid, methyl 2,4,6,8-tetramethyldecanoate, 2,4,6,8-tetramethylundecanol, 2,4,6,8-tetramethylundecanoic acid and 2,4,6,8-tetramethylundecanoic acid methyl ester. 5. The method according to any one of the preceding claims, characterized in that the compound having multiple methyl branched hydrocarbon chain is selected from 4,6-dimethyloctan-2-ol, 2, 4, 6-Trimethyloctanol, 2,4,6-Trimethyloctansäure, Methyl 2,4,6-trimethyloctanoate, 2,4,6,8-tetramethyldecanol, methyl 2,4,6,8-tetramethyldecanoate and 2,4,6,8-tetramethylundecanoic acid methyl ester. 6. The method according to any one of the preceding claims, characterized in that the compound is used with multiple methyl branched hydrocarbon chain as the racemate. 7. The method according to any one of claims 1 to 5, characterized in that the compound with multiple methyl branched hydrocarbon chain is exactly one enantiomer. 8. The method according to claim 7, characterized in that the compound with multiple methyl-branched hydrocarbon chain is selected from (2R, 4R, 6R) -4,6-dimethyloctan-2-ol, (2S, 4R, 6R) -4, 6-dimethyloctan-2-ol, (2R, 4R, 6R) -2,4,6-trimethyloctanol, (2R, 4R, 6R) -2,4,6-trimethyloctanoic acid, (2R, 4R, 6R) -2,4,6-trimethyloctanoic acid methyl ester, (2R, 4R, 6R, 8R) -2,4,6,8-tetramethyl decanol, (2R, 4R, 6R, 8R) - 2,4,6,8-tetramethyl decanoic acid methyl ester and (2R, 4R, 6R, 8R) - 2,4,6,8-tetramethylundecanoic acid methyl ester. 9. The method according to any one of the preceding claims, characterized in that the enzyme system is in the form of living cells. 10. The method according to claim 9, characterized in that the enzyme system is present in the form of an isolated from a microorganism enzyme system. 11. The method according to claim 10, characterized in that the enzyme system is a isolated from a microorganism soluble enzyme system. 12. The method according to any one of claims 9, 10 or 11, characterized in that an enzyme system from Bacillus megaterium is used. 13. The method according to claim 12, characterized in that an enzyme system from Bacillus megaterium strain ATCC 14945 is used. 14. The method according to any one of claims 12 or 13, characterized in that an enzyme system is used which is heterologously expressed in the form of a suitable vector in another organism. 15. The method according to any one of the preceding claims, characterized in that exactly one oxidation product is isolated. 16. The method according to any one of the preceding claims, characterized in that the isolated oxidation product is a ketone. 17. The method according to any one of claims 1 to 15, characterized in that the isolated oxidation product is an alcohol. 18. The method according to any one of the preceding claims, characterized in that as an oxidation product in the position (ω-1) oxidized compound is recovered. 19. The method according to any one of claims 1 to 17, characterized in that as an oxidation product in the position (ω-2) oxidized compound is recovered. 20. The method according to any one of claims 1 to 17, characterized in that as an oxidation product in the position (ω-3) oxidized compound is recovered. 21. The method according to any one of the preceding claims 1 to 20, characterized in that (i) an oxidation of a (Ii) all oxidation products are isolated and analyzed; and (iii) if the result is unsatisfactory, the oxidation is carried out using another enzyme system according to any one of claims 9 to 14, wherein steps (i) to (iii) are run through until essentially a desired oxidation product is isolated. 22. Oxidation product which is isolated according to one of the preceding claims.