Conversion of Farnesyl Acetone to Homofarnesyl Acetate by a Buyer-Billiger Monooxygenase

Abstract

A method for producing homofarnesyl acetate, comprising contacting farnesyl acetone with a Baeyer-Villiger monooxygenase (BVMO) enzyme in the presence of a cofactor, and optionally further comprising hydrolyzing the homofarnesyl acetate to homofarnesol.

Description

Technical Field

[0001] The present invention relates to a new process for the preparation of homofarnesol, especially (3E,7E)-homofarnesol, and its ethyl derivatives.

Background Art

[0002] Homofarnesol is an important intermediate for the production of (-)-Ambrox (3a,6,6,9a-tetramethyldodecahydronaphtho[2,1-b]furan), a fragrance component in high demand. This document describes various processes for the preparation of homofarnesol. For example, homofarnesol is synthesized in a lengthy process via homofarnesyl amide starting from Nerolidol (3,7,11-trimethyldodeca-1,6,10-trien-3-ol) (A.F. Barrero et al., J. Org. Chem. 1996, 61, 2215). Alternatively, homofarnesol may be prepared by decarbonylation of Nerolidol in the presence of a polar solvent and a palladium halide catalyst (WO92 / 06063). Another method for the production of homofarnesol starting from dihydrofuran via homogeraniol in five steps has been described by P. Kociensiki et al. (J. Org. Chem. 1989, 54, 1215). More recently, the synthesis of homofarnesol via Wittig olefination from geranylacetone, followed by ring opening of the cyclopropane ring and formyloxidation, has been described in the literature (WO2013 / 156398).

[0003] All the previously known methods have drawbacks such as, for example, the reagents used are expensive, the process conditions are not economically attractive (e.g., the temperature is below 0 °C), the reagents used are toxic, and / or the solvents used are flammable. Accordingly, there is a need to provide a new or improved method for making homofarnesol and ethyl homofarnesol and / or its precursors. SUMMARY OF THE INVENTION

[0004] According to a first aspect of the present invention, an acetate represented by formula (I)

Chemical formula

Chemical formula

[0005] According to a second aspect of the present invention, an alcohol represented by formula (III)

Chemical formula

[0006] According to a third aspect of the present invention, there is provided the use of an acetate represented by formula (I) obtained by and / or obtainable by the first aspect of the present invention as a precursor for the production of an alcohol represented by formula (III), which is optionally used as a substrate for the enzyme-mediated production of fragrance components.

[0007] Aspects of any aspect of the present invention may provide one or more of the following advantages: · An unusual and novel biological pathway to precursors of homofarnesol / ethyl homofarnesol, · Use of enzyme technology for the implementation of green chemistry and environmentally friendly synthetic processes.

[0008] Details, examples and preferences provided in connection with any one or more specific aspects of the invention described herein are further described herein and will be equally applicable to all aspects of the invention. Any combination of aspects, examples and preferences described herein is covered by the invention in all its possible variations, unless specifically indicated herein or clearly negated by context.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

[0010] Detailed Description The present invention is based on the surprising discovery that a ketone represented by formula (II) having a linear alkene chain undergoes oxidation in the presence of a Baeyer-Villiger monooxygenase (BVMO) enzyme, resulting in the formation of acetate, which can be used as a precursor for the preparation of alcohols such as homofarnesol or ethyl homofarnesol. BVMO enzymes are known to be able to catalyze the conversion of ketones to esters. However, these reported enzymes have only been described as acting in ecosystems on cyclic substrates (including terpenones and benzocondensed ketones), aliphatic ketones, and aliphatic hydroxyketones. Surprisingly, it has now been discovered that the conversion of polyunsaturated branched ketones, as defined by formula (II), to the acetate represented by formula (I) is also catalyzed by BVMO enzymes. There is no literature reporting that BVMO enzymes can act on polyunsaturated branched ketones.

[0011] Therefore, the acetate represented by formula (I)

Chemical formula

Chemical formula

[0012] The compound represented by formula (II) appears in the form of four different stereoisomers, for example, as a compound represented by formula (II) having a 5E,9E- or 5Z,9E- configuration.

[0013] In one embodiment, the method includes contacting a 5E,9E-compound represented by formula (II) with a BVMO enzyme in the absence of any other stereoisomer represented by formula (II).

[0014] In other aspects, the compound represented by formula (II) may be, for example, a mixture of stereoisomers. In one aspect, the mixture comprises a 5E,9E-compound represented by formula (II) and one or more other stereoisomers represented by formula (II).

[0015] In one aspect, the mixture comprises a 5E,9E-compound represented by formula (II) and a 5E,9Z-compound represented by formula (II).

[0016] In one aspect, the mixture comprises a 5E,9E-compound represented by formula (II) as a main component. The main component means that the weight percentage of the 5E,9E-compound present in the mixture is greater than the weight percentage of the 5Z,9E-compound represented by formula (II).

[0017] When R is methyl, the compound represented by formula (II) may be referred to as farnesyl acetone encompassing E,E-farnesyl acetone, E,Z-farnesyl acetone, Z,E-farnesyl acetone, Z,Z-farnesyl acetone, and mixtures thereof. When R is ethyl, the compound represented by formula (II) may be referred to as ethyl farnesyl acetone encompassing E,E-ethyl farnesyl acetone, E,Z-ethyl farnesyl acetone, Z,E-ethyl farnesyl acetone, Z,Z-ethyl farnesyl acetone, and mixtures thereof. The compound represented by formula (II) where R is methyl is commercially available. The compound represented by formula (II) where R is ethyl is novel per se. It may be synthesized from 5,9-dimethyldeca-4,8-dienal according to the procedure as described in Example 11.

[0018] As used herein, the term "BVMO (Baeyer-Villiger monooxygenase)" refers to a monooxygenase that can catalyze various oxidation reactions and encompasses Baeyer-Villiger oxidation for the production of the ester compound represented by formula (I) by oxidation of the ketone represented by formula (II). The BVMO enzyme may be a wild-type enzyme or a modified enzyme. As used herein, the term "wild-type" refers to the naturally occurring form of a substance, whether in relation to a polypeptide such as an enzyme, a polynucleotide such as a gene, an organism, a cell, or any other substance. As used herein, the term "modified" refers to a substance that is different from the wild-type, whether in relation to a polypeptide such as an enzyme, a polynucleotide such as a gene, an organism, a cell, or any other substance. Suitable alterations to a wild-type substance may result in the production of a modified substance, which includes alterations to genetic material and alterations to protein material. Alterations to genetic material may include any genetic modification known in the art that makes the material different from the wild-type. Examples of such genetic modifications include, but are not limited to, deletions, insertions, substitutions, fusions, etc., which may be made on the polynucleotide(s) containing the relevant gene(s) to be modified.

[0019] Regarding the object of the present invention, as long as the BVMO enzyme can be produced in a host cell and catalyze the reaction to produce an acetate represented by formula (I) having an ester group introduced into its chain from a ketone represented by formula (II) (for example, homofarnesyl acetate from farnesyl acetone), the BVMO can be, but is not particularly limited to, BVMO derived from microorganisms such as Pseudomonas sp., Rhodococcus sp., Brevibacterium sp., Comanonas sp., Acinetobacter sp., Arthrobacter sp., Brachymonas sp., Themobifida sp., Gordonia sp., Pseudooceanicola sp., more preferably BVMO derived from Pseudomonas sp., Brachymonas sp., Pseudooceanicola sp. or Acinetobacter sp., and most preferably BVMO derived from Acinetobacter sp., Pseudomonas sp. (for example, Pseudomonas veronii), Brachymonas sp. (for example, Brachymonas petroleovorans) or Pseudooceanicola sp. (for example, Pseudooceanicola batsensis). In a particular embodiment, the BVMO is derived from Acinetobacter sp. or Pseudomonas sp. (for example, Pseudomonas veronii). The nucleotide sequence of the BVMO coding gene can be obtained from a database such as NCBI's GenBank.

[0020] Enzymes may be defined by their functions according to the EC classification. The Enzyme Commission number (EC number) is a numerical classification system for enzymes based on the chemical reactions they catalyze. A BVMO suitable for performing the conversions described herein belongs, for example, to the EC 1.4.13 class (oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen accompanied by one donor, and incorporation of one atom of oxygen into the other donor).

[0021] The BVMO may be, for example, one or more BVMO enzymes used in the following examples, such as EW-103 (a mutant of the wild-type Acinetobacter sp. BVMO enzyme) obtained from EnzymeWorks, Inc., San Diego (USA), or MekA obtained from Gecco Biotech B.V.

[0022] The Baeyer-Villiger monooxygenase cofactor (BVMO cofactor) is a cofactor that assists the BVMO enzyme during the catalysis of the reaction. The BVMO cofactor used in the methods provided herein may be of any type suitable for assisting in the conversion of a ketone represented by formula (II) to an acetate represented by formula (I). The cofactor may be, for example, an inorganic molecule or an organic molecule. The BVMO cofactor may be selected, for example, from nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), or a combination thereof. Preferably, the BVMO cofactor is present in the reaction mixture at an initial molar concentration with respect to BVMO such that the BVMO enzyme is saturated with NAD(P)H. Thus, preferably, NAD(P)H is present in the reaction mixture at a concentration at least equal to the concentration of the BVMO enzyme.

[0023] In one aspect of the invention, NAD(P)H is present in the reaction mixture at an initial concentration between about 1 and 10 mg / ml, such as between 1 and 5 mg / ml, or between 1 and 4 mg / ml.

[0024] Generally, cofactors are expensive and thus cannot be used in stoichiometric amounts, and it is desirable to regenerate them. Furthermore, by regenerating cofactors, it is possible to increase the efficiency of the desired conversion or facilitate the separation of products, thereby further reducing the cost of the process. For this reason, cofactor regeneration systems are often used.

[0025] Glucose dehydrogenase (GDH) enzymes are common for the regeneration of NAD(P)H. For example, GDH enzymes are commercially available from Codexis Inc. (GDH-105, GDH-901, CDX-019), Johnson Matthey (GDH-101) or EnzymeWorks (GDH-EW). According to the literature, additional GDH enzymes are available, for example, glucose dehydrogenase from Bacillus subtilis (UniProtKB accession number: P12310, E.C. 1.1.1.47, (1986) J. Bacteriol. 1986, 166, 238 - 43). GDH enzymes can generally be used for cofactor regeneration, for example, in combination with glucose. Alternatively, alcohol dehydrogenase (ADH) enzymes can be used for cofactor regeneration when combined with, for example, isopropanol, provided that they do not interfere with the reaction catalyzed by certain BVMO enzymes.

[0026] Other enzymes that can be used in cofactor regeneration include, for example, phosphite dehydrogenase (PTDH) in combination with, for example, sodium phosphite. The Baeyer-Villiger monooxygenase may be present in the reaction mixture in any suitable form known in the art, such as, but not limited to, a cell-free extract, or may be contained within a host organism cell, and these may be in free form in solution / suspension, or in any suitable manner known in the art, such as, but not limited to, a purified form and / or an immobilized form (held on a membrane, or bound to / within a column). Preferably, the BVMO is present in the reaction mixture at a concentration necessary to produce the required amount of acetate represented by formula (I) that can be produced at the relative level of dissolved oxygen.

[0027] In one embodiment, the Baeyer-Villiger monooxygenase is present in the reaction mixture as a cell extract prepared from the cells in which it was produced, where the cells are preferably the bacterial host cells used to produce the BVMO enzyme. The cell extract may be obtained by any suitable means capable of lysing the host cells, including, but not limited to, sonication, RNAse / lysozyme treatment, freeze-thaw treatment, or alkali treatment. Preferably, the cell extract is then treated to remove cell debris before being used as a source of Baeyer-Villiger monooxygenase in the methods described herein. The cell lysate may be treated by any suitable means known in the art, including, but not limited to, filtration, centrifugation, or purification with salts, to obtain a clarified cell extract.

[0028] The cofactor regeneration system plays a role in regenerating the BVMO cofactor after being used to assist the conversion of the BVMO cofactor of the ketone represented by formula (II) to acetate represented by formula (I). The cofactor regeneration system may also, for example, regenerate the reduced form of the cofactor (e.g., NADH and / or NADPH). The cofactor regeneration system used in the methods described herein may be of any type suitable for regenerating the BVMO cofactor useful in converting the ketone represented by formula (II) to acetate represented by formula (I) (e.g., converting farnesylacetone to homofarnesylacetate) by the BVMO enzyme. Thus, a method for producing acetate represented by formula (I) from the ketone represented by formula (II) is provided in a further aspect, the method comprising contacting the ketone represented by formula (II) with a BVMO enzyme in the presence of a cofactor and a cofactor regeneration system.

[0029] In one particular embodiment, the cofactor regeneration system is an ADH / isopropanol cofactor regeneration system. In another particular embodiment, the cofactor regeneration system is a GDH / glucose cofactor regeneration system. In another particular embodiment, the cofactor regeneration system is a phosphite dehydrogenase / phosphite cofactor regeneration system. Using an ADH / isopropanol cofactor regeneration system has the advantage of no acidification of the reaction medium compared to the GDH / glucose system. The method for producing acetate represented by formula (I) from the ketone represented by formula (II) is carried out under conditions of time, temperature, pH and solubilizing agent that enable the conversion of the ketone represented by formula (II) to acetate represented by formula (I).

[0030] The pH of the reaction mixture may range from 4 to 9, preferably from 7 to 9 (including a pH of about 8.5), and may be maintained by addition of a buffer to the reaction mixture or by pH correction. Exemplary buffers for this purpose are Tris-HCl buffer or Glycine / NaOH buffer.

[0031] For the BVMO enzyme under consideration, the temperature is between about 15°C and about 60°C, for example from about 15°C to about 50°C, or from about 15°C to about 45°C, or from about 30°C to about 60°C, or from about 35°C to about 55°C. The temperature can be kept constant or varied during the bioconversion process. The methods provided herein may further include the purification of the compound represented by formula (I) and / or the separation from any unreacted compound represented by formula (II). For example, the compound represented by formula (I) may be purified by solvent extraction (e.g., methylbenzene, hexane, tert-butyl methyl ether (tBME)) and / or distillation.

[0032] In a further aspect of the invention, the compound represented by formula (I) may be further reacted. For example, the compound represented by formula (I) may be hydrolyzed to a compound represented by formula (III)

Chemical formula

[0033] In certain embodiments, the hydrolysis is carried out with a biocatalyst and optionally may be carried out in the same reaction broth. Alternatively, the compound represented by formula (I) may be chemically hydrolyzed, preferably after purification, in a methanol solution in the presence of, for example, potassium carbonate. The alcohol thus obtained may be used, for example, as a substrate for biocatalytic conversion.

[0034] In certain embodiments, the compound represented by formula (III) where R is methyl may be converted to (-)-Ambrox in the presence of the SHC (Squalene Hopene Cyclase) enzyme. In one aspect, the compound represented by formula (III) where R is ethyl may be converted to ethyl ambruticin (which is described in more detail in WO2021 / 110858). The examples described herein are illustrative of the disclosure and are not intended to be limiting. Different aspects of the disclosure are described in accordance with the disclosure. Many modifications and changes may be made to the techniques described and illustrated herein without departing from the spirit and scope of the disclosure. Therefore, it should be understood that the examples are illustrative only and do not limit the scope of the disclosure.

[0035] Examples Example 1: Gas chromatography (GC) analysis, calculation of conversion rate Inject 1 μl of the solvent phase (solvent extraction sample) into a 30 m × 0.32 mm × 0.25 μm Zebron ZB-5 column (split ratio 3). The column was developed at a constant flow rate (4 ml / min, H2) with a temperature gradient: 100 °C, 15 °C / min to 200 °C, 120 °C / min to 240 °C, and 4 minutes at 240 °C. Inlet temperature: 250 °C, detector temperature: 250 °C (Thermo Trace 1310 GC device)

[0036] The farnesyl acetone conversion rate was calculated from the recorded substrate and product peak areas as follows. Conversion rate (%): 100 × (product ピーク面積 / (product ピーク面積 + substrate ピーク面積 )

[0037] Example 2: BVMO reaction of farnesyl acetone using ethionamide monooxygenase (EthA) E. coli TOP10 transformed with a plasmid for the production of EthA (GenBank: AAK48336.1, UniProtKB P9WNF8, Fraaije et al. 2004, J. Biol. Chem. 279(5), 3354) was grown in 200 ml LB medium supplemented with 100 μg / ml ampicillin and 200 μg / ml FAD until an OD 650nmCultivation was carried out up to 0.75 (37 °C, 200 rpm). Induction of enzyme production was then caused by adding 0.2% arabinose. Farnesyl acetone (0.1%) was added to the culture, and incubation was continued at 30 °C for an additional 23 h. The culture was sampled, extracted with methyl-t-butyl ether (MTBE), and the substrate and product contents were analyzed by GC. The identity of the reaction product was confirmed by GC-MS analysis. The conversion rate of farnesyl acetone to homofarnesyl acetate was 2 - 5% in two sets of independent experiments.

[0038] Example 3: BVMO reaction of farnesyl acetone using cyclopentadecanone monooxygenase (CPDMO) CPDMO (GenBank AB232538.1, UniProtKB Q1T7B5) was produced from E. coli BL21(DE3) cells transformed with a plasmid derived from pJ401 for CPDMO production. A 200 ml LB medium culture (50 μg / ml kanamycin) was inoculated from an overnight preculture and incubated at 28 °C, 160 rpm for about 3.5 h until OD 650nm reached 0.500 - 0.600. For induction of enzyme production, IPTG was added up to 0.8 mM. The culture was incubated at 28 °C, 160 rpm for an additional 3 - 4 h. Cells were harvested by centrifugation and washed. The recovered cell pellet was suspended in 2.5 ml of reaction buffer (50 mM Tris-Cl, pH 9.0), and the cells were disrupted by sonication. Finally, 16.5 ml of 50 mM Tris-HCl, pH 9.0 was added to the sonicated material, resulting in a crude CPDMO enzyme preparation. To measure the enzyme activity, 10 μl of farnesyl acetone and 40 μl of 50 mM NADH were added to 950 μl of the crude CPDMO enzyme preparation. The reaction was incubated at 30 °C for 3 h, extracted with MTBE, and analyzed by GC. The conversion rate of farnesyl acetone to homofarnesyl acetate was approximately 7%.

[0039] Example 4: BVMO reaction of farnesyl acetone using BVMO enzyme from EnzymeWorks Eight buyer biliverdin monooxygenase enzymes available from EnzymeWorks, Inc, (http: / / www.enzymeworking.com), the enzyme supplier, were tested for their ability to convert farnesylacetone to homofarnesylacetate. The reaction mixture contained 2 g / l farnesylacetone, 25 mM glucose, 1 mM NADP, 2 g / l BVMO enzyme, and 2 g / l glucose dehydrogenase (GDH) in 100 mM glycine / NaOH buffer (pH 9.0). The reaction mixture was incubated at 30 °C for 24 h with gentle shaking (70 rpm). 0.5 ml of the reaction mixture was extracted with 1.5 ml of MTBE and analyzed by GC. With EW-103BVMO, the farnesylacetone conversion rate was approximately 50% in 24 h.

[0040] Example 5: BVMO reaction of farnesyl acetone using BVMO enzyme from Gecco - Biotech The BVMO enzymes (wild-type or mutant enzymes) supplied by Gecco Biotech (http: / / www.gecco-biotech.com / ) were applied to the conversion of farnesylacetone to homofarnesylacetate. The BVMO enzymes were provided as purified fusion proteins with phosphite dehydrogenase (PTDH). 20 mM farnesylacetone (about 5 g / l) was reacted in 50 mM Tris HCl (pH 7.5) buffer (1 ml reaction volume) in the presence of 20 μM BVMO, 1 mM NADP, and 100 mM Na2HPO3·5H2O at 24 °C for 20 h.

[0041] The results summarized in the following table showed that the farnesylacetone conversion rate was between 3% and 52% depending on the BVMO enzyme used. The abbreviations used in the table are: n.a.: not applicable, STMO: steroid monooxygenase, PAMO: phenylacetone monooxygenase, HAPMO: 4-hydroxyacetophenone monooxygenase, ACMO: acetone monooxygenase, CPDMO: cyclopentadecanone 1,2-monooxygenase.

Table 1

[0042] Example 6: Cofactor requirements of BVMO enzyme EW - 103 Using EW-103, it was investigated whether both NADPH and NADH can act as cofactors in the conversion of farnesylacetone. The reaction was carried out in 50 mM Tris-HCl buffer (pH 8.5) using 7.6 mM farnesylacetone, 1 mM cofactor, 2 mg / ml BVMO EW-103, 2 mg / ml GDH and 25 mM glucose (5 ml volume, Heidolph Synthesis 1, 30 °C, 900 rpm). In one reaction, NADP was replaced by NAD. Farnesylacetone conversion was supported by NADH as a cofactor, but surprisingly, after 2 h of incubation, it suddenly stopped at a conversion rate of about 20%. When NADPH was used as a cofactor, complete farnesylacetone conversion was obtained at approximately the same time. Two different glucose dehydrogenase (GDH) enzymes were used and the reaction was repeated at increased NAD concentrations (1, 2 and 5 mM). Regardless of the applied NAD concentration and the GDH enzyme used, farnesylacetone conversion stopped at about 20%, but complete conversion was achieved again when NADPH was the cofactor (see Figure 1).

[0043] Example 7: Cofactor regeneration system The use of glucose dehydrogenase (GDH) or alcohol dehydrogenase (ADH) when using glucose or isopropanol for cofactor regeneration can be envisaged. The sensitivity of EW-103 to isopropanol and acetone was tested to investigate the possibility of using ADH / isopropanol instead of GDH / glucose. The reaction was carried out in 50 mM Tris-HCl buffer (pH 8.5) (5 ml, Heidolph Synthesis 1, 30 °C, 900 rpm) using 7.6 mM farnesyl acetone (2 g / l), 1 mM NADP, 2 g / l BVMO EW-103, 2 g / l GDH and 25 mM glucose, and isopropanol or acetone was added up to 5% and 10% (v / v). Adding 5% isopropanol or acetone to the reaction mixture had no effect on the farnesyl acetone conversion rate. When isopropanol or acetone was added up to 10%, the farnesyl acetone conversion rate decreased. When the reaction mixture contained 10% isopropanol, the farnesyl acetone conversion rate was only about 64% after 24 h. EW-103 BVMO was less sensitive to the addition of acetone: the farnesyl acetone conversion rate was 80% after 24 h in the reaction containing 10% acetone (see Figure 2).

[0044] This result indicated the possibility of using the alcohol dehydrogenase (ADH) / isopropanol cofactor regeneration system. The ADH enzyme is available from many enzyme manufacturers and suppliers (for example, KRED-P2-H07 from Codexis Inc.).

[0045] The use of the ADH / isopropanol cofactor regeneration system was confirmed in a reaction carried out in 50 mM Tris-Cl buffer (pH 8.5) (total volume 150 mL) using 8 g / l farnesyl acetone (30.5 mM) in the presence of 1 mM NADPH, 1 g / L BVMO EW-103, 0.5 g / L ADH (Codexis KRED-P2-H07), and 35 mM isopropanol. The farnesyl acetone conversion rate was about 75% after 24 h. In this reaction configuration, using 1 g of EW-103 BVMO enzyme, about 6 g of farnesyl acetone was converted to homofarnesyl acetate. This conversion yield was higher than that in a similar experimental operation using the GDH / glucose cofactor regeneration system, where 1 g of BVMO EW-103 enzyme could only convert 3.6 g of farnesyl acetone.

[0046] Example 8: Reaction products of BVMO farnesyl acetone conversion As a product of the conversion of farnesylacetone by the BVMO enzyme used, homofarnesyl acetate was identified by GC-MS analysis. In some reactions, interestingly and surprisingly, the production of both homofarnesyl acetate and homofarnesol as reaction products was observed. This is thought to be the result of the hydrolysis of homofarnesyl acetate due to a BVMO-independent reaction catalyzed by, for example, lipase (present in the enzyme lyophilized product or cell extract used). This was not further investigated, but the oxidation step of farnesylacetone to homofarnesyl acetate and the subsequent hydrolysis of homofarnesyl acetate to homofarnesol can be catalyzed in a one-pot reaction combined with a BVMO-catalyzed reaction and, for example, a lipase-catalyzed hydrolysis of a suitable homofarnesyl acetate and applying appropriate reaction conditions.

[0047] Example 9: Process - related reaction conditions and conversion yields The conversion of farnesylacetone using EW-103 BVMO was carried out with 9 g / l of BVMO EW-103, 3.2 g / l of ADH KRED-P2-H07 and 1.5 g / l of NADPH (2 mM) in the presence of 27 g / l of isopropanol (0.44 M) with 106 g / l of substrate (0.4 M). The reaction was carried out with constant stirring in 50 mM Tris-Cl buffer (pH 8.5) at 30 °C. The reaction samples were extracted with MTBE and analyzed by GC. After a 48-hour reaction, the conversion rate of farnesylacetone was about 70%, corresponding to a conversion yield of 8 g of farnesylacetone per 1 g of BVMO enzyme.

[0048] Further reactions were carried out with 106 g / l of substrate (0.4 M) in the presence of 27 g / l of isopropanol (0.44 M) while varying other reaction parameters: the BVMO, ADH, and NADPH concentrations were set as follows: (1) 45 g / l, 10 g / l, 5 mM, or (2) 5 g / L, 10 g / L, 0.5 mM After a reaction of approximately 24 hours, the conversion rate of farnesyl acetone in (1) was 86.2%, and as judged from the reaction sample extracted with MTBE, it represents the conversion yield of 2 g of farnesyl acetone per 1 g of BVMO enzyme. In (2), as judged from the reaction sample extracted with MTBE, the conversion rate of farnesyl acetone was 59%, corresponding to a conversion yield in which approximately 12.5 g of farnesyl acetone was converted per 1 g of BVMO enzyme. This result indicates that it is highly likely to completely convert farnesyl acetone using BVMO enzyme (for example, BVMOEW-103) in about 24 hours, and by carefully setting the enzyme with respect to the substrate ratio, the conversion yield defined as the number of grams of substrate converted per 1 g of BVMO enzyme, and the productivity defined from the viewpoints of the farnesyl acetone conversion rate per 1 g of BVMO enzyme and the reaction time are increased.

[0049] Example 10: Conversion of ethyl farnesyl acetone using BVMO enzyme Ethyl farnesyl acetone (a mixture of 4 isomers) was subjected to BVMO oxidation using EnzymeWorks' BVMOEW-103. The reaction mixture (5 ml volume) contained 2 g / L of substrate, 2 g / L of BVMO enzyme, 0.5 g / L of NADP, 4.5 g / L of glucose, and 2 g / L of GDH in 50 mM glycine / NaOH buffer (pH 9.0). The reaction mixture was incubated at 30 °C and 650 rpm (Heidolph Synthesis 1 apparatus). The reaction mixture was sampled at 5, 12.5, and 48 h after the start of the reaction, extracted (0.6 ml of the reaction mixture with respect to 0.7 ml of MTBE), and the substrate / product content was analyzed by GC-FID. Four reaction products were recognized from the four isomer substrate peaks. GC-MS analysis confirmed that ethyl farnesyl acetone was converted to homoethyl farnesyl acetate. An overall conversion rate of 25 - 30% was observed.

[0050] As an example, when the reaction product, homoethyl farnesyl acetate, is further hydrolyzed using, for example, a lipase enzyme, ethyl homofarnesol is produced, which is a substrate for cyclization to Ethylambrofix using the Squalene Hopene Cyclase (SHC) enzyme.

[0051] Example 11: Ethyl farnesyl acetone A solution of ethylmagnesium bromide (3 M, 175 mL, 525 mmol, 1.2 eq) in diethyl ether was added dropwise to a solution of 5,9-dimethyldeca-4,8-dienal (E / Z mixture, 78.8 g, 437 mmol) in diethyl ether (200 mL) at -50 °C over 30 minutes. After the addition was complete, the cooling bath was removed and the mixture was stirred for 1 hour. Then, it was poured into 2 M aqueous HCl (200 mL) and the mixture was extracted with methyl t-butyl ether (MTBE, 150 mL). The organic layer was washed with water and diluted aqueous NaCl until pH neutral and dried over MgSO4. After filtration and removal of the solvent in a rotary evaporator, a colorless transparent liquid was obtained (79.2 g, 86%), which was dissolved in acetone (200 mL). After cooling to 0 °C, Jones reagent (4 M CrO3 in aqueous H2SO4, 94.1 mL) was added dropwise, during which the temperature rose to 43 °C and the mixture turned greenish-brown. After the addition was complete, the mixture was stirred at room temperature for 1 h, then an additional amount of Jones reagent (30 mL) was added and stirring was continued for 20 minutes, then 2-propanol (20 mL) was added. Workup and extraction were carried out as described above to yield a clear yellow liquid (74.8 g), which was distilled on a 10 cm Vigreux column at 91 - 95 °C (0.09 mbar) to yield 7,11-dimethyldodeca-6,10-dien-3-one (50.1 g, 64%, E / Z mixture) as a slightly yellow transparent oil.

[0052] A solution of 7,11-dimethyldodeca-6,10-dien-3-one (34.0 g, 163 mmol) in THF (100 mL) was added to a freshly prepared solution of vinylmagnesium bromide in THF (0.9 M, 228 mmol, 1.4 equiv) at -5 °C for 30 minutes. The resulting mixture was stirred at room temperature for 20 minutes and then hydrolyzed by adding a saturated aqueous solution of NH4Cl (150 mL). Workup was carried out as described above to yield a clear yellow liquid (37.5 g), which was purified by distillation on a 10 cm Vigreux column (distilled at 90 °C / 0.07 mbar) to give 3-ethyl-7,11-dimethyldodeca-1,6,10-trien-3-ol (12.0 g, 31%, colorless transparent liquid, E / Z ratio 5:4 by GC).

[0053] To this product (12.0 g, 50.8 mmol) were added 2-methoxypropene (7.33 g, 102 mmol, 2 equiv) and phosphoric acid (10 mg). The mixture was placed in an autoclave vessel and stirred at 170 °C for 3 h. After cooling to room temperature, the same amounts of 2-methoxypropene and phosphoric acid as above were added, and the mixture was stirred at 170 °C for 18 h. After cooling to room temperature, the mixture was concentrated in a rotary evaporator to yield a brown liquid (14.91 g), which was distilled in a Kugelrohr oven at 150 °C / 0.05 mbar to give a clear yellow liquid (12.66 g), which was further purified by flash chromatography on silica gel using heptane / MTBE (95:5) to give a colorless liquid (a mixture of E / Z isomers, 31 / 26 / 25 / 18% by GC) as 6-ethyl-10,14-dimethylpentadeca-5,9,13-trien-2-one (6.51 g, 46%).

[0054] 11H-NMR (CDCl3, 400 MHz): 4.88 - 5.37 (m, 3 H), 2.46 (d, J=7.8 Hz, 2 H), 2.29 (br d, J=7.8 Hz, 2 H), 2.15 (s, 3 H), 1.96 - 2.11 (m, 10 H), 1.67 - 1.73 (m, 5 H), 1.60 - 1.65 (m, 4 H), 0.94 - 1.03 (m, 3 H). 13 13C-NMR (CDCl3, 101 MHz): 208.8 (s), 208.7 (s), 142.3 (s), 142.2 (s), 142.1 (s), 135.3 (s), 135.2 (s), 135.1 (s), 135.0 (s), 131.5 (s), 131.3 (s), 131.3 (s), 124.7 (d), 124.3 (d), 124.2 (d), 124.0 (d), 122.0 (d), 121.6 (d), 121.5 (d), 44.1 (t), 39.7 (t), 39.7 (t), 36.8 (t), 36.5 (t), 32.0 (t), 32.0 (t), 31.9 (t), 30.6 (t), 30.3 (t), 30.0 (q), 29.9 (q), 29.5 (t), 26.9 (t), 26.8 (t), 26.7 (t), 26.7 (t), 26.6 (t), 26.6 (t), 25.7 (q), 25.7 (q), 23.4 (q), 23.4 (q), 23.1 (t), 23.1 (t), 22.7 (t), 22.2 (t), 22.1 (t), 17.7 (q), 17.6 (q), 16.0 (q), 16.0 (q), 14.1 (q), 13.2 (q), 13.2 (q), 12.8 (q). MS (EI, 70eV): 276 (M + , <1), 218(<1), 207(<1), 149(10), 136(15), 121(28), 107(10), 95(16), 81(36), 69(89), 55(15), 43(100).

Claims

**Claim 1** Formula (I) 【Chemical 1】 wherein R is methyl or ethyl and an acetate represented by Formula (II) [Chemical Formula 2] wherein R has the meaning given above and a method for producing from a ketone represented by, the method comprising contacting a compound represented by formula (II) with a Baeyer-Villiger monooxygenase (BVMO) enzyme in the presence of a cofactor. **Claim 2** The method according to claim 1, wherein the cofactor is selected from NADH and NADPH, or a combination thereof. **Claim 3** The method according to claim 1 or 2, wherein the method further comprises hydrolyzing the acetate represented by formula (I) to an alcohol represented by formula (III) 【Chemical Formula 3】 wherein R is methyl or ethyl .

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