Enzymatic method for the production of enriched MUSK fragrance

Abstract

Described are compositions and methods relating to an enzymatic method for making intermediates of (-)-(R)-3-methylcyclopentadecan-1-one (Leavo Muscone), a well-known musk fragrance compound. The compositions and methods involve the enzymatic conversion of 3-methylcyclopentadecane-1,5-diol into intermediate compounds in the production of enriched Leavo Muscone, which can be used directly to make fragrance ingredients for commercial purposes.

Description

ENZYMATIC METHOD FOR THE PRODUCTION OF ENRICHED MUSK FRAGRANCETECHNICAL FIELD

[0001] The present compositions and methods relate to an enzymatic method for making intermediates of (-)-(R)-3-methylcyclopentadecan-l-one (Leavo -Muscone), a well-known musk fragrance compound. The compositions and methods involve the enzymatic conversion of 3-methylcyclopentadecane-l,5-diol into intermediate compounds in the production of enriched Leavo-Muscone, which can be used directly to make fragrance ingredients for commercial purposes.BACKGROUND

[0002] Muscone (3 -methylcyclopentadecan- 1 -one) is an important physiologically active component of musk. (R)-Muscone was first discovered in 1906, isolated from the male musk deer, Moschus moschiferus. The non-natural (A>-enantiomer was described as having a poorer musk odor. As a key perfumery component, the (A)-enantiomer has been the target of a number of total syntheses. The formula of (R)-Muscone is depicted, below, as Formula V:Formula V

[0003] Multi-step chemical methods have been described for synthesizing Muscone. Enzymatic methods have also been described, which are more environmentally friendly and superior for producing desired enantiomers.

[0004] Direct enzymatic methods of enriched (-)-( / )-Muscone involve kinetic resolutions, implying the removal of unwanted (A) -enantiomer and loss of material. Desymmetrization is a method allowing to convert the complete starting material into desired enantiomer. Muscemor can be converted to Leavo-muscone, an even more valuable scent molecule that is a base fragrance ingredient in many perfumes.SUMMARY

[0005] The present compositions and methods relate to the production of perfume molecules and precursor molecules. Aspects and embodiments of the processes and compositions are described in the following, independently numbered paragraphs.1. In one aspect, a method for making a compound of Formula II and / or a compound of Formula III is provided,Formula IIFormula IIIcomprising contacting a compound of Formula I with an alcohol dehydrogenase.Formula I2. In some embodiments of the method of paragraph 1, contacting the compound of Formula I with an alcohol dehydrogenase is performed in the absence of a compound of Formula VI.Formula VI4. In some embodiments of the method of any preceding paragraph, the method provides a mixture comprising a compound of Formula II and a compound of Formula III.5. In some embodiments of paragraph 4, the method provides a mixture comprising a compound of Formula II and a compound of Formula III at a molar ratio of 1:1.4.6. In some embodiments, the method of any preceding paragraph further comprises treating a compound of Formula II and / or a compound of Formula in with acid to provide a compound of Formula IV.Formula IV7. In some embodiments, the method of paragraph 6 further comprises hydrogenating the compound of Formula IV to provide a compound of formula V.Formula V8. In another aspect, a composition prepared by the method of any of the previous paragraphs is provided.

[0006] These and other aspects and embodiments of present processes and compositions will be apparent from the description, including any accompanying Figures.DETAILED DESCRIPTIONI. Definitions

[0007] Prior to describing the present compositions and methods in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in the relevant art.

[0008] As used herein, the term “alcohol” refers to an organic compound in which a hydroxyl functional group (-OH) is bound to a saturated carbon atom.

[0009] As used herein, the term “alcohol dehydrogenase” (ADH) refers to a group of dehydrogenase enzymes (EC 1.1.1.1 ) that can convert alcohol groups to ketone groups or perform the reverse reaction. The term “alcohol dehydrogenase” may, for example, be used interchangeably with the terms “keto reductase” or “aldo-keto reductase” or “carbonyl reductase.”

[0010] As used herein, the singular articles “a,” “an,” and “the” encompass the plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety.

[0011] The following abbreviations / acronyms have the following meanings unless otherwise specified:ee enantiomeric excessEC enzyme commissionADH alcohol dehydrogenaseeq equivalentsNAD+P-nicotinamide adenine dinucleotideNADH P-nicotinamide adenine dinucleotide (reduced)NADP+ P-nicotinamide adenine dinucleotide phosphateNADPH P-nicotinamide adenine dinucleotide phosphate (reduced)GC gas chromatographyMCPD 3 -methylcyclopentadecane- 1,5 -di one (Formula VI)MDIOL 3 -methylcyclopentadecane- 1,5 -diol (Formula I)MEK methyl ethyl ketone (2-butanone)Hydroxy ketol 5-hydroxy-3-methylcyclopentadecan-l-one (Formula II) Ketol 14-methyl-16-oxabicyclo[10.3.1]hexadecan-l-ol (Formula III) BCE 14-methyl-16-oxabicyclo[10.3.1]hexadec-12 or 1-ene (Formula VII) Muscemor (E or Z)-3-methylcyclopentadec-4 or 5-en-l-one (Formula IV) Muscone 3-methylcyclopentadecan-l-one (Formula V)Pd / C palladium / carbonEtOH ethanol°C degrees Centigradeg gramg / L grams per literH2O waterhr hourkg kilogramM molarMEK Methyl Ethyl Ketonemg milligrammL or ml millilitermM millimolarPg microgrampL and pl microliterpM micromolarIL Enzymatic production of Formula V and intermediates from Formula I

[0012] The present compositions and methods are based on the finding that alcohol dehydrogenase (ADH; EC 1.1.1.1) can be used to convert compounds of Formula I to compounds of Formula II and Formula III, which are useful, for example, for providing intermediate compounds in the production of Formula V.Formula IFormula IIFormula IIIFormula V

[0013] Formula I may initially be prepared, for example, by contacting a compound of Formula VI with diatomic hydrogen in the presence of a metal catalyst, such as nickel, as is well known in the art.Formula VI

[0014] However, a feature of the present methods is that Formula I is directly contacted by ADH to enzymatically interconvert alcohol groups and ketone groups and to yield Formula II(5-hydroxy-3-methylcyclopentadecan-l-one; hydroxy ketol) and / or Formula III (14-methyl-16-oxabicy clo [10.3.1] hexadecan- 1 -ol ; ketol) .

[0015] The present process does not require that Formula VI is contacted with ADH to produce Formula II and / or Formula III. Certain embodiments of the methods require contacting Formula I with ADH in the absence of Formula VI, such that any trace amounts of Formula VI would not interfere with the present process. Formula I may be substantially free of Formula VI, measurably free of Formula VI or quantitatively free of Formula VI. In some embodiments, theratio of Formula VI to Formula I in the final metal-catalyzed reaction product is less than 5%, less than 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.05% or even less, on a molar basis, compared to the amount of Formula I.

[0016] In some embodiments, ADH produces from Formula I, a mixture of Formula II and Formula III. In some embodiments, ADH produces from Formula I, Formula II in the absence of Formula III. In some embodiments, ADH produces from Formula I, Formula III in the absence of Formula II.

[0017] Further according to the present processes, Formula VII (14-methyl-16-oxabicyclo[10.3.1]hexadec-12 or l-ene; BCE) may not be enzymatically produced from Formula I by alcohol dehydrogenase, although Formula VII may be produced by subsequent chemical manipulations.

[0018] Intermediate compounds Formula II and Formula III can be converted into Formula IV (E or Z, 3-methylcyclopentadec-4 or 5-en-l-one; Muscemor) by treatment with acid, and further converted to Formula V (3 -methylcyclopentadecan- 1 -one; Muscone) by hydrogenation (e.g., in the presence of a metal catalyst), using standard chemical processes.Formula V

[0019] These post-enzymatic conversion processes can be partial, essentially complete or complete, to produce desired fragrance molecules or precursor molecules.in. Selection of an ADH

[0020] Alcohol dehydrogenase (ADH; EC 1.1.1.1) is an enzyme family that catalyzes the oxidation and reduction of a wide variety of alcohols and carbonyl groups, generally including associated reverse reactions. ADH for use in the enzymatic conversion of Formula I to Formula II and Formula III may be a wild-type ADH enzyme that is occurs in nature. Alternatively, the ADH be an engineered variant of a wild-type ADH that includes one or more mutations known to affect activity, stability or expression.IV. Enzymatic reaction conditions

[0021] The optimum conditions (e.g., pH and temperature) for the enzymatic reaction with alcohol dehydrogenase (ADH) can be determined by a person of ordinary skill in the art.Exemplary conditions are described in WO2022017389 (Givaudan, CH). Enzymatic conversion may take place in the presence of an enzyme cofactor and a cofactor regeneration system. The cofactor may be NAD+ or NADP+, which generate NADH or NADPH, respectively, when an alcohol is converted to a carbonyl group. The bioconversion reaction may take place in an organic or aqueous solvent mixture. Reaction conditions may include a solubilizing agent (e.g., a surfactant, detergent, solubility enhancer, water miscible organic solvent and the like) in the bioconversion reaction.

[0022] The initial concentration of the compound Formula I as a substrate for the ADH-mediated enzymatic reaction may be about 10 mM or greater, about 20 mM or greater, about 30 mM or greater, about 40 mM or greater, about 50 mM or greater, about 100 mM or greater, about 150 mM or greater, about 200 mM or greater, about 250 mM or greater, about 300 mM or greater or about 400 mM or greater.

[0023] The initial concentration of ADH in the enzymatic reaction may be at least about 25 g / L, at least about 25 g / L or lower, at least about 20 g / L or lower, at least about 18 g / L or lower, at least about 16 g / L or lower, at least about 14 g / L or lower, at least about 12 g / L or lower or at least about 10 g / L or lower or even less.

[0024] Any number of phase separation, sedimentation, filtration, step vacuum concentration or other techniques may be use as needed during the production of Formula IV intermediates or Formula V.V. Uses for resulting fragrance molecules

[0025] Compounds produced by the present processes can be used as fragrance molecules (including fragrance molecule precursor molecules) for inclusion in fine perfumes and myriad other consumer products, including but not limited to soaps, shampoos, laundry and hand washing detergents and softening agents, air fresheners and the like. Formula V, and to a lesser extent Formula IV, are most commonly used as fragrance molecules, although Formula II and Formula III may independently be useful as primary fragrance molecules, in addition to being precursor molecules for Formula IV and Formula V. Fragrance molecules may be present in a diluent or carrier material and may be used in combination with auxiliary agents.

[0026] These and other aspects and embodiments of the present strains and methods will be apparent to the skilled person in view of the present description. The following examples are intended to further illustrate, but not limit, the present processes. Documents cited herein (including patents, patent applications, scientific publications, manufacturer's specifications and instructions.) are incorporated by reference in their entirety.EXAMPLESExample 1. Preparation of a mixture of Formula II and Formula III from Formula I using acetaldehyde for cofactor regeneration

[0027] Formula II and Formula III were prepared by enzymatic transformation of Formula I with ADH (PRO-KRED 384; Prozomix Limited, UK) using acetaldehyde for cofactor regeneration.

[0028] To a 100-ml round-bottom flask was added 45 ml of 100 mM potassium phosphate buffer pH 7.5. NAD+ (10 mg), and ADH (50 mg) was added to the mixture. A solution of Formula I (100 mg / ml) in dimethyl sulfoxide (5 mL) was added followed by acetaldehyde (600 pl, 0.2 M).The reaction mixture was stirred at room temperature for 22 hr, then extracted with 50 ml of ethyl acetate. The organic phase was analyzed by GC for the conversion into Formula II and Formula III of up to 43%.Example 2. Preparation of Formula II and Formula III from Formula I using MEK for cofactor regeneration

[0029] Formula II and Formula III were prepared by enzymatic transformation of Formula I with ADH (PRO-KRED 384; Prozomix Limited, UK) using MEK for cofactor regeneration.

[0030] To a 100-ml round-bottom flask was added 42 ml of 100 mM potassium phosphate buffer pH 8.5. NAD+(6 mg) and ADH (125 mg) were added, followed by a solution of Formula I (500 mg / ml) in MEK (2.5 ml). The reaction mixture was stirred at room temperature up to 72 hr, and then extracted with 50 ml of ethyl acetate. The organic phase was analyzed by GC for the conversion into Formula II and Formula III of up to 72%.Example 3. Preparation of Formula II and Formula III from Formula I using acetone for cofactor regeneration

[0031] Formula II and Formula III were prepared by enzymatic transformation of Formula I with ADH (PRO-KRED 384; Prozomix Limited, UK) using acetone for cofactor regeneration.

[0032] To a 100-ml round-botom flask was added 40 ml of 100 mM potassium phosphate buffer pH 8.5. NAD+ (6 mg) and ADH (125 mg) were added, followed by a solution of Formula I (250 mg / ml) in 50:50 acetone / toluene (5 ml). The reaction mixture was stirred at room temperature up to 72 hr, then extracted with 50 ml of ethyl acetate. The organic phase was analyzed by GC for the conversion into Formula II and Formula III of up to 95%.

[0033] In a 2-L jacketed reactor was added 425 ml of distilled water followed by 0.4 g of potassium phosphate monobasic and 8.5 g of potassium phosphate dibasic. Formula I (25 g) was prior dissolved in 50:50 acetone / toluene (50 ml) and added to the reaction mixture followed by NAD+ (0.25 g) and ADH (2.5 g). The biotransformation mixture was stirred at 25°C up to 72 hours.

[0034] The crude reaction composition was anaylzed by NMR indicating the formation of only Formula II and Formula III with a ratio of 1 : 1.4. After completion, the reaction was acidified to pH 3.0 following by the addition of 0.25 L of toluene. The resulting solution was heated to 60°C for 30 min before filtration on a bed of hyflo material to remove emulsion. The organic layer was collected, filtered and concentrated.Example 4. Preparation of Formula IV from Formula II and Formula III

[0035] In a 1-L reactor, 61g of Formula II and Formula III was dissolved in 540 ml of toluene.5.8 ml (0.4 eq) of 85% phosphoric acid was gently added in stirring mixture. The reaction mixture was heated to 93°C while removing water by azeotrope distillation. Completion was observed after overnight when conversion was over 95%. The reaction mixture was cooled to 30-35°C, 5% aqueous potassium hydroxide solution (120 mL) was gently added to the mixture over 15 min and let stirred for 30 min. The organic phase was collected and washed with aqueous sodium chloride solution (100 mL). The organic layer was finally concentrated resulting in 55.4 g of crude yielding to 97.6% of Formula IV.Example 5. Preparation of Formula V by Pd / C catalyzed hydrogenation of Formula IV

[0036] Formula V was prepared by Pd / C catalyzed hydrogenation of Formula IV:

[0037] In a 2 L autoclave, 380 g of crude Formula IV (1.37 mol), 3.8 g of Pd-C (palladium 10% on carbon) and 400 ml of 99% ethanol were loaded. The reaction was carried out at 12°C and at a hydrogen pressure of 25 bar. After completion (6 hr), the reaction was filtered, and the catalyst was washed with ethanol. The filtrate was concentrated yielding to 370 g crude Formula V at 98% molar yield of the R isomer with >80% ee.

Claims

CLAIMSWhat is claimed is:

1. A method for making a compound of Formula II and / or a compound of Formula III,Formula IIFormula IIIcomprising contacting a compound of Formula I with an alcohol dehydrogenase.Formula I2. The method of claim 1, wherein contacting the compound of Formula I with an alcohol dehydrogenase is performed in the absence of a compound of Formula VI.Formula VI4. The method of any preceding claim, wherein the method provides a mixture comprising a compound of Formula II and a compound of Formula III.

5. The method of claim 4, wherein the method provides a mixture comprising a compound of Formula II and a compound of Formula III at a molar ratio of 1 : 1.4.

6. The method of any preceding claim, further comprising treating a compound of Formula II and / or a compound of Formula III with acid to provide a compound of Formula IV.Formula IV7. The method of claim 6, further comprising hydrogenating the compound of Formula IV to provide a compound of formula V.Formula V8. A composition prepared by the method of any of the previous claims.

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