Methods for purifying (-)-ambrox

Abstract

A method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox, which method includes several washing steps, the product of the method, and uses of the product.

Description

[Technical Field]

[0001] Technical Field The present invention relates generally to a process for purifying crude flavor or fragrance or cosmetic ingredients or intermediates, and more particularly to a process for purifying crude (-)-ambrox. The invention also relates to the product of this process and various uses of the product. [Background technology]

[0002] background Formula (I) below: [ka] (-)-Ambrox, represented by the formula (I), is a commercially important perfume ingredient.

[0003] (-)-Ambrox is commercially known as Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlyn (Quest), Cetalox Laevo (Firmenich), Ambermor (Aromor), and / or Norambrenolide Ether (Pacific).

[0004] The (-)-Ambrox enantiomer, rather than the (+)-Ambrox enantiomer, provides desirable specific sensory benefits: the odor of the (-)-Ambrox enantiomer has been described as musky, woody, warm, or ambery, whereas the (+)-Ambrox enantiomer has a relatively weak odor note.

[0005] WO 2017 / 182542 (the contents of which are incorporated herein by reference) discloses a method for preparing (-)-ambrox by biotransformation of a 7E,3E / Z-homofarnesol mixture. Example 4 of WO 2017 / 182542 discloses a downstream process for purifying (-)-ambrox, which includes a heating step in which the biotransformation broth is heated to a temperature of about 80-85°C for about 15 minutes to inactivate the biocatalyst, after which the liquid (-)-ambrox is cooled to a temperature of about 20°C, where it recrystallizes. The (-)-ambrox crystals are separated from the biotransformation broth by filtration and washed with water on the filter to remove the remainder of the biotransformation broth. Upgrading the resulting crude (-)-ambrox to the desired chemical and olfactory qualities requires several successive steps, including dissolving the crystals in ethanol to clarify them, then bleaching the resulting solution, and finally recrystallizing the (-)-ambrox. This is a complex and labor-intensive process that uses multiple items of equipment. The process also uses flammable ethanol, which must be operated safely and precisely, and at the end of the process must either be discarded or upgraded for reuse.

[0006] It would therefore be desirable to provide new and improved methods for purifying (-)-ambrox. It would be particularly desirable to provide methods for simplifying the purification of (-)-ambrox, for example, by using less equipment, avoiding recrystallization, and avoiding the production of (-)-ambrox containing concentrates of mother liquor that must be reprocessed.

[0007] The same considerations are also applicable to the preparation of other fragrance ingredients and intermediates, crude flavor ingredients and intermediates, and crude cosmetic ingredients and intermediates, especially crystalline forms of fragrance ingredients and intermediates and crude flavor ingredients and intermediates made by bioconversion reactions (e.g., using SHC / HAC enzymes or other biocatalysts). Summary of the Invention

[0008] overview According to a first aspect of the present invention, there is provided a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: Washing with aqueous acid; Cleaning with an aqueous surfactant; and Optionally, washing with aqueous alkali Includes.

[0009] According to a specific embodiment of the first aspect of the present invention, there is provided a method for purifying crude (-)-ambrox, the method comprising: Washing with aqueous acid; Washing with an aqueous surfactant; and Optionally, washing with aqueous alkali Includes.

[0010] The washing steps of the first aspect of the invention may occur in any order suitable for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox.

[0011] According to a particular embodiment of the first aspect of the present invention, there is provided a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: (a) Washing crude flavor or fragrance or cosmetic ingredients or intermediates with aqueous acid; (b) washing the product of step (a) with aqueous alkali; (c) washing the product of step (b) with an aqueous detergent; Includes.

[0012] According to a specific embodiment of the first aspect of the present invention, there is provided a method for purifying crude (-)-ambrox, the method comprising: (a) washing crude (-)-ambrox with aqueous acid; (b) washing the product of step (a) with aqueous alkali; (c) washing the product of step (b) with an aqueous detergent; Includes.

[0013] According to a second aspect of the present invention, there is provided a flavour or fragrance or cosmetic ingredient or intermediate obtained by or obtainable by the method of the first aspect of the present invention (including any embodiment thereof).

[0014] According to a particular embodiment of the second aspect of the present invention, there is provided (-)-ambrox obtained by or obtainable by the method of the first aspect of the present invention (including any embodiment thereof).

[0015] According to a third aspect of the present invention, there is provided the use of a flavor or fragrance or cosmetic ingredient according to the second aspect of the present invention (including any embodiment thereof), for example (-)-ambrox, as a flavor or fragrance or cosmetic ingredient.

[0016] According to a fourth aspect of the present invention, there is provided a perfume composition comprising a perfume ingredient according to the second aspect of the present invention (including any embodiment thereof), for example (-)-ambrox.

[0017] According to a fifth aspect of the present invention there is provided a flavour composition comprising a flavour ingredient according to the second aspect of the present invention (including any embodiment thereof).

[0018] According to a sixth aspect of the present invention, there is provided a cosmetic composition comprising a cosmetic ingredient according to the second aspect of the present invention (including any embodiment thereof).

[0019] According to a seventh aspect of the present invention there is provided a household care, personal care, laundry care or air care composition comprising a perfume composition according to the fourth aspect of the present invention (including any embodiment thereof).

[0020] According to an eighth aspect of the present invention there is provided a food or beverage product comprising a flavour composition according to the fifth aspect of the present invention (including any embodiment thereof).

[0021] According to a ninth aspect of the present invention, there is provided a cosmetic product comprising the cosmetic composition of the sixth aspect of the present invention (including any embodiment thereof).

[0022] According to a tenth aspect of the present invention, there is provided a Nutsche-type agitating and drying filter configured to purify a crude flavor or fragrance or cosmetic ingredient or intermediate by the method of the first aspect of the present invention, wherein the crude flavor or fragrance or cosmetic ingredient or intermediate contains from about 20 wt % to about 30 wt % water, based on the wet weight of the crude flavor or fragrance or cosmetic ingredient or intermediate.

[0023] According to an eleventh aspect of the present invention, there is provided a process for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the process comprising washing the crude flavor or fragrance or cosmetic ingredient or intermediate in a Nutsche-type stirred-and-dried filter. The process may, for example, be according to the method of the first aspect of the present invention (including any embodiment thereof).

[0024] Certain embodiments of the present invention may provide one or more of the following advantages, for example compared to the process described in WO 2017 / 182542: • A simpler process for purifying crude flavor or fragrance or cosmetic ingredients or intermediates, such as crude (-)-ambrox; • A cheaper process for purifying crude flavor or fragrance or cosmetic ingredients or intermediates, such as crude (-)-ambrox; • A less labor-intensive process for purifying crude flavor or fragrance or cosmetic ingredients or intermediates, such as crude (-)-ambrox; • Greener and more environmentally friendly processes;

[0025] The following benefits can be achieved with a simpler, cheaper and less labor-intensive process: A reduction in the amount of equipment required to purify crude flavor or fragrance or cosmetic ingredients or intermediates, such as crude (-)-ambrox; • Avoids the need for recrystallization; ● Avoid or strongly reduce the use of ethanol; ●Reducing material loss • Avoid the production of flavor or fragrance or cosmetic ingredients or intermediates, such as crude (-)-ambrox, containing concentrates of mother liquor that must be reprocessed; • A high productivity process (e.g., >95 mol % based on crude starting material) that uses fewer steps and requires less time to purify (-)-ambrox; • Production of (-)-ambrox suitable for olfactory applications;

[0026] The simpler, cheaper, and less labor-intensive process described herein involves: • Ability to remove or reduce insoluble and / or particulate material; ●Can remove or reduce colored impurities; • The co-products of bioconversion reactions can be removed or reduced; ●Can remove or reduce odorous impurities

[0027] The details, examples, and preferences provided with respect to any one or more specific aspects of the invention as further described herein will apply equally to all aspects of the invention. Any combination of the embodiments, examples, and preferences described herein in all possible variations thereof is covered by the present invention unless otherwise indicated herein or clearly contradicted by context. [Brief explanation of the drawings]

[0028] Brief description of the figure [Figure 1] FIG. 1 shows an XRD pattern where the abscissa scales in degrees 2-theta and the ordinate is the intensity in counts. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description Provided herein is a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox, the method comprising: Washing with aqueous acid; Washing with an aqueous surfactant; and Optionally, washing with aqueous alkali Includes.

[0030] The washing steps of the first aspect of the invention may occur in any order suitable for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox. Each washing step refers to washing the crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox, or the product of a previous washing step, which may also be referred to as a crude flavor or fragrance or cosmetic ingredient or intermediate, such as crude (-)-ambrox.

[0031] For example, provided herein is a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: (a) Washing crude flavor or fragrance or cosmetic ingredients or intermediates with aqueous acid; (b) optionally washing the product of step (a) with aqueous alkali; and (c) washing the product of step (a) or the product of step (b), if present, with an aqueous detergent. Includes.

[0032] For example, provided herein is a method for purifying crude (-)-ambrox, the method comprising: (a) washing crude (-)-ambrox with aqueous acid; (b) optionally washing the product of step (a) with aqueous alkali; and (c) washing the product of step (a) or the product of step (b), if present, with an aqueous detergent. Includes.

[0033] For example, provided herein is a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: (a) Washing crude flavor or fragrance or cosmetic ingredients or intermediates with aqueous acid; (b) washing the product of step (a) with aqueous alkali; and (c) washing the product of step (b) with an aqueous detergent; Includes.

[0034] For example, provided herein is a method for purifying crude (-)-ambrox, the method comprising: (a) washing crude (-)-ambrox with aqueous acid; (b) washing the product of step (a) with aqueous alkali; and (c) washing the product of step (b) with an aqueous detergent; Includes.

[0035] For example, provided herein is a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: (a) Washing crude flavor or fragrance or cosmetic ingredients or intermediates with aqueous surfactants; (b) washing the product of step (a) with aqueous acid; and (c) optionally washing the product of step (b) with aqueous alkali. Includes.

[0036] For example, provided herein is a method for purifying crude (-)-ambrox, the method comprising: (a) washing crude (-)-ambrox with an aqueous surfactant; (b) washing the product of step (a) with aqueous acid; and (c) optionally washing the product of step (b) with aqueous alkali. Includes.

[0037] For example, provided herein is a method for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the method comprising: (a) washing a crude flavor or fragrance or cosmetic ingredient or intermediate with a composition comprising an aqueous acid and an aqueous surfactant; and (b) optionally washing the product of step (a) with aqueous alkali. Includes.

[0038] For example, provided herein is a method for purifying crude (-)-ambrox, the method comprising: (a) washing crude (-)-ambrox with a composition comprising an aqueous acid and an aqueous surfactant; and (b) optionally washing the product of step (a) with aqueous alkali. Includes.

[0039] Each of steps (a), (b), and / or (c) described herein may be preceded or followed by one or more additional method steps, e.g., one or more additional washing steps. For example, the method may include one or more water washes before or after each of steps (a), (b), and / or (c). When there are one or more additional method steps before or after each of steps (a), (b), and / or (c), the "product of step (a)" refers to the product of step (a) and any additional method steps performed prior to performing step (b), and the "product of step (c)" refers to the product of step (b) and any additional method steps performed prior to performing step (c).

[0040] Crude flavor or fragrance or cosmetic ingredients or intermediate starting materials "Crude flavor or fragrance or cosmetic ingredient or intermediate" means a fragrance ingredient or fragrance ingredient intermediate, or a flavor ingredient or flavor ingredient intermediate, or a cosmetic ingredient or cosmetic ingredient intermediate, and materials containing impurities.

[0041] As used herein, the term "perfume ingredient" refers to a compound that can be used in a composition to provide a desired odor.

[0042] As used herein, the term "flavor ingredient" refers to a compound that can be used in a composition to provide a desired taste sensation.

[0043] As used herein, the term "cosmetic ingredient" refers to a compound that can be used as an active ingredient in a cosmetic composition to provide a desired appearance when applied to a subject (e.g., a human subject), specifically to the subject's skin. This can include, for example, natural products, which can be crystalline.

[0044] As used herein, the term "perfume intermediate" refers to a compound that can be used to prepare a perfume ingredient.

[0045] As used herein, the term "flavor intermediate" refers to a compound that can be used to prepare a flavor ingredient.

[0046] As used herein, the term "cosmetic intermediate" refers to a compound that can be used to prepare a cosmetic ingredient.

[0047] As used herein, the term "impurities" refers to any material, solid or liquid, that is not a flavor, fragrance, or cosmetic ingredient or intermediate itself. This includes, for example, unreacted starting materials for the production of a flavor, fragrance, or cosmetic ingredient or intermediate, by-products of reactions that produce a flavor, fragrance, or cosmetic ingredient or intermediate, particulate material insoluble in solvents (such as water, water-miscible solvents, and organic solvents), cells, cell debris, and various impurities, such as impurities insoluble in ethanol (such as precipitated inorganic salts and proteins).

[0048] Thus, "purifying a flavor or fragrance or cosmetic ingredient or intermediate" refers to a process for removing and reducing the amount of impurities in a crude flavor or fragrance or cosmetic ingredient or intermediate.

[0049] The crude flavor or fragrance or cosmetic ingredient or intermediate may, for example, be in the form of a solid.

[0050] The crude flavor or fragrance or cosmetic ingredient or intermediate may, for example, be in crystalline form.

[0051] The crude flavor or fragrance or cosmetic ingredient or intermediate may be prepared by a bioconversion process, for example, using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) enzyme. Thus, the purification methods described herein may further include a step of preparing the crude flavor or fragrance or cosmetic ingredient or intermediate by a bioconversion process prior to the purification step described herein. The bioconversion broth resulting from a bioconversion process (e.g., as described herein) may generally include a solid phase containing the flavor or fragrance or cosmetic ingredient or intermediate and a liquid phase(s) containing water and / or an oily phase, which may contain, for example, residual unreacted starting materials and other oily or oil-soluble impurities or by-products.

[0052] The term "crude flavor or fragrance or cosmetic ingredient or intermediate" may refer to a material that is separated from the bioconversion broth in a preliminary purification step (e.g., by filtration) prior to the purification methods described herein.

[0053] The presently described purification methods may work particularly well on products where the impurities are in the form of solids deposited on the product surface, which may mean that they can be easily removed by the washing steps described herein.

[0054] The reaction conditions, and any preliminary purification steps, for making the crude flavor or fragrance or cosmetic ingredient or intermediate may follow, for example, the methods and preliminary purification steps described herein for the crude (-)-ambrox starting material (but using the correct starting material).

[0055] The crude flavor or fragrance or cosmetic ingredient or intermediate may be selected from, for example, (-)-ambrox, (-)-ambra-oxide, 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan, and (+)-amberketal. In particular, the crude flavor or fragrance ingredient or intermediate may be (-)-ambrox.

[0056] Crude (-)-Ambrox starting material "Crude (-)-ambrox" refers to a material containing (-)-ambrox and impurities. As used herein, the term "impurities" refers to any material, solid or liquid, that is not (-)-ambrox. This includes, for example, unreacted starting materials for the production of (-)-ambrox (e.g., homofarnesol), by-products of the reaction to produce (-)-ambrox (e.g., compounds represented by formula (II), (III), and / or (IV) as set forth in Table 2 herein), particulate material insoluble in solvents (e.g., water, water-miscible solvents, and organic solvents), cells, cell debris, and various impurities, such as impurities insoluble in ethanol (e.g., precipitated inorganic salts and proteins).

[0057] Therefore, "purifying (-)-ambrox" refers to a method for removing and reducing the amount of impurities in crude (-)-ambrox.

[0058] Crude (-)-ambrox may be prepared by a bioconversion process using the enzyme squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) to convert homofarnesol to (-)-ambrox (see further details below). The bioconversion broth resulting from a bioconversion process (e.g., as described herein) may generally include a solid phase containing (-)-ambrox and a liquid phase(s) containing water and / or an oily phase, which may contain residual unreacted homofarnesol and other oily or oil-soluble impurities or by-products. For example, some compounds represented by formulas (II), (III), and (IV) may be present in such an oily phase.

[0059] The term "crude (-)-ambrox" may refer to the material that is separated from the bioconversion broth in a preliminary purification step (e.g., by filtration) prior to the purification methods described herein.

[0060] The solid phase (including (-)-ambrox) obtained from the bioconversion process may be separated from the liquid phase of the bioconversion broth by filtration (such as centrifugal filtration) or decantation / sedimentation. By selecting a filter with an appropriate mesh size, it is also feasible to separate the solid form of (-)-ambrox from particulate material (e.g., cells or cell debris) present in the bioconversion broth. Decantation / sedimentation, like filtration, exploits the particle size and / or particle mesh differences between the particulate matter and the solid form of (-)-ambrox, allowing for their separation; the former remains suspended in the supernatant and can be discarded, while the latter can be isolated and recovered as a sediment for further purification steps. Suitable methods for isolating (-)-ambrox from the bioconversion broth containing cell debris are described in WO 2017 / 182542 and WO 2016 / 170106, the contents of which are incorporated herein by reference. For example, the biocatalyst may first be inactivated by heating or by raising the pH of the bioconversion broth to about 10-11 (alkalinization). Cells, cell debris, insoluble material, and / or other particulate material may then be removed from the (-)-ambrox by physical means, such as filtration, which may have a deodorizing effect, since cells and / or cell debris and / or insoluble material and / or other particulate material attached to the (-)-ambrox may cause unpleasant off-notes if they remain in the crude (-)-ambrox product.

[0061] The biocatalyst may be inactivated, for example, by raising the temperature of the bioconversion broth. The temperature may be raised to a temperature high enough to inactivate the bacteria but below the melting point of (-)-ambrox (i.e., below about 75-80°C). Thus, (-)-ambrox does not turn into a liquid. Rather, at this temperature (e.g., from about 50°C to about 60°C, such as about 55°C), (-)-ambrox becomes slightly soluble, reducing the average crystal size. The crystals then grow again during the cooling phase, which facilitates their separation from the liquid phase of the bioconversion broth by filtration.

[0062] For example, the temperature of the bioconversion broth may be increased to a temperature ranging from about 50° C. to about 60° C. For example, the temperature of the bioconversion broth may be increased to a temperature ranging from about 52° C. to about 58° C., or from about 54° C. to about 56° C. For example, the temperature of the bioconversion broth may be increased to a temperature of about 55° C.

[0063] Alternatively, the biocatalyst may be inactivated by raising the pH of the bioconversion broth to about 10-11, e.g., using sodium hydroxide. This may reduce energy consumption and time loss compared to, for example, inactivating the biocatalyst by heating, reduce flocculation (which may interfere with filtration), reduce odor issues, and / or leave behind primarily inorganic impurities that are insoluble in ethanol (which may be removed by filtration).

[0064] Thus, crude (-)-ambrox that undergoes the purification methods described herein has undergone biocatalytic inactivation and may be substantially or completely free of intact cells.

[0065] After the (-)-ambrox has been separated from the particulate material of the bioconversion broth (e.g., by filtration), any aqueous material present in the crude (-)-ambrox may be residue from the broth in which the cells were originally cultured. The crude (-)-ambrox may undergo an additional pre-purification wash step using, for example, water prior to the purification methods described herein. After the additional pre-purification wash step with water, any aqueous material present in the crude (-)-ambrox may be water.

[0066] Crude (-)-ambrox may contain, for example, less than or equal to about 99 wt% (-)-ambrox, based on the dry content of the crude (-)-ambrox. For example, crude (-)-ambrox may contain less than or equal to about 98 wt%, or less than or equal to about 97 wt%, or less than or equal to about 96 wt%, or less than or equal to about 95 wt% (-)-ambrox, based on the dry content of the crude (-)-ambrox.

[0067] The crude (-)-ambrox may contain, for example, greater than or equal to about 80 wt% (-)-ambrox, based on the dry content of the crude (-)-ambrox, such as greater than or equal to about 81 wt%, greater than or equal to about 82 wt%, greater than or equal to about 83 wt%, greater than or equal to about 84 wt%, greater than or equal to about 85 wt%, greater than or equal to about 86 wt%, or greater than or equal to about 87 wt%, based on the dry content of the crude (-)-ambrox. may contain greater than or equal to about 88 wt%, or greater than or equal to about 89 wt%, or greater than or equal to about 90 wt%, or greater than or equal to about 91 wt%, or greater than or equal to about 92 wt%, or greater than or equal to about 93 wt%, or greater than or equal to about 94 wt%, or greater than or equal to about 95 wt% (-)-ambrox.

[0068] For example, crude (-)-ambrox may contain from about 80 wt% to about 99 wt%, or from about 85 wt% to about 97 wt%, or from about 90 wt% to about 95 wt%, or from about 93 wt% to about 97 wt% (-)-ambrox, based on the dry content of the crude (-)-ambrox.

[0069] Crude (-)-ambrox may contain, for example, total impurities equal to or greater than about 1 wt.% based on the dry content of crude (-)-ambrox. For example, crude (-)-ambrox may contain total impurities equal to or greater than about 2 wt.%, or equal to or greater than about 3 wt.%, or equal to or greater than about 4 wt.%, or equal to or greater than about 5 wt.% based on the dry content of crude (-)-ambrox.

[0070] Crude (-)-ambrox may, for example, contain total impurities equal to or less than about 20 wt. % based on the dry content of crude (-)-ambrox. For example, crude (-)-ambrox may contain total impurities of less than or equal to about 19 wt%, or less than or equal to about 18 wt%, or less than or equal to about 17 wt%, or less than or equal to about 16 wt%, or less than or equal to about 15 wt%, or less than or equal to about 14 wt%, or less than or equal to about 13 wt%, or less than or equal to about 12 wt%, or less than or equal to about 11 wt%, or less than or equal to about 10 wt%, or less than or equal to about 9 wt%, or less than or equal to about 8 wt%, or less than or equal to about 7 wt%, or less than or equal to about 6 wt%, based on the dry content of the crude (-)-ambrox.

[0071] For example, crude (-)-ambrox may contain from about 1 wt % to about 20 wt %, or from about 2 wt % to about 15 wt %, or from about 3 wt % to about 10 wt % total impurities based on the dry content of crude (-)-ambrox.

[0072] The dry content of crude (-)-ambrox is the material remaining after the water content has been subtracted. For example, a water content of 25% as determined by Karl Fischer titration is equivalent to a dry content of 75%. The dry content is solid, although a small amount of oily residue may be present. The oily residue may be, for example, one or more compounds represented by formula (II), (III), and / or (IV) as set forth in Table 2 herein, or any unreacted homofarnesol.

[0073] The crude (-)-ambrox may, for example, contain water. Alternatively, the crude (-)-ambrox may be dry (i.e., free of any water). Removing water from the crude (-)-ambrox may be advantageous to avoid bacterial growth and off-note production in the crude material.

[0074] Crude (-)-ambrox may contain, for example, about 2 wt% or more of water based on the wet weight of the crude total (-)-ambrox. For example, crude (-)-ambrox may contain about 5 wt% or more, about 10 wt% or more, about 15 wt% or more, or about 20 wt% or more of water based on the wet weight of the crude total (-)-ambrox.

[0075] Crude (-)-ambrox may contain, for example, less than or equal to about 50 wt% water, based on the wet weight of the crude total (-)-ambrox. For example, crude (-)-ambrox may contain less than or equal to about 45 wt%, or less than or equal to about 40 wt%, or less than or equal to about 35 wt%, or less than or equal to about 30 wt%, or less than or equal to about 25 wt%, or less than or equal to about 20 wt% water, based on the wet weight of the crude total (-)-ambrox.

[0076] For example, crude (-)-ambrox may contain from about 2 wt% to about 50 wt%, or from about 10 wt% to about 25 wt%, or from about 20 wt% to about 25 wt% water based on the wet weight of the total crude (-)-ambrox.

[0077] The crude (-)-ambrox may, for example, comprise a total dry content equal to or greater than about 50 wt.% based on the wet weight of the crude total (-)-ambrox. For example, the crude (-)-ambrox may comprise a total dry content equal to or greater than about 55 wt.%, or equal to or greater than about 60 wt.%, or equal to or greater than about 65 wt.%, or equal to or greater than about 70 wt.% based on the wet weight of the crude total (-)-ambrox.

[0078] Crude (-)-ambrox may, for example, comprise a total dry content of less than or equal to about 100 wt%, or less than or equal to about 99 wt%, or less than or equal to about 98 wt%, or less than or equal to about 95 wt%, or less than or equal to about 90 wt%, or less than or equal to about 85 wt%, or less than or equal to about 80 wt%, or less than or equal to about 75 wt%, based on the wet weight of the crude total (-)-ambrox.

[0079] For example, crude (-)-ambrox may comprise a total dry content of from about 50 wt% to about 100 wt%, or from about 60 wt% to about 98 wt%, or from about 70 wt% to about 95 wt%, or from about 75 wt% to about 90 wt%, based on the wet weight of crude total (-)-ambrox.

[0080] For example, crude (-)-ambrox may contain about 70% to about 75% by weight dry material and about 20% to about 25% by weight water. The dry residue in crude (-)-ambrox may contain about 5% to about 10% by weight impurities.

[0081] The methods described herein are particularly suitable for purifying crude (-)-ambrox produced by a bioconversion process using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) enzyme, which converts E,E-homofarnesol to (-)-ambrox. The term "SHC / HAC enzyme" encompasses both wild-type SHC / HAC enzymes and variants of wild-type SHC / HAC enzymes. Suitable methods for preparing (-)-ambrox by a biocatalytic process using an SHC enzyme are described, for example, in WO 2016 / 170106, WO 2016 / 170099, WO 2017 / 182542, WO 2018 / 157021, and Eichhorn et al. (2018), Adv. Synth. Catal., 360:2339-2351, the contents of which are incorporated herein by reference. Any suitable bioconversion method may be used to produce crude (-)-ambrox. Suitable SHC / HAC enzymes include, but are not limited to, those disclosed in Tables 13 and 14 of WO 2016 / 170099, such as SHC / HAC enzyme 215G2 SHC.

[0082] Crude (-)-ambrox may be made by a process comprising culturing a host cell to produce an SHC / HAC enzyme and contacting the host cell and / or the SHC / HAC enzyme produced by the host cell with 3E,7E-homofarnesol under conditions suitable to promote conversion of 3E,7E-homofarnesol to (-)-ambrox.

[0083] The host cells may be cultured to produce sufficient biocatalyst, harvested, washed (and optionally stored (e.g., refrigerated, frozen, or lyophilized)) before initiating the bioconversion step.

[0084] In some embodiments, (-)-ambrox is in a solid form, as described in WO 2016 / 170106, WO 2016 / 170099, and / or WO 2017 / 182542, the contents of which are incorporated herein by reference. Due to its extremely low solubility in water, (-)-ambrox crystallizes in the broth during the bioconversion process, which occurs as soon as the E,E-homofarnesol conversion rate exceeds approximately 50%.

[0085] In some embodiments, following the bioconversion reaction (i.e., after the initial crystallization during the bioconversion process), no recrystallization step (i.e., a step involving solubilizing (-)-ambrox crystals and then recrystallizing (-)-ambrox) is required to purify the crude (-)-ambrox.

[0086] Homofarnesol may have the isomerism shown below. Table 1. Homofarnesol isomers [Table 1]

[0087] Although homofarnesol can exist as a mixture of four isomers, the (3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E) isomers, Figure 12 of WO 2016 / 170099 shows the reaction products ((-)-ambrox and compound (IV)) produced when EEH is used as the starting material (for biotransformation with wild-type SHC / HAC and / or SHC / HAC variants) and the reaction products ((-)-ambrox and compounds (II), (III), and (IV) (see Table 2)) produced when EE:EZ is used as the starting material. For easy reference, compounds (I) through (IV) are identified in Table 2 below. Scheme 2 of Eichhorn et al. (2018), Adv. Synth. Catal., 360:2339-2351 also demonstrates that the (E,Z) isomer (corresponding to the 3Z,7E homofarnesol isomer) produces only compounds (II) and (III), but not (-)-ambrox.

[0088] As used herein, a reference to (3E,7E)-homofarnesol is a reference to E,E-homofarnesol, which is also designated EEH.

[0089] The starting material for the processes described herein for preparing (-)-ambrox may be, for example, (3E,7E)-homofarnesol or a mixture containing (3E,7E)-homofarnesol, e.g., a mixture of homofarnesol isomers containing (3E,7E)-homofarnesol.

[0090] Preferably, the homofarnesol starting material comprises a mixture of (3E,7E) and (3Z,7E), referred to herein as the EE:EZ isomeric mixture. The EE:EZ isomeric mixture of homofarnesol has a CAS number of 35826-67-6. [ka]

[0091] For example, the weight ratio of EEH:EZH is approximately 100:00; 99:01; 98:02; 97:03; 96:04; 95:05; 94:06; 93:07; 92:08; 91:09; 90:10; 89:11; 88:12; 87:13; 86:14; 85:15; 84:16; 83:17; 82:18; 81:19; 80:20; 79:21; 78:22; 77:23; 76:24; 75:25; 74:26; 73:27; 72:28; 71:29; 70:30; 69:31 The homofarnesol starting material may have an EE:EZ weight ratio of 86:14. For example, the homofarnesol starting material may have an EE:EZ weight ratio of 80:20.

[0092] The number of homofarnesol isomers present can affect the reaction speed. SHC / HAC enzymes can sometimes convert E,E-homofarnesol to (-)-ambrox from a complex mixture of homofarnesol isomers (e.g., EE:EZ:ZE:ZZ). However, lower conversion rates can also be observed, which is consistent with the notion that homofarnesol isomers other than EEH may compete with EEH for access to the SHC / HAC enzyme active site and thus act as competitive inhibitors for the conversion of EEH to (-)-ambrox and / or as alternative substrates (see, e.g., Eichhorn et al. (2018) Adv. Synth. Catal. 360:2339-2351, the contents of which are incorporated herein by reference). Consequently, the homofarnesol substrate may contain an isomeric mixture of 2 to 4 isomers, preferably 2 isomers.

[0093] Not all of the homofarnesol (e.g., EEH) starting material may be converted to (-)-ambrox or a by-product of the reaction. Any unreacted homofarnesol may be removed, for example, by filtration, prior to purification of the crude (-)-ambrox. However, the crude (-)-ambrox may contain small amounts of homofarnesol (e.g., EEH).

[0094] For example, crude (-)-ambrox may contain equal to or greater than about 0.05 wt% homofarnesol, or equal to or greater than about 0.1 wt%, or equal to or greater than about 0.5 wt%, or equal to or greater than about 1 wt%, or equal to or greater than about 2 wt% homofarnesol, based on the dry content of crude (-)-ambrox.

[0095] For example, crude (-)-ambrox may contain less than or equal to about 5 wt% homofarnesol based on the dry content of the crude (-)-ambrox, e.g., crude (-)-ambrox may contain less than or equal to about 4 wt%, or less than or equal to about 3 wt% homofarnesol based on the dry content of the crude (-)-ambrox.

[0096] For example, crude (-)-ambrox may contain from about 0.05 wt% to about 5 wt%, or from about 0.1 wt% to about 4 wt%, or from about 0.5 wt% to about 2 wt% homofarnesol, based on the dry content of the crude (-)-ambrox.

[0097] The bioconversion methods described herein may produce (-)-ambrox of formula (I), along with one or more by-products, such as compounds of formulas (II), (III), and (IV) shown below: Table 2. Nomenclature for compounds of formulas (I), (II), (III), and (IV) [Table 2]

[0098] Thus, crude (-)-ambrox may contain one or more compounds of formula (II), (III), and / or (IV).

[0099] For example, crude (-)-ambrox may contain about 1 wt. % or more of the total compounds represented by formula (II), (III), and / or (IV), based on the dry content of the crude (-)-ambrox. For example, crude (-)-ambrox may contain about 2 wt. % or more, about 3 wt. % or more, about 4 wt. % or more, about 5 wt. % or more, about 6 wt. % or more, about 7 wt. % or more, or about 8 wt. % or more of the total compounds represented by formula (II), (III), and / or (IV), based on the dry content of the crude (-)-ambrox.

[0100] For example, crude (-)-ambrox may contain less than or equal to about 15 wt.% of the total compounds represented by formula (II), (III), and / or (IV), based on the dry content of the crude (-)-ambrox. For example, crude (-)-ambrox may contain less than or equal to about 14 wt.%, or less than or equal to about 13 wt.%, or less than or equal to about 12 wt.%, or less than or equal to about 11 wt.%, or less than or equal to about 10 wt.% of the total compounds represented by formula (II), (III), and / or (IV), based on the dry content of the crude (-)-ambrox.

[0101] For example, crude (-)-ambrox may contain from about 1 wt % to about 15 wt %, or from about 3 wt % to about 12 wt %, or from about 5 wt % to about 10 wt % of the total compounds represented by formula (II), (III), and / or (IV), based on the dry content of the crude (-)-ambrox.

[0102] On average, crude (-)-ambrox may contain, for example, about 73.5 wt. % (-)-ambrox, 2.5 wt. % (co-products - compounds (II), (III), and (IV) plus the homofarnesol starting material), 3 wt. % extraneous alcohol-insoluble material (not detected by GC analysis), and 21 wt. % water.

[0103] Crude (-)-ambrox typically contains 1.5-2.0 wt% of the co-product (III) (9-epi-ambrox) and 0.25-0.5% of each of the co-products (II) and (IV).

[0104] Crude (-)-Ambra-oxide starting material As used herein, the term "(-)-Ambra-oxide" refers to a compound represented by formula (X) below. (-)-Ambra-oxide may be produced, for example, by combining with one or more by-products represented by formulas (XI), (XII), or (XIII) below. [Table 3]

[0105] (-)-Ambra-oxide can be produced from (+)-Larixol as described in Bolster et al., Tetrahedron, 2002, 58(26), pages 5275-5285.

[0106] (-)-Ambra-oxide can also be produced by enzymatically converting E,E-bishomofarnesol (bisEEH) or a mixture of isomers of bishomofarnesol containing bisEEH to (-)-ambra-oxide or a mixture containing (-)-ambra-oxide using an SHC / HAC enzyme (which may be a wild-type SHC / HAC enzyme or a variant of a wild-type SHC / HAC enzyme).

[0107] "Crude (-)-ambra-oxide" refers to a material containing (-)-ambra-oxide and impurities. As used herein, the term "impurities" refers to any material, solid or liquid, that is not (-)-ambra-oxide. This includes, for example, unreacted starting materials for the production of (-)-ambra-oxide (e.g., bishomofarnesol), by-products of the reaction to produce (-)-ambra-oxide (e.g., compounds represented by formulas (XI), (XII), and / or (XIII)), particulate material insoluble in solvents (e.g., water, water-miscible solvents, and organic solvents), cells, cell debris, and various impurities, such as impurities insoluble in ethanol (e.g., precipitated inorganic salts and proteins).

[0108] Therefore, "purifying (-)-ambra-oxide" refers to a method for removing and reducing the amount of impurities in crude (-)-ambra-oxide.

[0109] Crude (-)-ambra-oxide may be prepared by a bioconversion process using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) enzyme (including wild-type and variants of the wild-type enzyme) to convert E,E-bishomofarnesol to (-)-ambra-oxide. The bioconversion broth resulting from a bioconversion process (e.g., as described herein) may generally include a solid phase containing (-)-ambra-oxide and a liquid phase(s) containing water and / or an oily phase, which may contain residual unreacted starting materials and other oily or oil-soluble impurities or by-products. The bioconversion process for the preparation of crude (-)-ambra-oxide may be carried out, for example, using the same methods as described above for (-)-ambrox.

[0110] The term "crude (-)-ambrox-oxide" may refer to the material separated from the bioconversion broth in a preliminary purification step (e.g., by filtration) prior to the purification methods described herein. Separation may be carried out, for example, using the same methods described for (-)-ambrox.

[0111] Starting material for crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan 3a-Ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan (the ethyl analog of ambrox, which has a melting point of approximately 61° C., approximately 10° C. lower than that of ambrox) can be synthesized by the method of formula (XX): [ka] It has the relative configuration of the structure shown in

[0112] The compound of formula (XX) contains a number of chiral carbon atoms, and therefore one or more stereoisomers (including enantiomers and diastereomers) of the compound of formula (XX) may also exist. The compound of formula (XX) may, for example, be prepared in combination with one or more stereoisomers of the compound of formula (XX).

[0113] 3a-Ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan can be synthesized by the reaction of SHC / HAC enzyme (which may be a wild-type SHC / HAC enzyme or a variant of the wild-type SHC / HAC enzyme) with the aid of the following formula (XXII): [ka] can be produced by enzymatically converting ethyl homofarnesol or a mixture of ethyl homofarnesol isomers represented by the formula:

[0114] "Crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan" means a material containing 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and impurities. As used herein, the term "impurities" refers to any material, solid or liquid, that is not 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan. This includes, for example, unreacted starting materials for the production of 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan (e.g., ethyl homofarnesol), by-products of the reaction to produce 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan (e.g., the compound represented by formula (XXI)), particulate material insoluble in solvents (such as water, water-miscible solvents, and organic solvents), cells, cell debris, and various impurities, such as impurities insoluble in ethanol (such as precipitated inorganic salts and proteins).

[0115] Therefore, "purifying 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan" refers to a method for removing and reducing the amount of impurities in crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan.

[0116] Crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan may be prepared by a bioconversion process using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) enzyme (including wild-type enzymes and variants of the wild-type enzyme) to convert ethyl homofarnesol to 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan. The bioconversion broth resulting from a bioconversion process (e.g., as described herein) may generally include a solid phase containing 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan and a liquid phase(s) containing water and / or an oily phase, which may contain residual unreacted starting materials and other oily or oil-soluble impurities or by-products. The bioconversion process for the preparation of crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan may be carried out, for example, using the same method as described above for (-)-ambrox.

[0117] The term "crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan" may refer to material separated from the bioconversion broth in a preliminary purification step (e.g., by filtration) prior to the purification methods described herein. Separation may be carried out, for example, using the same methods described for (-)-ambrox.

[0118] Crude (+)-Amberketal Starting Material (+)-Amberketal is represented by the formula (XXX) below. [ka] wherein R is methyl.

[0119] The compound of formula (XXX) contains a number of chiral carbon atoms, and therefore one or more stereoisomers (including enantiomers and diastereomers) of the compound of formula (XXX) may also exist. The compound of formula (XXX) may, for example, be prepared in combination with one or more stereoisomers of the compound of formula (XXX).

[0120] (+)-Amberketal can be synthesized by the reaction of SHC / HAC enzyme (which may be a wild-type SHC / HAC enzyme or a variant of the wild-type SHC / HAC enzyme) with the following formula (XXXI): [ka] hydroxyfarnesyl acetone or a mixture of hydroxyfarnesyl acetone isomers represented by the formula:

[0121] Compounds of formula XXX also have the formula (XXXIa): [ka] wherein R is H, methyl, or ethyl. The compound of formula (I) may also be produced by a method comprising contacting the compound with a squalene-hopene cyclase (SHC) enzyme or enzyme variant.

[0122] "Crude (+)-amberketal" refers to a material containing (+)-amberketal and impurities. As used herein, the term "impurities" refers to any material, solid or liquid, that is not (+)-amberketal. This includes, for example, unreacted starting materials for the production of (+)-amberketal (e.g., hydroxyfarnesylacetone), by-products of the reaction producing (+)-amberketal, particulate material insoluble in solvents (such as water, water-miscible solvents, and organic solvents), cells, cell debris, and various impurities, such as impurities insoluble in ethanol (such as precipitated inorganic salts and proteins).

[0123] Therefore, "purifying (+)-amberketal" refers to a method for removing impurities in crude (+)-amberketal and reducing the amount of impurities.

[0124] Crude (+)-amberketal may be prepared by a bioconversion process using a squalenehopene cyclase / homofarnesol ambrox cyclase (SHC / HAC) enzyme (including wild-type and variants of the wild-type enzyme) to convert hydroxyfarnesyl acetone to (+)-amberketal. The bioconversion broth resulting from a bioconversion process (e.g., as described herein) may generally include a solid phase containing (+)-amberketal and a liquid phase(s) containing water and / or an oily phase, which may contain residual unreacted starting materials and other oily or oil-soluble impurities or by-products. The bioconversion process for the preparation of crude (+)-amberketal may be carried out, for example, using the same methods as those described above for (-)-amberx.

[0125] The term "crude (+)-amberketal" may refer to material that is separated from the bioconversion broth in a preliminary purification step (e.g., by filtration) prior to the purification methods described herein. Separation may be carried out, for example, using the same methods described for (-)-ambrox.

[0126] To wash The methods provided herein include multiple washing steps. By "washing" is meant that a solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox, is contacted with a liquid to remove one or more impurities. For example, in step (a), the liquid may be an aqueous acid. For example, in step (b), the liquid may be an aqueous alkali. For example, in step (c), the liquid may be an aqueous surfactant.

[0127] Contacting the solid material with a liquid can dissolve one or more impurities in the liquid so that they can be separated from the solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as the solid material containing (-)-ambrox. In particular, contacting the solid material with an aqueous acid can dissolve one or more impurities in the aqueous acid so that they can be separated from the solid material containing (-)-ambrox.

[0128] Contacting the solid material with an aqueous surfactant may cause one or more impurities to become suspended in the liquid so that they can be separated from the solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox. Contacting the solid material with aqueous alkali may, for example, neutralize any residual acid present from the aqueous acid wash step.

[0129] Washing may occur by dispensing the liquid over the surface of the solid material containing the flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox. The liquid may then be passed through the solid material containing the flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox, and removed, for example, naturally or using pressure or vacuum.

[0130] Washing may include, for example, mixing the liquid with a solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox. This may help maximize contact between the liquid and the solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox. The liquid may then be separated from the solid material containing a flavor or fragrance or cosmetic ingredient or intermediate, such as a solid material containing (-)-ambrox, by filtration.

[0131] In particular, any filter cake resulting from each washing step may be resuspended in the subsequent washing step to improve the efficiency of the subsequent washing step. All washing steps may occur, for example, in a single piece of equipment.

[0132] The methods described herein may further include, for example, drying the purified flavor or fragrance or cosmetic ingredient or intermediate after the washing step, for example, drying the purified (-)-ambrox after the washing step. The drying step may occur, for example, in the same equipment as the washing step.

[0133] Washing and / or drying may occur, for example, in a Nutsche-type agitator-drying filter. This is a single piece of equipment that can perform both filtering and drying functions and includes impellers (e.g., blades) that act to agitate the material within the vessel. The impellers can perform several operations through motions parallel and / or perpendicular to the shaft. For example, the impellers can continue to fluidize the slurry contents until most of the mother liquor has been filtered, help maintain the cake uniform and free of cracks, help reslurry the filter cake, and / or help move the filter cake toward the discharge outlet. A typical unit consists of a concave vessel with a perforated plate. The entire vessel can be maintained at the desired temperature using a limpet jacket, a coated bottom plate, and an agitator (blade and shaft) through which a heat transfer medium can flow. The vessel can be made completely leak-proof against the application of vacuum or pressure. The drying function of the Nutsche type agitator-drying filter may be performed by applying a vacuum, which removes the waste liquid and makes the cake ready for any subsequent washing steps.

[0134] The use of Nutsche-type agitated-drying filters may offer advantages over other filters, such as belt filters. In particular, the use of Nutsche-type agitated-drying filters may improve the washing and resuspension of solid materials. For example, the use of Nutsche-type agitated-drying filters may reduce the amount of residual water and / or other liquid materials (and impurities present in the residual water and / or other liquid materials) in the washed product compared to other types of filters (e.g., belt filters).

[0135] Each washing step (e.g., aqueous acid wash, any aqueous alkali wash, aqueous surfactant wash, and / or any optional water wash step) may use a weight ratio of washing liquid (e.g., aqueous acid, aqueous alkali, aqueous surfactant, water) to dry crude material (e.g., dried crude (-)-ambrox) of at least about 1:1. For example, each washing step may use a weight ratio of washing liquid to dry crude material (e.g., dried crude (-)-ambrox) of at least about 2:1 or at least about 3:1. The dried crude material (e.g., dried crude (-)-ambrox) may be the product of any previous washing steps that have already been performed.

[0136] Each washing step (e.g., aqueous acid wash, any aqueous alkali wash, aqueous detergent wash, and / or any optional water wash step) may use a weight ratio of washing liquid (e.g., aqueous acid, aqueous alkali, aqueous detergent, water) to dry crude material (e.g., dried crude (-)-ambrox) equal to or less than about 10:1. For example, each washing step may use a weight ratio of washing liquid to dry crude material (e.g., dried crude (-)-ambrox) equal to or less than about 8:1, or at least about 6:1, or equal to or less than about 5:1. The dried crude material (e.g., dried crude (-)-ambrox) may be the product of any previous washing steps that have already been performed.

[0137] For example, each washing step (e.g., aqueous acid wash, optional aqueous alkali wash, aqueous surfactant wash, and / or any optional water wash step) may use a weight ratio of washing liquid (e.g., aqueous acid, aqueous alkali, aqueous surfactant, water) to dried crude material (e.g., dried crude (-)-ambrox) ranging from about 1:1 to about 10:1, or from about 2:1 to about 6:1, or from about 3:1 to about 5:1. The dried crude material (e.g., dried crude (-)-ambrox) may be the product of any previous washing steps that have already been performed.

[0138] The temperature of the washing liquid used in each washing step may be, for example, equal to or higher than about 5°C. The temperature of the washing liquid used in each washing step may be, for example, above room temperature (i.e., above about 22°C). For example, the temperature of the aqueous surfactant may be above room temperature. This may help, for example, to improve the efficiency of the washing step. For example, the temperature of the washing liquid used in each washing step may be equal to or higher than about 30°C, or equal to or higher than about 35°C, or equal to or higher than about 40°C. The temperature of the washing liquid in each washing step may be, for example, below the melting point of the flavor or fragrance or cosmetic ingredient or intermediate, such as (-)-ambrox (approximately 72°C to 75°C). For example, the temperature of the washing liquid in each washing step may be equal to or lower than about 75°C, or equal to or lower than about 70°C, or equal to or lower than about 65°C, or equal to or lower than about 60°C.

[0139] Aqueous Acid Wash The acid may be, for example, any suitable acid. In particular, the acid may be any acid suitable for dissolving one or more impurities and thus separating the impurities from a solid flavor or fragrance or cosmetic ingredient or intermediate (e.g., solid (-)-ambrox). For example, the acid may be a weak acid or a strong acid. Examples of strong acids include, but are not limited to, sulfuric acid or nitric acid at 0.5 to 1.0 wt %.

[0140] The acid may be, for example, a mineral acid, a sulfonic acid, a carboxylic acid, or a halogenated carboxylic acid. The acid may be, for example, a compound containing an acidic phenolic group. In particular, the acid may be, for example, a weak acid. A weak acid is an acid that only partially dissociates in aqueous solution, as opposed to a strong acid that completely dissociates in aqueous solution.

[0141] Strong and weak acids have pK a It may be identified by determining the value of the ion (A - and H + ) pK of the acid (HA) a The value may be determined using the following equation: pK a = - log 10 K a K a = [A-] [H+] / [HA] pK less than 0 in aqueous solution a Acids with a pK value of 0 or greater in aqueous solution are considered strong acids. a Acids with a value of 0.1 are considered weak acids.

[0142] Examples of weak acids include, for example, oxalic acid (HO2C2O2H), sulfurous acid (H2SO3), phosphoric acid (H3PO4), nitrous acid (HNO2), boric acid (H3BO3), benzoic acid (C6H5COOH), acetic acid (CH3COOH), propionic acid (CH3CH3COOH), monochloroacetic acid (ClCH2COOH), dichloroacetic acid (Cl2CHCOOH), trichloroacetic acid (CCl3CO2H), monobromoacetic acid (BrCH2COOH), dibromoacetic acid (Br2CHCOOH). ), difluoroacetic acid (F2CHCOOH), trifluoroacetic acid (F3CCOOH), formic acid (HCOOH), lactic acid (CH3CH(OH)COOH), succinic acid ((CH2)2(CO2H)2), uric acid (C5H4N4O3), malic acid (C4H6O5), maleic acid (HO2CCHCHCO2H), tartaric acid (HOOC-CHOH-CHOH-COOH), gluconic acid (HOCH2-(CHOH)4-COOH), and citric acid (C6H8O7).

[0143] The acid may be, for example, an organic acid. The acid may be, for example, a carboxylic acid. Examples of organic acids and carboxylic acids are oxalic acid (HO2C2O2H), benzoic acid (C6H5COOH), acetic acid (CH3COOH), propionic acid (CH3CH3COOH), haloacetic acids (e.g., monochloroacetic acid (ClCH2COOH), dichloroacetic acid (Cl2CHCOOH), trichloroacetic acid (CCl3CO2H), monobromoacetic acid (BrCH2COOH), dibromoacetic acid (Br2CHCOOH), difluoroacetic acid (C2H2 These include F2O2), trifluoroacetic acid (C2HF3O2), formic acid (HCOOH), lactic acid (CH3CH(OH)COOH), succinic acid ((CH2)2(CO2H)2), uric acid (C5H4N4O3), malic acid (C4H6O5), maleic acid (HO2CCHCHCO2H), tartaric acid (HOOC-CHOH-CHOH-COOH), gluconic acid (HOCH2-(CHOH)4-COOH), and citric acid (C6H8O7).

[0144] The acid may be, for example, a weak organic acid. The acid may be, for example, a weak carboxylic acid. The acid may be, for example, an organic carboxylic acid. The acid may be, for example, a weak organic carboxylic acid. In some embodiments, the acid is citric acid.

[0145] The aqueous acid may have a concentration equal to or greater than about 0.01 wt%, for example. For example, the aqueous acid may have a concentration equal to or greater than about 0.1 wt%, or equal to or greater than about 0.5 wt%, or equal to or greater than about 1.0 wt%, or equal to or greater than about 1.5 wt%, or equal to or greater than about 2.0 wt%, or equal to or greater than about 2.5 wt%.

[0146] The aqueous acid may have a concentration of, for example, less than or equal to about 15.0 wt %, such as less than or equal to about 12.5 wt %, or less than or equal to about 10.0 wt %, or less than or equal to about 7.5 wt %, or less than or equal to about 5.0 wt %, or less than or equal to about 4.0 wt %, or less than or equal to about 3.0 wt %.

[0147] For example, the aqueous acid may have a concentration ranging from about 0.01 wt% to about 15.0 wt%, or from about 0.5 wt% to about 10.0 wt%, or from about 1.0 wt% to about 7.5 wt%, or from about 2.0 wt% to about 3.0 wt%.

[0148] The aqueous acid may have a concentration equal to or greater than about 0.01 wt%. For example, the aqueous acid may have a concentration equal to or greater than about 0.1 wt%, or equal to or greater than about 0.5 wt%, or equal to or greater than about 1.0 wt%, or equal to or greater than about 1.5 wt%, or equal to or greater than about 2.0 wt%, or equal to or greater than about 2.5 wt%. The aqueous acid may have a concentration equal to or less than about 5.0 wt%. For example, the aqueous acid may have a concentration equal to or less than about 4.5 wt%, or equal to or less than about 4.0 wt%, or equal to or less than about 3.5 wt%, or equal to or less than about 3.0 wt%. For example, the aqueous acid may have a concentration ranging from about 0.01 wt% to about 5.0 wt%, or from about 0.5 wt% to about 4.0 wt%, or from about 1.0 wt% to about 3.0 wt%, or from about 2.0 wt% to about 3.0 wt%.

[0149] The temperature of the aqueous acid can be, for example, equal to or greater than about 5° C. For example, the temperature of the aqueous acid can be equal to or greater than about 10° C., or equal to or greater than about 15° C., or equal to or greater than about 17° C.

[0150] The temperature of the aqueous acid can be, for example, equal to or less than about 50° C. For example, the temperature of the aqueous acid can be equal to or less than about 45° C., or equal to or less than about 40° C., or equal to or less than about 35° C., or equal to or less than about 30° C., or equal to or less than about 25° C., or equal to or less than about 23° C., or equal to or less than about 22° C., or equal to or less than about 21° C.

[0151] For example, the temperature of the aqueous acid may range from about 17°C to about 50°C, or from about 17°C to about 40°C, or from about 17°C to about 30°C, or from about 17°C to about 25°C, or from about 18°C ​​to about 22°C, or from about 20°C to about 22°C.

[0152] The temperature of the aqueous acid may be, for example, equal to or greater than about 17° C. For example, the temperature of the aqueous acid may be equal to or greater than about 18° C., or equal to or greater than about 19° C., or equal to or greater than about 20° C. The temperature of the aqueous acid may be, for example, equal to or less than about 23° C. For example, the temperature of the aqueous acid may be equal to or less than about 22° C., or equal to or less than about 21° C. For example, the temperature of the aqueous acid may range from about 17° C. to about 23° C., or from about 18° C. to about 22° C., or from about 20° C. to about 22° C.

[0153] The aqueous acid wash may be repeated one or more times, for example, prior to the next step (e.g., washing with aqueous alkali). In other words, the aqueous alkali wash may be performed two or more times prior to the next step (e.g., washing with aqueous alkali). When washing with aqueous acid is repeated, there may be no other intermediate steps between two or more washes with aqueous acid.

[0154] When washing with aqueous acid is repeated, at least about 90 wt. %, e.g., at least about 95 wt. %, or at least about 98 wt. %, or at least about 99 wt. %, or 100 wt. % of the aqueous acid from the preceding (e.g., first) wash with aqueous acid may be separated (e.g., by filtration) from the solid material containing the flavor or fragrance or cosmetic ingredient or intermediate (e.g., the solid material containing (-)-ambrox) prior to commencing the next (e.g., second) wash with aqueous acid.

[0155] Aqueous alkaline cleaning The alkali may be, for example, any suitable alkali. In particular, the alkali may be any alkali suitable for neutralizing any residual acid from the acid wash step. For example, the alkali may be a weak alkali or a strong alkali. In particular, the alkali may be, for example, a strong alkali, which is an alkali that completely dissociates in aqueous solution, as opposed to a weak alkali, which only partially dissociates in aqueous solution.

[0156] Strong and weak alkalis have pK b It may be identified by determining the value of OH. - and B + ) pK of alkali (e.g., BOH) b The value may be determined using the following equation: pK b = - log 10 K b K b = [B + ] [OH - ] / [BOH] pK less than 0 in aqueous solution b Alkalis with a pK value of 0 or greater in aqueous solution are considered strong alkalis. b Alkalis with a value of 0.1 are considered to be weakly alkaline.

[0157] The alkali may be, for example, a hydroxide (ie, it releases hydroxide ions when it dissociates in aqueous solution) or a carbonate or hydrogen carbonate (bicarbonate). Examples of alkalis include, for example, lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca(OH)2), lithium carbonate (Li2CO3), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), calcium carbonate (CaCO3), lithium bicarbonate (LiHCO3), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), and calcium bicarbonate (Ca(HCO3)2). In some embodiments, the alkali is sodium hydroxide.

[0158] The aqueous alkali may have a concentration equal to or greater than about 0.01 wt%, for example, equal to or greater than about 0.1 wt%, or equal to or greater than about 0.5 wt%, or equal to or greater than about 1.0 wt%, or equal to or greater than about 1.5 wt%, or equal to or greater than about 2.0 wt%, or equal to or greater than about 2.5 wt%.

[0159] The aqueous alkali may have a concentration of, for example, less than or equal to about 15.0 wt%. For example, the aqueous alkali may have a concentration of less than or equal to about 12.5 wt%, or less than or equal to about 10.0 wt%, or less than or equal to about 7.5 wt%, or less than or equal to about 5.0 wt%, or less than or equal to about 4.0 wt%, or less than or equal to about 3.0 wt%.

[0160] For example, the aqueous alkali may have a concentration ranging from about 0.01 wt% to about 15.0 wt%, or from about 0.5 wt% to about 10.0 wt%, or from about 1.0 wt% to about 7.5 wt%, or from about 2.0 wt% to about 3.0 wt%.

[0161] The aqueous alkali may have a concentration equal to or greater than about 0.01 wt%. For example, the aqueous alkali may have a concentration equal to or greater than about 0.1 wt%, equal to or greater than about 0.5 wt%, equal to or greater than about 1.0 wt%, equal to or greater than about 1.5 wt%, equal to or greater than about 2.0 wt%, or equal to or greater than about 2.5 wt%. The aqueous alkali may have a concentration equal to or less than about 5.0 wt%. For example, the aqueous alkali may have a concentration equal to or less than about 4.5 wt%, equal to or less than about 4.0 wt%, equal to or less than about 3.5 wt%, or equal to or less than about 3.0 wt%. For example, the aqueous alkali may have a concentration ranging from about 0.01 wt% to about 5.0 wt%, or from about 0.5 wt% to about 4.0 wt%, or from about 1.0 wt% to about 3.0 wt%, or from about 2.0 wt% to about 3.0 wt%.

[0162] The temperature of the aqueous alkali may be, for example, equal to or greater than about 5° C. For example, the temperature of the aqueous alkali may be equal to or greater than about 10° C., or equal to or greater than about 15° C., or equal to or greater than about 20° C., or equal to or greater than about 25° C., or equal to or greater than about 30° C., or equal to or greater than about 35° C., or equal to or greater than about 36° C., or equal to or greater than about 37° C., or equal to or greater than about 38° C., or equal to or greater than about 40° C.

[0163] The temperature of the aqueous alkali may be, for example, equal to or less than about 75° C. For example, the temperature of the aqueous alkali may be equal to or less than about 70° C., or equal to or less than about 65° C., or equal to or less than about 60° C., or equal to or less than about 55° C., or equal to or less than about 50° C., or equal to or less than about 45° C., or equal to or less than about 44° C., or equal to or less than about 43° C., or equal to or less than about 42° C., or equal to or less than about 41° C.

[0164] For example, the temperature of the aqueous alkali may range from about 5°C to about 75°C, or from about 15°C to about 70°C, or from about 20°C to about 60°C, or from about 25°C to about 50°C, or from about 35°C to about 45°C, or from about 38°C to about 42°C.

[0165] The temperature of the aqueous alkali may be, for example, equal to or greater than about 35°C. For example, the temperature of the aqueous alkali may be equal to or greater than about 36°C, or equal to or greater than about 37°C, or equal to or greater than about 38°C, or equal to or greater than about 39°C, or equal to or greater than about 40°C. The temperature of the aqueous alkali may be, for example, equal to or less than about 45°C. For example, the temperature of the aqueous alkali may be equal to or less than about 44°C, or equal to or less than about 43°C, or equal to or less than about 42°C, or equal to or less than about 41°C. For example, the temperature of the aqueous alkali may range from about 35°C to about 45°C, or from about 38°C to about 42°C.

[0166] The aqueous alkaline washing may be repeated one or more times, for example, prior to the next step (e.g., washing with an aqueous surfactant). In other words, the aqueous alkaline washing may be performed two or more times prior to performing the next step (e.g., washing with an aqueous surfactant). When repeated washing with aqueous alkali, there may be no other intermediate steps between two or more washings with aqueous alkali.

[0167] When washing with aqueous alkali is repeated, at least about 90 wt. %, e.g., at least about 95 wt. %, or at least about 98 wt. %, or at least about 99 wt. %, or 100 wt. % of the aqueous alkali from the preceding (e.g., first) wash with aqueous alkali may be separated (e.g., by filtration) from the solid material containing the flavor or fragrance or cosmetic ingredient or intermediate (e.g., the solid material containing (-)-ambrox) prior to commencing the next (e.g., second) wash with aqueous alkali.

[0168] Aqueous surfactant cleaning The surfactant may be, for example, any suitable surfactant. For example, the surfactant may be any surfactant suitable for separating one or more impurities from a flavor or fragrance or cosmetic ingredient or intermediate, such as (-)-ambrox. For example, the surfactant may be an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, or a nonionic surfactant.

[0169] In particular, the surfactant may be, for example, an anionic surfactant. Anionic surfactants are surfactants that contain an anionic functional group at their head. Anionic surfactants include, for example, sulfonates, phosphates, sulfates, and carboxylates.

[0170] Examples of anionic surfactants include, for example, alkyl sulfates (e.g., ammonium lauryl sulfate, sodium dodecyl sulfate (SDS)), alkyl ether sulfates (e.g., sodium lauryl ether sulfate (SLES), sodium myristyl sulfate), alkyl carboxylates (e.g., stearates such as sodium stearate), alkyl benzene sulfonates, alkyl sulfonates, alkyl ether sulfonic acids, salts of fluorinated fatty acids, silicones, fatty alcohol sulfates, polyoxyethylene fatty alcohol ether sulfates, alpha-olefin sulfonates, polyoxyethylene fatty alcohol phosphate ethers, alkyl alcohol amides, alkyl sulfonic acid acetamides, alkyl succinic acid sulfonates, amino alcohol alkyl benzene sulfonates, naphthenates, alkyl phenol sulfonates, dioctyl sodium sulfosuccinate, perfluorooctane sulfonates, perfluorobutane sulfonates, alkyl-aryl ether phosphates, and alkyl ether phosphates.

[0171] In some embodiments, the surfactant is sodium dodecyl sulfate (SDS). Examples of nonionic surfactants include, for example, sophorolipids, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid ethoxylates, amine ethoxylates and / or fatty acid amides, poloxamers, fatty acid esters of polyhydroxy compounds, fatty acid esters of glycerol, fatty acid esters of sorbitol, fatty acid esters of sucrose, alkyl polyglucosides, and amine oxides.

[0172] The aqueous surfactant may have a concentration of, for example, about 0.01 wt% or greater. For example, the aqueous surfactant may have a concentration of about 0.05 wt% or greater, about 0.1 wt% or greater, about 0.2 wt% or greater, about 0.3 wt% or greater, about 0.4 wt% or greater, or about 0.5 wt% or greater.

[0173] The aqueous surfactant may have a concentration of, for example, about 10.0 wt% or less. For example, the aqueous surfactant may have a concentration of, for example, about 9.0 wt% or less, or about 8.0 wt% or less, or about 7.0 wt% or less, or about 6.0 wt% or less, or about 5.0 wt% or less, or about 4.0 wt% or less, or about 3.0 wt% or less, or about 2.0 wt%.

[0174] For example, the aqueous surfactant may have a concentration ranging from about 0.01 wt% to about 10.0 wt%, or from about 0.05 wt% to about 8.0 wt%, or from about 0.1 wt% to about 6.0 wt%, or from about 0.5 wt% to about 5.0 wt%, or from about 0.5 wt% to about 1.0 wt%. The aqueous surfactant may have a concentration equal to or greater than about 0.05 wt%. For example, the aqueous surfactant may have a concentration equal to or greater than about 0.1 wt%, or equal to or greater than about 0.2 wt%, or equal to or greater than about 0.3 wt%, or equal to or greater than about 0.4 wt%, or equal to or greater than about 0.5 wt%. The aqueous surfactant may have a concentration equal to or less than about 5.0 wt%, for example. For example, the aqueous surfactant may have a concentration of about 4.0 wt% or less, or about 3.0 wt% or less, or about 2.0 wt% or less, or about 1.0 wt% or less, For example, the aqueous surfactant may have a concentration ranging from about 0.05 wt% to about 5.0 wt%, or from about 0.2 wt% to about 4.0 wt%, or from about 0.3 wt% to about 3.0 wt%, or from about 0.4 wt% to about 2.0 wt%, or from about 0.5 wt% to about 1.0 wt%.

[0175] The temperature of the aqueous detergent may be, for example, equal to or greater than about 5°C. For example, the temperature of the aqueous surfactant may be equal to or greater than about 10°C, or equal to or greater than about 15°C, or equal to or greater than about 20°C, or equal to or greater than about 25°C, or equal to or greater than about 30°C, or equal to or greater than about 35°C, or equal to or greater than about 40°C, or equal to or greater than about 45°C, or equal to or greater than about 50°C, or equal to or greater than about 55°C, or equal to or greater than about 60°C, or equal to or greater than about 62°C, or equal to or greater than about 64°C, or equal to or greater than about 65°C, or equal to or greater than about 66°C, or equal to or greater than about 68°C, or equal to or greater than about 70°C.

[0176] The temperature of the aqueous detergent may be, for example, equal to or less than about 75° C. For example, the temperature of the aqueous detergent may be equal to or less than about 74° C., or equal to or less than about 73° C., or equal to or less than about 72° C. For example, the temperature of the aqueous surfactant may range from about 5°C to about 75°C, or from about 35°C to about 75°C, or from about 50°C to about 72°C, or from about 60°C to about 75°C.

[0177] The temperature of the aqueous detergent may be, for example, equal to or greater than about 60°C. For example, the temperature of the aqueous detergent may be equal to or greater than about 62°C, or equal to or greater than about 64°C, or equal to or greater than about 65°C, or equal to or greater than about 66°C, or equal to or greater than about 68°C, or equal to or greater than about 70°C. The temperature of the aqueous detergent may be, for example, equal to or less than about 75°C. For example, the temperature of the aqueous detergent may be equal to or less than about 74°C, or equal to or less than about 73°C, or equal to or less than about 72°C. For example, the temperature of the aqueous detergent may range from about 60°C to about 80°C, or from about 65°C to about 75°C, or from about 68°C to about 72°C.

[0178] Washing with an aqueous surfactant may, for example, be repeated one or more times, in other words, washing with an aqueous surfactant may be carried out two or more times. When washing with an aqueous detergent is repeated, there may be no other intermediate steps between two or more washes with an aqueous detergent.

[0179] When washing with an aqueous surfactant is repeated, at least about 90 wt. %, e.g., at least about 95 wt. %, or at least about 98 wt. %, or at least about 99 wt. %, or 100 wt. % of the aqueous surfactant from the preceding (e.g., first) wash with aqueous surfactant may be separated (e.g., by filtration) from the solid material containing the flavor or fragrance or cosmetic ingredient or intermediate (e.g., the solid material containing (-)-ambrox) prior to commencing the next (e.g., second) wash with aqueous surfactant.

[0180] Additional (optional) steps The methods described herein may further include one or more additional steps before and / or after the aqueous acid wash, the optional aqueous alkali wash, and the aqueous detergent wash. For example, the methods described herein may further include one or more washing steps before and / or after the optional aqueous alkali wash and the aqueous detergent wash.

[0181] For example, the methods described herein may further include washing with water between one or more of washing with an aqueous acid, washing with any aqueous alkali, and washing with an aqueous detergent. For example, the methods described herein may further include washing with water prior to washing with an aqueous acid, washing with any aqueous alkali, and / or washing with an aqueous detergent. For example, the methods described herein may further include washing with water after washing with any aqueous alkali. For example, the methods described herein may further include washing with water after washing with an aqueous acid. For example, the methods described herein may further include washing with water after washing with an aqueous detergent.

[0182] For example, the methods described herein may include, as an initial step, washing the crude flavor or fragrance or cosmetic ingredient or intermediate with water prior to the purification methods described herein. For example, the methods described herein may include, as an initial step, washing the crude (-)-ambrox with water prior to the purification methods described herein.

[0183] The temperature of the water for each additional water washing step may be, for example, equal to or greater than about 5° C. For example, the temperature of the water for each additional water washing step may be, for example, equal to or greater than about 10° C., or equal to or greater than about 15° C., or equal to or greater than about 20° C., or equal to or greater than about 25° C., or equal to or greater than about 30° C., or equal to or greater than about 35° C., or equal to or greater than about 40° C., or equal to or greater than about 45° C., or equal to or greater than about 50° C., or equal to or greater than about 55° C., or equal to or greater than about 60° C.

[0184] The temperature of the water for each additional water washing step may be, for example, equal to or less than about 75°C, for example, the temperature of the water for each additional water washing step may be, for example, equal to or less than about 74°C, or equal to or less than about 73°C, or equal to or less than about 72°C, or equal to or less than about 71°C, or equal to or less than about 70°C.

[0185] For example, the temperature of the water for each additional water wash step may range from about 5°C to about 75°C, or from about 20°C to about 72°C, or from about 25°C to about 70°C, or from about 40°C to about 70°C, or from about 60°C to about 70°C.

[0186] For example, the method described herein may further include washing with water between washing with aqueous alkali and washing with aqueous surfactant. In other words, the product of the aqueous alkali wash may be washed with water one or more times before washing with aqueous surfactant. For example, the product of the aqueous alkali wash may be washed with water twice before washing with aqueous surfactant.

[0187] The temperature of the water used to wash the product of the aqueous alkaline wash prior to the aqueous surfactant wash may be, for example, equal to or greater than about 17° C. For example, the water temperature may be equal to or greater than about 18° C., or equal to or greater than about 19° C., or equal to or greater than about 20° C.

[0188] The temperature of the water used to wash the product of the aqueous alkaline wash prior to the aqueous detergent wash may be, for example, equal to or less than about 23° C. For example, the water temperature may be equal to or less than about 22° C., or equal to or less than about 21° C. For example, the temperature of the water used to wash the product of an aqueous alkaline wash prior to an aqueous surfactant wash may range from about 17°C to about 23°C, or from about 18°C ​​to about 22°C, or from about 20°C to about 22°C.

[0189] For example, the method described herein may further comprise washing with water after washing with aqueous surfactant.In other words, the product of washing with aqueous surfactant may be washed with water one or more times.For example, the product of washing with aqueous surfactant may be washed with water two or more times, three or more times, or four or more times.For example, the product of washing with aqueous surfactant may be washed with water until the filtrate is clear.

[0190] For example, the product of washing with an aqueous detergent may be washed one or more times, e.g., two times, with water having a temperature equal to or greater than about 50° C. and / or equal to or less than about 70° C., followed by one or more times, e.g., two times, with water having a temperature equal to or greater than about 17° C. and / or equal to or less than about 23° C. Water having a temperature equal to or greater than about 50° C. and / or equal to or less than about 70° C. may have a temperature equal to or greater than about 52° C., or equal to or greater than about 54° C., or equal to or greater than about 55° C., or equal to or greater than about 56° C., or equal to or greater than about 58° C., or equal to or greater than about 60° C. Water having a temperature equal to or greater than about 50° C. and / or equal to or less than about 70° C. may have a temperature equal to or less than about 68° C., or equal to or less than about 66° C., or equal to or less than about 65° C., or equal to or less than about 64° C., or equal to or less than about 62° C. For example, water having a temperature equal to or greater than about 50° C. and / or equal to or less than about 70° C. may range from about 50° C. to about 70° C., or from about 55° C. to about 65° C., or from about 58° C. to about 62° C. Water having a temperature equal to or greater than about 17° C. and / or equal to or less than about 23° C. may have a temperature equal to or greater than about 18° C., or equal to or greater than about 19° C., or equal to or greater than about 20° C., for example. Water having a temperature equal to or greater than about 17°C and / or equal to or less than about 23°C may, for example, have a temperature equal to or less than about 22°C, or equal to or less than about 21°C.Water having a temperature equal to or greater than about 17°C and / or equal to or less than about 23°C may have a temperature ranging, for example, from about 17°C to about 23°C, or from about 18°C ​​to about 22°C, or from about 20°C to about 22°C.

[0191] The water used in any washing step may be, for example, distilled water. A final washing step (e.g., immediately prior to any drying step) may, for example, use a mixture of alcohol and water, such as a mixture of ethanol and water, for example a 50:50 w / w mixture of ethanol and water.

[0192] After the washing steps, the product may be dried to form a refined flavor or fragrance or cosmetic ingredient or intermediate. For example, the product of the aqueous surfactant washing (and any further optional steps) may be dried to form a refined flavor or fragrance or cosmetic ingredient or intermediate. For example, the product may be dried when all washing steps are complete.

[0193] After the washing steps, the product may be dried to form purified (-)-ambrox. For example, the product of the aqueous detergent wash (and any further optional steps) may be dried to form purified (-)-ambrox. For example, the product may be dried when all washing steps are complete.

[0194] Refined flavor or fragrance or cosmetic ingredients or intermediate products The products of the processes described herein may be referred to as refined flavor or fragrance or cosmetic ingredients or intermediates. Provided herein are refined flavor or fragrance or cosmetic ingredients or intermediates obtained or obtainable by the processes described herein (including all aspects thereof).

[0195] Purified (-)-ambrox product The product of the methods described herein may also be referred to as purified (-)-ambrox. Provided herein is purified (-)-ambrox obtained or obtainable by the methods described herein (including all aspects thereof). In some embodiments, the purified (-)-ambrox is in solid form, as described in WO 2017 / 182542, the contents of which are incorporated herein by reference.

[0196] Purified (-)-ambrox may contain, for example, about 98.0 wt% or greater (-)-ambrox based on dry material. For example, purified (-)-ambrox may contain about 98.5 wt% or greater, or about 99.0 wt% or greater (-)-ambrox based on dry material. Purified (-)-ambrox may contain, for example, less than about 99.5 wt% (-)-ambrox based on dry solid material. For example, purified (-)-ambrox may contain about 98.0 wt% to about 99.5 wt%, or about 98.5 wt% to about 99.5 wt%, or about 99.0 wt% to about 99.5 wt% (-)-ambrox based on dry solid material.

[0197] The compounds of formulae (II), (III), and (IV) and unreacted homofarnesol may be present in purified (-)-ambrox in olfactory-acceptable amounts. This means that the compounds of formulae (II), (III), and (IV) are present in amounts below their odor detection thresholds or in amounts that do not contribute to the olfactory characteristics of (-)-ambrox in a manner that affects its odor characteristics. Purified (-)-ambrox containing olfactory-acceptable amounts of any such compounds is identifiable to the skilled perfumer as possessing the odor characteristics of commercial-grade (-)-ambrox (e.g., AMBROFIX™) obtained by a synthetic procedure from sclareol (ex-sclareol), and is available from Givaudan.

[0198] Purified (-)-ambrox may contain, for example, greater than or equal to about 0.1 wt. % or greater than or equal to about 0.2 wt. % of the compound of formula (II) based on the dry material. Purified (-)-ambrox may contain, for example, less than or equal to about 0.4 wt. % or less than or equal to about 0.3 wt. % of the compound of formula (II) based on the dry material.

[0199] Purified (-)-ambrox may contain, for example, greater than or equal to about 0.1 wt. % or greater than or equal to about 0.2 wt. % of the compound of formula (III) based on the dry material, or less than or equal to about 0.4 wt. % or less than or equal to about 0.3 wt. % of the compound of formula (III) based on the dry material.

[0200] Purified (-)-ambrox may contain, for example, greater than or equal to about 0.1 wt. % or greater than or equal to about 0.2 wt. % of the compound of formula (IV) based on the dry solids. Purified (-)-ambrox may contain, for example, less than or equal to about 0.8 wt. % or less than or equal to about 0.6 wt. % or less than or equal to about 0.4 wt. % of the compound of formula (IV) based on the dry solids.

[0201] Purified (-)-ambrox may contain, for example, greater than or equal to about 0.3 wt.%, or greater than or equal to about 0.4 wt.%, or greater than or equal to about 0.5 wt.% of the total compounds of formulas (II), (III), and (IV) based on the dry material. Purified (-)-ambrox may contain, for example, less than or equal to about 1.2 wt.%, or less than or equal to about 1.0 wt.%, or less than or equal to about 0.8 wt.% of the total compounds of formulas (II), (III), and (IV) based on the dry material.

[0202] Purified (-)-ambrox may contain, for example, about 0.1 wt% or more, about 0.2 wt% or more, about 0.3 wt% or more, or about 0.4 wt% or more of unreacted homofarnesol based on the dry material. Purified (-)-ambrox may contain, for example, about 0.8 wt% or less, about 0.7 wt% or less, or about 0.6 wt% or less of unreacted homofarnesol compound based on the dry material. The wt% of each compound in purified (-)-ambrox may be determined by gas chromatography.

[0203] The methods described herein may provide (-)-ambrox in a yield equal to or greater than about 95 mol%, based on the crude starting material. For example, the methods described herein may provide (-)-ambrox in a yield equal to or greater than about 96 mol%, or equal to or greater than about 97 mol%, or equal to or greater than about 98 mol%, based on the crude starting material. For example, the methods described herein may provide (-)-ambrox in a yield equal to or less than about 100 mol%, or equal to or less than about 99 mol%, based on the crude starting material.

[0204] Purified (-)-ambrox may be analyzed, for example, by X-ray diffraction (XRD). Powder XRD data can be collected in a straightforward manner using diffractometer instruments that are well known in the art.

[0205] Powder XRD patterns may be obtained using a STOE STADI PX-ray diffractometer. System description: The diffractometer was used in transmission mode (flat sample holder, curved germanium (111) monochromator, and CuKal radiation 1.54060 Å) using a position-sensitive detector. The generator voltage was 40 kV and the current was 40 mA. Detector: Mythen IK. Experimental parameters: Pattern measurements were taken between approximately 4° and 26° 2θ. The accuracy of the determined diffraction angles is approximately + / - 2° 2θ.

[0206] Purified (-)-ambrox may, for example, exhibit a powder X-ray diffraction pattern having at least one of the following peaks at diffraction angles 2 theta: approximately 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / - 0.2°. Purified (-)-ambrox in solid form may be characterized, for example, by a powder X-ray diffraction pattern exhibiting the following peaks at diffraction angles 2 theta: approximately 15.6, 16.2, 16.7, 17.0, 17.4, and 18.3 + / - 0.2°.

[0207] Purified (-)-ambrox in solid form may be characterized, for example, by a powder X-ray diffraction pattern substantially as depicted in Figure 1 below. The shape of the crystals can be determined by microscopy according to methods well known in the art.

[0208] The average diameter measurement of the crystals may be determined by laser granulometry. Laser granulometry is a technique well known in the art. The average particle size may be determined on any particle size analyzer known for such purposes, for example, on a CILAS 1180 No. 516 instrument. Measurements may be made according to ISO Standard 13320-1 (revised 2009) using the Fraunhofer method with water as the carrier liquid and an Obscuration Index of 24.

[0209] Purified (-)-ambrox in solid form may, for example, comprise elongate crystals having an average diameter between 10 and 400 microns, e.g., between 40 microns and 400 microns, e.g., between 100 microns and 400 microns. Purified (-)-ambrox in solid form may comprise elongated crystals having a length measured along their longest dimension of, for example, 20 to 600 microns, such as 40 to 500 microns, for example 100 to 400 microns, such as greater than 100 microns, for example greater than 200 microns, for example greater than 300 microns.

[0210] Purified (-)-ambrox contains (-)-ambrox as the predominant compound, but may also contain other compounds that may or may not impart pleasant olfactory notes to the biotransformation mixture and thus may contribute, in a positive or negative way, to the sensory characteristics of the purified (-)-ambrox end product. Consequently, sensory analysis of purified (-)-ambrox may be performed using well-established sensory tests utilized by skilled experts (e.g., perfumers), so that the tests can help determine whether a chemically related target product is also an olfactorily related end product compared to a reference product.

[0211] Purified (-)-Ambrox products may be tested for their olfactory purity, quality, and sensory profile against a commercially available reference of (-)-Ambrox. (-)-Ambrox materials may also be tested by experts in applied studies to determine whether the material meets specifications for its organoleptic profile. Various uses of (-)-Ambrox include, but are not limited to, fine fragrance or consumer products such as fabric care, toiletries, beauty care, and cleaning products, including essentially all products in which currently available Ambrox ingredients are used commercially, including, but not limited to, the following products: Ambrox (Firmenich), Ambroxan (Henkel), Ambrofix (Givaudan), Amberlin (Quest), Cetalox Levo (Firmenich), Ambermor (Aromor), and Norambrenolide Ether (Pacific).

[0212] A reference product for the purified (-)-ambrox described herein may be, for example, (-)-ambrox in solid form, produced according to the methods as described in WO 2017 / 182542 (e.g., Example 4 of WO 2017 / 182542). A reference product for the purified (-)-ambrox described herein may be, for example, ex sclareolide (-)-ambrox flakes, produced according to the method as described in WO 2018 / 154048.

[0213] "Sclareolide" is used synonymously with "(3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyl-1,4,5,5a,7,8,9,9b-octahydrobenzo[e]benzofuran-2-one." Dextrorotatory or (+)-sclareolide has the following structural formula: [ka] It has.

[0214] Sclareolide can be obtained, for example, by the cyclase-catalyzed conversion of (3E / 7E)-homofarnesic acid, which is then chemically reduced (e.g., with LiAlH4 or NaBH4) to form ambrox-1,4-diol [Mookherjee et al.; Perfumer and Flavorist (1990), 15:27]. Ambrox-1,4-diol may then be chemically converted to (-)-ambrox using various processes (see, e.g., U.S. Pat. No. 5,274,134). A reference product for the purified (-)-ambrox described herein may be, for example, (-)-ambrox made by any method known to those skilled in the art.

[0215] For example, (-)-ambrox was first discovered after chemical transformation of components of clary sage (Salvia sclarea L.) (see M. Hinder, M. Stoll, Hlv. Chim. Acta 1950, 33, 1308-1312) and was later identified as the essential odorant of alcoholic ambergris tinctures (see B.D. Mookherjee, R.R. Patel, Proceedings 7th Int. Congr. Ess. Oils 1977, Kyoto, Japan, Paper No. 136).

[0216] Ambrox has traditionally been produced by semisynthesis from (-)-sclareol (9.1.1, below), a diterpene diol isolated from clary sage, by various companies on a total scale of >100 t / a: [ka]

[0217] Avoca, a major manufacturer of sclareolide (9.1.2), has replaced the chemical oxidation of (-)-sclareol (9.1.1) to (-)-sclareolide (9.1.2) with a biocatalytic process in Cryptococcus albidus (see MI Farbood, JA Morris, AE Downey, US Pat. Appl. To IFF, US 4970163, prior to 28 August 1989).

[0218] Direct conversion of sclareol to sclareodiol (9.1.4), a more advanced intermediate for 9.1.3, is achieved by Hyphozyma roseonigra, but this microorganism has low tolerance to the antimicrobial activity of (-)-sclareol (9.1.1) (see X. Wang, X. Zhang, Q. Yao, D. Hua, J. Qing, Braz. J. Microbiol. 2018, 49S, 160-165). [ka]

[0219] Aiming to produce 9.1.3 (ambrofix / ambrox / ambroxan) independently from plant-sourced materials, Schalk et al. transferred the biogenetic pathway of sclareol into E. coli (see M. Schalk, L. Pastore, M.A. Mirata, S.Khim, M.Schouwey, F.Deguerry, V.Pineda, L.Rocci, L.Daviet, J.Am.Chem.Soc. 2012, 134, 18900-18903). Biosynthesis from geranylgeranyl diphosphate (GGPP) involved two diterpene cyclases (see scheme below). The corresponding gene was functionally expressed in an E. coli strain genetically modified for GGPP overproduction, which allowed the production of (-)-sclareol (9.1.1) at 1.5 g / L. Based on these results, an industrial fermentation process in yeast was developed, which resulted in a new quality of ambrox, named Ambrox Super (not to be confused with "Superambrox", which is 5(6)-dehydro-ambrox) (see R.L. Snowden, Chem.Biodiv.2008,5,958-969). [ka] GGPP = geranylgeranyl diphosphate SsLPS: Salvia sclarea labdadienyl diphosphate synthase SsScS: Salvia sclareol synthase

[0220] Use of the purified product Further provided herein is the use of the refined flavor components described herein as flavor ingredients. The flavor ingredients may be used in consumer food and beverage product applications. Thus, also provided herein are flavor compositions comprising the purified flavor ingredients described herein. A "flavor composition" can be, for example, any composition comprising the purified flavor ingredients. The flavor composition can further comprise, for example, other flavor ingredients, carrier materials, diluents, or combinations thereof.

[0221] Also provided herein are food or beverage products comprising the flavor compositions as described herein. Further provided herein is the use of the purified cosmetic ingredient described herein as a cosmetic ingredient. The cosmetic ingredient can be used in a cosmetic product.

[0222] Thus, also provided herein are cosmetic compositions containing the purified cosmetic ingredients described herein. A "cosmetic composition" can be, for example, any composition containing a purified cosmetic ingredient. The cosmetic composition can further include, for example, other cosmetic ingredients, carrier materials, diluents, or combinations thereof.

[0223] Also provided herein is a cosmetic product comprising the flavor ingredient or flavor composition as described herein. The cosmetic product may be, for example, a skin care product (e.g., a moisturizer), a makeup product (e.g., a foundation, a concealer, a lipstick, an eye shadow, an eyeliner), a hair care product (e.g., a shampoo, a conditioner, a dye), an oral hygiene product (e.g., a toothpaste, a mouthwash), or a deodorant.

[0224] Further provided herein is the use of the purified (-)-ambrox described herein as a flavor ingredient. Further provided herein is the use of the purified perfume ingredients described herein (e.g., purified (-)-ambrox described herein) as perfume ingredients. The perfume ingredients can be used in both consumer product applications and fine fragrance applications.

[0225] Thus, also provided herein are perfume compositions comprising the purified perfume ingredients described herein (e.g., purified (-)-ambrox described herein). A "perfume composition" can be, for example, any composition comprising a purified perfume ingredient (e.g., purified (-)-ambrox) and a substrate.

[0226] As used herein, "substrate" encompasses all known fragrance ingredients selected from the wide range of natural products and synthetic molecules currently available, such as essential oils, alcohols, aldehydes and ketones, ethers and acetals, esters and lactones, macrocycles and heterocycles, and / or the like, in admixture with one or more ingredients or excipients conventionally used in conjunction with odorants in fragrance compositions (e.g., carrier materials, diluents, and other adjuvants commonly used in the art).

[0227] Fragrance ingredients known in the art are readily available commercially from major fragrance manufacturers. Non-limiting examples of such ingredients include: essential oils and extracts, such as bead onion, costus root oil, oakmoss absolute, geranium oil, tree moss absolute, basil oil, fruit oils such as bergamot oil and mandarin oil, myrtle oil, palmarosa oil, patchouli oil, petitgrain oil, jasmine oil, rose oil, sandalwood oil, absinthe oil, lavender oil and / or ylang-ylang oil; alcohols, such as cinnamic alcohol ((E)-3-phenylprop-2-en-1-ol); cis-3-hexenol ((Z)-hex-3-en-1-ol); citronellol (3,7-dimethyloct-6-en-1-ol); dihydromyrcenol (2,6-dimethyloct-7-en-2-ol); Ebanol™ ((E)-3-methyl-5-(2,2,3-trimethylcyclopenta[4-methyl-2 ... -3-en-1-yl)pent-4-en-2-ol; eugenol (4-allyl-2-methoxyphenol); ethyl linalool ((E)-3,7-dimethylnona-1,6-dien-3-ol); farnesol ((2E,6Z)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol); geraniol ((E)-3,7-dimethylocta-2,6-dien-1-ol); Super Lily of the Valley (Super Muguet™ ((E)-6-ethyl-3-methyloct-6-en-1-ol); Linalool (3,7-dimethylocta-1,6-dien-3-ol); Menthol (2-isopropyl-5-methylcyclohexanol); Nerol (3,7-dimethyl-2,6-octadien-1-ol); Phenylethyiol (2-phenylethanol); Rhodinol™ (3,7-dimethyloct-6-en-1-ol); Sandalore™ (3 -methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; terpineol (2-(4-methylcyclohex-3-en-1-yl)propan-2-ol); or Timberol™ (1-(2,2,6-trimethylcyclohexyl)hexan-3-ol); 2,4,7-trimethylocta-2,6-dien-1-ol, and / or [1-methyl-2(5-methylhex-4-en-2-yl)cyclopropyl]-methanol; - aldehydes and ketones, such as anisaldehyde (4-methoxybenzaldehyde); alpha-amyl cinnamaldehyde (2-benzylideneheptanal); Georgywood™ (1-(1,2,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone); hydroxycitronellal (7-hydroxy-3,7-dimethyloctanal); Iso E Super Super® (1-(2,3,8,8-tetramethyl-1,2,3,4,5,6,7,8-octahydronaphthalen-2-yl)ethanone); Isoraldeine® ((E)-3-methyl-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one); 3-(4-isobutyl-2-methylphenyl)propanal; maltol; methyl cedryl ketone; methyl ionone; verbenone; and / or vanillin; ethers and acetals, such as, for example, Ambrox® (3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1H-benzo[e][1]benzofuran); geranyl methyl ether ((2E)-1-methoxy-3,7-dimethylocta-2,6-diene); rose oxide (4-methyl-2-(2-methylprop-1-en-1-yl)tetrahydro-2H-pyran); and / or Spirambrene® (2',2',3,7,7-pentamethylspiro[bicyclo[4.1.0]heptane-2,5'-[1,3]dioxane]); macrocycles, such as ambrettolide ((Z)-oxacycloheptadecan-10-en-2-one); ethylene brassylate (1,4-dioxacycloheptadecan-5,17-dione); and / or Exaltolide® (16-oxacyclohexadecan-1-one); and - heterocycles, e.g., isobutylquinoline (2-isobutylquinoline) Includes.

[0228] As used herein, "carrier material" means a material that is practically neutral from the odorant's point of view, ie, a material that does not significantly modify the odorant's organoleptic properties.

[0229] "Diluent" means any diluent conventionally used in conjunction with odorants, such as diethyl phthalate (DEP), dipropylene glycol (DPG), isopropyl myristate (IPM), triethyl citrate (TEC), and alcohol (e.g., ethanol).

[0230] The term "auxiliary agent" refers to an ingredient that may be employed in a perfume composition for reasons not specifically related to the olfactory performance of the composition. For example, an auxiliary agent, such as an antioxidant adjuvant, may be an ingredient that acts as an aid in processing a perfume ingredient(s) or a composition containing the ingredient(s), or may improve the handling or storage of a fragrance ingredient or a composition containing the same. The antioxidant may be selected from, for example, Tinogard® TT (BASF), Tinogard® Q (BASF), tocopherol (including its isomers, CAS 59-02-9; 364-49-8; 18920-62-2; 121854-78-2), 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT, CAS 128-37-0), and the related phenol, hydroquinone (CAS 121-31-9).

[0231] It may also be an ingredient that provides additional benefits such as imparting color or texture, or that imparts lightfastness or chemical stability to one or more ingredients contained in the perfume composition.

[0232] It is not possible to give an exhaustive description of the nature and type of adjuvants commonly used in perfume compositions containing them, but it should be mentioned that said ingredients are well known to those skilled in the art.

[0233] Also provided herein is a consumer product comprising a perfume composition as described herein (including any embodiment thereof). The consumer product may be, for example, a household care (e.g., cleaning), personal care (e.g., cosmetics), laundry care, or air care composition.

[0234] The following numbered paragraphs define specific aspects of the present invention: 1. A process for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the process comprising: Washing with aqueous acid; Washing with an aqueous surfactant; and Optionally, washing with aqueous alkali Includes:

[0235] 2. The method of paragraph 1, wherein the crude flavor or fragrance or cosmetic ingredient or intermediate is in solid form. 3. The process of paragraph 1 or 2, wherein the crude flavor or fragrance or cosmetic ingredient or intermediate is made by a bioconversion process. 4. The method of any preceding paragraph, wherein the crude flavor or fragrance or cosmetic ingredient or intermediate is made by a bioconversion process using an SHC / HAC enzyme.

[0236] 5. The method of any preceding paragraph, wherein the method is for purifying crude (-)-ambrox, crude (-)-ambra-oxide, crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan, or crude (+)-amberketal. 6. The method is as follows: Washing crude flavor or fragrance or cosmetic ingredients or intermediates with aqueous acid; washing the product of step (a) with aqueous alkali; washing the product of step (b) with an aqueous surfactant; The method of any preceding paragraph, including

[0237] 7. The method of any preceding paragraph wherein the acid is a weak acid. 8. The method of any preceding paragraph, wherein the acid is an organic acid. 9. The method of any preceding paragraph wherein the acid is citric acid. 10. The method of any preceding paragraph, wherein the aqueous acid has a concentration equal to or greater than about 0.01 wt % and / or equal to or less than about 15.0 wt %, for example, from about 0.1 wt % to about 5.0 wt %.

[0238] 11. The method of any preceding paragraph, wherein the temperature of the aqueous acid ranges from about 17°C to about 50°C, e.g., from about 17°C to about 23°C. 12. The method of any preceding paragraph, wherein washing with aqueous acid is repeated one or more times prior to washing with aqueous alkali. 13. The method of any preceding paragraph, wherein the alkali is a strong alkali. 14. The method of any preceding paragraph wherein the alkali is a hydroxide. 15. The method of any preceding paragraph wherein the alkali is sodium hydroxide.

[0239] 16. The method of any preceding paragraph, wherein the aqueous alkali has a concentration equal to or greater than about 0.01 wt % and / or equal to or less than about 15.0 wt %, for example, from about 0.1 wt % to about 5.0 wt %. 17. The method of any preceding paragraph, wherein the temperature of the aqueous alkali ranges from about 5°C to about 75°C, e.g., from about 35°C to about 45°C. 18. The method of any preceding paragraph, wherein washing with aqueous alkali is repeated one or more times prior to washing with aqueous surfactant.

[0240] 19. The method of any preceding paragraph, wherein the surfactant is an anionic surfactant. 20. The method of any preceding paragraph, wherein the surfactant is sodium dodecyl sulfate (SDS). 21. The method of any preceding paragraph, wherein the aqueous surfactant has a concentration equal to or greater than about 0.01 wt% and / or equal to or less than about 10.0 wt%, for example, from about 0.1 wt% to about 5.0 wt%. 22. The method of any preceding paragraph, wherein the temperature of the aqueous surfactant ranges from about 5°C to about 75°C, e.g., from about 60°C to about 70°C.

[0241] 23. The method of any preceding paragraph, wherein washing with an aqueous surfactant is repeated one or more times. 24. The method of any preceding paragraph, further comprising one or more steps of washing with water prior to or after washing with aqueous acid, washing with aqueous alkali, and / or washing with aqueous surfactant. 25. The method of any preceding paragraph, wherein the aqueous alkali washed product is washed with water prior to washing with aqueous surfactant. 26. The method of paragraph 24 or 25, wherein the temperature of the water ranges from about 5°C to about 75°C, e.g., from about 17°C to about 23°C.

[0242] 27. The method of paragraph 25 or 26, wherein the aqueous alkali washed product is washed two or more times with water prior to washing with aqueous surfactant. 28. The method of any preceding paragraph, wherein the aqueous surfactant washed product is washed with water. 29. The method of paragraph 28, wherein the aqueous surfactant washed product is washed with water having a temperature ranging from about 50°C to about 70°C, followed by washing with water having a temperature ranging from about 17°C to about 23°C.

[0243] 30. The method of paragraph 28 or 29, wherein the aqueous surfactant washed product is washed two or more times with water having a temperature ranging from about 50°C to about 70°C. 31. The method of any of paragraphs 28-31, wherein the aqueous surfactant washed product is washed two or more times with water having a temperature ranging from about 17°C to about 23°C. 32. The method of any preceding paragraph, wherein a crude flavor or fragrance or cosmetic ingredient or intermediate, e.g., crude (-)-ambrox, is separated from the bioconversion broth prior to the purification method.

[0244] 33. The process of paragraph 31, wherein separation of the crude flavor or fragrance or cosmetic ingredient or intermediate, e.g., crude (-)-ambrox, occurs by physical means, e.g., by filtration. 34. The method of paragraph 33, wherein the bioconversion broth is subjected to a heat inactivation step or an alkalinization step prior to separation. 35. The method of paragraph 34, wherein alkalinization occurs by raising the pH of the bioconversion broth to a pH of about 10 to about 11. 36. The method of paragraph 34, wherein heat inactivation occurs by raising the temperature of the bioconversion broth to a maximum temperature of 55°C.

[0245] 37. The method of any preceding paragraph, wherein all cleaning steps occur in a single piece of equipment. 38. The method of any preceding paragraph, wherein all washing steps occur in a Nutsche-type stirred-dry filter. 39. The method of paragraph 38, wherein the crude flavor or fragrance or cosmetic ingredient or intermediate, e.g., crude (-)-ambrox, contains from about 20 wt. % to about 30 wt. % water, based on the wet weight of the crude (-)-ambrox. 40. A flavor or fragrance or cosmetic ingredient or intermediate obtained by or obtainable by the process of any of paragraphs 1 to 39, for example (-)-ambrox.

[0246] 41. Use of a flavor or fragrance or cosmetic ingredient as referred to in paragraph 40, such as (-)-ambrox, as a flavor or fragrance or cosmetic ingredient. 42. A perfume composition comprising a fragrance ingredient of paragraph 40, such as (-)-ambrox. 43. A flavor composition comprising the flavor ingredient of paragraph 40. 44. A cosmetic composition comprising the cosmetic ingredient of paragraph 40. 45. A household care, personal care, laundry care, or air care composition containing a perfume composition of paragraph 42. 46. ​​A food or beverage product containing the flavor composition of paragraph 43. 47. A cosmetic product comprising the cosmetic composition of paragraph 44.

[0247] 48. A Nutsche-type stirred and dried filter configured to purify a crude flavor or fragrance ingredient or intermediate, such as crude (-)-ambrox, by the method of any one of paragraphs 1 to 39, wherein the crude flavor or fragrance ingredient or intermediate, such as crude (-)-ambrox, contains from about 20 wt. % to about 30 wt. % water, based on the wet weight of the crude flavor or fragrance ingredient or intermediate, such as crude (-)-ambrox. 49. A process for purifying a crude flavor or fragrance ingredient or intermediate, the process comprising washing the crude flavor or fragrance ingredient or intermediate in a Nutsche-type stirred and dried filter. 50. The process is in accordance with any of paragraphs 1 to 39 of paragraph 47.

[0248] example Example 1 Preparation of crude (-)-ambrox For the avoidance of doubt, all references to WT SHC and SHC variants are to WT AacSHC (SEQ ID NO: 1) and variants thereof.

[0249] SEQ ID NO: 1: Amino acid sequence of Alicyclobacillus acidocaldarius SHC (AacSHC) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR The 215G2 SHC variant encompasses the mutations M132R, A224V, and I432T compared to WT AacSHC.

[0250] Biocatalytic Production Method 1 SHC plasmid preparation The gene encoding the squalene hopene cyclase 215G2 SHC from Alicyclobacillus acidocaldarius was inserted into the plasmid pET-28a(+) under the control of an IPTG-inducible T7 promoter for protein production in Escherichia coli. The plasmid map of pET-28a(+) (5369 bp) is shown in Figure 5 of WO 2016 / 170099. The cloning and expression regions of the pET-28a(+) plasmid are shown in Figure 21 of WO 2016 / 170099. The plasmid was transformed into E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0251] Erlenmeyer flask culture Either complex (LB) or minimal media was used for protein production. M9 is an example of a minimal medium that has been used successfully.

[0252] Medium preparation The initial selected minimal medium was prepared for a 350 ml culture as follows: 35 ml citric acid / phosphate stock (133 g / L KH2PO4, 40 g / L (NH4)2HPO4, 17 g / L citric acid.H2O, adjusted to pH 6.3) was added to 307 ml H2O, and the pH was adjusted to 6.8 with 32% NaOH as required. After autoclaving, 0.850 ml 50% MgSO4, 0.035 ml trace element solution (composition in the next section), 0.035 ml thiamine solution, and 7 ml 20% glucose were added.

[0253] SHC Biocatalyst Production (Biocatalyst Production) For small-scale biocatalyst production (wild-type SHC or SHC variants), a 350 ml culture (medium supplemented with 50 μg / ml kanamycin) was inoculated from a preculture of E. coli strain BL21(DE3) containing the SHC production plasmid. Cells were grown under constant agitation (250 rpm) until the OD reached approximately 0.5 (OD 650nm ) at 37°C.

[0254] Protein production was then induced by adding IPTG to a concentration of 300 μM, followed by an additional 5–6 h of incubation with constant shaking. The resulting biomass was finally collected by centrifugation and washed with 50 mM Tris-HCl buffer (pH 7.5). Cells were stored as pellets at 4°C or -20°C until further use. Regardless of the medium used, 2.5–4 grams of cells (wet weight) were typically obtained from 1 liter of culture.

[0255] Fermentations were prepared and carried out in 750 ml Infors HT reactors. Deionized water was added to the fermenter. The reactor was equipped with all required probes (pO2, pH, sampling, antifoam), C+N feed bottles and sodium hydroxide bottles and autoclaved.

[0256] After autoclaving, add the following to the reactor: 20ml 10x Phosphate / Citrate Buffer 14ml 50% glucose 0.53ml MgSO4 solution 2ml (NH4)2SO4 solution 0.020ml trace element solution 0.400ml thiamine solution 0.200ml Kanamycin Stock

[0257] The running parameters set were: pH = 6.95, pO2 = 40%, T = 30°C, stirring at 300 rpm. Cascade: rpm fixed at 300, min 300, max 1000, flow rate l / min fixed at 0.1, min 0, max 0.6. Antifoam control: 1:9.

[0258] Fermenter OD of 0.4-0.5 650nmThe culture was inoculated from a seed culture of up to 1000 ml. This seed culture was grown in LB medium (plus kanamycin) at 37°C and 220 rpm for 8 h. Fermentation was initially run in batch mode for 11.5 h, after which C+N feeding was initiated with a feed solution (sterile glucose solution (143 ml H2O + 35 g glucose) to which the following was added after sterilization: 17.5 ml (NH4)2SO4 solution, 1.8 ml MgSO4 solution, 0.018 ml trace element solution, 0.360 ml thiamine solution, and 0.180 ml kanamycin stock. The feed was performed at a constant flow rate of approximately 4.2 ml / h. Glucose and NH4 were added to assess the availability of C- and N-sources during the culture. + Glucose levels usually remain very low.

[0259] Cultures were grown for a total of approximately 25 hours and typically reached an OD of 40-45. 650nm SHC production was then initiated in the fermentor by adding IPTG to a concentration of approximately 1 mM (IPTG was added in pulses or over a 3-4 hour period using an injection syringe), and the temperature was set to 40°C and pO2 to 20%. Induction of SHC production continued at 40°C for 16 h. At the end of induction, cells were harvested by centrifugation, washed with 0.1 M citric acid / sodium citrate buffer (pH 5.4), and stored as pellets at 4°C or -20°C until further use.

[0260] In general, the specific activity of the biocatalysts produced was higher when minimal medium was used compared to complex medium, with all other conditions remaining unchanged. Induction was successfully carried out at 30°C or 37°C. It was noted that biocatalysts with higher specific activity were obtained when induction was performed at 40-43°C. Fermentation typically produced between 20 and 30 grams of cells (wet weight).

[0261] Biotransformation at 125g / l E,E-homofarnesol (EEH) A typical EEH biotransformation reaction contained 125 g / L EEH, 250 g / L wet weight cells (producing 215G2 SHC), and 1.3% SDS in 0.1 M succinate / NaOH buffer (pH 5.4).

[0262] The reaction is incubated at 35° C. under constant agitation. The wet weight cell concentration of the cell suspension used to prepare the bioconversion can be determined, for example, by centrifuging an aliquot of this cell suspension at 10° C. and 17,210 g for 10 min to calculate the volume of cell suspension required to set the cell concentration in the EEH bioconversion reaction to 250 g / L. EEH conversion is usually complete within 72 hours of reaction time.

[0263] Purification of crude (-)-ambrox The crude (-)-ambrox contained 70-75 wt% dry material and 20-25 wt% water. The dry material contained 5-10 wt% chemical and insoluble impurities.

[0264] The crude (-)-ambrox was purified by the following procedure: 1) Two washes with 2.5 wt% aqueous citric acid at room temperature; 2) Two washes with 0.1 wt% aqueous sodium hydroxide at 40°C; 3) Two washes with room temperature water; 4) two washes with 0.5 wt% sodium dodecyl sulfate (SDS) at 70°C; 5) Two washes with 60°C water; 6) Wash twice or three times with room temperature water (until the filtrate is clear);

[0265] Highly pure (>99% by GC analysis) olfactory-grade (-)-ambrox was obtained in an average yield of 95 mol% based on the crude starting material (believed to be due to mechanical losses during transfer from the wash reactor to the Buchner filter and vice versa).

[0266] Example 2 Crude (-)-ambrox was obtained by the same process described for Example 1. 10 kg of crude (-)-ambrox, containing 77.9 wt% (-)-ambrox, 18.2 wt% water, and 3.9 wt% chemical and insoluble impurities, was placed in a 50 liter stainless steel Nutsche-type stirred filter fitted with a two-blade bottom entry impeller and a 5 micron filter cloth. The crude (-)-ambrox was purified by the following procedure: 1) Two washes of 20 kg each of 2.5 wt% aqueous oxalic acid at ambient temperature, 2) Two washes of 20 kg each of warm (40°C) 0.1 wt% aqueous sodium hydroxide; 3) Two rinses with 20 kg of water at ambient temperature, 4) One wash with 20 kg of warm (40°C) 0.5 wt% aqueous sodium dodecyl sulfate (SDS); 5) Rinse twice with 20 kg of warm (40°C) water each. 6) Three rinses with 20 kg of water at ambient temperature, 7) Two final rinses with 20 kg each of 50 wt % aqueous ethanol at ambient temperature.

[0267] The slurry was agitated for 30 minutes between each wash and 15 minutes between each rinse, then the impeller was stopped and the waste aqueous liquors were completely drained from the filter by suction before the next wash or rinse.

[0268] After a final aqueous alcohol rinse, the cake was dried in the filter with intermittent stirring at 5 mbar pressure and 40-45° C. for 12 hours, yielding 7.4 kg of 99.2 GC% pure olfactory grade (−)-ambrox in 94 mol% yield based on crude starting material.

[0269] The above broadly describes certain aspects of the present invention without limiting it. Variations and modifications that will be readily apparent to those skilled in the art are intended to be within the scope of the present invention as defined in and by the appended claims.

Claims

1. 1. A process for purifying a crude flavor or fragrance or cosmetic ingredient or intermediate, the process comprising: Washing with aqueous acid; Washing with an aqueous surfactant; and Wash with aqueous alkali at temperatures below 75°C Including, the crude flavor or fragrance or cosmetic ingredient or intermediate is in solid form and is produced by a bioconversion process (e.g., a bioconversion process using SHC / HAC enzymes); The process wherein the crude flavor or fragrance or cosmetic ingredient or intermediate is selected from crude (-)-ambrox, crude (-)-ambra-oxide, crude 3a-ethyl-6,6,9a-trimethyldodecahydronaphtho[2,1-b]furan, and crude (+)-amberketal.

2. Here's how: (a) washing a crude flavor or fragrance or cosmetic ingredient or intermediate with an aqueous acid; (b) washing the product of step (a) with aqueous alkali; (c) washing the product of step (b) with an aqueous surfactant.

2. The method of claim 1, comprising:

3. 3. The method according to claim 1 or 2, wherein the acid is a weak acid and / or an organic acid and / or citric acid.

4. 4. The method of any one of claims 1 to 3, wherein the temperature of the aqueous acid ranges from 17°C to 50°C (e.g., 17°C to 23°C).

5. 5. The method according to any one of claims 1 to 4, wherein the alkali is a strong alkali and / or hydroxide and / or sodium hydroxide.

6. 6. The method of any one of claims 1 to 5, wherein the temperature of the aqueous alkali ranges from 5°C to 75°C (e.g., 35°C to 45°C).

7. The method according to any one of claims 1 to 6, wherein the surfactant is an anionic surfactant (e.g., sodium dodecyl sulfate (SDS)).

8. 8. The method of any one of claims 1 to 7, wherein the temperature of the aqueous surfactant ranges from 5°C to 75°C (e.g., 60°C to 70°C).

9. 9. The method of any one of claims 1 to 8, further comprising one or more steps of washing with water, prior to or after washing with aqueous acid, washing with aqueous alkali, and / or washing with aqueous surfactant.

10. 10. The method according to any one of claims 1 to 9, wherein all washing steps occur in a Nutsche-type stirred and dried filter.

11. The method of claim 10, wherein the crude flavor or fragrance ingredient or intermediate (e.g., crude (-)-ambrox) contains from 20 wt. % to 30 wt. % water based on the wet weight of the crude flavor or fragrance ingredient or intermediate (e.g., crude (-)-ambrox).

12. 1) purifying a crude flavor or fragrance or cosmetic ingredient or intermediate by the method of any one of claims 1 to 11; and 2) preparing a household care, personal care, laundry care, or air care composition using the product obtained in 1); 1. A method for making a household care, personal care, laundry care, or air care composition, comprising:

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