Solid form of (-)-ambrox formed by bioconversion of homofarnesol in the presence of biocatalyst

Abstract

To provide a solid form of (-)-Ambrox, as well as methods of preparing and purifying the same.SOLUTION: There is provided a solid form of a compound of the formula (I), wherein the solid form is characterized by at least one of the following characteristics: it exhibits an x-ray diffraction pattern having at least one of the following peaks at diffraction angles 2 θ of about 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / -0.2°; it comprises elongate crystals having an average diameter of between about 10 to about 400 microns measured by laser granulometry; it comprises elongate crystals having a length measured along their longest dimension of greater than 100 microns; and / or it has an L* value of 90 or more; an a* value of less than 1 and greater the -1; and a b* value of less than 8, wherein L*, a* and b* values represent the CIELAB system's chromaticity coordinates.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a solid form of (-)-Ambrox and methods for preparing and purifying it. [Background technology]

[0002] Background of the Invention AMBROFIX™ is a compound of the general formula (I): [ka] It is the trade name of (-)-Ambrox, which has the formula (I) and is marketed by Givaudan, the proprietary distributor of (-)-Ambrox.

[0003] AMBROFIX™ is a very important molecule in the perfumer's palette. It delivers a very strong, very substantial, and very stable ambery note for use in any perfumed product. AMBROFIX™ is available from Givaudan and is the most suitable material for obtaining an authentic ambergris odor note.

[0004] Currently, AMBROFIX™, like other mass-produced forms of (-)-Ambrox, is produced synthetically from naturally occurring starting materials. The supply and quality of the specific starting materials depend on climatic conditions and socio-economic factors. Furthermore, because the starting materials may be extracted from natural sources in small yields, they are almost certainly commercially available at prices that would make their use increasingly uneconomical on an industrial scale. Therefore, a more cost-effective process that can be industrialized is needed if mass-produced industrial supplies of AMBROFIX™ are to remain available at reasonable cost.

[0005] An industrially scalable biotechnological route to (−)-ambrox would be attractive because it is potentially less complex, greener, and more environmentally responsible than a fully synthetic chemical procedure.

[0006] A potentially viable substrate for bioconversion to provide (-)-ambrox is homofarnesol. In their seminal paper, Neumann et al. (Biol. Chem. Hoppe-Seyler Vol. 367 pp. 723-726 (1986)) discussed the feasibility of enzymatically catalyzed conversion of homofarnesol to (-)-ambrox using the enzyme squalenehopene cyclase (SHC). The homofarnesol employed was a mixture of the molecule's four geometric isomers. Of the four isomers, only the 7E,3E geometric isomer (using conventional nomenclature) could be cyclized, resulting in very low yields of the desired (-)-ambrox.

[0007] JP 2009-60799 (Kao) discloses a synthesis by which SHC acts on homofarnesol substrate to produce (-)-ambrox. The substrate is a mixture of all four geometric isomers (3Z,7Z; 3E,7Z; 3Z,7E; and 3E,7E). The document only discloses the preparation of (-)-ambrox from homofarnesol using a liquid extract containing SHC prepared from a recombinant microorganism expressing the SHC gene. The homofarnesol mixture is converted to (-)-ambrox and its 9-epi stereoisomer, and purification can be achieved by distillation or column chromatography. Kao does not describe a process for converting homofarnesol to (-)-ambrox using substantially whole or intact microorganisms that produce SHC, nor does it provide any technical teaching regarding downstream processing of the complex mixture resulting from such a process that would yield (-)-ambrox in olfactory pure form.

[0008] To the applicant's knowledge, the prior art does not describe any viable and industrially scalable process for providing (-)-ambrox in olfactorily pure form, comprising the SHC-catalyzed bioconversion of homofarnesol. Furthermore, even if homofarnesol bioconversion were to be achieved on an industrial scale, a cost-effective source of highly pure 3E,7E-homofarnesol would need to be available. However, although synthetic routes to homofarnesol have been described in the literature (see, e.g., US 2013 / 0273619), to the applicant's knowledge, there are no cost-effective, industrial-scale sources of pure 7E,3E-homofarnesol currently available.

[0009] There is a need to provide an economically feasible and industrially scalable route to the valuable fragrance ingredient (-)-ambrox. In co-pending patent applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT / EP2014 / 072882 (published as WO2015 / 059290), applicants describe an efficient method for preparing 7E,3E / Z-homofarnesol mixtures enriched in the 7E,3E geometric isomer. The 7E,3E / Z-homofarnesol mixture is prepared from beta-farnesene, preserving the isomeric information contained in the starting material, such that the homofarnesol double bond at the 7-position is locked in the E-configuration. However, this elegant chemistry still results in a 3E / Z isomeric mixture. Pure 7E,3E-homofarnesol remains synthetically challenging and can only be achieved by economically uneconomical purification of the isomeric mixture.

[0010] Despite developments in the biocatalytic production of (-)-ambrox from homofarnesol, there remains a need to provide an efficient means of separating and purifying (-)-ambrox from the bioconversion medium, which contains particulate matter and other materials, such as cell debris and possible by-products, unreacted substrate, and any solvents or other reagents employed in the bioconversion process. Summary of the Invention

[0011] In addressing the shortcomings of the prior art, applicants have surprisingly discovered that a crystalline form of (-)-ambrox can be formed during biocatalytic processes. More specifically, applicants have discovered that crystals of (-)-ambrox form in bioconversion media.

[0012] The surprising and unexpected manner in which (-)-ambrox crystallizes in bioconversion media, as well as the physical characteristics of the crystals formed, are particularly relevant in terms of the isolation and purification of an essentially colorless and olfactorily pure form of (-)-ambrox, which exhibits an authentic ambergris odor note, from the bioconversion media. In cases where the biocatalyst is a microbial biocatalyst, the finding that (-)-ambrox has been obtained in olfactorily pure quality is particularly surprising in light of the fact that microbial biocatalysts present in bioconversion media can have highly unpleasant, even offensive, off-note characteristics. The surprising finding that (-)-ambrox crystals form outside the microbial catalyst in the bioconversion media allows for particularly efficient separation of (-)-ambrox from the foul-smelling media.

[0013] Thus, in a first aspect, the present invention provides a solid form of a compound of formula (I): [ka] wherein said solid form is characterized by at least one of the following features: it exhibits an X-ray diffraction pattern having at least one of the following peaks at diffraction angles 2θ: about 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / - 0.2°; it comprises elongated crystals having an average diameter between 10 and 400 microns, more specifically between 40 and 400 microns, and even more specifically between 100 and 400 microns, as measured by laser granulometry; it comprises elongated crystals having a length measured along its longest dimension of 20 to 600 microns, more particularly 40 to 500 microns, and even more particularly 100 to 400 microns, preferably greater than 100 microns, more particularly greater than 200 microns, and even more particularly greater than 300 microns, as measured by laser granulometry; and It is substantially colorless as that term is defined hereinafter.

[0014] In a more specific embodiment, the solid form of the compound of Formula (I) is characterized by a powder X-ray diffraction pattern exhibiting an X-ray diffraction pattern with peaks at the following diffraction angles 2θ: about 15.6, 16.2, 16.7, 17.0, 17.4, and 18.3 + / - 0.2 degrees. In an even more particular embodiment, the solid form of the compound of formula (I) is characterized by a powder X-ray diffraction pattern substantially as depicted in Figure 1 below.

[0015] To the applicant's knowledge, a solid form of (-)-ambrox formed by a biocatalytic process has not previously been described or suggested in the prior art. Thus, in another of its aspects, the present invention provides a solid form of a biotransformation product according to formula (I). In an embodiment of the present invention, the solid form of the biotransformation product according to formula (I) has at least one of the characteristics mentioned herein above.

[0016] The powder X-ray diffraction data referred to above can be collected in a straightforward manner using diffraction measurement equipment well known in the art. The methodology and instrumentation used to measure the powder X-ray diffraction patterns referred to herein are described in more detail in the examples.

[0017] The shape of the crystals was determined by microscopy according to methods well known in the art and need not be further detailed here. The characteristic size and shape of individual crystals obtained according to the present invention are shown below in FIG.

[0018] The average diameter of the crystals was determined by laser granulometry, a technique well known in the art. The average particle size can be determined by any particle size analyzer known for such purposes, for example, the CILAS 1180 No. 516 instrument. Measurements can be performed according to ISO standard 13320-1 (revised 2009) using a Fraunhofer with water as the carrier liquid and an obscuration index of 24.

[0019] As mentioned above, the fact that (-)-ambrox appears in crystalline form in the biotransformation medium, and the physical characteristics of the crystals, i.e., their size and density, are particularly relevant in terms of the efficiency of the isolation of (-)-ambrox and ultimately its clarity and olfactory purity.

[0020] More specifically, it was possible to efficiently separate the crystals from the bioconversion medium containing particulate matter, e.g., cell debris, as well as any by-products, impurities, etc., by means of a size separation step, i.e., a separation step based on physical characteristics such as size, shape, and / or density of the crystals, more specifically, a filtration or decantation step.

[0021] Furthermore, efficient separation of the crystals, not only from particulate and other materials, but also from the multicomponent, dark bioconversion medium, also enables isolation of (-)-ambrox from impurities, including any by-products that may be formed, which may include structural isomers and stereoisomers, such as the stereo or structural isomers referred to herein below as compounds (II), (III), and (IV), which, unlike (-)-ambrox, do not crystallize in the bioconversion medium.

[0022] Thus, the manner in which (-)-ambrox crystallizes, as well as the size separation step, allows applicants to obtain (-)-ambrox as an essentially colorless and olfactorily pure product.

[0023] Thus, in another of its aspects, the present invention provides a method for preparing a solid form of (-)-ambrox, comprising the steps of: I) forming crystalline (-)-ambrox in a bioconversion medium by means of a biocatalytic process; and II) Isolation of (-)-ambrox from biotransformation media The method includes:

[0024] The bioconversion medium from which crystalline (-)-ambrox can be separated by means of the present invention may contain all manner of impurities, e.g., particulate matter, by-products, etc. In particular, when the bioconversion is carried out using a microbial biocatalyst, crystalline (-)-ambrox can be separated from the bioconversion medium containing cells and cell debris.

[0025] In a particular embodiment of the invention, the separation step is a size separation step which not only allows the separation of crystalline material from the liquid phase(s), but also allows the separation of the crystals from any particulate matter, such as cells or cell debris, due to the physical characteristics of the crystals, such as their size, shape and / or density.

[0026] In more particular aspects, the size separation step is a filtration step, a decantation step, or a combination of filtration and decantation. The (-)-ambrox may also be pelletized instead of or in addition to filtration and / or decantation. Preferably, the (-)-ambrox crystals are melted prior to filtration and / or decantation and / or pelletization.

[0027] In a specific embodiment of the present invention, prior to the separation step, ii) the bioconversion medium can be heated to melt the (-)-ambrox crystals before being slowly cooled to recrystallize the (-)-ambrox. To this end, the bioconversion medium can be heated to a temperature of at least 55°C. Heating can be carried out gradually over a period ranging from about 15 minutes to about 2 hours, before being slowly cooled to a temperature of about 20°C or less, e.g., to 4°C, at a rate of several degrees per hour, more specifically, at a rate of about 5°C per hour. After this cooling, the bioconversion medium can be maintained at 4°C for 8 hours. The recrystallization step allows for the formation of larger and more uniform crystals, which helps to further improve the efficiency of the separation step.

[0028] Surprisingly, it was found that the addition of salts to the bioconversion medium prior to recrystallization aided crystal growth, which further increased the crystal size and produced a more uniform distribution of crystal sizes. Specific salts include inorganic salts, and in particular calcium chloride, sodium chloride, magnesium chloride, potassium chloride, and lithium chloride.

[0029] The increased size and improved uniformity of the crystals can facilitate subsequent size separation steps, as smaller or broken crystals formed during the bioconversion process are eliminated.

[0030] The size separation step can be carried out by filtration employing a filter having a mesh size large enough to allow particulate matter contained in the bioconversion medium, such as cells and cell debris, to pass through with the filtrate, but small enough to capture all or substantially all of the (-)-ambrox crystals as a retentate. The (-)-ambrox crystals can be filtered through a suitable filter bed having a mesh size between 10 and 100 microns or greater, preferably greater than about 50 microns, 60 microns, 70 microns, 80 microns, 90 microns or 100 microns.

[0031] Regarding the means for carrying out the filtration, any of the known filtration techniques and equipment can be adopted. However, preference is given to techniques that can be industrially scaled up. In this respect, centrifugal filtration is particularly preferred. Filtration centrifugation devices are widely known in the art (see, for example, Chapter 1 of "Separation and Purification Techniques in Biotechnology" by Frederick J. Dechow Noves; publication ISBN No. 0-8155-1197-3). The device may be a disc centrifuge or any of its variations, including a cylindrical centrifuge.

[0032] The filtration step can be carried out in a continuous screen centrifuge, such as a Siebtechnic H250. The mesh size can be any of the sizes mentioned hereinabove, and particularly about 100 microns or larger. For efficiency of the filtration process, the centrifuge can be operated between 500 g and 2500 g, and more particularly at about 2000 g, and even more particularly at 2028 g. The rate at which the bioconversion medium is fed into the centrifuge can be optimized to provide the most efficient filtration possible and can include rates of 300-800 kg / hr, and more particularly about 400 kg / hr, and even more particularly about 430 kg / hr.

[0033] Alternatively, the size separation step may be carried out by means of a decantation step. In the decantation process, the bioconversion medium is fed into a suitable decantation device and subjected to gravity acceleration therein. According to Stoke's Law, smaller, less dense particulate matter in the bioconversion medium, such as cells or cell debris, remains suspended in the supernatant containing other impurities of the bioconversion process, while the relatively large, dense (-)-ambrox crystals sink as sediment onto a bed provided for them. Separation can be performed statically, or it can be accelerated by applying a force of up to 6000 g, more specifically about 500-1500 g, and more specifically about 1000 g, and even more specifically 1170 g, to the bioconversion medium.

[0034] The supernatant may be removed and discarded, or alternatively, may be subjected to a further decantation step to separate and recover any crystalline (-)-ambrox remaining in the supernatant. Decantation equipment is generally known in the art. A specific equipment suitable for use in the present invention is a Guinard Decanter, e.g., Model D1LC20HC. The bioconversion medium can be fed into the equipment at any desired rate optimized to provide efficient throughput, e.g., up to 500 kg / hour, and more specifically, about 300-400 kg / hour.

[0035] Filtration and decantation may be employed individually as steps in the particle size separation process, or a combination of filtration and decantation steps may be used. The (-)-Ambrox crystals separated from the reaction medium following the size separation step may be washed to remove any particulate matter, such as cells or cell debris, and any other remaining impurities, such as by-products, solvent, unreacted substrate, etc.

[0036] Solvents useful for washing the crystals include any solvent in which (-)-ambrox is insoluble or very slightly soluble at the temperature at which the washing step is carried out. More specifically, (-)-ambrox is considered to be insoluble or very slightly soluble in solvents in which the solubility of (-)-ambrox is 10% by weight or less at 5°C.

[0037] Washing may be carried out using water, a water-miscible solvent, or a mixture thereof. Suitable water-miscible solvents include lower alkanols such as ethanol. More specifically, the crystals are washed with water, for example, 3 parts water to 1 part crystal. Multiple washings may be performed. After washing, the filtrate, or optionally the supernatant, can be discarded.

[0038] Recovery of (-)-ambrox can be achieved by its mechanical removal from a filter or decanter apparatus or belt filter (e.g., modifications 7, 8, 9, and 20), collected, and dried. In this form, (-)-ambrox can be used in perfumery applications without further purification or polishing. More typically, however, (-)-ambrox may be dissolved in a solvent and subjected to further purification steps as described in more detail below before being used in perfumery applications.

[0039] The (-)-ambrox obtained from the separation step can be dissolved in a suitable solvent, including a lower alkanol such as toluene or ethanol, for example 96% ethanol. The (-)-Ambrox solution may be subjected to a filtration step over a filter having a mesh size suitable to remove and separate from the (-)-Ambrox any remaining particulate matter, such as cells or cell debris, that may not have been removed during the particle size separation step, followed by washing. Suitable mesh sizes may start between 0.22 and 1 micron and may be up to 150 microns. Any filtration technique and device known in the art to be suitable for such purposes may be employed in accordance with the present invention.

[0040] Ethanol can be used to dissolve the crystals. The amount of ethanol used is preferably sufficient to dissolve (-)-ambrox at about 25°C, typically about 1 part crystal to 4 parts ethanol, and the solution is filtered through a 0.22 micron filter (e.g., KDS15) at 1 bar pressure and ambient temperature. In addition, it may be finally filtered through a 0.22 micron filter as a polishing filtration step.

[0041] Filtration at this stage to remove any remaining particulate matter, such as cells or cell debris, can have a deodorizing effect on (-)-Ambrox, since residual cells or cell debris, if any, can cause very unpleasant off-notes in the finished (-)-Ambrox product. Due to the fixative nature of (-)-Ambrox, the off-notes can be particularly persistent, and their removal is particularly important for the purpose of obtaining olfactorily pure (-)-Ambrox.

[0042] The (-)-ambrox can be subjected to an optional bleaching step to remove any residual color that may be present despite efficient crystal separation from the bioconversion medium. For fragrance applications, even lightly colored (-)-ambrox is undesirable as a raw material, so achieving the maximum possible clarity of the (-)-ambrox is particularly important if the product is to be valuable for fragrance applications, and particularly for fine fragrance or scented cosmetic applications.

[0043] To this end, a solution of solubilized (-)-ambrox crystals can be contacted with a bleaching agent such as activated montmorillonite clay and / or activated carbon. Suitable bleaching agents include TONSIL FF, more specifically TONSIL 412FF, and / or animal black or Norit. Optionally, as an alternative to adding bleach to a solution of (-)-ambrox, the solution can be concentrated and the solid residue distilled under reduced pressure.

[0044] The bleaching agent may be added to a refluxing solution (80-85°C) of (-)-ambrox. For this purpose, the bleaching agent may be added as a suspension in a suitable solvent, such as ethanol. Contact of the bleaching agent with the solution may be for as long as 30 minutes. The bleaching agent is then removed by filtration, for example, using a 25 micron filter.

[0045] Bleaching agents may not be effective at reducing the color of dark reaction mixtures, much less to the extent required or suitable for perfumery applications. However, due to the efficient isolation and purification of (-)-ambrox from the bioconversion medium described herein, the solution of (-)-ambrox is already substantially colorless, and it is not difficult to obtain the desired bleaching effect for this solution. As a result, despite being the product of a biocatalytic process, it is possible to obtain a form of (-)-ambrox with the desired brightness and hue expected if the product is to be employed in perfumery applications.

[0046] According to the method of the present invention, the deodorized and decolorized (-)-ambrox solution can be provided in solid form by solvent removal. Solvent removal can be effected by recrystallization or by evaporation. The recovered (-)-ambrox can be obtained in its recrystallized form or as a solid residue, which can be ground, depending on the manner in which solvent removal is carried out.

[0047] The solvent can be removed by evaporation and the remaining solids can be recovered, for example by grinding, and stored for use in perfumery applications. Alternatively, the residual solid may be dissolved in a suitable recrystallization solvent. Suitable solvents for this purpose are water-miscible alkanols such as ethanol, or mixtures of said alkanols with water. The recrystallization solvent can be heated to about 75-80°C for 15 minutes before slowly cooling to about 10-15°C. The resulting crystals can be collected by filtration and optionally dried under vacuum (e.g., 0.5 bar).

[0048] As a result of the process according to the invention, (-)-ambrox can be obtained in a solid form that is substantially colorless and olfactorily pure.

[0049] Thus, in another of its aspects, the present invention provides a substantially colorless solid form of (-)-ambrox having an L* value of 90 or greater; an a* value of less than 1 and greater than -1; and a b* value of less than 8, wherein the L*, a*, and b* values ​​represent the CIELAB L*a*b* chromaticity coordinates. The solid forms of (-)-Ambrox according to the present invention exhibit a high degree of lightness or brightness and a low degree of yellowness, which is important for perfume applications, and particularly for perfumes intended for use in fine fragrances or cosmetics, because visual aesthetics as well as odor are important and the scented product must not discolor as a result of the incorporation of the perfume ingredient.

[0050] (-)-Ambrox according to the invention has an L* value, indicating lightness, of 90 or more, and a b* value, indicating a blue-yellow hue, of 8 or less. The L* value is preferably 92 or more, or even 93 or more. The b* value is preferably 5 or less, or even 4 or less.

[0051] The L* value defines the lightness of an object and is expressed as a value between 0 and 100. An L* value of 100 indicates the brightest state (complete white), and an L* value of 0 indicates the darkest state (complete black). The b* value defines the blue-yellow hue of an object. The higher the b* value, the more yellow it is. The lower the b* value, the more blue it is.

[0052] The L* and b* values ​​can be expressed by Lab chromaticity coordinates according to the color difference display method. The L* and b* values ​​can be measured using any suitable commercially available spectrophotometer, such as a Minolta CM3500d.

[0053] The spectrophotometer should be turned on for at least one hour before measurements are taken. The glass container provided for it should be half-filled with the solid product to be measured, taking care to ensure that the bottom of the container is completely covered by the product. The filled container should then be placed in the sample stand provided for it. The sample key on the instrument should be pressed and the L*a*b* values ​​read from the display panel. Before taking any readings, the instrument should be calibrated for zero and 100% reflectance by placing the black and white objects provided for it over the instrument's optical sensor window.

[0054] In an embodiment of the present invention, the solid form of (-)-ambrox is the crystalline form characterized above. In an embodiment of the present invention, the solid form of (-)-ambrox is a biotransformation product. In an embodiment of the present invention, the solid form of (-)-ambrox is a biotransformation product formed according to the biotransformation process as described herein.

[0055] In yet another aspect of the present invention, there is provided a form of (-)-ambrox obtained by a biocatalytic process as described herein. In a more particular embodiment, the biocatalytic process is a microbial biocatalytic process. In yet another aspect of the present invention there is provided the use of a form of (-)-ambrox as defined herein in fragrance or flavour applications.

[0056] The principles, use, and implementation of the present invention may be further explained and understood with reference to the accompanying detailed description and drawings. Before describing specific embodiments of the invention in more detail, however, it should be understood that the invention is not limited in principle to the details set forth hereinafter. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description of the Invention In accordance with the present invention, the crystalline form of (-)-ambrox is obtained by a bioconversion process. The exact nature of the bioconversion (e.g., the nature of the biocatalyst used, the substrate, the reaction conditions for the bioconversion of the substrate, etc.) is not critical, provided that the conditions are such that the biocatalyst can convert the substrate to produce (-)-ambrox in a form that is crystallized in the bioconversion medium.

[0058] In an embodiment of the present invention, a substrate consisting of a mixture of 7E,3E / Z-homofarnesol undergoes a bioconversion process whereby the homofarnesol mixture is enzymatically cyclized in the presence of a recombinant microorganism expressing an enzyme, specifically a squalenehopene cyclase (SHC) biocatalyst, capable of bioconverting homofarnesol to (-)-ambrox, to produce a reaction mixture from which (-)-ambrox can be isolated in a substantially colorless and olfactorily pure form by surprisingly efficient downstream processing.

[0059] In one aspect of the present invention, there is provided an enzyme-catalyzed cyclization of homofarnesol to provide a reaction mixture comprising (-)-ambrox, wherein the homofarnesol comprises a mixture of 7E,3E / Z-geometric isomers of homofarnesol, and the reaction is carried out in the presence of a recombinant microorganism producing the enzyme, more particularly a substantially whole or intact recombinant microorganism producing the enzyme.

[0060] The cyclization reaction is carried out in the presence of an SHC biocatalyst that is capable of bioconverting homofarnesol to (-)-ambrox. The SHC biocatalyst is a wild-type or mutant enzyme, or a microorganism, preferably a recombinant E. coli microorganism, expressing a gene encoding the SHC enzyme. The SHC biocatalyst can be used in any form, including, but not limited to, purified SHC enzyme, a crude extract containing the SHC enzyme, or immobilized SHC enzyme (e.g., on a support), or the biocatalyst can be a microorganism that produced or produces SHC, such as intact recombinant whole cells and / or fragmented cells or membrane fractions containing the SHC enzyme.

[0061] In a particular embodiment of the present invention, the homofarnesol mixture is enriched in the 7E,3E-geometric isomer. In a more specific embodiment, the homofarnesol mixture is at least 55 / 45 by weight of 7E,3E / 7E,3Z. In a more specific embodiment, the homofarnesol mixture is at least 70 / 30 by weight of 7E,3E / 7E,3Z. In an even more specific embodiment, the homofarnesol mixture is at least 80 / 20 by weight of 7E,3E / 7E,3Z.

[0062] In an even more specific embodiment, the homofarnesol mixture is at least 90 / 10 by weight of 7E,3E / 7E,3Z. In an even more specific embodiment, the homofarnesol mixture is at least 95 / 5 by weight of 7E,3E / 7E,3Z. In a specific embodiment of the present invention, the homofarnesol mixture consists of the 7E,3E / Z-geometric isomer of homofarnesol, and is free of other geometric isomers.

[0063] Those skilled in the art will understand that the terms 7E, 7Z, 3E, or 3Z used in reference to homofarnesol refer to the orientation of the double bond at the 7- and 3-positions of homofarnesol, respectively. The 7E,3E-homofarnesol compound has CAS No. 459-89-2, while the 7E,3Z-homofarnesol compound has CAS No. 138152-06-4. Use of the term 7E,3E / Z-homofarnesol refers to a mixture of the compounds.

[0064] Methods for obtaining homofarnesol mixtures useful as substrates in cyclization reactions in accordance with the methods of the present invention are described in the above-referenced co-pending applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT / EP2014 / 072882 (published as WO2015 / 059290), which are incorporated herein by reference in their entireties.

[0065] In general terms, these publications describe the synthesis of homofarnesol mixtures by converting farnesene, more specifically alpha-farnesene and / or beta-farnesene, into the corresponding cyclopropanated farnesene derivatives using an organic solution of an N-alkyl-N-nitrosourea. The cyclopropanated derivatives then undergo ring-opening and rearrangement in the presence of a Bronsted acid to provide homofarnesol mixtures selective for the 7E,3E geometric isomer. The use of farnesene as the starting material is particularly preferred because it ensures that the E-configuration of the double bond at the 7-position of homofarnesol is fixed.

[0066] The particular reaction conditions which form particular embodiments of the present invention are described in the co-pending applications as well as in the examples herein below and need not be further detailed here.

[0067] The cyclization of homofarnesol to provide a reaction mixture containing (-)-ambrox can be catalyzed by a squalenehopene cyclase (SHC). The SHC can be a wild-type enzyme (e.g., SEQ ID NO: 1) or a mutant thereof (e.g., SEQ ID NO: 2 or SEQ ID NO: 4). The SHC can be obtained from Alicyclobacillus acidocaldarius, Zymomonas mobilis, or Bradyrhizobium japonicum (as described in Example 3b of US 2012 / 0135477 A1).

[0068] However, the enzymes can also be produced by recombinant means, using techniques generally known in the art. The term "recombinant" as used with respect to enzymes refers to enzymes produced by recombinant DNA techniques, i.e., produced from cells transformed with an exogenous DNA construct encoding the desired enzyme. Thus, the term "recombinant DNA" encompasses recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote (or into the genome of a homologous cell at a location other than its natural chromosomal site).

[0069] The nucleic acid molecule is operably linked to an expression control sequence that allows expression in a prokaryotic and / or eukaryotic host cell. As used herein, "operably linked" means that the expression control sequence is incorporated into a genetic construct so as to effectively control the expression of the coding sequence of interest.

[0070] The transcriptional / translational control elements referred to above include, but are not limited to, inducible and non-inducible, constitutive, cell cycle-regulated, metabolically-regulated promoters, enhancers, operators, silencers, repressors, and other elements known to those of skill in the art that drive or otherwise regulate gene expression. Such control elements include, but are not limited to, control elements that direct constitutive expression or allow inducible expression, such as, for example, the CUP-1 promoter, tet repressors employed in, for example, tet-on or tet-off systems, lac systems, and trp system control elements.

[0071] For example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of protein expression within the concentration range of 100 μM to 1.0 mM. This compound is a molecular mimic of allolactose, a lactose metabolite that initiates transcription of the lac operon, and is therefore used to induce protein expression when genes are under the control of the lac operator.

[0072] Similarly, nucleic acid molecules can form part of hybrid genes encoding additional polypeptide sequences, such as sequences that function as markers or reporters. Examples of marker and reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures involved in practicing the present disclosure, those skilled in the art will be aware of additional useful reagents, such as additional sequences that can function as markers or reporters.

[0073] The recombinant polynucleotide can encode an SHC enzyme, such as wild-type SHC or a mutant thereof, which can be inserted into a vector for expression and optional purification. One type of vector is a plasmid that represents a circular double-stranded DNA loop into which additional DNA segments can be ligated.

[0074] Some vectors can control the expression of genes to which they are operatively linked. These vectors are called "expression vectors." Expression vectors suitable for DNA recombinant technology are usually plasmid-type. Typically, expression vectors contain genes such as wild-type SHC or its mutant forms. Since plasmids are the most commonly used vector type, the terms "plasmid" and "vector" are used interchangeably herein.

[0075] Such vectors can include DNA sequences that are not naturally occurring in the host cell, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other genes or DNA sequences that one wishes to introduce into a non-recombinant host, including, but not limited to:

[0076] It will be understood that typically, the genome of a recombinant host is expanded by stable introduction of one or more recombinant genes. However, self- or replicating plasmids or vectors can also be used within the scope of the present disclosure. Furthermore, the present disclosure can be practiced using low copy number, e.g., single copy, or high copy number plasmids or vectors.

[0077] In preferred embodiments, vectors of the present disclosure include plasmids, phagemids, phages, cosmids, artificial bacterial and artificial yeast chromosomes, knockout or knock-in constructs, synthetic nucleic acid sequences, or cassettes, some of which may be produced in the form of linear polynucleotides, plasmids, megaplasmids, synthetic or artificial chromosomes such as plant, bacterial, mammalian, or yeast artificial chromosomes.

[0078] Preferably, the protein encoded by the introduced polynucleotide is produced intracellularly upon introduction of the vector. Various gene substrates can be incorporated into plasmids. The plasmids are often standard cloning vectors, such as bacterial multicopy plasmids. The substrates can be incorporated into the same or different plasmids. Often, at least two different types of plasmids with different types of selectable markers are used, allowing for the selection of cells containing at least two types of vectors.

[0079] Typically, bacteria or yeast cells can be transformed with any one or more of the following nucleotide sequences, as is well known in the art. For in vivo recombination, the gene to be recombined with the genome or other genes is used to transform the host using standard transformation techniques. In a preferred embodiment, DNA providing an origin of replication is included in the construct. The origin of replication can be appropriately selected by one skilled in the art. Depending on the nature of the gene, a supplementary origin of replication may not be required if a sequence already exists in the gene or genome that can act as an origin of replication by itself.

[0080] Bacterial or yeast cells can be transformed by foreign or heterologous DNA when such DNA is introduced inside the cell. The transforming DNA may or may not be integrated, i.e., covalently linked into the genome of the cell. In prokaryotes and yeast, for example, the transforming DNA may be maintained as an episomal element such as a plasmid.

[0081] With respect to eukaryotic cells, a stably transfected cell is one in which the transfected DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.

[0082] Generally, the introduced DNA is not originally endogenous to the host that is the recipient of the DNA, although it is within the scope of the present disclosure to isolate a DNA segment from a given host and subsequently introduce one or more additional copies of that DNA into the same host, for example, to enhance production of a gene product or alter the expression pattern of a gene. In some cases, the introduced DNA will modify or even replace an endogenous gene or DNA sequence, for example, by homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

[0083] The present disclosure also features a recombinant host. The term "recombinant host" is also referred to as "genetically modified host cell" or "transgenic cell" and refers to a host cell that contains heterologous nucleic acid or whose genome has been expanded by at least one integrated DNA sequence. The host cell of the present disclosure can be genetically modified by a polynucleotide or vector as outlined above.

[0084] Host cells that can be used for the purposes of the present disclosure include, but are not limited to, prokaryotic cells such as bacteria (e.g., Escherichia coli and Bacillus subtilis) that can be transformed by recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosome, or cosmid DNA expression vectors containing the polynucleotide molecules of the present disclosure; and simple eukaryotic cells such as yeast (e.g., Saccharomyces and Pichia) that can be transformed by recombinant yeast expression vectors containing the polynucleotide molecules of the present disclosure.

[0085] Depending on the host cell and the respective vector used to introduce the polynucleotides of this disclosure, the polynucleotides may be integrated, for example, into a chromosome or mitochondrial DNA, or may be maintained extrachromosomally, for example, in an episome, or may only be transiently contained within the cell.

[0086] The term "cell" or production cell, as used herein, particularly with reference to genetic engineering and the introduction of one or more genes or clusters of genes into a cell, is understood to refer to any prokaryotic or eukaryotic cell. Both prokaryotic and eukaryotic host cells are contemplated for use in accordance with the present disclosure, including bacterial host cells such as E. coli or Bacillus species, yeast host cells such as S. cerevisiae, insect host cells such as Spodoptora frugiperda, or human host cells such as HeLa and Jurkat.

[0087] Specifically, the cells are eukaryotic cells, preferably fungal, mammalian, or plant cells, or prokaryotic cells. Suitable eukaryotic cells include, for example, without limitation, mammalian cells, yeast cells, or helminth cells, including insect cells (including Sf9), amphibian cells (including melanophore cells), or Caenorhabditis cells (including Caenorhabditis elegans). Suitable mammalian cells include, for example, without limitation, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293 T-Rex™ cells, or other transfectable eukaryotic cell lines. Suitable bacterial cells include, without limitation, Escherichia coli.

[0088] Preferably, prokaryotes such as E. coli, Bacillus, Streptomyces, or mammalian cells such as HeLa cells or Jurkat cells, or plant cells such as Arabidopsis may be used. Preferably, the cell is an Aspergillus species or a fungal cell, which may preferably be selected from the group consisting of the genera Saccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces, Yarrowia, Pichia, and Aspergillus. Preferably, the E. coli host cell is an E. coli host cell recognized by industry and regulatory agencies (including, but not limited to, E. coli K12 host cells or E. coli BL21 host cells as demonstrated in the examples).

[0089] One preferred host cell for use in the present disclosure is Escherichia coli, which can be recombinantly prepared as described herein. Thus, the recombinant host can be a recombinant E. coli host cell. For E. coli, there is a library of available mutants, plasmids, detailed computer models of metabolism, and other information, allowing for the rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to create recombinant E. coli microorganisms.

[0090] In one embodiment, the recombinant E. coli microorganism comprises a nucleotide sequence encoding an SHC gene, or a functional equivalent / homologue thereof, including but not limited to a variant, homolog, mutant, derivative, or fragment thereof.

[0091] Another preferred host cell for use in the present disclosure is S. cerevisiae, which is widely used as a chassis organism in synthetic biology. Therefore, the recombinant host can be S. cerevisiae. For S. cerevisiae, there are libraries of available mutants, plasmids, detailed computer models of metabolism, and other information, allowing for the rational design of various modules to enhance product yield. Methods for generating recombinant S. cerevisiae microorganisms are known.

[0092] The cells are cultured in a conventional manner. The culture medium contains a carbon source, at least one nitrogen source, and inorganic salts, to which vitamins are added. The components of this medium can be those conventionally used to culture the microbial species in question.

[0093] Carbon sources used in the present methods include any molecule that can be metabolized by the recombinant host cell to promote growth and / or production of (-)-ambrox. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., found in molasses), fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose, or other glucose-containing polymers.

[0094] In embodiments employing yeast as a host, suitable carbon sources include, for example, sucrose, fructose, xylose, ethanol, glycerol, and glucose. The carbon source can be provided to the host organism throughout the culture period, or alternatively, the organism can be grown in the presence of another energy source, e.g., protein, for a period of time and then provided with the carbon source only during the fed-batch phase.

[0095] The suitability of a recombinant host cell microorganism for use in the methods of the present disclosure can be determined by simple testing procedures using well-known methods. For example, the microorganism to be tested can be grown in a rich medium (e.g., LB medium, Bactotryptone Yeast Extract medium, nutrient medium, etc.) under pH, temperature, and aeration conditions commonly used for microbial growth.

[0096] Once a recombinant microorganism (i.e., recombinant host cell) that produces the desired product of biotransformation has been selected, the product is typically produced on a large scale by a production host cell strain in a suitable expression system and fermentation, e.g., by microbial production in cell culture.

[0097] In one embodiment of the present disclosure, a defined minimal medium such as M9A is used for cell culture. The components of M9A medium include 14 g / L KH2PO4, 16 g / L K2HPO4, 1 g / L Na3.2H2O citrate, 7.5 g / L (NH4)2SO4, 0.25 g / L MgSO4.7H2O, 0.015 g / L CaCl2.2H2O, 5 g / L glucose, and 1.25 g / L yeast extract. In another embodiment of the present disclosure, a rich medium such as LB was used. The components of LB (Luria-Bertani) medium include 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl. Other examples of mineral media and M9 mineral media are described in, for example, US Pat. No. 6,524,831 B2 and and US2003 / 0092143A1.

[0098] The recombinant microorganism can be grown in a batch, fed-batch, or continuous process, or a combination thereof. Typically, the recombinant microorganism is grown in a fermentor at a defined temperature in the presence of a suitable nutrient source, e.g., a carbon source, for a desired period of time to bioconvert homofarnesol to (-)-ambrox in the desired amount.

[0099] Recombinant host cells can be cultured in any suitable manner, for example, by batch culture or fed-batch culture. As used herein, the term "batch culture" refers to a culture method in which culture medium is neither added nor removed during the culture. As used herein, the term "fed-batch" refers to a culture method in which culture medium is added during the culture, but the culture medium is not removed.

[0100] One embodiment of the present disclosure provides a method for producing (-)-ambrox in a cell-based system, the method comprising producing wild-type SHC or a variant thereof in the cell-based system under suitable conditions, feeding homofarnesol to the cell-based system, converting homofarnesol to (-)-ambrox using the wild-type SHC or a variant thereof produced using the cell-based system, recovering ambrox from the cell-based system, and isolating (-)-ambrox from the system. Expression of other nucleotide sequences can serve to enhance the method. The bioconversion method can include expression of additional other nucleotide sequences in the cell-based system. Expression of other nucleotide sequences can enhance the bioconversion pathway to make (-)-ambrox.

[0101] A further aspect of the present disclosure is a bioconversion method for making (-)-ambrox, the method comprising growing host cells containing a wild-type SHC or a variant thereof, producing the wild-type SHC or variant thereof in the host cells, supplying homofarnesol (e.g., EEH) to the host cells, incubating the host cells under appropriate pH, temperature, and solubilizing agent conditions to promote conversion of homofarnesol to ambrox, and recovering (-)-ambrox. The production of wild-type SHC or variant thereof in the host cells provides a method for making (-)-ambrox when homofarnesol is added to the host cells under suitable reaction conditions. The conversion achieved can be enhanced by adding more biocatalyst and SDS to the reaction mixture.

[0102] Recombinant host cell microorganisms can be cultured in a number of ways to provide suitable quantities of cells expressing wild-type SHC or mutant enzymes for the subsequent bioconversion step. Because applicable microorganisms for the bioconversion step vary widely (e.g., yeast, bacteria, and fungi), culture conditions will, of course, be tailored to the specific requirements of each species, which are well known and documented.

[0103] Any method known in the art for growing cells of a recombinant host microbial organism can be used to produce cells that can be used in the subsequent bioconversion step of the present disclosure. Typically, cells are grown to a certain density (as measurable as optical density (OD)) to produce sufficient biomass for the bioconversion reaction. The culture conditions selected not only affect the amount of cells (biomass) obtained, but the quality of the culture conditions also affects how well the biomass becomes a biocatalyst.

[0104] Recombinant host cell microorganisms that express the wild-type SHC or mutant gene and produce the wild-type SHC or mutant enzyme are referred to as biocatalysts that are suitable for use in bioconversion reactions. In some embodiments, the biocatalyst is a recombinant whole cell that produces the wild-type SHC or mutant, or it can be in suspension or immobilized format.

[0105] In one embodiment, the biocatalyst is produced in sufficient quantity (to generate sufficient biomass), harvested, washed (and optionally stored (e.g., frozen or lyophilized)) prior to the bioconversion step.

[0106] In a further embodiment, the cells are produced in sufficient quantities (to generate sufficient biocatalyst) and then the reaction conditions are adjusted for the bioconversion reaction without the need to harvest and wash the biocatalyst. This one-step (or "one-pot") method is advantageous because it simplifies the process while reducing costs. The culture medium used to grow the cells is also suitable for use in the bioconversion reaction if the reaction conditions are adjusted to promote the bioconversion reaction.

[0107] The disclosed bioconversion methods are carried out under time, temperature, pH, and solubilizing agent conditions to effect conversion of homofarnesol feedstock to (-)-ambrox. The pH of the reaction mixture may be within the range of 4 to 8, preferably 5 to 6.5, and more preferably 4.8 to 6.0 for SHC mutant enzymes, and within the range of about pH 5.0 to about pH 7.0 for wild-type SHC enzymes, and may be maintained by the addition of a buffer to the reaction mixture.

[0108] An exemplary buffer for this purpose is citrate buffer. Alternatively, tap water or deionized water, supplemented with or without 0.5% or 0.9% NaCl, can be used as a buffer substitute when adjusted to a pH that provides optimal biocatalytic activity, including but not limited to a pH range of 5.0 to 8.0.

[0109] Thus, in another of its aspects, the present invention provides a solid form of (-)-ambrox formed or obtainable by the bioconversion process disclosed herein, wherein the bioconversion reaction is carried out in a medium using tap water or deionized water as a buffer substitute and in a pH range that ensures optimal biocatalytic activity, preferably in the pH range of 5.0 to 8.0.

[0110] The preferred temperature is between about 15° C. and about 45° C., preferably between about 20° C. and about 40° C., but it can be higher in thermophilic organisms, up to 55° C., especially when wild-type enzymes from thermophilic microorganisms are used. The temperature may be kept constant or may be varied during the bioconversion process.

[0111] It may be useful to include a solubilizing agent (e.g., surfactant, detergent, solubility enhancer, water-miscible organic solvent, etc.) in the bioconversion reaction. Examples of surfactants include, but are not limited to, Triton X-100, Tween 80, taurodeoxycholic acid, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS).

[0112] Applicants have selected and identified SDS as a particularly useful solubilizing agent from a long list of other, less useful solubilizing agents. In particular, Applicants have identified SDS as a significantly superior solubilizing agent, for example, to Triton X-100, in terms of reaction rate and yield for the bioconversion of homofarnesol to (-)-ambrox.

[0113] Without wishing to be bound by theory, the use of SDS in recombinant microbial host cells may be advantageous because SDS may favorably interact with the host cell membrane to make the SHC enzyme (which is a membrane-bound enzyme) more accessible to the homofarnesol substrate. Additionally, the inclusion of SDS at appropriate levels in the reaction mixture may improve the properties of the emulsion (homofarnesol in water) and / or improve access of the homofarnesol substrate to the SHC enzyme within the host cell, while simultaneously preventing disruption (e.g., denaturation / inactivation of wild-type SHC or mutant enzymes).

[0114] The concentration of solubilizing agent (e.g., SDS) used in a bioconversion reaction is affected by the amount of biomass and the concentration of the substrate (EEH), i.e., there is some interdependence between the concentration of solubilizing agent (e.g., SDS), the amount of biomass, and the concentration of the substrate (EEH).

[0115] For example, as the concentration of the homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilizing agent (e.g., SDS) are required for efficient bioconversion. For example, if the solubilizing agent (e.g., SDS) concentration is too low, suboptimal homofarnesol conversion may be observed. On the other hand, if the solubilizing agent (e.g., SDS) concentration is too high, there may be a risk that the biocatalyst will be affected by either destruction of intact microbial cells and / or denaturation / inactivation of the SHC / HAC enzymes.

[0116] The selection of a suitable concentration of SDS in relation to the amount of biomass and the substrate (EEH) concentration is within the knowledge of one of ordinary skill in the art. For example, predictive models for determining suitable SDS, substrate (EEH), and biomass concentrations are available to one of ordinary skill in the art.

[0117] The temperature of the bioconversion reaction for the wild-type SHC enzyme is about 45-60°C, preferably 55°C. The pH range for the bioconversion reaction for the wild-type SHC enzyme is from about 5.0 to 7.0, more preferably from about 5.6 to about 6.2, and even more preferably about 6.0.

[0118] The temperature of the bioconversion reaction for the SHC mutant enzyme is from about 34°C to about 50°C, preferably about 35°C. The pH of the bioconversion reaction for the SHC mutant enzyme is about 4.8 to 6.4, preferably about 5.2 to 6.0.

[0119] Preferably, the solubilizing agent used in the biotransformation reaction is SDS. The [SDS] / [cell] ratio is in the range of about 10:1 to 20:1, preferably about 15:1 to 18:1, preferably about 16:1, when the ratio of biocatalyst to EEH homofarnesol is about 2:1.

[0120] The SDS concentration in the biotransformation reaction for the SHC mutant enzyme is in the range of about 1-2%, preferably in the range of about 1.4-1.7%, and even more preferably about 1.5%, when the homofarnesol concentration is about 125 g / L EEH and the biocatalyst concentration is 250 g / L (corresponding to an OD (650 nm) of about 175). The ratio of biocatalyst to EEH homofarnesol substrate is in the range of about 0.5:1 to 2:1, in some embodiments 2:1, preferably about 1:1 or 0.5:1.

[0121] In some embodiments, (-)-ambrox is produced using a biocatalyst to which a homofarnesol substrate is added. The substrate can be added by feeding using known means (e.g., a peristaltic pump, an infusion syringe, etc.). Homofarnesol is an oil-soluble compound and is provided in an oil format. Given that the biocatalyst is present in the aqueous phase, the bioconversion reaction can be considered a two-phase system when homofarnesol is added to the bioconversion reaction mixture. This is true even when a solubilizing agent (e.g., SDS) is present.

[0122] Further details of suitable bioconversion process conditions are disclosed in the examples herein below.

[0123] The bioconversion process produces a bioconversion medium containing the desired (-)-ambrox and also numerous by-products. More specifically, the medium contains, in addition to (-)-ambrox, a complex mixture of by-products, including a novel structural isomer of (-)-ambrox as set forth in formula (II), as well as known stereoisomers of (-)-ambrox as set forth in formulas (III) and (IV). [ka]

[0124] While not intending to be bound by any particular theory, applicants believe that the compound of formula (II) is formed by cyclization of the 7E,3Z-geometric isomer of homofarnesol, which has a detection threshold of >500 ng / l and is described as being virtually odorless.

[0125] As noted above, the applicant believes that the compound of formula (II) is a novel molecule and as such forms a further aspect of the present invention. Perfume ingredients and perfume compositions consisting of or comprising compound (II), and scented articles containing same, form further aspects of the present invention.

[0126] The use of compounds of formula (II) as perfume ingredients in perfume applications such as fine perfumes or functional perfume compositions such as personal care, household care and fabric care compositions forms a further additional aspect of the present invention. A mixture of (-)-ambrox and an olfactory acceptable amount of compound (II) forms yet another aspect of the present invention.

[0127] The term "olfactorily acceptable amount" as used herein in relation to the compound of formula (II), or any of the other by-products (III) or (IV), or indeed any substance present as an impurity in (-)-ambrox formed according to the process of the present invention, is understood to mean that the compound or substance is present in a mixture with (-)-ambrox in an amount below its odor detection threshold or in an amount that does not contribute to the olfactory properties of (-)-ambrox in such a way as to affect its olfactory characteristics.

[0128] (-)-Ambrox containing an olfactory acceptable amount of any such compound or substance will be identifiable to the skilled perfumer as having the odor characteristics of commercial grades of (-)-Ambrox, such as AMBROFIX™, obtained by synthetic procedures ex-sclareol and available from Givaudan.

[0129] In a preferred embodiment of the present invention, the reaction mixture is free or substantially free of unreacted homofarnesol.

[0130] Applicant has discovered that homofarnesol is a strong solvent for (-)-ambrox as well as for the by-products of the bioconversion process. Thus, in the presence of significant amounts of homofarnesol, (-)-ambrox and the by-products remain dissolved together in an intractable crude mixture, from which separation and ultimate isolation of (-)-ambrox in olfactory-pure form is difficult and time-consuming. It has been found that reducing the level of unreacted homofarnesol in the mixture of (-)-ambrox with compounds (II), (III), and (IV) significantly facilitates downstream processing and isolation / purification of (-)-ambrox.

[0131] As will be appreciated by those skilled in the art, downstream processing is a critical step in the production of useful compounds formed by bioconversion processes. As part of a compound's synthesis, it can affect the physical characteristics of that compound. In the case of preparing perfume ingredients by biotechnological methods, it is desirable to be able to separate the target compound from the reaction mixture in an olfactory-pure form so that the desired odor characteristics of the target compound are not distorted by the odor contribution of the complex mixture of impurities and by-products that may be present in the fermentation medium or biocatalyst.

[0132] Thus, the present invention provides a method for isolating and purifying (-)-ambrox from a biotransformation medium containing one or more of compounds (II), (III) and (IV). In yet another aspect of the present invention, there is provided a method for improving or enhancing the odor of (-)-ambrox, comprising the steps of separating and purifying (-)-ambrox from a bioconversion medium containing one or more of compounds (II), (III) and (IV).

[0133] In its isolated and purified form, (-)-ambrox should contain none of compounds (II), (III), or (IV), or if it contains any of said compounds, each should be present in an olfactory-acceptable amount.

[0134] The bioconversion medium obtained from the bioconversion processes described herein generally comprises a solid phase containing crude (-)-ambrox, and a liquid phase(s) consisting of water, and an oil phase which may contain any residual homofarnesol and any oily or oil-soluble impurities or by-products. One or more of by-products (II), (III), and (IV) may be present in such oil phase.

[0135] The solid phase may be separated from the liquid phase(s) by filtration, such as centrifugal filtration, or by decantation. Furthermore, and with respect to separation by filtration, it is also possible to separate the solid form of (-)-ambrox from particulate matter, such as cellular material and / or debris, in the bioconversion medium by selecting a filter with an appropriate mesh size. Decantation, like filtration, exploits the particle size difference between this particulate matter and the solid form of (-)-ambrox to separate them; the former remains suspended in the supernatant and can be discarded, while the latter can be isolated as a precipitate, recovered, and optionally subjected to further purification steps.

[0136] Once the (-)-ambrox has been separated from the particulate matter, e.g., cellular material and / or debris, and the liquid phase(s), it may be washed before being subjected to further work-up procedures to isolate the (-)-ambrox from any impurities, such as compounds (II), (III) and (IV).

[0137] In a specific embodiment of the present invention, the method for isolating and purifying (-)-ambrox comprises the step of selectively crystallizing (-)-ambrox from a mixture that may contain one or more of compounds (II), (III) or (IV) and any other impurities formed in the bioconversion medium.

[0138] The term "selectively crystallizing" refers to a process step whereby (-)-ambrox is caused to crystallize from the solvent, while by-products such as compounds (II), (III), and (IV), or any other impurities, remain dissolved in the crystallization solvent to the extent that the isolated crystalline material contains only (-)-ambrox, or, if it contains any of compounds (II), (III), or (IV), they are present only in olfactory-acceptable amounts.

[0139] Selective crystallization can occur when (-)-ambrox crystallizes from the bioconversion medium, while any impurities, such as by-products (II), (III), and (IV), that may be present in the bioconversion medium, remain in the oil phase. In such cases, any of compounds (II), (III), and (IV) present in the bioconversion medium can be separated from the crystalline (-)-ambrox by decantation and / or filtration and washing during the same process step in which (-)-ambrox is separated from any particulate matter, such as cells and cell debris.

[0140] The (-)-ambrox crystallized from the bioconversion medium can be separated by filtration and / or decantation in the manner described above. The crystals can then be dissolved in a suitable solvent, and the solution can be further processed in the manner also described above. In particular, the solution can be microfiltered to remove any remaining particulate matter, such as cells or cell debris; decolorized by passing it through a suitable bleaching agent; and / or selectively crystallized from the solvent to separate the crystalline (-)-ambrox from any residues of by-products, such as compounds (II), (III), or (IV), or any other remaining impurities.

[0141] Crystallization can be carried out in a suitable organic solvent. The choice of solvent is based on considerations such as the difference in solubility between room temperature and high temperature or boiling solvents; as well as the need for a recoverable amount of crystals in the cooled solvent. Usually, the compound to be separated is dissolved in a relatively polar solvent, and then a less polar solvent is added to bring the dissolved compound to its solubility limit, thereby initiating crystallization. In industrial processes, this is also related to issues of cost as well as handling safety. Suitable solvents include, but are not limited to, methanol, acetone, petroleum ether, hexane, t-butyl methyl ether, THF, and ethyl acetate. A preferred solvent includes ethyl alcohol. A combination of two solvents may also be used.

[0142] In a particularly preferred embodiment of the present invention, selective crystallization is carried out by dissolving a mixture containing (-)-ambrox and one or more of compounds (II), (III) and (IV) in warm methanol and selectively crystallizing (-)-ambrox by slowly adding a non-solvent such as water to a cooled ethanol solvent.

[0143] Given the close structural relationship between (-)-ambrox and the by-product compounds (II), (III), and (IV), which are each a structural isomer and two stereoisomers of (-)-ambrox, it was remarkable that (-)-ambrox could be selectively crystallized from such a mixture to provide (-)-ambrox in olfactorily pure form in high yield. One skilled in the art would reasonably expect that one or more compounds would crystallize under the same or substantially similar conditions as (-)-ambrox, making downstream processing much more complex, time-consuming, and expensive than is actually found.

[0144] The surprisingly facile manner in which (-)-ambrox can be separated from mixtures containing compounds (II), (III) and / or (IV) by crystallization represents a distinct advantage of the present invention. The ease with which (-)-ambrox could be isolated by crystallization could be contrasted with the observation that (-)-ambrox could not be recovered in such an easy manner and in such high yields from mixtures containing (II), (III) and / or (IV) by other purification techniques such as rectification or by solvent extraction because the boiling points of (-)-ambrox and the by-products (II), (III) and (IV) are so close.

[0145] The term "olfactorily pure" as used in connection with (-)-ambrox is intended to mean that the (-)-ambrox is free of compounds (II), (III), or (IV), or any other substances found in the reaction mixture, or that if such compounds or substances are present, they are present in an amount that is olfactorily acceptable as that term is defined herein.

[0146] In an embodiment of the invention, (-)-ambrox in olfactorily pure form contains less than 5% by weight of any of compounds (II), (III) or (IV). In more specific embodiments, (-)-ambrox in olfactory pure form contains less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05% by weight of each of compounds (II), (III), or (IV).

[0147] The quality of separation of (-)-ambrox by selective crystallization from a mixture containing compounds (II), (III), and / or (IV) can be affected by the composition of the mixture from which it is separated. More specifically, the quality of separation of (-)-ambrox by crystallization from a mixture of compounds (II), (III), and / or (IV) was improved when the weight ratio of (-)-ambrox to the other compounds (II), (III), and / or (IV) in the mixture was greater than 70:30, more specifically 80:20, more specifically 90:10, even more specifically 95:5, and even more specifically 97:3.

[0148] Furthermore, the quality of separation of (-)-ambrox by crystallization can be affected by the amount of unreacted homofarnesol present in the mixture from which it is separated. More specifically, the quality of separation is improved when the level of unreacted homofarnesol is less than 30% by weight, more specifically less than 20% by weight, more specifically less than 10% by weight, more specifically less than 5% by weight, and more specifically less than 3% by weight, more specifically less than 2% by weight, and more specifically less than 1% by weight, based on the weight of the mixture from which (-)-ambrox is crystallized and separated.

[0149] Preferably, the reagents and reaction conditions used in the bioconversion process of the present invention are such that the reaction proceeds to 100% or substantially 100% conversion of homofarnesol, such that no unreacted homofarnesol remains in the bioconversion medium. However, if unreacted homofarnesol is present, it can be separated from (-)-ambrox and other by-products, although this is economically disadvantageous, for example, by distillation or by washing the (-)-ambrox crystals with a suitable solvent.

[0150] Thus, in a specific embodiment of the present invention, there is provided a method for isolating and purifying (-)-ambrox from a mixture containing one or more of compounds (II), (III) and (IV), wherein the mixture is free or substantially free of homofarnesol. In a more specific embodiment, isolation and purification of (-)-ambrox from a mixture containing one or more of compounds (II), (III), and (IV) and free of, or substantially free of, homofarnesol is achieved by selective crystallization of (-)-ambrox.

[0151] The (-)-ambrox obtained by the process of the present invention is obtained in olfactory pure form. Olfactory pure (-)-ambrox forms another aspect of the present invention. The crystalline form of (-)-ambrox forms yet another aspect of the present invention.

[0152] The (-)-ambrox formed according to the methods of the present invention may be mixed with one or more additional perfume ingredients to form fragrance compositions suitable for use in perfumery products, including use in fine perfumery and consumer products such as personal care, fabric care and household care products.

[0153] Thus, in another aspect, the present invention provides a fragrance composition comprising (-)-ambrox and at least one other fragrance ingredient, said fragrance composition containing an olfactory-acceptable amount of one or more of compounds (II), (III) or (IV). [Brief explanation of the drawings]

[0154] For a better understanding of the present invention, reference is made to the accompanying drawings, in which: [Figure 1] FIG. 1 shows an X-ray diffraction pattern where the abscissa scales in degrees 2θ and the ordinate is the intensity in counts. [Figure 2] FIG. 2 shows a microscope image of the single crystal, clearly showing the elongated shape of the crystal and its length along its long dimension of over 330 microns (338.21 microns).

[0155] [Figure 3] FIG. 3 compares the relative amounts of (-)-ambrox and its isomers (II), (III), and (IV) in the bioconversion medium; the toluene extract; the crystal morphology; and the filtrate after crystal recovery. [Figure 4] Figure 4 shows a schematic of the downstream process for producing (-)-ambrox. The resulting (-)-ambrox extract can be subjected to further deodorizing or decolorizing steps, which are described in more detail herein below.

[0156] The invention will now be further illustrated with reference to the following examples. Example 1: Preparation of homofarnesol General analytical conditions: Nonpolar GC / MS: 50°C / 2 min, 20°C / min 200°C, 35°C / min 270°C. GC / MS Agilent 5975C MSD with HP 7890A Series GC system. Nonpolar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm x 0.25 mm x 12 m. Carrier gas: Helium. Injector temperature: 230°C. Split: 1:50. Flow: 1.0 ml / min. Transfer line: 250°C. MS-quad: 106°C. MS source: 230°C.

[0157] A) Preparation of MNU in THF A solution of urea (175 g, 2.9 mol) and methylamine hydrochloride (198 g, 2.9 mol) in water (400 ml) is heated to reflux (105 °C) under stirring for 3.5 hours. At 40 °C, NaNO (101 g, 1.45 mol) dissolved in water (200 ml) is added. After 15 minutes, THF (1000 ml) is added, resulting in a clear biphasic mixture. Concentrated HSO (110 g, 1.1 mol) is added at 0-5 °C and stirred within 1.5 hours. After an additional 0.5 hours at 0-5 °C, two clear phases separate at 25 °C. The organic phase (A) (1065 ml, theoretically 1.35 M) can be stored at 0-5 °C for several days or immediately proceeded to the cyclopropanation reaction vessel.

[0158] After phase separation, the aqueous phase is extracted twice with THF (2 × 1 L). This yields 1100 ml of Phase B and 1075 ml of Phase C. Phase A yields 51% conversion of the terminal alkene to cyclopropane in the subsequent cyclopropanation reaction, while Phase B yields <0.5% cyclopropane and Phase C yields no detectable conversion. We conclude that >99% of the MNU is extracted after the initial phase separation. Therefore, the aqueous phase is typically discarded after the initial phase separation (from organic Phase A) following treatment with concentrated aqueous KOH and acetic acid.

[0159] B) Preparation of E-Δ farnesene using MNU in THF [ka] 1.35 M N-methyl-N-nitrosourea in THF (136 mL, 184 mmol) was added dropwise at 0°C to a vigorously stirred mixture of E-beta-farnesene (CAS 18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 mL, 40%) at 0-5°C. After the addition of 4 mL of MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) predissolved in 0.5 mL of dichloromethane was added. The remaining MNU solution was added over 4 h at 0-5°C. GC at this stage showed 28% unconverted E-beta-farnesene, 65% of the desired monocyclopropane (shown above), and 3% of the biscyclopropanated compound 5.

[0160] After 16 h at 25°C, acetic acid (100 ml) is added at 0-5°C, followed by tert-butyl methyl ether (250 ml). After phase separation, the organic phase is washed with 2 M HCl (250 ml), and the aqueous phase is extracted with tert-butyl methyl ether (250 ml). The combined organic layers are washed with water (2 x 100 ml), 10% aqueous NaOH (2 x 100 ml), and water (2 x 100 ml), dried over MgSO, filtered, and concentrated to give 26.9 g of a slightly yellow liquid containing 9% E-beta-farnesene, 82% of the desired monocyclopropane compound, and 6% of the biscyclopropanated by-product.

[0161] The desired compound can be further isolated by distillation purification. Addition of 1 g of KCO (1 g) and distillation through a 30 cm steel coil column at 40-60 mbar yields 147 g of monocyclopropane compound (68% corr) at 135-145 °C. Pooling of fractions yields 92 g of monocyclopropane compound with 100% purity.

[0162] Analytical data for E-Δ farnesene: 1H-NMR (CDCl3, 400 MHz): 5.1 (2 m, 2 H), 4.6 (2 H), 2.2 (2 H), 2.1 (4 H), 2.0 (2 H), 1.7 (s, 3 H), 1.6 (2 s, 6 H), 1.3 (1 H), 0.6 (2 H), 0.45 (2 H) ppm. 13C-NMR (CDCl3, 400 MHz): 150.9 (s), 135.1 (s), 131.2 (s), 124.4 (d), 124.1 (d), 106.0 (t), 39.7 (t), 35.9 (t), 26.7 (t), 25.7 (q), 17.7 (q), 16.0 (d), 6.0 (t) ppm. GC / MS: 218 (2%, M+), 203 (5%, [M - 15]+), 175 (11%), 147 (31%), 134 (15%), 133 (20%), 121 (12%), 107 (55%), 95 (16%), 93 (30%), 91 (20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67 (22%), 55 (20%), 53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915 (m), 2854 (m), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018 (m), 875 (s), 819 (m), 629 (w). Analytical calculated values ​​for C16H26: C, 88.00; H, 12.00. Result: C, 87.80; H, 12.01.

[0163] C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol ((7E)-homofarnesol) A mixture of (E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane (E-Δfarnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internal standard), and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube is heated under stirring at 150° C. After 18 h and complete conversion (by GC), the mixture is poured into water (50 ml) and toluene (50 ml).

[0164] The phases are separated, and the aqueous phase is extracted with toluene (50 ml). The combined organic layers are washed with concentrated aqueous Na2CO3 (50 ml) and concentrated NaCl (2 x 50 ml), dried over MgSO4, filtered, and evaporated under reduced pressure to give a brownish resin (1.35 g). This is mixed with 30% aqueous KOH (4.3 ml) and stirred at 25 °C for 2 hours. GC analysis reveals the formation of 96% (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to an internal standard. The E / Z ratio is 68:22. The analytical data for the E isomer are consistent with those from the literature. See, for example, P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

[0165] Example 2 SHC plasmid preparation and biocatalyst production SHC plasmid preparation The gene encoding Alicyclobacillus acidocaldarius squalene-hopene cyclase (AacSHC) (GenBank M73834, Swissprot P33247) was inserted into the plasmid pET-28a(+), where it is under the control of an IPTG-inducible T7 promoter for protein production in E. coli. The plasmid was transformed into E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0166] Erlenmeyer flask culture For protein production, either rich media (LB medium) or minimal media was used, with M9 being an example of a minimal medium that has been used successfully.

[0167] Medium preparation The default minimal medium was prepared as follows for a 350 ml culture: 307 ml H2O was added to 35 ml citrate / phosphate stock (133 g / L KH2PO4, 40 g / L (NH4)2HPO4, 17 g / g citric acid and 17 g / g H2O, pH adjusted to 6.3), and the pH was adjusted to 6.8 with 32% NaOH as needed. 0.850 ml of 50% MgSO4 was autoclaved, followed by the addition of 0.035 ml of trace element solution (composition in the next section), 0.035 ml of thiamine solution, and 7 ml of 20% glucose.

[0168] SHC Biocatalyst Production (Biocatalyst Production) For small-scale biocatalyst production (wild-type SHC or SHC mutants), 350 ml cultures (medium supplemented with 50 μg / ml kanamycin) were inoculated from precultures of E. coli strain BL21(DE3) containing the SHC production plasmid. Cells were grown at 37°C with constant agitation (250 rpm) to an optical density (OD) of approximately 0.5. 650nm ) was grown.

[0169] 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. The cells were stored as pellets at 4°C or -20°C until further use. Typically, 2.5–4 grams of cells (wet weight) were obtained from 1 liter of culture, regardless of the medium used.

[0170] Fermentations were prepared and carried out in a 750 ml InforsHT reactor. 168 ml of deionized water was added to the fermentation vessel. The reactor was equipped with all necessary probes (pO2, pH, sampling, antifoam), C+N feed and sodium hydroxide bottles and autoclaved. After autoclaving, the following components were added 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.

[0171] The reaction conditions are set as follows: pH = 6.95, pO2 = 40%, T = 30°C, stirring at 300 rpm. Cascade: rpm set point at 300, min 300, max 1000, flow L / min set point 0.1, min 0, max 0.6. Antifoam control: 1:9.

[0172] From the seed culture, an OD of 0.4–0.5 650nm The fermentor was inoculated to a concentration of 0.01%. This seed culture was grown in LB medium (+ kanamycin) at 37°C and 220 rpm for 8 hours. The fermentation was initially run in batch mode for 11.5 hours, after which a C+N feed was initiated with the feed solution (sterilized glucose solution (143 ml H2O + 35 g glucose), which, after sterilization, had been supplemented with 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 / hour. The glucose and NH4 + Measurements were taken externally to assess the availability of C and N sources in the culture. Normally, glucose levels remained very low.

[0173] Cultures were grown for a total of approximately 25 hours, at which point they typically reached an OD of 40-45. 650nm SHC production was then initiated by adding IPTG to the fermentor (either as an IPTG pulse or using an injection syringe over 3-4 hours) to a final concentration of approximately 1 mM, and setting the temperature to 40°C and pO2 to 20%. Induction of SHC production continued for 16 hours at 40°C. At the end of induction, cells were collected 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.

[0174] Result 1a In general, the specific activity of the produced biocatalysts was higher when minimal medium was used compared to rich medium, all other conditions remaining unchanged. Induction was successful at 30 or 37°C. It was noted that biocatalysts with higher specific activity were obtained when induction was performed at 40-43°C.

[0175] Result 1b Table 1 below shows the culture volume, optical density, and amount of cells at both the start and end of induction, as well as the amount of biomass (wet weight) collected for the two examples.

[0176] [Table 1]

[0177] Wild-type SHC amino acid sequence (SEQ ID NO: 1) (GenBank M73834, Swissprot P33247) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0178] Mutant F601Y SHC amino acid sequence (SEQ ID NO: 2) - Mutation with respect to SEQ ID NO: 1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0179] Mutant F605W SHC nucleotide sequence (SEQ ID NO: 3)

[0180] Mutant F605W SHC amino acid sequence (SEQ ID NO: 4) - Mutation with respect to SEQ ID NO: 1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQADGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR

[0181] Example 3a Biotransformation of 7E,3E / Z-homofarnesol mixture The biotransformation was carried out using the following reaction conditions: The reaction (150.1 g total volume) was carried out in an Infors HT 750 ml fermenter containing 146 g / L total homofarnesol using a homofarnesol substrate of 86:14 7E,3E:7E,3Z mixture, 250 g / L cells (formed according to the method of Example 2, fermentation), and 1.55% SDS in 0.1 M citric acid / sodium citrate buffer, pH 5.4. The reaction was carried out at 35°C with constant agitation (900 rpm), and pH control was performed using 10 to 40% citric acid in water.

[0182] The reaction mixture was subjected to the isolation and purification steps described in Example 4 below.

[0183] Example 3b Biotransformation of 7E,3E / Z-homofarnesol mixture The biotransformation was carried out using the following reaction conditions: Reactions (total volume 2.5 ml) were carried out in 11 ml glass reaction vessels on a Heidolph Synthesis 1 apparatus in 0.1 M citric acid / sodium citrate buffer at 50°C and pH 6.0 with vigorous shaking (800 rpm). Reactions were performed with 1 g / l E,E-homofarnesol (from a homofarnesol stock with an EE:EZ ratio of 86:14) and wild-type SHC at an OD of 30 according to the method described in Example 2. 650nm The reaction mixture contained 100 ml of E,E-homofarnesol, 0.12% SDS, and 1 ml of 100 ml of E,E-homofarnesol. Approximately 48 hours after the start of the reaction, the conversion of E,E-homofarnesol was approximately 60%.

[0184] When the reaction was cooled to room temperature, Ambrofix crystals appeared upon microscopic analysis of a sample taken from the reaction mixture. 650nm Further addition of cells, equivalent to a 10 increase in HCl and an additional 24 h of incubation, allowed complete E,E-homofarnesol conversion. Microscopic observation of a sample of the reaction mixture indicated the presence of an increased number of Ambrofix crystals.

[0185] Reactions were also carried out in Infors HT 750 ml fermenters in 0.1 M citric acid / sodium citrate buffer at pH 6.0 in a total volume of 150.1 g. Reactions contained 1 g / L E,E-homofarnesol, 0.12% SDS, and wild-type SHC-producing cells at 118 g / L wet weight and were incubated at 50°C with vigorous shaking (700 rpm). Approximately 30 hours after the start of the reaction, conversion of E,E-homofarnesol was approximately 85%. Microscopic examination of a sample of the reaction mixture allowed identification of Ambrofix crystals.

[0186] Homofarnesol was again added to the equivalent of 1 g / L and the reaction was carried out for approximately another 50 hours; cells were also added to the equivalent of 32 g / L (wet weight). After a total reaction time of approximately 66 hours, the conversion of E,E-homofarnesol was approximately 85%. Microscopic examination of a sample of the reaction mixture allowed the observation of an increasing number of Ambrofix crystals.

[0187] Example 4 A typical downstream process is described below in FIG. 4 for reference. In the first step, the bioconversion medium is heated to a temperature of about 80-85°C for about 15 minutes to melt the (-)-ambrox crystals. The (-)-ambrox, which is liquid at this stage, is recrystallized by cooling the reaction medium at a rate of about 5°C per hour to a temperature of 20°C.

[0188] In the second step, after (-)-ambrox has crystallized, the crystals are separated from the bioconversion medium by filtration. Filtration is carried out in a continuous screen centrifuge (Siebtechnic H250) with a sieve size of 100 microns. The centrifuge is operated at an acceleration of 2028 G and a feed rate of 430 kg / h. Due to the significant size difference between the crystals and the cell debris, the majority of the crystals are retained on the sieve and can be washed with water and mechanically collected by means of knives provided for this purpose.

[0189] In the third step, the filtrate from the second step is fed into a continuous decanter set up to separate the cellular debris retained in the supernatant from any crystals that passed through the filter in step 2, which settle as sediment in the decanter apparatus. The decanter is operated at an acceleration of 1170 G and a feed rate of 370 kg / hr. The crystals collected in the decanter are washed with water and combined with the crystals obtained in step 2. The combined crystals are washed and statically decanted to remove any remaining cellular debris in the supernatant, and the washed crystals are prepared for further processing.

[0190] In the fourth step, the washed crystals are resolubilized with ethanol (96% technical grade) in an amount of 4 parts ethanol to 1 part crystal. The solution is filtered through a submicron filter (0.6-1.0 micron, KDS15) at ambient temperature and 1 bar pressure before being filtered again through a 0.22 micron filter.

[0191] The (-)-Ambrofix extract thus formed can be subjected to further deodorizing and decolorizing steps as described below. The ethanol solution was concentrated to dryness under vacuum. The concentrate was redissolved in industrial-grade denatured ethanol, and bleach (Tonsil 412FF) plus diatomaceous earth filter agent (CELATOM FW 50) was added under stirring. The mixture was refluxed at 80-85°C for 30 minutes under stirring before being cooled to 55-65°C. The mixture was filtered through a 25-micron filter to remove solids.

[0192] Excess denatured alcohol was removed from the clear, decolorized solution by atmospheric distillation. Water was then added to the hot solution, and the resulting mixture was stirred for 15 minutes at 75-80°C. The solution was slowly cooled to -10--15°C, allowing (-)-ambrox to crystallize. The crystallized (-)-ambrox was filtered off and dried in a vacuum oven (50-60°C; 1-5 mbar).

[0193] Example 5a Downstream processing: Comparison of solid-liquid separation and toluene extraction as a means of selectively isolating (-)-ambrox from bioconversion media. 200 ml of the inactivated bioconversion medium was extracted with MTBE and analyzed by gas chromatography.

[0194] solid-liquid separation 200 ml of inactivated bioconversion medium was centrifuged (Sorvall GS3, 5000 rpm, 10 min, 10°C) to separate the solids from the liquid phase. This resulted in an 80 ml solid pellet separating from approximately 120 ml of liquid supernatant. The supernatant was removed, extracted with MTBE, and analyzed by gas chromatography. Similarly, the pellet was extracted with MTBE, and the MTBE extract was analyzed by gas chromatography.

[0195] Toluene extraction 200 ml of bioconversion medium was extracted 6x with 45 ml toluene. The organic phase was collected and filtered to remove any cell debris. The toluene was stripped off, and the residue was dissolved in MTBE before analysis by gas chromatography.

[0196] analysis The GC analysis results are depicted below in Figure 3. The results reveal that the solid phase collected by centrifugation contained extremely high levels of (-)-ambrox and very small amounts of by-products II, III, and IV. On the other hand, the toluene extract was not enriched in (-)-ambrox compared to the crude biotransformation medium. The results suggest that (-)-ambrox is crystallized from the biotransformation medium, while structurally related compounds (II), (III), and (IV) remain in the liquid phase. The residues of (II), (III), and (IV) found in the analysis of the solid phase were residues that could be removed simply by thorough washing of the solid phase.

[0197] From this experiment it can be concluded that a particle size separation step (such as filtration and / or decantation) not only allows for the separation of solid forms of (-)-ambrox from cellular debris, but it can also be used to completely or substantially separate solid (-)-ambrox from structurally related by-products such as compounds (II), (III) and (IV).

[0198] Example 5b Sensory analysis Objective: To perform a sensory analysis of (-)-ambrox and compounds (II), (III) and (IV) formed in the crude and crystallized material. Biotransformation of E,E-homofarnesol leads to (-)-ambrox and compound (IV). Biotransformation of E,Z-homofarnesol leads to the macrocyclic ether compound (II) and the epi-ambrox compound (III). The crude mixture of (-)-ambrox contains the desired (-)-ambrox, compounds (II), (III) and (IV) present in amounts of 87.1 wt%, 2.8 wt%, 2.5 wt%, and 7.6 wt%, respectively.

[0199] When the crude mixture was selectively crystallized (laboratory scale), the crystallized material had the same constituents as the crude mixture when analyzed by gas chromatography, but present in amounts of 99.1%, 0.1%, 0.1%, and 0.7% by weight, respectively. Residues (II), (III), and (IV) are believed to be oily residues adhering to the (-)-ambrox crystals.

[0200] The sensory analysis results were as follows: (-)-Ambrox: Odor threshold 0.2ng / l. Compound (IV): Weak, IsoE, woody, GC detection threshold 5-10ng. Compound (II): "Odorless" (GC threshold >500ng). Compound (III): GC threshold approximately 10x higher than (-)-ambrox (approximately 2 ng).

[0201] conclusion Sensory analysis of the three by-products (compounds II, III, and IV) shows a weaker odor than that from (-)-ambrox. In fact, the odor of epi-ambrox (compound III) is about 10 times weaker than that of (-)-ambrox, suggesting that it is essentially odorless.

[0202] Sensory analysis demonstrated that removal of one or more by-product compounds from (-)-ambrox can improve the odor of the remaining compound (e.g., ( ) ambrox), even if the removed compound is in fact an odorless compound itself. That is, an improvement in the odor of ambrox in terms of olfactory purity, as determined by trained perfumers (using recognized benchmarks for acceptable olfactory purity), was observed in the absence of compounds II, III, and IV.

[0203] Example 6 X-ray characterization of the solid form of (-)-ambrox formed by a microbial fermentation process Powder X-ray diffraction patterns were obtained using a STOE STADI PX-ray diffractometer.

[0204] System description: The diffractometer was used in transmission mode (flat sample holder, curved germanium(III) 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 1K. Experimental parameters: Pattern measurements were performed between approximately 4° and 26° 2θ. The accuracy of the determined diffraction angles was approximately + / - 0.2° 2θ.

Claims

1. Formula (I) 【Chemistry 1】 1. A solid form of a compound represented by the formula: It exhibits an X-ray diffraction pattern having at least one of the following peaks at diffraction angles 2θ: about 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / - 0.2°; It comprises elongated crystals having an average diameter of at least about 10 to about 400 microns as measured by laser granulometry; It comprises elongated crystals having a length along their longest dimension greater than 100 microns; and / or it has an L* value of 90 or greater; an a* value less than 1 and greater than −1; and a b* value less than 8, wherein the L*, a*, and b* values ​​represent the CIELAB L*a*b* chromaticity coordinates; The solid form is characterized by at least one of the following:

2. 2. The solid form of claim 1, wherein the X-ray diffraction pattern exhibits peaks at all of the following diffraction angles 2θ: about 15.6, 16.2, 16.7, 17.0, 17.4, and 18.3 + / - 0.2 degrees.

3. 3. The solid form of claim 1 or 2, characterized by an X-ray diffraction pattern substantially as depicted in Figure 1.

4. The solid form of any one of claims 1 to 3, which is the product of a bioconversion process.

5. 5. The solid form of claim 4, wherein the bioconversion process is an enzyme-catalyzed cyclization reaction of homofarnesol, which comprises a mixture of 7E,3E and 7E,3Z geometric isomers of homofarnesol, and the reaction is carried out in the presence of a biocatalyst.

6. 7. The solid form of claim 6, wherein the biocatalyst is a recombinant microorganism expressing a gene encoding the enzyme, or an isolated enzyme, or an immobilized enzyme.

7. 7. The solid form of claim 6, wherein the enzyme is a wild-type squalenehopene cyclase or a mutant form of a wild-type squalenehopene cyclase.

8. 10. A method for preparing a solid form of (-)-ambrox as defined in any one of claims 1 to 7 formed by a bioconversion process, comprising the following steps: I) forming crystalline (-)-ambrox in a bioconversion medium by means of a biocatalytic process; and II) Separating (-)-Ambrox from the biotransformation medium The method comprising:

9. 9. The method of claim 8, wherein the separation step is a filtration step, a decantation step, or a combination of both a filtration step and a decantation step.

10. 10. The method of claim 8 or 9, wherein prior to the separation step, the bioconversion medium is heated to a temperature of at least 80°C to melt the crystalline (-)-ambrox; and then slowly cooled so that the (-)-ambrox is recrystallized from the bioconversion medium.

11. 11. The method of any one of claims 8 to 10, wherein the recovered (-)-ambrox crystals are solubilized in a solvent and, before being rendered into a solid form by removing the solvent by evaporation or recrystallization, the solution is filtered through a submicron filter to remove any residual particulate matter that was present in the bioconversion medium.

12. A solid form of (-)-ambrox formed by the method of any one of claims 8 to 11.

13. 13. The solid form of claim 12, having an L* value of 90 or greater; an a* value less than 1 and greater than −1; and a b* value less than 8, wherein the L*, a*, and b* values ​​represent CIELAB L*a*b* chromaticity coordinates.

14. Use of the solid form of (-)-ambrox according to any one of claims 1 to 7, 12 and 13 as a perfume ingredient.

15. A perfume composition comprising a solid form of (-)-ambrox as defined in any one of claims 1 to 7, 12 and 13 dissolved or dispersed in the composition.

16. 16. A household care, personal care, laundry care or air care composition comprising the fragrance composition of claim 15.

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