Solid forms of (-)-Ambroxan formed by biotransformation of homofarnesol in the presence of a biocatalyst

Abstract

A solid form of (‑)‑Ambroxan formed through a biotransformation process.

Description

[0001] The present application is a divisional application of the Chinese patent application with application number 201780024917.7, application date April 20, 2017, and invention name “Solid form of (-)-Ambroxan formed by biotransformation of homofarnesol in the presence of a biocatalyst”. Field of the Invention

[0002] The present invention relates to solid forms of (-)-ambrox and methods of preparing and purifying them. Background of the Invention

[0003] AMBROFIX TM is the proprietary trade name of Givaudan's (-)-Ambroxan, which has the formula (I)

[0004]

[0005] AMBROFIX TM AMBROFIX is an important molecule in the perfumer's palette of ingredients. It delivers an extremely powerful, highly substantial and highly stable amber note suitable for all fragrance applications. AMBROFIX is available from Givaudan TM It is the most suitable material to obtain the true ambergris scent.

[0006] Currently, AMBROFIX TM Like other commercial forms of (-)-Ambroxol, it is produced by synthetic chemistry from raw materials of natural origin. The availability and quality of certain raw materials depend on climatic conditions as well as socio-economic factors. Furthermore, since the raw materials can be extracted from natural resources in moderate quantities, their prices are likely to increase, making their use on an industrial scale uneconomical. Therefore, if AMBROFIX is to continue to be available at a reasonable cost, TM In order to achieve commercial industrial supply, a more cost-effective production and purification method that can be industrialized is needed.

[0007] An industrially scalable technological route to the bio-formation of (-)-ambroxan would be attractive as it would potentially be less complex, more environmentally friendly and more environmentally friendly than fully synthetic chemical approaches.

[0008] A potentially useful substrate for bioconversion attempts to provide (-)-Ambroxol is homofarnesol. In their seminal paper, Neumann et al. (Biol. Chem. Hoppe-Seyler, Vol. 367, pp. 723-726, (1986)) discussed the feasibility of converting homofarnesol to (-)-Ambroxol under enzymatic catalysis using an enzyme such as squalene Hopene cyclase (SHC). The homofarnesol used is a mixture of four geometric isomers of the molecule. Of the four isomers, only the 7E, 3E geometric isomer (using conventional nomenclature) can be cyclized, and then only with very low yields of the desired (-)-Ambroxol.

[0009] JP2009-60799 (Kao) discloses a synthetic method in which SHC acts on a homofarnesol substrate to produce (-)-Ambroxol. The substrate is a mixture of all four geometric isomers (3Z, 7Z; 3E, 7Z; 3Z, 7E; and 3E, 7E). The reference only discloses a method for preparing (-)-Ambroxol from homofarnesol using a liquid extract containing SHC prepared using a recombinant microorganism expressing the SHC gene. The homofarnesol mixture is converted into (-)-Ambroxol and its 9-epi stereoisomer, and can be purified by distillation or column chromatography. Kao does not describe a method in which homofarnesol is converted into (-)-Ambroxol using a substantially full or intact microorganism that produces SHC, and furthermore, it does not provide any technical guidance related to the downstream processing of the complex reaction mixture obtained by such a method that can produce (-)-Ambroxol in olfactory pure form.

[0010] To the best of the applicant's knowledge, the prior art does not describe any viable, industrially scalable process involving the SHC-catalyzed biotransformation of homofarnesol to give (-)-Ambroxan in olfactorily pure form.

[0011] Furthermore, if bioconversion of homofarnesol is to be achieved on an industrial scale, a cost-effective source of high-purity 3E,7E-homofarnesol should be available. However, although synthetic pathways for synthesizing homofarnesol are described in the literature (see, e.g., US2013 / 0273619), to the knowledge of the applicant, there is currently no cost-effective, industrial-scale source of pure 7E,3E-homofarnesol currently available.

[0012] There remains a need to provide an economically viable and industrially scalable route to the valuable fragrance ingredient (-)-Ambroxan.

[0013] In co-pending patent applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT / EP2014 / 072882 (published as WO2015 / 059290), the applicants describe an efficient process for preparing a 7E,3E / Z-homofarnesol mixture enriched in the 7E,3E geometric isomer. The 7E,3E / Z-homofarnesol mixture is prepared from β-farnesene and retains the isomeric information contained in this starting material, such that the homofarnesol at the 7-position is fixed in the E-configuration. However, even this refined chemical composition still produces a 3E / Z isomeric mixture. Pure 7E,3E-homofarnesol remains synthetically challenging and can only be achieved by purification of the isomeric mixture, which is economically disadvantageous.

[0014] Despite the developments related to the biocatalytic production of (-)-Ambroxan from homofarnesol, there remains a need to provide an efficient means of isolating and purifying (-)-Ambroxan from bioconversion media which contain particulate material and other material, such as cell debris, as well as possible by-products, unreacted substrate and any solvents or other reagents used in the bioconversion process. SUMMARY OF THE INVENTION

[0015] In addressing the deficiencies of the prior art, the applicants surprisingly discovered that crystalline forms of (-)-Ambroxol can be formed in a biocatalytic process. More specifically, the applicants discovered that crystals of (-)-Ambroxol form in a biotransformation medium.

[0016] The surprising and unexpected manner in which (-)-Ambroxan crystallizes in the bioconversion medium and the physical properties of the crystals formed are particularly relevant in terms of the isolation and purification of (-)-Ambroxan in olfactory pure form, substantially colorless and exhibiting true ambergris notes from the bioconversion medium. The finding that (-)-Ambroxan is obtained in olfactory pure quality is particularly surprising in case the biocatalyst is a microbial biocatalyst, in view of the fact that the microbial biocatalyst present in the bioconversion medium may have extremely unpleasant or even offensive off-odor characteristics. The surprising finding that (-)-Ambroxan crystals are formed outside the microbial catalyst in the bioconversion medium enables a particularly effective separation of (-)-Ambroxan from malodorous odor media.

[0017] Thus, the present invention provides in a first aspect a solid form of a compound of formula (I)

[0018]

[0019] wherein the solid form is characterized by at least one of the following features:-

[0020] It exhibits a powder X-ray diffraction pattern having at least one of the following peaks at diffraction angles 2θ of about 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / - 0.2°;

[0021] It comprises elongated crystals having an average diameter of 10-400 micrometers, more particularly 40-400, and more particularly 100-400, as determined by laser granulometry;

[0022] It comprises elongated crystals having a length along their longest dimension measured by laser granulometry of 20 to 600 microns, more particularly 40 to 500 and more particularly 100 to 400, preferably greater than 100, more particularly greater than 200 and more particularly greater than 300 microns; and

[0023] It is essentially colorless, as that term is defined hereinafter.

[0024] In a more specific embodiment, the solid form of the compound of Formula (I) is characterized by a powder X-ray diffraction pattern exhibiting the following peaks at diffraction angles 2θ of about 15.6, 16.2, 16.7, 17.0, 17.4 and 18.3 + / - 0.2°.

[0025] In a more specific embodiment, the solid form of the compound of formula (I) is characterized by being substantially as follows Figure 1 Powder X-ray diffraction pattern depicted in .

[0026] To date, to the best of the applicant's knowledge, solid forms of (-)-Ambroxan formed by biocatalytic processes have not been described or suggested in the prior art.

[0027] Thus, the present invention provides, in another aspect thereof, a solid form of the bioconversion product of formula (I).

[0028] In one embodiment of the present invention, the solid form of the bioconversion product of formula (I) has at least one of the characteristics mentioned above.

[0029] The powder x-ray diffraction data referred to above can be collected in a straightforward manner using diffractometer equipment well known in the art.The methods and instruments for measuring the powder X-ray diffraction patterns referred to herein are described in more detail in the Examples.

[0030] The shape of the crystals is determined by microscopic examination according to methods well known in the art and need not be described in further detail here.

[0031] The characteristic size and shape of a single crystal obtained according to the present invention are shown below Figure 2 middle.

[0032] The average diameter measurement of the crystals is measured by laser granulometry. Laser granulometry is a well-known technique in the art. The average particle size can be measured on any particle size analyzer known for this purpose, such as CILAS 1180 No.516 instrument. The Fraunhofer method can be used to measure according to ISO Standard 13320-1 (2009 revised edition), wherein water is used as the carrier liquid and the obscuration index (Obscuration Index) is 24.

[0033] As mentioned above, the fact that (-)-Ambroxan appears in crystalline form in the biotransformation medium as well as the physical characteristics of the crystals, namely their size and density, are particularly relevant with regard to the isolation efficiency of (-)-Ambroxan and ultimately its clarity and olfactory purity.

[0034] More specifically, the crystals can be effectively separated from the bioconversion medium including particulate material (e.g., cell debris) and any by-products, impurities, etc. by a particle separation step, which is a separation step utilizing the physical properties of the crystals (e.g., size, shape and / or density), more specifically a filtration or decantation step.

[0035] Furthermore, the efficient separation of the crystals not only from the particles and other materials but also from the multi-component and highly colored biotransformation medium enables the effective separation of (-)-Ambroxan from impurities, including any by-products that may be formed, which may include structural (constituent) isomers and stereoisomers, such as those stereo or constitutional isomers hereinafter referred to as compounds (II), (III) and (IV), which, unlike (-)-Ambroxan, do not crystallize in the biotransformation medium. Thus, as a result of the manner in which (-)-Ambroxan crystallizes and the particle isolation step, enables Applicants to obtain (-)-Ambroxan as a substantially colorless and olfactory pure product.

[0036] Therefore, the present invention provides in another aspect thereof a process for preparing a solid form of (-)-Ambroxan, the process comprising the steps of:-

[0037] I) forming crystalline (-)-Ambroxan in a bioconversion medium by a biocatalytic process; and

[0038] II) separating the crystalline (-)-Ambroxan from the biotransformation medium.

[0039] The bioconversion medium from which crystalline (-)-Ambroxan may be isolated according to the present invention may contain various impurities, such as particulate material, by-products, etc. In particular, when a microbial biocatalyst is used for the bioconversion, crystalline (-)-Ambroxan may be separated from the bioconversion medium containing cells and cell debris.

[0040] In a particular embodiment of the invention, the separation step is a particle isolation step, which allows not only the separation of the crystalline material from the liquid phase, but also the separation of the crystals from any particulate material, such as cells or cell fragments, due to the physical properties of the crystals, such as their size, shape and / or density.

[0041] In a more specific embodiment, the particle isolation step is a filtration step, a decantation step or a combination of filtration and decantation.

[0042] Alternatively or in addition to filtering and / or decanting, (-)-Ambroxol may be precipitated. Preferably, (-)-Ambroxol is melted prior to filtering and / or decanting and / or precipitating.

[0043] In a particular embodiment of the invention, prior to separation step ii), the bioconversion medium may be heated to melt the (-)-Ambrox crystals and then gradually cooled to allow recrystallization of the (-)-Ambrox. For this purpose, the bioconversion medium may be heated to a temperature of at least 55°C. The heating may be performed gradually over a period of about 15 minutes to about 2 hours and then slowly cooled to a temperature of about 20°C or lower, for example to 4°C, at a rate of a few degrees per hour, more particularly about 5°C per hour. After this cooling, the bioconversion medium is maintained at 4°C during 8 hours.

[0044] The recrystallization step enables the formation of larger and more uniform crystals, which helps to further improve the efficiency of the separation step.

[0045] Surprisingly, it was found that adding salts to the bioconversion medium prior to recrystallization aided crystal growth, which further increased the size of the crystals and produced a more uniform crystal size distribution. Specific salts include inorganic salts and in particular calcium chloride, sodium chloride, magnesium chloride, potassium chloride and lithium chloride.

[0046] The increased size and improved uniformity of the crystals can facilitate subsequent particle isolation steps, as smaller or broken crystals formed during bioconversion are eliminated.

[0047] The particle isolation step may be carried out by filtration, wherein the filter used has a mesh size large enough to allow particulate material, such as cells and cell debris, contained in the bioconversion medium to pass through the filter with the filtrate, while the mesh size is small enough to capture all or substantially all of the (-)-Ambrox crystals as the retentate.

[0048] The (-)-Ambroxol crystals may be filtered using a suitable filter bed having a mesh size of 10-100 microns or greater, preferably greater than about 50, 60, 70, 80, 90 or 100 microns.

[0049] As for the way of filtering, any known filtering technology and equipment can be used. However, it is preferred to provide an industrially scalable technology. Centrifugal filtration is particularly suitable in this regard.

[0050] Filtration centrifugation devices are widely known in the art (see, for example, "Separation and Purification Techniques in Biotechnology", Chapter 1, Frederick J. Dechow Noves; Publications ISBN No. 0-8155-1197-3. The device may be any variation thereof, including a separation plate centrifuge or a cylinder type centrifuge.

[0051] Can use continuous screen centrifuge to carry out filtering step, for example Siebtechnic H250 equipment.Screen size can be any size related to above and particularly about 100 microns or larger.Aspect the efficiency of filtering process, centrifuge can be with 500-2500g and more specifically about 2000g, still more specifically 2028g operation.Can optimize the speed of the bioconversion culture medium that feeds into centrifuge so that the possibility of most effective filtering is provided, and can comprise 300-800kg / hour and more specifically about 400kg / hour, still more specifically the speed of about 430kg / hour.

[0052] Alternatively, the particle separation step may be performed by a decantation step.

[0053] During the decantation process, the bioconversion medium is fed into a suitable decantation apparatus and subjected to gravity acceleration therein. According to Stoke's Law, smaller and less dense particulate material such as cells or cell debris in the bioconversion medium remain suspended in the supernatant containing other impurities of the bioconversion process, while relatively large and dense (-)-Ambroxol crystals settle as precipitate on a bed provided therefor. Separation can be performed statically or can be promoted by subjecting the bioconversion medium to a force of up to 6000 g, more specifically about 500-1500 g, and more specifically about 1000 g and still more specifically 1170 g.

[0054] The supernatant may be separated and discarded, or alternatively may be subjected to a further decantation step to separate and recover any crystalline (-)-Ambroxan remaining in the supernatant.

[0055] Decantation equipment is generally known in the art. A specific equipment suitable for use in the present invention is a Guinard Decanter, such as model D1LC20HC. The bioconversion medium can be fed into the equipment at any desired optimized rate to provide an effective processing capacity, such as up to 500 kg / hour and more specifically about 300-400 kg / hour.

[0056] Filtration and decantation may be used alone as steps in the particle isolation process, or a combination of filtration and decantation steps may be used. The (-)-Ambroxol crystals separated from the reaction medium of the particle isolation step may be washed to remove any residual particulate material, such as cells or cell debris, and any other residual impurities, such as by-products, solvents, unreacted substrate, etc.

[0057] The solvent used for washing the crystals includes any solvent in which (-)-Ambroxan is insoluble or extremely poorly soluble at the temperature at which the washing step is carried out. More specifically, it is considered that (-)-Ambroxan is insoluble or extremely poorly soluble in a solvent in which the solubility of (-)-Ambroxan is 10 wt% or less at 5°C.

[0058] Washing is carried out using water, a water-miscible solvent or a mixture thereof. Suitable water-miscible solvents include lower alkanols, such as ethanol.

[0059] More specifically, the crystals are washed with water, for example 3 masses of water to 1 mass of crystals. Multiple washes may be performed.

[0060] After washing, the filtrate or, as the case may be, the supernatant may be discarded.

[0061] Recovery of (-)-Ambroxan may be achieved by mechanical removal from a filter or decanting apparatus or belt filter (e.g. Modifications 7, 8, 9 and 20), collection and drying. (-)-Ambroxan may be used in this form for fragrance applications without further purification or polishing.

[0062] More typically, however, (-)-Ambroxan may be dissolved in a solvent and subjected to further purification steps as set out in more detail below before use in fragrance applications.

[0063] The (-)-Ambroxan obtained from the separation step may be dissolved in a suitable solvent. Suitable solvents include toluene or a lower alkanol, such as ethanol, for example 96% ethanol.

[0064] The (-)-Ambroxol solution may be subjected to a filtration step on a filter having a mesh size suitable for removing and separating from the (-)-Ambroxol any residual particulate material such as cells or cell debris that may not have been removed during the particle isolation step and subsequent washing process. Suitable mesh sizes may start from 0.22-1 micron and go up to 150 microns. Any filtration technique and equipment known in the art to be suitable for such purposes may be used in accordance with the present invention.

[0065] Ethanol may be used to dissolve the crystals. The amount of ethanol used is preferably sufficient to dissolve the (-)-Ambroxol at about 25°C, typically about 1 part crystals per 4 parts ethanol, and the solution is filtered through a 0.22 micron filter (e.g. KDS 15) at 1 bar pressure and ambient temperature. Additionally, a final filtration using a 0.22 micron filter may be used as a fine filtration step.

[0066] Filtering at this stage to remove any residual particulate material such as cells or cell fragments may have a deodorizing effect on the (-)-Ambroxan. This is because residual cells or cell fragments may be responsible for extremely unpleasant off-odors if retained in the final (-)-Ambroxan product. Off-odors may linger particularly due to the immobilizing nature of (-)-Ambroxan and thus their removal is particularly important for the purpose of obtaining olfactorily pure (-)-Ambroxan.

[0067] (-)-Ambroxan may be subjected to an optional bleaching step to remove any residual coloring matter which may still be present despite the effective separation of the crystals from the bioconversion medium. For perfumery applications, even slightly colored (-)-Ambroxan is undesirable as a raw material and it is therefore particularly important to purify (-)-Ambroxan to the greatest possible extent, provided that the product is valuable for perfumery applications and in particular fine perfumery applications or perfumed cosmetics.

[0068] For this purpose, the solubilized (-)-Ambroxan crystal solution may be contacted with a bleaching agent, such as activated montmorillonite clay and / or activated carbon. Suitable bleaching agents include TONSIL FF, more particularly TONSIL 412 FF and / or animal carbon black or Norit.

[0069] Optionally, as an alternative to adding a bleaching agent to the (-)-Ambroxol solution, the solution may be concentrated and the solid residue distilled under reduced pressure.

[0070] A bleaching agent may be added to the (-)-Ambroxol refluxing solution (80-85° C.). For this purpose, the bleaching agent may be added as a suspension in a suitable solvent, such as ethanol. The contact of the bleaching agent and the solution may be for a period of about 30 minutes. Thereafter, the bleaching agent is removed by filtration, for example using a 25 micron filter.

[0071] Bleaching agents may not necessarily be effective in reducing the color of a highly colored reaction mixture, and not necessarily to the extent that is desired or appropriate for a fragrance application. However, due to the efficient isolation and purification of (-)-Ambroxan from the bioconversion medium described herein, the (-)-Ambroxan solution is already substantially colorless, and obtaining the desired bleaching effect on the solution is less difficult. As a result, a form of (-)-Ambroxan can be obtained that, despite being the product of a biocatalytic process, has the desired brightness and color that is desirable if the product is to be used in a fragrance application.

[0072] According to the process of the present invention, a deodorized and decolorized (-)-Ambroxan solution can be provided in solid form by removing the solvent. The solvent removal can be achieved by recrystallization or by evaporation. Recovered (-)-Ambroxan can be obtained in a recrystallized form or as a solid residue which can be comminuted depending on the manner in which the solvent was removed.

[0073] The solvent can be removed by evaporation and the remaining solid can be recovered, for example by comminution, stored and used in fragrance applications.

[0074] Alternatively, the residual solid can be dissolved in a suitable recrystallization solvent. Suitable solvents for this purpose are alkanols miscible with water, such as ethanol, or a mixture of the alkanol and water. The recrystallization solvent can be heated to about 75-80° C. for 15 minutes and then progressively cooled to about 10-15° C. The crystals obtained can be recovered by filtration, and optionally vacuum dried (e.g., 0.5 bar).

[0075] As a result of the process of the invention, (-)-Ambroxan can be obtained in solid form which is essentially colorless and olfactorily pure.

[0076] Accordingly, the present invention provides, in another aspect thereof, a substantially colorless solid form of (-)-Ambroxol 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 chromaticity coordinates of the CIELAB system.

[0077] The solid forms of (-)-Ambroxol of the present invention exhibit high brightness or brilliance and low yellowness. This is important for fragrance applications and in particular for fine fragrances or fragrances intended for use in cosmetics, as visual beauty and fragrance are highly valuable and the perfumed product should not be discolored as a result of the introduction of the fragrance ingredient.

[0078] The (-)-Ambroxan of the present invention has an L* value indicating brightness of 90 or more and a b* value indicating a bluish-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.

[0079] The L* value is a value specifying the brightness of a substance and is represented by a value from 0 to 100. An L* value of 100 represents the lightest state (completely white) and an L* value of 0 represents the darkest state (completely black).

[0080] The b* value specifies the blue-yellow color of a substance. The larger the b* value, the yellower it is. The smaller the b* value, the bluer it is.

[0081] The L* value and the b* value can be expressed by Lab chromaticity coordinates according to the color difference indication method. The L* value and the b* value can be measured using any suitable commercially available spectrophotometer such as Minolta CM3500d.

[0082] Before taking the measurement, the spectrophotometer should be powered on for at least 1 hour. The glass container provided for this purpose 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. Thereafter, the filled container should be placed in the sample platform provided for this purpose. The sample key on the instrument should be pressed and the L*a*b* value read from the display panel.

[0083] Before taking any readings, the instrument should be calibrated for 0 and 100% reflection by placing black and white objects equipped for this purpose on the windows of the instrument's optical sensor.

[0084] In one embodiment of the present invention, the solid form of (-)-Ambroxol is the crystalline form characterized above.

[0085] In one embodiment of the present invention, the solid form of (-)-Ambroxan is a biotransformation product.

[0086] In one embodiment of the present invention, the solid form of (-)-Ambroxan is a bioconversion product formed according to a bioconversion process as described herein.

[0087] Another aspect of the present invention provides a form of (-)-Ambroxan obtainable by a biocatalytic process as described herein.

[0088] In a more specific embodiment, said biocatalytic process is a microbial biocatalytic process.

[0089] According to another aspect of the invention there is provided the use of a form of (-)-Ambroxan as defined herein in a perfume or fragrance application.

[0090] The principles, uses and embodiments of the present invention can be further illustrated and understood with reference to the accompanying detailed description and drawings. However, before explaining the specific embodiments of the present invention in more detail, it should be understood that the present invention is not limited to the details described below. DETAILED DESCRIPTION OF THE INVENTION

[0091] According to the present invention, the crystalline form of (-)-Ambroxan is obtained by a bioconversion process. The precise 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, as long as the conditions allow the biocatalyst to convert the substrate to produce (-)-Ambroxan in a crystalline form in the bioconversion medium.

[0092] In one embodiment of the present invention, a substrate consisting of a 7E,3E / Z-homofarnesol mixture is subjected to a bioconversion process whereby the homofarnesol mixture is enzymatically cyclized in the presence of a recombinant microorganism expressing an enzyme, in particular a squalene-hopaene cyclase (SHC) biocatalyst capable of bioconverting homofarnesol into (-)-ambroxol, thereby obtaining a reaction mixture from which (-)-ambroxol can be isolated in a substantially colorless and olfactorily pure form using surprisingly efficient downstream processing.

[0093] One aspect of the present invention provides an enzyme-catalyzed cyclization of homofarnesol to obtain a reaction mixture comprising (-)-ambroxol, wherein the homofarnesol comprises a 7E,3E / Z-geometric isomer mixture of homofarnesol, and wherein the reaction is carried out in the presence of a recombinant microorganism, which is a recombinant microorganism that produces an enzyme, more specifically a substantially complete or intact recombinant microorganism that produces an enzyme.

[0094] The cyclization reaction is carried out in the presence of an SHC biocatalyst capable of bioconverting homofarnesol into (-)-Ambroxan.

[0095] The SHC biocatalyst is a wild-type or variant enzyme or a microorganism expressing a gene encoding the SHC enzyme, preferably a recombinant E. coli microorganism. The SHC biocatalyst can be used in any form, such as, but not limited to, a purified SHC enzyme, a crude extract containing the SHC enzyme, or an immobilized SHC enzyme (e.g., on a carrier), or the biocatalyst can be a microorganism with a produced or producing SHC enzyme, such as intact recombinant whole cells and / or fragmented cells or membrane fractions containing the SHC enzyme.

[0096] In a specific embodiment of the present invention, the homofarnesol mixture is enriched in the 7E,3E-geometric isomer.

[0097] In a more specific embodiment, the homofarnesol mixture is at least 55 / 45 by weight 7E,3E / 7E,3Z.

[0098] In a more specific embodiment, the homofarnesol mixture is at least 70 / 30 by weight 7E,3E / 7E,3Z.

[0099] In a still more specific embodiment, the homofarnesol mixture is at least 80 / 20 by weight 7E,3E / 7E,3Z.

[0100] In a still more specific embodiment, the homofarnesol mixture is at least 90 / 10 by weight 7E,3E / 7E,3Z.

[0101] In a still more specific embodiment, the homofarnesol mixture is at least 95 / 5 by weight 7E,3E / 7E,3Z.

[0102] In a more specific embodiment, the homofarnesol mixture consists of the 7E,3E / Z-geometric isomer and is free of other homofarnesol geometric isomers.

[0103] It will be understood by those skilled in the art that the term 7E, 7Z, 3E or 3Z used in conjunction with homofarnesol refers to the orientation of the double bonds at the 7-position and 3-position 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. The use of the term 7E,3E / Z-homofarnesol refers to a mixture of compounds.

[0104] Methods for obtaining a homofarnesol mixture used as a substrate in the cyclization reaction of the process of the present invention are cited in the above-mentioned pending applications PCT / EP2014 / 072891 (published as WO2015 / 059293) and PCT / EP2014 / 072882 (published as WO2015 / 059290), which are introduced as references in their entirety. In general, they describe the synthesis of a homofarnesol mixture by converting farnesene, more specifically α-farnesene and / or β-farnesene, into its corresponding cyclopropanated farnesene derivative using an organic solution of N-alkyl-N-nitrosourea. The cyclopropanated derivative is then subjected to a ring opening and rearrangement reaction in the presence of a Bronsted acid to obtain a homofarnesol mixture, which is selective for the 7E, 3E geometric isomers. The use of farnesene as a starting material is particularly preferred because it ensures that the E-configuration of the double bond at the 7-position of homofarnesol is fixed.

[0105] Specific reaction conditions that form particular embodiments of the present invention are set forth in the co-pending applications and in the examples below and need not be described in further detail here.

[0106] The cyclization of homofarnesol to obtain a reaction mixture containing (-)-ambroxol can be catalyzed by squalene-hopene cyclase (SHC). SHC can be a wild-type enzyme (e.g., SEQ ID No. 1) or a variant thereof (e.g., SEQ ID No. 2 or SEQ ID No. 4). SHC can be obtained from Alicyclobacillus acidocaldarius (Bacillus acidocaldarius), Zymomonas mobilis, or Bradyrhizobium japonicum (as exemplified in Example 3b of US2012 / 0135477A1).

[0107] However, the enzymes may also be produced by recombinant means using techniques generally known in the art.

[0108] The term "recombinant" as used in relation to the production of an enzyme shall refer to an enzyme produced by recombinant DNA technology, i.e., produced by cells transformed with an exogenous DNA construct encoding the desired enzyme. Thus, the term "recombinant DNA" includes recombinant DNA introduced into a vector, introduced into an autonomously replicating plasmid or virus, or introduced into the genomic DNA of a prokaryote or eukaryote (or into the genome of a homologous cell at a location other than a non-native chromosomal location).

[0109] Nucleic acid molecules are operably linked to expression control sequences, thereby allowing expression in prokaryotic and / or eukaryotic host cells. As used herein, "operably linked" refers to the introduction of a genetic construct so that the expression control sequence effectively controls the expression of the coding sequence of interest. The transcription / translation regulatory elements referred to above include, but are not limited to, promoters, enhancers, operators, silencers, repressors, and other elements known to those skilled in the art and driven by inducible and non-inducible, constitutive, cell cycle regulated, metabolically regulated. Otherwise, it is to regulate gene expression. Such regulatory elements include, but are not limited to, regulatory elements that guide constitutive expression or regulatory elements that allow inducible expression, such as CUP-1 promoters, such as tet-repressors used, for example, tet-repressors in tet-on or tet-off systems, lac systems, trp system regulatory elements. As an example, isopropyl β-D-1-thiogalactopyranoside (IPTG) is an effective inducer of protein expression in a concentration range of 100 μM to 1.0 mM. This compound is a molecular mimetic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and is therefore used to induce protein expression where genes are under the control of the lac operon.

[0110] Similarly, nucleic acid molecules can form part of a hybrid gene encoding another polypeptide sequence, for example, a sequence used as a marker or reporter. Examples of markers 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 standard methods related to the implementation of the present disclosure, those skilled in the art will know other useful reagents, for example, other sequences that can function as markers or reporter molecules.

[0111] The recombinant polynucleotide may encode an SHC enzyme, such as wild-type SHC or a variant thereof, which may be inserted into a vector for expression and optional purification. One type of vector is a plasmid, which represents a circular double-stranded DNA loop into which additional DNA segments are linked. Certain vectors can control the expression of genes to which they are functionally linked. These vectors are referred to as "expression vectors". In general, expression vectors suitable for DNA recombinant techniques are of the plasmid type. Typically, an expression vector comprises a gene, such as wild-type SHC or a variant thereof. In this specification, the terms "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used type of vector.

[0112] Such vectors may include DNA sequences, which include but are not limited to DNA sequences naturally present in host cells, DNA sequences that are not usually transcribed into RNA or translated into proteins ("expressed"), and other genes or DNA sequences that are desired to be introduced into non-recombinant hosts. It is understood that, typically, the genome of the recombinant host is enhanced by the stable introduction of one or more recombinant genes. However, autonomous or replicating plasmids or vectors may also be used within the scope of the present disclosure. Moreover, the present disclosure may be implemented using low copy numbers, such as single copy or high copy number plasmids or vectors.

[0113] In a preferred embodiment, the vectors of the present disclosure comprise plasmids, phagemids, bacteriophages, cosmids, artificial bacterial and artificial yeast chromosomes, knockout or knockin constructs, synthetic nucleic acid sequences or cassettes, and a subset can be produced in the form of linear polynucleotides, plasmids, megaplasmids, synthetic or artificial chromosomes such as plant, bacterial, mammalian or yeast artificial chromosomes.

[0114] Preferably, the protein encoded by the introduced polynucleotide is produced in the cell after introduction into the vector. A variety of gene substrates can be introduced into plasmids. Plasmids are typically standard cloning vectors, such as bacterial multicopy plasmids. The substrates can be introduced into the same or different plasmids. At least two different types of plasmids with different types of selection markers are usually used to select cells containing at least two types of vectors.

[0115] Typically, bacteria or yeast cells can be transformed with any one or more of the following nucleotide sequences well known in the art. For in vivo recombination, genes with genome or other genetic recombination are used to transform the host using standard transformation techniques. In suitable embodiments, DNA providing a replication origin is included in the construct. The replication origin can be appropriately selected by the technician. Depending on the nature of the gene, if the sequence is already present in a gene or genome that can serve as a replication origin itself, a supplementary replication origin may not be needed.

[0116] When such DNA is introduced into cells, bacteria or yeast cells can be transformed by exogenous or heterologous DNA. The transforming DNA may or may not be integrated, i.e., covalently linked to the cell genome. For example, in prokaryotes and yeast, the transforming DNA may be maintained on free elements such as plasmids. For eukaryotic cells, stably transformed cells are cells in which the transfected DNA has been integrated into the chromosome, so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones composed of daughter cell groups containing the transforming DNA.

[0117] Typically, the introduced DNA is not initially resident in a host that is a recipient of the DNA, but it is also within the scope of the present disclosure to isolate a DNA fragment from a given host and subsequently introduce one or more other copies thereof, i.e., the DNA enters the same host, e.g., to enhance the production of a gene product or to alter the expression pattern of a gene. In some cases, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms, plant cells, and plants.

[0118] The present disclosure also relates to recombinant hosts. The term "recombinant host", also referred to as a "genetically modified host cell" or a "transgenic cell", refers to a host cell that contains a heterologous nucleic acid or whose genome has been enhanced by at least one introduced DNA sequence. The host cells of the present disclosure can be genetically modified using a polynucleotide or vector as outlined above.

[0119] Host cells useful for the purposes of the present disclosure include, but are not limited to, prokaryotic cells, such as bacteria (e.g., E. coli and Bacillus subtilis), which can be transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, bacterial artificial chromosomes, or cosmid DNA expression vectors containing the polynucleotide molecules of the present disclosure; simple eukaryotic cells such as yeast (e.g., Saccharomyces and Pichia), which can be transformed with, for example, recombinant yeast expression vectors containing the polynucleotide molecules of the present invention. Depending on the host cell and the corresponding vector used to introduce the polynucleotide, the polynucleotide can be integrated into, for example, the chromosome or mitochondrial DNA, or can be maintained extrachromosomally, for example, additionally, or can be only transiently contained in the cell.

[0120] The term "cell" as used herein, particularly in relation to genetic engineering and the introduction of one or more genes or assembled gene clusters into a cell or production cell, should be understood to refer to any prokaryotic or eukaryotic cell. Prokaryotic and eukaryotic host cells are contemplated for use according to the present disclosure, including bacterial host cells such as Escherichia coli or Bacillus sp, yeast host cells such as Saccharomyces cerevisiae, insect host cells such as Spodoptora frugiperda or human host cells such as HeLa and Jurkat.

[0121] Specifically, the cell is a eukaryotic cell, preferably a fungus, a mammalian or plant cell, or a prokaryotic cell. Suitable eukaryotic cells include, for example but not limited to, mammalian cells, yeast cells or insect cells (including Sf9), amphibian cells (including melanocytes), or worm cells, including Caenorhabditis cells (including Caenorhabditis elegans). Suitable mammalian cells include, for example but not limited to, COS cells (including Cos-1 and Cos-7), CHO cells, HEK293 cells, HEK293T cells, HEK293T-RexTM cells or other transfectable eukaryotic cell lines. Suitable bacterial cells include, but are not limited to, Escherichia coli.

[0122] Preferably, prokaryotes such as E. coli, Bacillus, Streptomyces or mammalian cells such as HeLa cells or Jurkat cells, or plant cells such as Arabidopsis can be used.

[0123] Preferably, the cell is an Aspergillus sp. or a fungal cell, which may preferably be selected from the group consisting of Saccharomyces, Candida, Kluyveromyces, Hansenula, Schizosaccharomyces, Yarrowia, Pichia and Aspergillus.

[0124] Preferably, the E. coli host cell is an E. coli host cell approved 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).

[0125] A preferred host cell for use with the present disclosure is E. coli, which can be recombinantly prepared as described herein. Thus, the recombinant host can be a recombinant E. coli host cell. There are mutant libraries, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing rational design of various modules to improve product yields. Methods similar to the above-mentioned Saccharomyces spp. can be used to prepare recombinant E. coli microorganisms.

[0126] In one embodiment, the recombinant E. coli microorganism comprises a nucleotide sequence encoding an SHC gene or a functional equivalent / homolog thereof, including but not limited to variants, homologous mutants, derivatives or fragments thereof.

[0127] Another preferred host cell for use with the present disclosure is Saccharomyces cerevisiae, which is a widely used substrate organism in synthetic biology. Thus, the recombinant host can be Saccharomyces cerevisiae. There are mutant libraries, plasmids, detailed computer models of metabolism and other information available for Saccharomyces cerevisiae, allowing for the rational design of various modules to improve product yields. Methods for preparing recombinant Saccharomyces cerevisiae microorganisms are known.

[0128] The cell culture is carried out in a conventional manner. The culture medium contains a carbon source, at least one nitrogen source and inorganic salts, and vitamins are added thereto. The components of the culture medium can be the components commonly used to cultivate the microorganism species.

[0129] The carbon source used in the present method includes any molecule that can be metabolized by a recombinant host cell to promote the growth and / or production of (-)-Ambroxan. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, glycerol, glucose, cellulose, starch, cellobiose or other glucose-containing polymers.

[0130] In embodiments using yeast as a host, for example, carbon sources such as sucrose, fructose, xylose, ethanol, glycerol and glucose are suitable. The carbon source can be provided to the host organism throughout the culture period, or alternatively, the organism can be grown for a period of time in the presence of another energy source (e.g., protein) and then only provided during the fed-batch phase.

[0131] The suitability of the recombinant host cell microorganism for the method of the present disclosure can be determined by using a simple test procedure of well-known methods. For example, the microorganism to be tested can be bred under the pH, temperature and aeration conditions generally used for microbial propagation in a rich culture medium (e.g., LB culture medium, Bacto-tryptone yeast extract culture medium, nutrient medium, etc.). Once the recombinant microorganism (i.e., recombinant host cell) producing the required bioconversion product is selected, the product is typically produced by an expression system and fermentation suitable for example, by the microbial production large-scale production host cell system in cell culture.

[0132] In one embodiment of the present disclosure, a defined minimal medium such as M9A is used for cell culture. The composition of M9A medium comprises: 14 g / L KH 2 PO 4 , 16g / LK 2 HPO 4 , 1g / L Na 3 Citric acid.2H 2 O, 7.5 g / L (NH 4 ) 2 SO 4 、0.25g / L MgSO 4 .7H2 O, 0.015 g / L CaCl 2 .2H 2 O, 5 g / L glucose and 1.25 g / L yeast extract).

[0133] In another embodiment of the present disclosure, a nutrient-rich medium such as LB (Luria-Bertani) is used. The ingredients of LB include: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl).

[0134] Other examples of mineral media and M9 mineral media are disclosed in, for example, US 6,524,831 B2 and US 2003 / 0092143 A1.

[0135] 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 in the presence of a suitable nutrient source (e.g., a carbon source) at a defined temperature for a desired period of time in order to bioconvert homofarnesol to the desired amount of (-)-Ambroxan.

[0136] The 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" is a culture method in which medium is neither added nor removed during the culture period. As used herein, the term "fed-batch" refers to a culture method in which medium is added during the culture period, but the medium is not removed.

[0137] One embodiment of the present disclosure provides a method for producing (-)-Ambroxan in a cell system, the method comprising producing wild-type SHC or a variant thereof in a cell system under suitable conditions, feeding homofarnesol to the cell system, converting homofarnesol to (-)-Ambroxan using SHC or a variant produced using the cell system, collecting (-)-Ambroxan from the cell system and isolating (-)-Ambroxan from the system. Expression of other nucleotide sequences can be used to enhance the method. The bioconversion method can include additional expression of other nucleotide sequences in the cell system. Expression of other nucleotide sequences can enhance the bioconversion pathway for preparing (-)-Ambroxan.

[0138] Another embodiment of the present disclosure is a bioconversion method for preparing (-)-Ambroxol, comprising growing a host cell comprising a wild-type SHC or variant gene, producing the wild-type SHC or variant enzyme in the host cell, supplying homofarnesol (e.g., EEH) to the host cell, incubating the host cell under pH, temperature, and solubilizing agent conditions suitable for promoting conversion of homofarnesol to Ambroxol, and collecting the (-)-Ambroxol. Production of the wild-type SHC or variant enzyme in the host cell provides a method for preparing (-)-Ambroxol, where homofarnesol is added to the host cell under suitable reaction conditions. The conversion rate can be increased by adding more biocatalyst and SDS to the reaction mixture.

[0139] Recombinant host cell microorganisms can be cultured in a variety of ways to provide a suitable amount of cells expressing wild-type SHC or variant enzymes for subsequent bioconversion steps. Since microorganisms suitable for bioconversion steps vary widely (e.g., yeast, bacteria, and fungi), the culture conditions can of course be adjusted according to the specific requirements of each species, and these conditions are well known and documented. Any method known in the art for culturing recombinant host cell microorganism cells can be used to produce cells that can be used in the subsequent bioconversion steps of the present disclosure. Typically, cells are grown to a specific density (measurable as optical density (OD)) to produce sufficient biomass for bioconversion reactions. The selected culture conditions not only affect the amount of cells (biomass) obtained, but the quality of the culture conditions also affects how the biomass becomes a biocatalyst. Recombinant host cell microorganisms that express wild-type SHC or variant genes and produce wild-type SHC or variant enzymes are referred to as biocatalysts, which are suitable for bioconversion reactions. In some embodiments, the biocatalyst is a recombinant whole cell that produces wild-type SHC or variant enzymes or it can be in a suspended or fixed form.

[0140] In one embodiment, the biocatalyst is produced in sufficient quantity (to generate sufficient biomass), harvested and washed (and optionally stored (eg, frozen or lyophilized)), and then subjected to the bioconversion step.

[0141] In another embodiment, the cells are produced in sufficient quantities (to produce sufficient biocatalyst), and then the reaction conditions are adjusted, without the need to harvest and wash the biocatalyst for use in the bioconversion reaction. This one-step (or "single pot") process 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, provided that the reaction conditions are adjusted to promote the bioconversion reaction.

[0142] The bioconversion process of the present disclosure is carried out under conditions of time, temperature, pH and solubilizing agent to provide conversion of homofarnesol feedstock to (-)-ambroxol. The pH of the reaction mixture can be 4-8, preferably 5-6.5, more preferably 4.8-6.0 for the SHC variant enzyme, and in the range of about pH 5.0 to about pH 7.0 for the wild-type SHC enzyme, and can be maintained by adding a buffer to the SHC enzyme. An exemplary buffer for this purpose is a citric acid 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 pH, which provides optimal biocatalyst activity, including but not limited to a pH range of 5.0 to 8.0.

[0143] Thus, another aspect of the present invention provides (-)-Ambroxan in solid form formed or obtainable by the bioconversion process disclosed herein, wherein the bioconversion reaction is carried out in a culture medium using tap water or deionized water as a buffer substitute and within a pH range that ensures optimal biocatalytic activity, preferably a pH range of 5.0-8.0.

[0144] Preferred temperatures are from about 15° C. to about 45° C., preferably from about 20° C. to about 40° C., but can be higher for thermophilic organisms, up to 55° C., especially if wild-type enzymes from thermophilic microorganisms are used. The temperature can be kept constant or can be varied during the bioconversion process.

[0145] It may be useful to include a solubilizing agent (e.g., a surfactant, a detergent, a solubility enhancer, a water-miscible organic solvent, etc.) in the biotransformation reaction. Examples of surfactants include, but are not limited to, Triton X-100, Tween 80, tauro-hydroxycholate, sodium taurodeoxycholate, sodium dodecyl sulfate (SDS), and / or sodium lauryl sulfate (SLS).

[0146] Applicants selected and identified SDS as a particularly useful solubilizing agent from a long list of other less useful solubilizing agents. Specifically, Applicants identified SDS as a better solubilizing agent than, for example, Triton X-100 in terms of reaction rate and yield of the bioconversion reaction of homofarnesol to (-)-Ambroxol.

[0147] Without wishing to be bound by theory, the use of SDS with recombinant microbial host cells may be advantageous because SDS can advantageously interact with the host cell membrane to make the SHC enzyme (which is a membrane-bound enzyme) more accessible to the homofarnesol substrate. In addition, 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 preventing disruption (e.g., denaturation / inactivation of wild-type SHC or variant enzymes).

[0148] The concentration of the solubilizing agent (e.g., SDS) used in the bioconversion reaction is affected by the amount of biomass and the concentration of the substrate (EEH). That is, there is a certain degree of interdependence between the concentration of the solubilizing agent (e.g., SDS), the amount of biomass, and the concentration of the substrate (EEH). As an example, as the concentration of the homofarnesol substrate increases, sufficient amounts of biocatalyst and solubilizing agent (e.g., SDS) are required to carry out an effective bioconversion reaction. For example, if the concentration of the solubilizing agent (e.g., SDS) is too low, suboptimal homofarnesol conversion may be observed. On the other hand, for example, if the concentration of the solubilizing agent (e.g., SDS) is too high, there may be a risk that the biocatalyst is affected by the destruction of intact microbial cells and / or the denaturation / inactivation of the SHC / HAC enzyme.

[0149] It is within the knowledge of those skilled in the art to select a suitable concentration of SDS in the context of the amount of biomass and the concentration of substrate (EEH). For example, those skilled in the art can obtain a predictive model to determine suitable SDS, substrate (EEH) and biomass concentrations.

[0150] The temperature of the bioconversion reaction of the wild-type SHC enzyme is about 45-60°C, preferably 55°C.

[0151] The pH range of the bioconversion reaction of the wild-type SHC enzyme is about 5.0 to 7.0, more preferably about 5.6 to about 6.2, even more preferably about 6.0.

[0152] The temperature of the bioconversion reaction of the SHC variant enzyme is about 34°C to about 50°C, preferably about 35°C.

[0153] The pH of the bioconversion reaction of the SHC variant enzyme is about 4.8-6.4, preferably about 5.2-6.0.

[0154] Preferably, the solubilizing agent used in the bioconversion reaction is SDS.

[0155] When the ratio of biocatalyst to EEH homofarnesol is about 2:1, the ratio of [SDS] / [cells] is about 10:1-20:1, preferably about 15:1-18:1, preferably about 16:1.

[0156] When the homofarnesol concentration is about 125 g / l EEH and the biocatalyst concentration is 250 g / l (corresponding to an OD of about 175 (650 nm)), the SDS concentration in the bioconversion reaction is about 1-2%, preferably about 1.4-1.7%, even more preferably about 1.5% for the SHC variant enzyme.

[0157] The ratio of biocatalyst to EEH homofarnesol substrate is in the range of about 0.5:1-2:1, in some embodiments 2:1, preferably about 1:1 or 0.5:1.

[0158] In some embodiments, (-)-Ambroxan is produced using a biocatalyst to which a homofarnesol substrate is added. The substrate can be added by feeding using known methods (e.g., peristaltic pumps, infusion syringes, etc.). Homofarnesol is an oil-soluble compound and is provided in the form of an oil. Given that the biocatalyst is present in the aqueous phase, when homofarnesol is added to the bioconversion reaction mixture, the bioconversion reaction can be considered a two-phase system. This is true even in the presence of a solubilizing agent (e.g., SDS).

[0159] Further details of suitable bioconversion process conditions are disclosed in the Examples listed below.

[0160] The bioconversion process produces a bioconversion medium containing the desired (-)-Ambroxan as well as a number of by-products. More particularly, in addition to (-)-Ambroxan, the medium also contains a complex mixture of by-products including the novel (-)-Ambroxan structural isomer of formula (II) and the known stereoisomers of (-)-Ambroxan of formula (III) and (IV)

[0161]

[0162] Applicants believe, although not wishing to be bound by any particular theory, that the compound of formula (II) is formed by cyclization of the 7E,3Z-geometric isomer of homofarnesol. It is described as being virtually odorless with a detection threshold of >500 ng / l.

[0163] As stated above, the applicants believe that the compounds of formula (II) are novel molecules and therefore form a further aspect of the present invention.

[0164] Perfume ingredients and perfume compositions consisting of or comprising compound (II), and perfume articles containing them form further aspects of the invention.

[0165] The use of compounds of formula (II) as perfume ingredients in perfume applications, such as fine perfume or functional perfume compositions, such as personal care, home care and fabric care compositions, forms a further aspect of the present invention.

[0166] Mixtures of (-)-Ambroxol and an olfactorily acceptable amount of Compound (II) form a further aspect of the invention.

[0167] The term "olfactory acceptable amount" as used herein in relation to the compound of formula (II) or any other by-product (III) or (IV) or indeed any material which may be present as an impurity in the (-)-Ambrox formed according to the process of the present invention is to be understood as the presence of the compound or material in a mixture with (-)-Ambrox in an amount below its odour detection threshold or in an amount which does not contribute to the olfactory characteristics of (-)-Ambrox in a manner which affects the olfactory characteristics of (-)-Ambrox. (-)-Ambrox containing any such compound or material in an olfactory acceptable amount may be recognised by a skilled perfumer as having the properties of a commercial grade of (-)-Ambrox such as AMBROFIX TM Odor characteristics, AMBROFIX TM Ex-sclareol is obtained by synthetic methods and is available from Givaudan.

[0168] In a preferred embodiment of the present invention, the reaction mixture is free or substantially free of unreacted homofarnesol.

[0169] Applicants have discovered that homofarnesol is an effective solvent for (-)-Ambroxol as well as the by-products of the above-mentioned bioconversion process. Thus, in the presence of appreciable amounts of homofarnesol, (-)-Ambroxol and the by-products remain dissolved together in a crude, intractable mixture from which separation and ultimate isolation of (-)-Ambroxol in olfactory pure form is difficult, time-consuming and expensive. It has been found that reducing the level of unreacted homofarnesol in a mixture with (-)-Ambroxol and compounds (II), (III) and (IV) significantly facilitates downstream processing and separation / purification of (-)-Ambroxol.

[0170] As will be appreciated by those skilled in the art, downstream processing is a key operation in the preparation of useful compounds formed by bioconversion processes. As part of the synthesis of a compound, it can affect the physical properties of the compound. In the case of fragrance ingredients prepared by biotechnological methods, it is desirable that the target compound can be isolated 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 a complex mixture of contaminants and by-products that may be present in the fermentation medium or the biocatalyst.

[0171] Thus, the present invention provides a method for isolating and purifying (-)-Ambroxan from a biotransformation medium comprising one or more of compounds (II), (III) and (IV).

[0172] Another aspect of the present invention provides a method for improving or enhancing the flavor of (-)-Ambroxan, the method comprising the steps of isolating and purifying (-)-Ambroxan from a bioconversion medium comprising one or more of compounds (II), (III) and (IV).

[0173] In isolated and purified form, (-)-Ambroxan will not contain any of compounds (II), (III) or (IV), or, if it does contain any of said compounds, each will be present in an olfactorily acceptable amount.

[0174] The bioconversion medium obtained from the bioconversion process described above typically comprises a solid phase containing crude (-)-Ambroxan and a liquid phase consisting of water and an oil phase, the oil phase may contain any residual homofarnesol and any other oily or oil-soluble impurities or by-products. One or more of the by-products (II), (III) and (IV) may be present in such an oil phase.

[0175] The solid phase may be separated from the liquid phase by filtration, such as centrifugal filtration or decantation. In addition, with respect to separation by filtration, by selecting a filter with an appropriate mesh size, the solid form of (-)-Ambroxol may also be separated from particulate material, such as cellular material and / or debris, in the bioconversion medium. Decantation, similar to filtration, exploits the difference in particle size between such particulate matter and the solid form of (-)-Ambroxol to allow their separation, with the former remaining suspended in the supernatant, which may be discarded, while the latter may be separated as a precipitate to be recovered and optionally subjected to further purification steps.

[0176] Once (-)-Ambroxan is separated from the particulate material (e.g. cellular material and / or debris) and the liquid or aqueous phase, it may be washed before further work-up procedures to separate (-)-Ambroxan from any remaining impurities such as compounds (II), (III) and (IV).

[0177] In a particular embodiment of the present invention, the method for isolating and purifying (-)-Ambroxan comprises the step of selectively crystallizing (-)-Ambroxan from a mixture which may contain one or more of compounds (II), (III) or (IV) and any other impurities formed in the biotransformation medium.

[0178] The phrase "selective crystallization" refers to a process step in which (-)-Ambroxan is crystallized from a solvent while by-products, such as compounds (II), (III) and (IV) or any other impurities are dissolved in the crystallization solvent, to the extent that the isolated crystalline material contains only (-)-Ambroxan, or if it contains any by-products, such as compounds (II), (III) or (IV), or any other impurities, they are present only in olfactory acceptable amounts.

[0179] When (-)-Ambroxol is crystallized from the bioconversion medium, selective crystallization may occur, while any impurities that may be present in the bioconversion medium, such as by-products (II), (III) or (IV), remain in the oil phase. In this case, any compounds (II), (III) and (IV) present in the bioconversion medium may be separated from the crystallized (-)-Ambroxol by decantation and / or filtration and washing in the same process steps as the separation of (-)-Ambroxol from any particulate matter, such as cells and cell debris.

[0180] The (-)-Ambroxan crystallized from the bioconversion medium may be isolated by filtration and / or decantation in the manner described above. Thereafter, the crystals may be dissolved in a suitable solvent and the solution further processed also in the manner described above. In particular, the solution may be microfiltered to remove any residual particulate material 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 (-)-Ambroxan from any residues of by-products such as compounds (II), (III) or (IV) or any other residual impurities.

[0181] Crystallization can be carried out in a suitable organic solvent. The choice of solvent is based on a variety of considerations, such as the difference in solubility at room temperature and at elevated temperatures or in boiling solvents; and the need to recover a large amount of crystals in a cold solvent. Typically, the compound to be separated is dissolved in a relatively polar solvent, and then a relatively less polar solvent is added to allow the dissolved compound to reach its solubility limit, at which point crystallization begins. In addition, in industrial processes, cost issues and handling safety are also concerns. Suitable solvents include, but are not limited to, methanol, acetone, petroleum ether, hexane, tert-butyl methyl ether, THF and ethyl acetate. Preferred solvents include ethanol. Solvent pairs can also be used.

[0182] In a particularly preferred embodiment of the present invention, the selective crystallization is carried out by dissolving a mixture containing (-)-Ambroxan and one or more compounds (II), (III) and (IV) in warm ethanol and allowing (-)-Ambroxan to selectively crystallize by slowly adding a non-solvent (such as water) to the cooled ethanol solution.

[0183] Given the close structural relatedness of (-)-Ambroxol to the byproduct compounds (II), (III) and (IV), which are respectively a constitutional isomer and two stereoisomers of (-)-Ambroxol, it is noteworthy that (-)-Ambroxol can be selectively crystallized from this mixture to provide (-)-Ambroxol in olfactory pure form and in high yield. One skilled in the art would reasonably expect that one or more of the compounds would crystallize under the same or substantially similar conditions as (-)-Ambroxol, making downstream processing far more complicated, time consuming and expensive than is found.

[0184] A particular advantage of the present invention is that (-)-Ambroxol can be isolated in a surprisingly simple manner from a mixture containing compounds (II), (III) and / or (IV) by crystallization.

[0185] The ease with which (-)-Ambroxan can be isolated by crystallization can be contrasted with the observation that (-)-Ambroxan cannot be recovered in such a simple manner and in such high yields from a mixture comprising (II), (III) and / or (IV) by other purification techniques, for example by distillation (due to the very similar boiling points of (-)-Ambroxan and the by-products (II), (III) and (IV)) or by solvent extraction.

[0186] The term "olfactorily pure" in relation to (-)-Ambroxan means that (-)-Ambroxan is free of compounds (II), (III) or (IV) or any other material in the reaction mixture, or if such compounds or materials are present, they are present in olfactorily acceptable amounts, as such terms are defined herein.

[0187] In one embodiment of the invention, (-)-Ambroxan in olfactory pure form comprises less than 5% by weight of any of compounds (II), (III) or (IV).

[0188] In more specific embodiments, (-)-Ambroxan in olfactory pure form comprises 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).

[0189] The quality of (-)-Ambroxan separated from a mixture of compounds (II), (III) and / or (IV) by selective crystallization may be affected by the composition of the mixture from which they are separated. More specifically, the quality of the mixture of (-)-Ambroxan separated from compounds (II), (III) and / or (IV) by crystallization is improved when the weight ratio of (-)-Ambroxan to the other compounds (II), (III) and (IV) in the mixture is greater than 70:30, more particularly 80:20, more particularly 90:10, more particularly 95:5, more particularly 97:3.

[0190] Furthermore, the quality of the separation of (-)-Ambroxol by crystallization may be affected by the amount of unreacted homofarnesol present in the separated mixture. More particularly, the separation quality is improved when the level of unreacted homofarnesol is below 30% by weight, more particularly below 20% by weight, more particularly below 10% by weight, still more particularly below 5% by weight and still more particularly below 3% by weight, still more particularly below 2% by weight and still more particularly below 1% by weight, based on the weight of the mixture from which (-)-Ambroxol is crystallized.

[0191] Preferably, the reagents and reaction conditions used in the bioconversion process of the present invention are such that the reaction is carried out with 100% homofarnesol conversion, or substantially so, so that no unreacted homofarnesol is left in the bioconversion medium. However, if unreacted homofarnesol is present, although it is economically disadvantageous, it can be separated from (-)-Ambroxan and other by-products, for example, by distillation or by washing the crystals of (-)-Ambroxan with a suitable solvent.

[0192] Therefore, in a particular embodiment of the present invention, there is provided a method for isolating and purifying (-)-Ambroxan from a mixture comprising one or more of compounds (II), (III) and (IV), which mixture is free or substantially free of homofarnesol.

[0193] In a more specific embodiment, the separation and purification of (-)-Ambroxan from a mixture comprising one or more of compounds (II), (III) and (IV) and free or substantially free of homofarnesol is carried out by selective crystallization of (-)-Ambroxan.

[0194] The (-)-Ambroxan obtained according to the process of the present invention is obtained in olfactory pure form. Odorlessly pure (-)-Ambroxan forms a further aspect of the present invention.

[0195] (-)-Ambroxan in crystalline form forms a further aspect of the present invention.

[0196] The (-)-Ambroxan formed according to the process of the present invention may be mixed with one or more additional perfume ingredients to form a perfume composition for use in fragrance applications, including fine perfumes, and in consumer products such as personal care, fabric care and home care products.

[0197] Thus, the present invention provides, in another aspect thereof, a fragrance composition comprising (-)-Ambroxan and at least one further fragrance ingredient, wherein the fragrance composition comprises an olfactorily acceptable amount of one or more of compounds (II), (III) or (IV). BRIEF DESCRIPTION OF THE DRAWINGS

[0198] For a better understanding of the present invention, reference is made to the accompanying drawings, in which:-

[0199] Figure 1 X-ray diffraction patterns are shown, with the abscissa scale being 2-theta degrees and the ordinate being intensity in counts.

[0200] Figure 2 A microscopic image of a single crystal is shown which clearly shows the elongated shape of the crystal and a length of over 330 microns (338.21 microns) along its long dimension.

[0201] Figure 3 The relative amounts of (-)-Ambroxan and its isomers (II), (III) and (IV) in the biotransformation medium; the toluene extract; the crystalline form; and the filtrate after crystal collection were compared.

[0202] Figure 4 An overview of the downstream processes leading to (-)-Ambroxan is presented. The (-)-Ambroxan extract obtained may be subjected to further deodorization or decolorization steps, as described in more detail below.

[0203] The invention is further illustrated with reference to the following examples.

[0204] Embodiment 1:

[0205] Preparation of Homofarnesol

[0206] General analysis conditions:

[0207] Non-polar GC / MS: 50°C / 2min, 20°C / min 200°C, 35°C / min 270°C. GC / MS Agilent 5975C MSD with HP 7890A Series GC system. Non-polar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22mm x0.25mm x 12m. Carrier gas: Helium. Injector temperature: 230°C. Split ratio 1:50. Flow rate: 1.0ml / min. Transfer line: 250°C. MS-quadrupole: 106°C. MS-source: 230°C.

[0208] A) Preparation of MNU in THF

[0209] A solution of urea (175 g, 2.9 mol) and methylamine hydrochloride (198 g, 2.9 mol) in water (400 ml) was heated to reflux (105 °C) for 3.5 h with stirring. NaNO dissolved in water (200 ml) was added at 40 °C. 2 (101 g, 1.45 mol). After 15 min, THF (1000 ml) was added to give a clear 2-phase mixture. Concentrated H 2 SO 4 (110 g, 1.1 mol) and stirred for 1.5 h. After another 0.5 h at 0-5 ° C, the two clear phases were separated at 25 ° C. The organic phase (A) (1065 ml, theoretical 1.35 M) was stored at 0-5 ° C for a few days or immediately transferred to the cyclopropanation reactor.

[0210] After phase separation, the aqueous phase was extracted twice with THF (2 x 1 l). This gave 1100 ml of phase B and 1075 of phase C. While phase A gave 51% conversion of the terminal olefin to cyclopropane in the subsequent cyclopropanation reaction, phase B gave <0.5% cyclopropane and phase C gave no detectable conversion. We conclude that >99% of MNU was extracted after the first phase separation. Therefore the aqueous phase was usually discarded after the first phase separation (from the organic phase A) after treatment with concentrated KOH and acetic acid.

[0211] B) Preparation of E-Δ-farnesene using MNU in THF

[0212]

[0213] At 0°C, N-methyl-N-nitrosourea 1.35M THF solution (136 ml, 184 mmol) was added dropwise to a rapidly stirred mixture of E-β-farnesene (CAS 18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 ml, 40%) at 0-5°C. After adding 4 ml of MNU solution, Pd(acac) pre-dissolved in 0.5 ml of dichloromethane was added. 2 (7.4 mg, 0.024 mmol, 0.02%). The remaining MNU solution was added over 4 hours at 0-5°C. GC at this stage showed 28% unconverted E-β-farnesene, 65% of the desired monocyclopropane (as shown above) and 3% of the dicyclopropanated compound 5. After 16 h at 25°C, acetic acid (100 ml) was added at 0-5°C, followed by tert-butyl methyl ether (250 ml). After phase separation, the organic phase was washed with 2M HCl (250 ml) and the aqueous phase was extracted with tert-butyl methyl ether (250 ml). The combined organic layers were washed with water (2×100 ml), 10% aqueous NaOH (2×100 ml) and water (2×100 ml), filtered through MgSO 4 Drying, filtration and concentration gave 26.9 g of a light yellow liquid containing 9% E-β-farnesene, 82% of the desired monocyclopropane compound and 6% of a dicyclopropanated byproduct.

[0214] The desired compound can be further isolated by purification by distillation.

[0215] Add 1g K 2 CO 3 (1 g) and distilled at 135-145°C at 40-60 mbar on a 30 cm steel coil column to give 147 g of monocyclopropane compound (68% corr). Pooling of fractions gave 92 g of monocyclopropane compound with a purity of 100%.

[0216] Analytical data of E-Δfarnesene:

[0217] 1 H-NMR (CDCl 3 , 400MHz): 5.1 (2m, 2H), 4.6 (2H), 2.2 (2H), 2.1 (4H), 2.0 (2H), 1.7 (s, 3H), 1.6 (2s, 6H), 1.3 (1H), 0.6 (2H), 0.45 (2H)ppm. 13C-NMR(CDCl 3, 400MHz): 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%), 9 5 (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). Calculated value C 16 H 26 : C, 88.00; H, 12.00. Found: C, 87.80; H, 12.01.

[0218] C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-triene-1-ol ((7E)-homofarnesol)

[0219] A mixture of (E)-(6,10-dimethylundecyl-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 was heated at 150 ° C with stirring. After 18 h and complete conversion (according to GC), the mixture was poured into water (50 ml) and toluene (50 ml). The phases were separated and the aqueous phase was extracted with toluene (50 ml). The combined organic layers were washed with concentrated Na 2 CO 3 (50 ml) and concentrated NaCl (2 x 50 ml), washed with MgSO 4 Drying, filtration and evaporation under reduced pressure gave a brown resin (1.35 g), which was mixed with 30% aqueous KOH solution (4.3 ml) and stirred at 25° C. for 2 h. GC analysis showed 96% formation of (7E)-4,8,12-trimethyltrideca-3,7,11-triene-1-ol according to the internal standard. E / Z ratio 68:22. The analytical data of the E-isomer were consistent with those in the literature, see, for example, P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

[0220] Example 2

[0221] SHC plasmid preparation and biocatalyst production

[0222] SHC plasmid preparation

[0223] The gene encoding the squalene-hopaene cyclase (AacSHC) of Alicyclobacillus acidocaldarius (GenBank M73834, Swissprot P33247) was inserted into the plasmid pET-28a(+) where it was used for protein production in E. coli under the control of the IPTG-inducible T7-promoter. The plasmid was transformed into the E. coli strain BL21(DE3) using a standard heat shock transformation protocol.

[0224] Erlenmeyer flask culture

[0225] For protein production, rich medium (LB medium) or minimal medium is used. M9 is an example of a minimal medium, which has been successfully used.

[0226] Medium preparation

[0227] The minimal medium selected as default was prepared for a 350 ml culture as follows: 35 ml of a citric acid / phosphate stock solution (133 g / l KH 2 PO 4 , 40g / l(NH 4 ) 2 HPO 4 , 17g / g citric acid.H 2 O, pH adjusted to 6.3) and 307 ml H 2 O, adjust pH to 6.8 with 32% NaOH as needed. After autoclaving, add 0.850 ml 50% MgSO 4 , 0.035 ml trace element solution (composition in the next section), 0.035 ml thiamine solution and 7 ml 20% glucose.

[0228] SHC Biocatalyst Production (biocatalyst production)

[0229] For small-scale biocatalyst production (wild-type SHC or SHC variants), 350 ml culture (medium supplemented with 50 μg / ml kanamycin) was inoculated from a preculture of E. coli strain BL21 (DE3) containing the SHC production plasmid. The cells were grown at 37 °C to approximately 0.5 (OD 650nm )’s optical density.

[0230] Protein production was then induced by adding IPTG to a concentration of 300 μM, followed by incubation for another 5-6 hours under 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 a pellet at 4°C or -20°C until further use. Typically, 2.5 to 4 grams of cells (wet weight) were obtained from 1 liter of culture, regardless of the medium used.

[0231] The fermentation was prepared and carried out in a 750 ml InforsHT reactor. 168 ml of deionized water was added to the fermentation vessel. The reaction vessel was equipped with all the required probes (pO 2 , pH, sampling, defoamer), C+N feed and sodium hydroxide bottles and autoclave. After autoclaving, the following ingredients were added to the reactor:-

[0232] 20ml 10x phosphate / citrate buffer

[0233] 14ml 50% glucose

[0234] 0.53 ml MgSO 4 Solution

[0235] 2ml(NH 4 ) 2 SO 4 Solution

[0236] 0.020ml trace element solution

[0237] 0.400ml thiamine solution

[0238] 0.200 ml kanamycin stock solution

[0239] The reaction conditions were set as follows: pH = 6.95, pO 2 = 40%, T = 30°C, stirring at 300 rpm. Cascade: rpm set point at 300, min 300, max 1000, flow rate l / min set point 0.1, min 0, max 0.6. Defoamer control: 1:9.

[0240] The fermenter was inoculated from the seed culture to an OD of 650nm The inoculum culture was grown in LB medium (+kanamycin) at 37°C and 220 rpm for 8 h. The fermentation was first carried out in batch mode for 11.5 h, with the feed solution (sterile glucose solution (143 ml H 2 O + 35g glucose) for C + N feed, add 17.5ml (NH 4 )2 SO 4 Solution, 1.8ml MgSO 4 solution, 0.018 ml trace element solution, 0.360 ml thiamine solution, 0.180 ml kanamycin stock solution. The feed was run at a constant flow rate of approximately 4.2 ml / h. Glucose and NH 4 + Measurements are performed externally to assess the availability of C- and N-sources in the culture. Typically glucose levels are kept very low.

[0241] The cultures were grown for approximately 25 hours in total, where they typically reached an OD of 40-45. 650nm SHC production was then initiated by adding IPTG to a final concentration of approximately 1 mM in the fermenter (either as an IPTG pulse or using an infusion syringe over 3-4 hours), setting the temperature to 40°C and adjusting the pO 2 The induction of SHC production was set to 20%. The induction was continued for 16 h at 40°C. At the end of the induction, the cells were collected by centrifugation, washed with 0.1 M citric acid / sodium citrate buffer, pH 5.4, and stored at 4°C or -20°C until further use.

[0242] Results Ia

[0243] In general, all other conditions being constant, the specific activity of the produced biocatalyst is higher when minimal medium is used compared to rich medium.

[0244] Induction was successfully performed at 30 or 37° C. It was noted that higher specific activity of the biocatalyst was obtained when induction was performed at 40-43° C.

[0245] Results Ib

[0246] Table 1 below shows the culture volume, optical density and the cell mass at the start and end of induction and the amount of biomass collected (wet weight) for two examples.

[0247] Table 1

[0248]

[0249] OD at inoculation 650nm : 0.45 (Example 1) and 0.40 (Example 2). Starting volume: 205 ml.

[0250] Wild-type SHC amino acid sequence (SEQ ID No. 1) (GenBank M73834, Swissprot P33247)

[0251] MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0252] Variant F601Y SHC amino acid sequence (SEQ ID No. 2) - Variant related to SEQ ID No. 1

[0253] MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR

[0254] Variant F605W SHC nucleotide sequence (SEQ ID No.3)

[0255]

[0256] Variant F605W SHC amino acid sequence (SEQ ID No. 4) - variant related to SEQ ID No. 1

[0257] MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALYPGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGK RMPLNIYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIRALDWLLERQAGDGSWGGIQPPWFYALIALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTG LAVLALRAAGLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFRWIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVE YLKREQKPDGSWFGRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGGRAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR

[0258] Example 3a

[0259] Biotransformation of 7E,3E / Z-homofarnesol mixture

[0260] The biotransformation was carried out using the following reaction conditions:

[0261] The reaction (150.1 g total volume) was carried out in an InforsHT 750 ml fermentor containing 146 g / l total homofarnesol using homofarnesol substrate (which was a mixture of 7E,3E:7E,3Z 86:14), 250 g / l cells (formed, fermented according to the method of Example 2) and 1.55% SDS in 0.1 M citric acid / sodium citrate buffer pH 5.4. The reaction was carried out at 35° C. under constant stirring (900 rpm) using 10-40% aqueous citric acid solution for pH control.

[0262] The reaction mixture was subjected to separation and purification as exemplified in Example 4 below.

[0263] Example 3b

[0264] Biotransformation of 7E,3E / Z-homofarnesol mixture

[0265] The biotransformation was carried out using the following reaction conditions:

[0266] The reaction (total volume 2.5 ml) was carried out in an 11 ml glass reaction vessel on a Heidolph Synthesis 1 apparatus at 50°C and pH 6.0 in 0.1 M citric acid / sodium citrate buffer with vigorous shaking (800 rpm). The reaction system contained 1 g / l E,E-homofarnesol (from a homofarnesol stock solution with a ratio of EE:EZ of 86:14), wild-type SHC enzyme produced as described in Example 2 to an OD of 650nm The reaction mixture was stirred for 2 h at 30 °C for 1 h and 0.12% SDS for 30 ng / mL. About 48 h after the start of the reaction, the conversion of E,E-homofarnesol was about 60%. When the reaction was cooled to room temperature, Ambrofix crystals appeared in the microscopic analysis of a sample taken from the reaction mixture. Further addition of an amount of OD 650nm A 10-fold increase in cells and a further incubation for 24 hours resulted in complete conversion of E,E-homofarnesol. Microscopic observation of a sample of the reaction mixture indicated the presence of an increased number of ambroxol crystals.

[0267] The reaction was also carried out in an InforsHT 750 ml fermenter in a total volume of 150.1 g, pH 6.0, in 0.1 M citric acid / sodium citrate buffer. The reaction system contained 1 g / l E,E-homofarnesol, 0.12% SDS and cells producing 118 g / l wet weight of wild-type SHC and was incubated at 50°C with vigorous shaking (700 rpm). About 30 hours after the start of the reaction, the conversion of E,E-homofarnesol was about 85%. Microscopic observation of a sample of the reaction mixture allowed the identification of ambroxol. Further homofarnesol was added to the equivalent of 1 g / l and the reaction was continued for about another 50 hours; cells equivalent to 32 g / l (wet weight) were also added. The total reaction time was about 66 hours and the conversion of E,E-homofarnesol was about 85%. By microscopic observation of the reaction mixture, an increasing number of ambroxol crystals could be observed.

[0268] Example 4

[0269] The general downstream processes are as follows Figure 4 It is cited for reference.

[0270] In the first step, the bioconversion medium is heated to a temperature of about 80-85°C for a period of about 15 minutes to melt the (-)-Ambroxol crystals. The (-)-Ambroxol, which is liquid at this stage, is recrystallized by cooling the reaction mixture to a temperature of 20°C at a rate of about 5°C / hour.

[0271] In the second step, after the crystallization of (-)-Ambrox, the crystals are separated from the bioconversion medium by filtration. The filtration is carried out in a continuous sieve centrifuge (Siebtechnic H250) with a mesh size of 100 microns. The centrifuge is operated at an acceleration of 2028G and a feed rate of 430kg / hour. Due to the significant size difference between the crystals and the cell debris, most of the crystals are retained on the sieve and can be washed with water and mechanically collected by a cutter provided for this purpose.

[0272] In the third step, the filtrate from the second step was added to a continuous decanter, which was set to separate the cell debris retained in the supernatant from any crystals that passed through the filter in step 2, which settled as precipitate in the settler. The decanter was operated at 1170G with a feed rate of 370 kg / hour. The crystals collected in the decanter were washed with water and combined with the crystals obtained in step 2. The combined crystals were washed and statically decanted to remove any residual cell debris in the supernatant, and the washed crystals were ready for further processing.

[0273] In the fourth step, the washed crystals were redissolved with ethanol (96% technical grade) in the amount of 1 mass of crystal to 4 mass of ethanol. The solution was filtered with a submicron filter (0.6 to 1.0 micron, KDS15) at ambient temperature and 1 bar pressure, and then filtered again through a 0.22 micron filter.

[0274] The (-)-Ambroxan extract thus formed may be subjected to further deodorization and decolorization steps as described below.

[0275] The ethanol solution was concentrated to dryness in vacuo. The concentrate was redissolved in industrial grade denatured ethanol and bleach (Tonsil 412FF) and diatomaceous earth filter (CELATOM FW 50) were added under stirring. The mixture was stirred and refluxed at 80-85°C for 30 minutes and then cooled to 55-60°C. The mixture was filtered through a 25 micron filter to remove solid matter.

[0276] Excess denatured alcohol is removed from the clear bleaching solution by atmospheric distillation. Water is then added to the hot solution and the resulting mixture is stirred at 75-80°C for 15 minutes. (-)-Ambroxol is crystallized by gradually cooling the solution to -10 to -15°C. The crystallized (-)-Ambroxol is filtered off and dried in a vacuum oven (50-60°C; 1-5 mbar).

[0277] Example 5a

[0278] Downstream processing: solid-liquid separation as a means of selectively isolating (-)-Ambroxan from biotransformation media Comparison of extraction with toluene

[0279] 200 ml of the inactivated biotransformation medium were extracted with MTBE and analyzed by gas chromatography.

[0280] Solid-liquid separation

[0281] 200 ml of inactivated biotransformation medium was centrifuged to separate the solid from the liquid phase (Sorvall GS3, 5000 rpm, 10 min, 10° C.). This separated approximately 80 ml of solid particles from approximately 120 ml of liquid supernatant. The supernatant was removed, extracted with MTBE and analyzed by gas chromatography. Similarly, the precipitate was extracted with MTBE and the MTBE extract was analyzed by gas chromatography.

[0282] Toluene extraction

[0283] 200 ml of biotransformation medium was extracted with 6 x 45 ml of toluene. The organic phase was collected and filtered to remove any cell debris. The toluene was stripped out and the residue was dissolved in MTBE and analyzed by gas chromatography.

[0284] analyze

[0285] The GC analysis results are as follows Figure 3 As shown. It can be clearly seen from the results that the solid phase collected by the centrifuge contains extremely high levels of (-)-Ambroxan, as well as extremely small amounts of by-products II, III and IV. On the other hand, the toluene extract is not enriched in (-)-Ambroxan compared to the crude bioconversion medium. The results show that while (-)-Ambroxan crystallizes from the bioconversion medium; structurally related compounds (II), (III) and (IV) remain in the liquid phase. The (II), (III) and (IV) residues found in the solid phase analysis are the only residues that can be removed by thorough washing of the solid phase. It can be concluded from this experiment that not only does the particle separation step (such as filtration and / or decantation) allow the separation of the solid form of (-)-Ambroxan from cell debris, but it can also be used to completely or substantially separate solid (-)-Ambroxan from structurally related by-products, such as compounds (II), (III) and (IV).

[0286] Example 5b

[0287] Sensory analysis

[0288] Objective: To investigate the formation of (-)-Ambroxan and compound (II) in crude and crystalline materials. (III) and (IV) sensory analysis

[0289] Biotransformation of E,E-homofarnesol yields (-)-ambroxol and compound (IV).

[0290] Biotransformation of E,Z-homofarnesol yields macrocyclic ether compound (II) and epi-ambroxol compound (III).

[0291] The crude mixture of (-)-Ambroxol contained the desired (-)-Ambroxol, compounds (II), (III) and (IV) present in amounts of 87.1 wt%, 2.8 wt%, 2.5 wt% and 7.6 wt%, respectively.

[0292] When the crude mixture was selectively crystallized (laboratory scale), the crystallized material had the same composition as the crude mixture when analyzed by gas chromatography, but they were present in amounts of 99.1 wt%, 0.1 wt%, 0.1 wt% and 0.7 wt%, respectively. The residues of (II), (III) and (IV) are believed to be oily residues attached to the (-)-Ambrox crystals.

[0293] The sensory analysis results are as follows:

[0294] (-)-Ambroxan: Odor threshold 0.2ng / l.

[0295] Compound (IV): Weak, IsoE, costus, GC-detection threshold 5-10 ng.

[0296] Compound (II): "odorless" (GC-threshold > 500 ng).

[0297] Compound (III): GC-threshold about 10x higher than (-)-Ambrox (about 2 ng).

[0298] Sensory analysis of the three by-products (Compounds II, III and IV) showed that the odor was weaker than that of (-)-Ambroxol. In fact, the odor of epi-Ambroxol (Compound III) was about 10 times weaker than that of (-)-Ambroxol, suggesting that it has essentially no odor.

[0299] The sensory analysis showed that the removal of one or more by-product compounds from (-)-Ambroxan can improve the odor of the remaining compound (i.e. (-)-Ambroxan), even if the removed compound itself is actually an odorless compound. That is, in the absence of Compounds II, III, and IV, an enhancement of the Ambroxan aroma in terms of olfactory purity as determined by a trained perfumer (using recognized benchmarks of acceptable olfactory purity) was observed.

[0300] Example 6

[0301] X-ray characterization of the solid form of (-)-Ambroxan formed by a microbial fermentation process

[0302] Powder X-ray diffraction patterns were acquired using a STOE STADI P X-ray diffractometer.

[0303] System description: The diffractometer (flat sample holder, curved Ge(111) monochromator and CuKa1 radiation 1.54060 Å) was used in transmission mode by using a position sensitive detector. Generator voltage was 40 kV and current was 40 mA. Detector: Mythen1K.

[0304] Experimental parameters: Pattern measurements were performed between 2θ = approximately 4° and 26°. The accuracy of the determined diffraction angles was approximately + / - 0.2° 2θ.

Claims

1. A fragrance composition comprising a solid form of (-)-Ambroxol of formula (I) wherein the solid form exhibits an X-ray diffraction pattern having the following peaks at 15.6, 16.2, 16.7, 17.0, 17.4, 18.3 + / - 0.2 o The peak at the diffraction angle 2θ, and wherein the solid form contains greater than 0% to less than 5% by weight of any compound (II) or (IV) at least one other flavor ingredient, and One or more of the compounds (II) or (IV) in an olfactory acceptable amount 2. A process for preparing a solid form of (-)-Ambroxol of formula (I), wherein the solid form is olfactorily pure and wherein the solid form of (-)-Ambroxan is formed by a bioconversion process comprising the steps of: I) forming crystalline (-)-Ambroxan in a bioconversion medium by a biocatalytic process; and II) separating the crystalline (-)-Ambroxan from the bioconversion medium, wherein the separation step is a filtration step, a decantation step, or a combination of a filtration step and a decantation step; wherein the bioconversion medium obtained from the bioconversion process comprises a solid phase containing crude (-)-Ambroxan, a liquid phase consisting of water and an oil phase, wherein the oil phase contains residual homofarnesol and other oily or oil-soluble impurities or by-products (II) and (IV) present in the oil phase; wherein the bioconversion process is an enzyme-catalyzed cyclization of homofarnesol comprising a mixture of 7E,3E and 7E,3Z geometric isomers of homofarnesol, wherein the cyclization reaction is carried out in the presence of an SHC biocatalyst capable of bioconverting the homofarnesol mixture enriched in the 7E,3E isomer into (-)-Ambroxol; wherein in the presence of appreciable amounts of homofarnesol, (-)-Ambroxol and by-products remain co-soluble; and wherein reducing the level of unreacted homofarnesol mixed with (-)-Ambroxol and compounds (II) and (IV) results in crystallization of (-)-Ambroxol and facilitates downstream processing and isolation / purification of (-)-Ambroxol in solid form from a biotransformation medium; and wherein the solid form contains from greater than 0% by weight to less than 5% by weight of any compound (II) or (IV) 3. The method according to claim 2, wherein prior to the separation step, the bioconversion medium is heated to a temperature of at least 55°C to melt the crystalline (-)-Ambroxan; and then slowly cooled to allow the (-)-Ambroxan to recrystallize from the bioconversion medium.

4. A process according to claim 2, wherein the recovered (-)-Ambroxol crystals are dissolved in a solvent and the solution is filtered through a submicron filter to remove any residual particulate material present in the bioconversion medium and then rendered into solid form by evaporation of the solvent or recrystallization.

5. The method according to claim 2, wherein the enzyme is wild-type squalene-hopene cyclase, or a variant of wild-type squalene-hopene cyclase.

6. The process according to claim 2, wherein the crystals are washed with water, a water-miscible solvent or a mixture thereof.

7. A process according to claim 2, wherein the recovery of (-)-Ambroxol is achieved by mechanically removing it from a filter or a decanting device or a belt filter, collecting it and drying it.

8. A process according to claim 2, wherein the solubilised solution of (-)-Ambroxan crystals is contacted with a bleaching agent.

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