Process for isolating and purifying ambrox

Abstract

A method of isolating and purifying ()-Ambrox from a reaction mixture comprising ()-Ambrox and one or more of the compounds (II), (III) and (IV) ##STR00001##

Claims

1. A method of isolating and purifying ()-Ambrox comprising the step of selectively crystallizing ()-Ambrox from a reaction mixture comprising one or more of the compounds (II), (III) and (IV) ##STR00005##

2. A method of improving or enhancing the odour of ()-Ambrox comprising the step of separating ()-Ambrox from a mixture comprising one or more of the compounds (II), (III) or (IV) ##STR00006## by selective crystallization of ()-Ambrox from the mixture, such that after the step of separating, ()-Ambrox contains none, or only olfactory acceptable amounts, of the compounds (II), (III) or (IV).

3. The method according to claim 1, wherein the reaction mixture is free, or is substantially free, of homofarnesol.

4. The method according to claim 1, wherein the crystallizing solvent is selected from the group consisting of water, methanol, acetone, petroleum ether, hexane, t-butyl methyl ether, THF and ethyl acetate ethanol, toluene and mixtures thereof.

5. The method according to claim 4, wherein the crystallizing solvent is an ethanol water mixture.

6. The method according to claim 1, wherein the reaction mixture is formed as a result of an enzyme-catalyzed cyclization of homofarnesol comprising a mixture of 7E,3E and 7E,3Z homofarnesol geometric isomers of homofarnesol, wherein the reaction is carried out in the presence of a recombinant microorganism expressing the gene encoding the enzyme.

7. The method according to claim 6, wherein the reaction mixture of 7E,3E and 7E,3Z homofarnesol is enriched in the 7E,3E geometric isomer.

8. The method according to claim 6, wherein the reaction mixture of 7E,3E and 7E,3Z homofarnesol consists of 7E,3E and 7E,3Z homofarnesol and no other geometric isomers of homofarnesol.

9. The method according to claim 6, wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least 80:20.

10. The method according to claim 6, wherein the enzyme is a wild-type squalene hopene cyclase or a variant of the wild-type squalene hopene cyclase.

11. A perfume ingredient consisting of ()-Ambrox and olfactory acceptable amounts of one or more of the compounds (II), (III) or (IV) ##STR00007##

12. The perfume ingredient according to claim 11, comprising crystalline ()-Ambrox.

13. A perfume composition comprising ()-Ambrox and at least one other perfume ingredient, wherein said perfume composition contains olfactory acceptable amounts of one or more of the compounds (II), (III) or (IV) ##STR00008##

14. The method according to claim 2, wherein the reaction mixture is free, or is substantially free, of homofarnesol.

15. The method according to claim 2, wherein the crystallizing solvent is selected from the group consisting of water, methanol, acetone, petroleum ether, hexane, t-butyl methyl ether, THF and ethyl acetate ethanol, toluene and mixtures thereof.

16. The method according to claim 15, wherein the crystallizing solvent is an ethanol water mixture.

17. The method according to claim 2, wherein the mixture is formed as a result of an enzyme-catalyzed cyclization of homofarnesol comprising a mixture of 7E,3E and 7E,3Z homofarnesol geometric isomers of homofarnesol, wherein the reaction is carried out in the presence of a recombinant microorganism expressing the gene encoding the enzyme.

18. The method according to claim 17, wherein the reaction mixture of 7E,3E and 7E,3Z homofarnesol is enriched in the 7E,3E geometric isomer.

19. The method according to claim 17, wherein the reaction mixture of 7E,3E and 7E,3Z homofarnesol consists of 7E,3E and 7E,3Z homofarnesol and no other geometric isomers of homofarnesol.

20. The method according to claim 17, wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least 80:20.

21. The method according to claim 17, wherein the enzyme is a wild-type squalene hopene cyclase or a variant of the wild-type squalene hopene cyclase.

22. The method according to claim 9, wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least 90:10.

23. The method according to claim 9, wherein the weight ratio of the 7E,3E isomer to 7E,3Z isomer is at least 95:5.

Description

EXAMPLES

Example 1

(1) Preparation of Homofarnesol

(2) General Analytical Conditions:

(3) Non-polar GC/MS: 50 C./2 min, 20 C./min 200 C., 35 C./min 270 C. GC/MS Agilent 5975C MSD with HP 7890A Series GC system. Non-polar column: BPX5 from SGE, 5% phenyl 95% dimethylpolysiloxane 0.22 mm0.25 mm12 m. Carrier Gas: Helium. Injector temperature: 230 C. Split 1:50. Flow: 1.0 ml/min. Transfer line: 250 C. MS-quadrupol: 106 C. MS-source: 230 C.

(4) A) Preparation of MNU in THF

(5) A solution of urea (175 g, 2.9 mol) and methylamine hydrochloride (198 g, 2.9 mol) in water (400 ml) is heated at reflux (105 C.) for 3.5 h under stirring. At 40 C. NaNO2 (101 g, 1.45 mol) dissolved in water (200 ml) is added. After 15 min THF (1000 ml) is added which results in a transparent 2-phase mixture. Conc. H2SO4 (110 g, 1.1 mol) is added at 0-5 C. and stirring within 1.5 h. After another 0.5 h at 0-5 C. the two transparent phases are separated at 25 C. The organic phase (A) (1065 ml, theoretically 1.35 M) is stored for a few days at 0-5 C. or forwarded immediately to the cyclopropanation reactor.

(6) After phase separation the water phase is extracted twice with THF (21 l). This gives 1100 ml of phase B and 1075 of phase C. Whereas phase A gives a 51% conversion of a terminal alkene to a cyclopropane in a subsequent cyclopropanation reaction, phase B gives <0.5% cyclopropane and phase C gives no detectable conversion. We conclude that >99% MNU is extracted after the first phase separation. Usually the water phase is therefore discarded after the first phase separation (from organic phase A) after treatment with conc. aqueous KOH and acetic acid.

(7) B) Preparation of E--Farnesene Using MNU in THF

(8) ##STR00004##

(9) N-Methyl-N-nitroso urea 1.35 M in THF (136 ml, 184 mmol) is added dropwise at 0 C. to a rapidly stirred mixture of E-beta-Farnesene (CAS 18794-84-8) (25 g, 122 mmol) and aqueous KOH (50 ml, 40%) at 0-5 C. After the addition of 4 ml of the MNU solution, Pd(acac)2 (7.4 mg, 0.024 mmol, 0.02%) pre-dissolved in 0.5 ml dichloromethane is added. The remaining MNU solution is added over 4 h at 0-5 C. A GC at this stage showed 28% unconverted E-beta-Farnesene, 65% of the desired monocyclopropane (shown above) and 3% of a biscyclopropanated compound 5. After 16 h at 25 C. acetic acid (100 ml) is added at 0-5 C., then tert-butyl methyl ether (250 ml). After phase separation the organic phase is washed with 2M HCl (250 ml) and the aqueous phase extracted with tert-butyl methyl ether (250 ml). The combined organic layers are washed with water (2100 ml), aqueous 10% NaOH (2100 ml) and water (2100 ml), dried over MgSO.sub.4, filtered and concentrated to give 26.9 g of a slightly yellow liquid which contains 9% E-beta-Farnesene, 82% of the desired monocyclopropane compound and 6% of a biscyclopropanated side product.

(10) The desired compound could be further isolated by distillative purification.

(11) Addition of 1 g K.sub.2CO.sub.3 (1 g) and distillation over a 30 cm steel coil column at 40-60 mbar gives 147 g monocyclopropane compound (68% corr) at 135-145 C. The fractions are pooled to give 92 g monocyclopropane compound of 100% purity.

(12) Analytical data of E- Farnesene:

(13) 1H-NMR (CDCl3, 400 MHz): 5.1 (2 m, 2 H), 4.6 (2 H), 2.2 (2 H), 2.1 (4 H), 2.0 (2 H), 1.7 (s, 3 H), 1.6 (2 s, 6 H), 1.3 (1 H), 0.6 (2 H), 0.45 (2 H) ppm. 13C-NMR (CDCl3, 400 MHz): 150.9 (s), 135.1 (s), 131.2 (s), 124.4 (d), 124.1 (d), 106.0 (t), 39.7 (t), 35.9 (t), 26.7 (t), 25.7 (q), 17.7 (q), 16.0 (d), 6.0 (t) ppm. GC/MS: 218 (2%, M+), 203 (5%, [M15]+), 175 (11%), 147 (31%), 134 (15%), 133 (20%), 121 (12%), 107 (55%), 95 (16%), 93 (30%), 91 (20%), 82 (11%), 81 (33%), 79 (42%), 69 (100%), 67 (22%), 55 (20%), 53 (21%), 41 (75%). IR (film): 3081 (w), 2967 (m), 2915 (m), 2854 (m), 1642 (m), 1439 (m), 1377 (m), 1107 (w), 1047 (w), 1018 (m), 875 (s), 819 (m), 629 (w). Anal. calcd. for C16H26: C, 88.00; H, 12.00. Found: C, 87.80; H, 12.01.

(14) C) Preparation of (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol ((7E)-homofarnesol)

(15) A mixture of (E)-(6,10-dimethylundeca-1,5,9-trien-2-yl)cyclopropane (E- Farnesene) (1 g, 4.6 mmol), dodecane (0.2 g, 1.15 mmol, internal standard) and L-(+)-tartaric acid (1 g, 6.9 mmol) in a pressure tube is heated under stirring at 150 C. After 18 h and complete conversion (according to GC) the mixture is poured on water (50 ml) and toluene (50 ml). The phases are separated and the aqueous phase extracted with toluene (50 ml). The combined organic layers are washed with conc. aqueous Na.sub.2CO.sub.3 (50 ml) and conc. NaCl (250 ml), dried over MgSO.sub.4, filtered and evaporated under reduced pressure to give a brownish resin (1.35 g) which is mixed with 30% aqueous KOH (4.3 ml) and stirred at 25 C. for 2 h. GC analysis reveals formation of 96% (7E)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol according to the internal standard. E/Z ratio 68:22. The analytical data of the E-isomer are consistent with the ones from the literature, see for example P. Kocienski, S. Wadman J. Org. Chem. 54, 1215 (1989).

Example 2

(16) SHC Plasmid Preparation and Biocatalyst Production

(17) SHC Plasmid Preparation

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

(19) Erlenmeyer Flask Cultures

(20) For protein production were used either rich medium (LB medium) or minimal media. M9 is one example of minimal media, which were successfully used.

(21) Media Preparation

(22) The minimal medium chosen as default was prepared as follows for 350 ml culture: to 35 ml citric acid/phosphate stock (133 g/l KH.sub.2PO.sub.4, 40 g/l (NH.sub.4).sub.2HPO.sub.4, 17 g/g citric acid.H.sub.2O with pH adjusted to 6.3) was added 307 ml H.sub.2O, the pH adjusted to 6.8 with 32% NaOH as required. After autoclaving 0.850 ml 50% MgSO.sub.4, 0.035 ml trace elements solution (composition in next section) solution, 0.035 ml Thiamin solution and 7 ml 20% glucose were added.

(23) SHC Biocatalyst Production (Biocatalyst Production)

(24) Small scale biocatalyst production (wild-type SHC or SHC variants), 350 ml culture (medium supplemented with 50 g/ml kanamycin) were inoculated from a pre-culture of the E. coli strain BL21(DE3) containing the SHC production plasmid. Cells were grown to an optical density of approximately 0.5 (OD.sub.650 nm) at 37 C. with constant agitation (250 rpm).

(25) Protein production was then induced by the addition of IPTG to a concentration of 300 M followed by incubation for a further 5-6 hours with constant shaking. The resulting biomass was finally collected by centrifugation, washed with 50 mM Tris-HCl buffer pH 7.5. The cells were stored as pellets at 4 C. or 20 C. until further use. In general 2.5 to 4 grams of cells (wet weight) were obtained from 1 litre of culture, independently of the medium used.

(26) The fermentation was prepared and run in 750 ml InforsHT reactors. To the fermentation vessel was added 168 ml deionized water. The reaction vessel was equipped with all required probes (pO.sub.2, pH, sampling, antifoam), C+N feed and sodium hydroxide bottles and autoclaved. After autoclaving, the following ingredients are added to the reactor: 20 ml 10 phosphate/citric acid buffer 14 ml 50% glucose 0.53 ml MgSO.sub.4 solution 2 ml (NH.sub.4).sub.2SO.sub.4 solution 0.020 ml trace elements solution 0.400 ml thiamine solution 0.200 ml kanamycin stock

(27) The reaction conditions are set as follows: pH=6.95, pO.sub.2=40%, T=30 C., Stirring at 300 rpm. Cascade: rpm setpoint at 300, min 300, max 1000, flow l/min set point 0.1, min 0, max 0.6. Antifoam control: 1:9.

(28) The fermenter was inoculated from a seed culture to an OD.sub.650 nm of 0.4-0.5. This seed culture was grown in LB medium (+Kanamycin) at 37 C., 220 rpm for 8 h. The fermentation was run first in batch mode for 11.5 h, where after was started the C+ N feed with a feed solution (sterilized glucose solution (143 ml H.sub.2O+ 35 g glucose) to which had been added after sterilization: 17.5 ml (NH.sub.4).sub.2SO.sub.4solution, 1.8 ml MgSO.sub.4 solution, 0.018 ml trace elements solution, 0.360 ml Thiamine solution, 0.180 ml kanamycin stock. The feed was run at a constant flow rate of approx. 4.2 ml/h. Glucose and NH.sub.4.sup.+ measurements were done externally to evaluate availability of the C- and N-sources in the culture. Usually glucose levels stay very low.

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

(30) Results Ia

(31) In general, with all other conditions unchanged the specific activity of the produced biocatalyst was higher when a minimal medium was used compared with a rich medium.

(32) Induction was carried out successfully at 30 or 37 C. It was noted that when the induction was done at 40-43 C., a biocatalyst of higher specific activity was obtained.

(33) Results Ib

(34) The following Table 1 shows for two examples the culture volume, optical density and amount of cells both at induction start and induction end as well as the amount of biomass collected (wet weight).

(35) TABLE-US-00001 TABLE 1 cells cells Volume.sub.induction start calculated Volume.sub.induction end collected (ml) OD.sub.650 nm induction start (g) (ml) OD.sub.650 nm, .sub.induction end (g) Example 1 273 40 10.9 342 55 28 Example 2 272 44 12.0 341 57 23 OD.sub.650 nm at inoculation: 0.45 (Example 1) and 0.40 (Example 2). Starting volumes: 205 ml.

(36) TABLE-US-00002 WildtypeSHCaminoacidsequence(SEQIDNo.1)(GenBankM73834,SwissprotP33247) MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALY PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLN IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIR ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAA GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFR WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWF GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGG RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDFYLGYTMYRHVFPTLALGRYKQAIERR VariantF601YSHCaminoacidsequence(SEQIDNo.2)-variantwithrespectto SEQIDNo.1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALY PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLN IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIR ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAA GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFR WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWF GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGG RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGYPGDFYLGYTMYRHVFPTLALGRYKQAIERR VariantF605WSHCnucleotidesequence(SEQIDNo.3) ATGGCTGAGCAGTTGGTGGAAGCGCCGGCCTACGCGCGGACGCTGGATCGCGCGGTGGAGTATCTCCTCTCCTGCCAAAAG GACGAAGGCTACTGGTGGGGGCCGCTTOTGAGCAACGTCACGATGGAAGCGGAGTACGTCCTCTTGTGCCACATTCTCGAT CGCGTCGATCGGGATCGCATGGAGAAGATCCGGCGGTACCTGTTGCACGAGCAGCGCGAGGACGGCACGTGGGCCCTGTAC CCGGGTGGGCCGCCGGACCTCGACACGACCATCGAGGCGTACGTCGCGCTCAAGTATATCGGCATGTCGCGCGACGAGGAG CCGATGCAGAAGGCGCTCCGGTTCATTCAGAGCCAGGGCGGGATCGAGTCGTCGCGCGTGTTCACGCGGATGTGGCTGGCG CTGGTGGGAGAATATCCGTGGGAGAAGGTGCCCATGGTCCCGCCGGAGATCATGTTCCTCGGCAAGCGCATGCCGCTCAAC ATCTACGAGTTTGGCTCGTGGGCTCGGGCGACCGTCGTGGCGCTCTCGATTGTGATGAGCCGCCAGCCGGTGTTCCCGCTG CCCGAGCGGGCGCGCGTGCCCGAGCTGTACGAGACCGACGTGCCTCCGCGCCGGCGCGGTGCCAAGGGAGGGGGTGGGTGG ATCTTCGACGCGCTCGACCGGGCGCTGCACGGGTATCAGAAGCTGTCGGTGCACCCGTTCCGCCGCGCGGCCGAGATCCGC GCCTTGGACTGGTTGCTCGAGCGCCAGGCCGGAGACGGCAGCTGGGGCGGGATTCAGCCGCCTTGGTTTTACGCGCTCATC GCGCTCAAGATTCTCGACATGACGCAGCATCCGGCGTTCATCAAGGGCTGGGAAGGTCTAGAGCTGTACGGCGTGGAGCTG GATTACGGAGGATGGATGTTTCAGGCTTCCATCTCGCCGGTGTGGGACACGGGCCTCGCCGTGCTCGCGCTGCGCGCTGCG GGGCTTCCGGCCGATCACGACCGCTTGGTCAAGGCGGGCGAGTGGCTGTTGGACCGGCAGATCACGGTTCCGGGCGACTGG GCGGTGAAGCGCCCGAACCTCAAGCCGGGCGGGTTCGCGTTCCAGTTCGACAACGTGTACTACCCGGACGTGGACGACACG GCCGTCGTGGTGTGGGCGCTCAACACCCTGCGCTTGCCGGACGAGCGCCGCAGGCGGGACGCCATGACGAAGGGATTCCGC TGGATTGTCGGCATGCAGAGCTCGAACGGCGGTTGGGGCGCCTACGACGTCGACAACACGAGCGATCTCCCGAACCACATC CCGTTCTGCGACTTCGGCGAAGTGACCGATCCGCCGTCAGAGGACGTCACCGCCCACGTGCTCGAGTGTTTCGGCAGCTTC GGGTACGATGACGCCTGGAAGGTCATCCGGCGCGCGGTGGAATATCTCAAGCGGGAGCAGAAGCCGGACGGCAGCTGGTTC GGTCGTTGGGGCGTCAATTACCTCTACGGCACGGGCGCGGTGGTGTCGGCGCTGAAGGCGGTCGGGATCGACACGCGCGAG CCGTACATTCAAAAGGCGCTCGACTGGGTCGAGCAGCATCAGAACCCGGACGGCGGCTGGGGCGAGGACTGCCGCTCGTAC GAGGATCCGGCGTACGCGGGTAAGGGCGCGAGCACCCCGTCGCAGACGGCCTGGGCGCTGATGGCGCTCATCGCGGGCGGC AGGGCGGAGTCCGAGGCCGCGCGCCGCGGCGTGCAATACCTCGTGGAGACGCAGCGCCCGGACGGCGGCTGGGATGAGCCG TACTACACCGGCACGGGCTTCCCAGGGGATTGGTACCTCGGCTACACCATGTACCGCCACGTGTTTCCGACGCTCGCGCTC GGCCGCTACAAGCAAGCCATCGAGCGCAGGTGA VariantF605WSHCaminoacidsequence(SEQIDNo.4)-variantwithrespectto SEQIDNo.1 MAEQLVEAPAYARTLDRAVEYLLSCQKDEGYWWGPLLSNVTMEAEYVLLCHILDRVDRDRMEKIRRYLLHEQREDGTWALY PGGPPDLDTTIEAYVALKYIGMSRDEEPMQKALRFIQSQGGIESSRVFTRMWLALVGEYPWEKVPMVPPEIMFLGKRMPLN IYEFGSWARATVVALSIVMSRQPVFPLPERARVPELYETDVPPRRRGAKGGGGWIFDALDRALHGYQKLSVHPFRRAAEIR ALDWLLERQAGDGSWGGIQPPWFYALTALKILDMTQHPAFIKGWEGLELYGVELDYGGWMFQASISPVWDTGLAVLALRAA GLPADHDRLVKAGEWLLDRQITVPGDWAVKRPNLKPGGFAFQFDNVYYPDVDDTAVVVWALNTLRLPDERRRRDAMTKGFR WIVGMQSSNGGWGAYDVDNTSDLPNHIPFCDFGEVTDPPSEDVTAHVLECFGSFGYDDAWKVIRRAVEYLKREQKPDGSWF GRWGVNYLYGTGAVVSALKAVGIDTREPYIQKALDWVEQHQNPDGGWGEDCRSYEDPAYAGKGASTPSQTAWALMALIAGG RAESEAARRGVQYLVETQRPDGGWDEPYYTGTGFPGDWYLGYTMYRHVFPTLALGRYKQAIERR

Example 3

(37) Bioconversion of 7E, 3E/Z-Homofarnesol Mixture

(38) Bioconversion was undertaken using the following reaction conditions:

(39) The reaction (150.1 g total volume) run in 0.1 M citric acid/sodium citrate buffer pH 5.4 in an InforsHT 750 ml fermenter contained 146 g/l total homofarnesol using a homofarnesol substrate, which was a mixture of 7E,3E:7E,3Z of 86:14, 250 g/l cells (formed in accordance with the method of Example 2, fermentation) and 1.55% SDS. The reaction was run at 35 C. with constant stirring (900 rpm), pH control was done using 10 to 40% citric acid in water.

(40) The reaction mixture was subjected to isolation and purification steps as set forth in Example 4, below.

Example 4

(41) Downstream Processing Procedure

(42) A reaction mixture formed from the bioconversion of 7E, 3E/Z-homofarnesol (86:14 3E:3Z) was subjected to steam distillation. The distillate was collected as a biphasic mixture. The organic phase was retained and the aqueous phase discarded. The composition of the organic phase was analysed by GC and the results shown in the Table 2 below (see crude).

(43) The organic phase was then concentrated to dryness. Ethanol was then added to the crude, dried product and the mixture warmed until the product was dissolved. At room temperature water is slowly added and ()-Ambrox crystallizes under occasional stirring and cooling in an ice bath.

(44) Table 1 shows the GC analytics results for the crystallized product. The data show a strong enrichment of ()-Ambrox, with practically no by-products (a), (b) or (c) being found in the crystallized sample.

(45) It should be noted that in Table 2, a, b and c refer to compound (II), compound (IV) and compound (III) respectively. EZH and EEH refer to 7E,3Z-homofarnesol and 7E,3E-homofarnesol respectively.

(46) TABLE-US-00003 TABLE 1 Peak area (GC) ()-Ambrox a b c ()-Ambrox EZH EEH (%) Crude 215073 190376 588769 6751605 13429 14184 86.9 Crystallized 10088 8951 64625 9032941 0 0 99.1

Example 5

(47) Extraction of the solid phase of the reaction broth:

(48) Given that ()-Ambrox is not soluble in water and is not liquid at temperatures below approx. 75 C., these properties were taken as possible advantages to extract the product from the solid phase of the biotransformation using either water-miscible solvents (e.g. ethanol) and water-immiscible solvents (e.g. toluene).

(49) 200 ml reaction broth was centrifuged to separate the solid from the liquid (aqueous) phase (Sorvall GS3, 5000 rpm, 10 min, 10 C.). This separated approx. 80 ml solid pellet from approx. an 120 ml liquid phase. Analysis (Gas chromatography) of the aqueous phase after MTBE extraction showed that it contained not more than approx. 0.3% of the ()-Ambrox initially present in the 200 ml reaction broth. Toluene and ethanol 99% were used for extracting ()-Ambrox from the solid phase.

(50) Toluene Extraction:

(51) 80 ml solid phase was extracted 6 with 45 ml toluene (approx. solid phase volume, vigorous shaking for 30 s, centrifugation (Sorvall GS3, 5000 rpm, 10 min, 10 C.). The solvent phase was analyzed with GC for its ()-Ambrox content. Over 99.5% of ()-Ambrox initially present in the reaction broth was extracted with 6 extractions representing a total toluene vol. of 1.35 the initial whole reaction broth volume (200 ml) or 3.4 the vol. of the solid phase.

(52) Ethanol Extraction:

(53) 80 ml solid phase was extracted (Infors Multifors HT, 35 C., 1000 rpm, 30 min) with approx. 160 ml (2 vol.) ethanol 99%, followed by centrifugation. ()-Ambrox did not crystallize during the extraction procedure. After 4 washes (total 640 ml ethanol, i.e. 3.2 the initial whole reaction broth volume or 8 the volume of the solid phase), about 99% of ()-Ambrox initially present in the reaction broth was recovered. Sufficient ethanol is required in the first extraction step to prevent ()-Ambrox crystallization (solubility in ethanol). When only 1 or vol of the solid phase was used in the first extraction step, a sticky paste was obtained, which was difficult to handle and ()-Ambrox crystallized as needles on the pellet during centrifugation. Temperature appeared as not being the factor responsible for this crystallization (extraction and centrifugation tested at room temperature and approx. 35 C.-40 C.).

(54) The ()-Ambrox concentration in the ethanol phase as well as the ethanol/water ratio of the liquid phase (residual moisture of the solid phase) appeared to be responsible for crystal formation. It was however noted that it was possible to reduce the volume of ethanol to 1 vol of the solid phase.

(55) As ()-Ambrox is not in the liquid phase at room temperature, it separates with the biomass and can be extracted with an organic solvent (e.g. a water-miscible solvent (e.g. ethanol) or a water-immiscible solvent (e.g. toluene). The centrifugation step that separates the ()-Ambrox into the solid phase of the reaction mixture is advantageous because it reduces the amount of solvent required to extract ()-Ambrox.

Example 6

(56) Sensory Analysis

(57) Purpose: to carry out a sensory analysis of ()-Ambrox and the compounds (II), (III) and (IV) formed in the crude material and in the crystallised material.

(58) Biotransformation of E,E-homofarnesol results in ()-Ambrox, and compound (IV).

(59) Biotransformation of E,Z-homofarnesol results in the macrocyclic ether compound (II) and epi-Ambrox compound (III).

(60) A crude mixture of ()-Ambrox comprises the desired ()-Ambrox, compound (II), (III) and (IV) present in an amount of 87.1 wt %, 2.8 wt %, 2.5 wt % and 7.6 wt % respectively.

(61) When a crude mixture is selectively crystallised (lab scale), the crystallised material has the same components as the crude mixture, but they are present in an amount of 99.1 wt %, 0.1 wt %, 0.1 wt % and 0.7 wt % respectively.

(62) The Sensory Analytical Results were as follows:

(63) ()-Ambrox: Odour Threshold 0.2 ng/l.

(64) Compound (IV): weak, IsoE, woody, GC-detection threshold 5-10 ng.

(65) Compound (II): odorless (GC-threshold >500 ng).

(66) Compound (III): GC-threshol about 10 higher than ()-Ambrox (circa 2 ng).

(67) The sensory analysis of the 3 by-products (compounds II, III and IV) indicates a weaker odour than that from ()-Ambrox. In fact, the epi-ambrox (Compound III) odor is about 10 fold weaker than ()-Ambrox suggesting that it is essentially odorless.

Example 7

(68) Ambrox Recovery by Steam Extraction

(69) Resulting Purity of the Crude (Steam Extracted) and Crystallized ()-Ambrox

(70) The biotransformation of EE:EZ-homofarnesol 86:14 provided a reaction mixture that was steam extracted. The steam distillate was collected as a biphasic mixture. The organic phase was retained and the aqueous phase discarded. The composition of the organic phase was analysed by GC and the results shown in the Table below (see crude). The organic phase was then concentrated to dryness. Ethanol was then added to the crude, dried product and the mixture warmed until the product was dissolved. At room temperature water is slowly added and ()-Ambrox crystallizes under occasional stirring and cooling in an ice bath.

(71) The tabulated data also shows the GC analytics results for products obtained after the steam extraction/distillation step (crude) and the crystallized product (()-Ambrox). The references in the Table to EZH and EEH refer to (3Z,7E)-homofarnesol and 7E,3E-homofarnesol respectively.

(72) The tabulated data below indicates that the particular starting material (EEH:EZH 86:14) produces the desired end product ()-Ambrox and a very specific mixture of by-products (II, IV and III) using the WT SHC enzyme or a SHC derivative. The data for the selective crystallization show a strong enrichment of () Ambrox, with practically no by-products (II), (IV) or (III) being found in the crystallized sample. Accordingly, this EE:EZ mixture provides an olfactively pure ()-Ambrox product, which is selectively crystallised in a relatively straightforward and cost-effective matter.

(73) TABLE-US-00004 TABLE shows the GC analytics results for the crystallized product. Peak area (GC) Ambrox (II) (IV) (III) Ambrox EZH EEH (%) Crude 215073 190376 588769 6751605 13429 14184 86.9 Crystallized 10088 8951 64625 9032941 0 0 99.1

(74) Steam extraction/filtration are environmentally friendly methods for isolating ()-Ambrox because it offers a convenient solvent-free isolation of ()-Ambrox with an associated inactivation of the biocatalyst.

(75) The ()-Ambrox produced using the bioconversion reaction may be extracted using solvent from the whole reaction mixture (e.g. using a water-immiscible solvent or by steam extraction/distillation or by filtration) or from the solid phase (e.g. using a water miscible solvent) using methods which are known to those skilled in the art.