PRODUCTION OF GUAIENE AND ROTUNDONE

Abstract

A process for producing rotundone from α-guaiene, in particular by oxidation of the C(3) position, wherein the α-guaiene is produced from a precursor by a sesquiterpene synthase. The sesquiterpene synthase is produced in a microorganism.

Claims

1. A fragrance or flavor ingredient comprising rotundone in an amount of about 10 to about 95 wt. %.

2. The fragrance or flavor ingredient according to claim 1, wherein the rotundone is in an amount of about 20 to about 70 wt. %.

3. The fragrance or flavor ingredient according to claim 1, wherein the rotundone is in an amount of about 10 to about 50 wt. %.

4. A process of making the fragrance or flavor ingredient of claim 1 comprising producing the rotundone from α-guaiene, wherein the α-guaiene is produced from a precursor by a sesquiterpene synthase, and wherein the sesquiterpene synthase is produced in a microorganism.

5. The process of claim 4, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 70%.

6. The process of claim 4, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 80%.

7. The process of claim 4, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 90%.

8. The process of claim 4, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 95%.

9. A consumer product comprising the fragrance or flavor ingredient according to claim 1.

10. A method of using the fragrance or flavor ingredient of claim 1 to impart at least one of a peppery or woody aroma.

11. A fragrance or flavor ingredient comprising rotundone and ketone 5: ##STR00004##

12. The fragrance or flavor ingredient according to claim 11, wherein the weight ratio of rotundone to ketone 5 ranges from about 20:1 to about 1:20.

13. The fragrance or flavor ingredient according to claim 11, wherein the weight ratio of rotundone to ketone 5 ranges from about 10:1 to about 2:1.

14. The fragrance or flavor ingredient according to claim 11, wherein the weight ratio of rotundone to ketone 5 ranges from about 5:1 to about 3:1.

15. A process of making the fragrance or flavor ingredient of claim 11 comprising producing the rotundone from α-guaiene, wherein the α-guaiene is produced from a precursor by a sesquiterpene synthase, and wherein the sesquiterpene synthase is produced in a microorganism.

16. A consumer product comprising the fragrance or flavor ingredient according to claim 11.

17. A method of using the fragrance or flavor ingredient of claim 11 to impart at least one of a peppery or woody aroma.

18. A process for producing rotundone from α-guaiene, wherein the α-guaiene is produced from a precursor by a sesquiterpene synthase, and wherein the sesquiterpene synthase is produced in a microorganism.

19. The process according to claim 18, wherein rotundone is produced from α-guaiene by oxidation of the C(3) position.

20. The process according to claim 18, wherein the microorganism is cultured under conditions suitable to produce α-guaiene.

21. The process according to claim 18, wherein the sesquiterpene synthase is isolated from the microorganism prior to production of α-guaiene.

22. The process according to claim 18, wherein the microorganism is a recombinant microorganism.

23. The process according to claim 18, wherein the precursor is an acyclic precursor.

24. The process according to claim 23, wherein the precursor is farnesyl pyrophosphate.

25. The process according to claim 18, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 70%.

26. The process according to claim 25, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 80%.

27. The process according to claim 26, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 90%.

28. The process according to claim 27, wherein the sesquiterpene synthase has a sequence identity with SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 6 of at least 95%.

29. The process according to claim 18, wherein rotundone is produced from α-guaiene by an oxidation selected from the group consisting of transition metal catalysis, organocatalysis, chromium oxidation, selenium oxidation, manganese oxidation, aerial oxidation, enzymatic oxidation, electrochemical oxidation and combinations thereof.

30. The process according to claim 29, wherein rotundone is produced from α-guaiene by organocatalysis.

31. The process according to claim 30, wherein rotundone is produced from α-guaiene by organocatalysis comprising the steps of: (a) forming a mixture containing α-guaiene, an organocatalyst, preferably selected from the group consisting of N-hydroxyphthalimide and tetrachloro-N-hydroxyphthalimide, and an oxidant in a solvent; and (b) effecting oxidation of the C(3) position of α-guaiene.

32. The process according to claim 29, wherein the rotundone is produced from α-guaiene by transition metal catalysis and the transition metal is selected from the group consisting of iron, copper, vanadium, manganese, molybdenum, cobalt, ruthenium, palladium, iridium, rhodium, titanium, chromium, gold, osmium and combinations thereof.

33. The process according to claim 32, wherein the rotundone is produced from α-guaiene by iron porphyrin catalysis comprising the steps of: (a) forming a mixture containing α-guaiene and an iron (III)-X porphyrin complex catalyst in a solvent; (b) introducing molecular oxygen into the mixture; and (c) effecting production of rotundone oxidation of the C(3) position of α-guaiene.

Description

ANALYTICAL METHODS EMPLOYED

[0091] Polar GCMS:

[0092] 35° C./2 min, 10° C./min to 50° C., 2.5° C./min to 240° C., 240° C./5 min. Thermo Scientific TSQ8000evo+Trace 1310 system. Polar column: Varian VF-WAX (polar, PEG phase). Column dimensions: 30 m length, 0.25 mm ID, 0.25 μm film thickness. Injector: splitless. Flow: 1.2 mL/min. Carrier gas: Helium. Injection volume: 1 μl. Injector temperature: 230° C. Transfer line: 250° C. MS-quadrupol: 160° C. MS-source: 230° C. Ionization mode: Electron Impact (EI) at 70 eV.

[0093] By this method, the products obtained were identified. Minor and unidentified byproducts were neglected and their percentages (usually <10%) are not given. Apart from α-guaiene 1 and rotundone 2, side products included rotundol 3, epoxy-guaiene 4, ketone 5, hydroxy-rotundone 6, and corymbolon 7.

##STR00003##

[0094] Components 1-7 as defined herein below are literature-known apart from ketone 5 which was isolated and its structure confirmed.

[0095] Nonpolar GC:

[0096] 100° C./2 min, 15° C./min to 240° C., 240° C./5 min. Thermo Focus GC. Nonpolar column: Agilent Technologies J&W Scientific DB-5 (nonpolar, 5% phenylmethylpolysiloxane). Column dimensions: 30 m length, 0.32 mm ID, 0.25 μm film thickness. Injector: Split. Injector temperature: 240° C. Detector: FID. Detector temperature: 270° C. Injection volume: 1 μl. Carrier gas: Helium. Split ratio: 1/42.3. Pressure: 70 kPa. Integrator: Hewlett Packard.

[0097] By this method, substrate purity, conversion and GC-purities of rotundone 2 after distillation were determined (% rpa).

[0098] Source of α-Guaiene

[0099] α-Guaiene was isolated from Clearwood™ by repeated distillation with purities of 37% to 85%. The purities were determined by GC and NMR with internal standard anisaldehyde. The other constituents were seychellene 33%), α-patchoulene 25%), and γ-patchoulene 5%). The purity was determined by GC and NMR with internal standard anisaldehyde.

[0100] Clearwood™ (CAS 1450625-49-6) is a perfumery ingredient in the patchouli, woody family and commercially available from Firmenich. This mixture of sesquiterpenes, which contains about 14 wt.-% of α-guaiene 1, is obtained by fermentation of sugars (IP.com Technical Disclosure IPCOM000233341D).

[0101] Examples for the biotechnological production of α-guaiene 1 are also given in Plant Physiol. 2010, 154, 1998-2007; Arch. Biochem. Biophys. 2006, 454, 123-136 and WO 2005/052163 A2.

Preparation of Rotundone 2 from α-Guaiene 1 (Iron Porphyrin Catalysis)

[0102] Chloro(tetraphenylporphyrinato)iron(III) (17 mg, 0.024 mmol) and imidazole (6.7 mg, 0.1 mmol) were added to α-guaiene 1 (61% ex Clearwood™, 1 g, 3 mmol) in a 1:1 ethanol/water mixture (20 mL) under stirring. Oxygen was bubbled into the greenish turbid mixture at 45° C. After 30 min the oxygen inlet was replaced by an oxygen balloon. After 5 hours GC indicated complete conversion to a mixture containing of, rotundone 2 (24%), epoxy-guaiene 4 (3%), ketone 5 (6%), hydroxy-rotundone 6 (4%), seychellene 8 (37%). The dark-brown mixture was evaporated partially under reduced pressure and the residue extracted with tert-butyl methyl ether. The combined organic layers were dried over MgSO.sub.4, filtered and evaporated. The residual brown oil (1.22 g) was bulb-to-bulb distilled at 150-230° C./0.03 mbar, giving 0.88 g rotundone 2 of 32% GC-purity (43% corr. yield) as a red-brown oil and 0.18 g of a brown residue.

Preparation of Rotundone 2 from α-Guaiene 1 (Organocatalysis)

[0103] In a 100 mL round-bottom two-necked flask equipped with a condenser, α-guaiene 1 (37% ex Clearwood™, 1.022 g, 1.85 mmol) and NHPI (0.082 g, 0.5 mmol) were dissolved in acetonitrile (30 mL) and water (15 mL) under positive nitrogen flow. The solution was heated to 50° C., and solid NaClO.sub.2 (0.678 g, 7.5 mmol) was added in portions. The reaction mixture was then allowed to stir at 50° C. for 21 h. Then, the solution was allowed to cool to room temperature and poured onto NaOH (aqueous, 2M). The product was extracted with MTBE and washed with brine. The combined organic layers were dried over MgSO.sub.4, filtered and evaporated. GCMS of the residue (0.92 g) indicated complete conversion to a mixture containing rotundone 2 (6%), rotundol 3 (1%), epoxy-guaiene 4 (1%), ketone 5 (1%), hydroxy-rotundone 6 (2%), seychellene (40%), α-patchoulene (11%), α-patchoulenone (2%) and β-patchoulenone (2%) The residual brown oil (0.92 g) was bulb-to-bulb distilled at 160-220° C./0.01 mbar, giving 0.92 g rotundone 2 of 17% GC-purity (33% corr. yield) as a red-brown oil and 90 mg of a brown residue.

Olfactory Description of Rotundone 2

[0104] (from iron porphyrin catalysis as described herein above, purified by distillation, dilution 1% in ethanol on blotter dipped freshly, after 4 h and after 1 week)

TABLE-US-00001 Time Olfactory Description 2 minutes fresh, woody, cedarwood, terpenic, wood fiber, slightly minty and terpinol 4 hour warm, woody, spicy, cedar wood like, wood fiber, sawdust, straw 1 week cedar wood like, wood fiber, sawdust, straw

[0105] Synthesis and Purification of Ketone 5

[0106] Oxygen was bubbled into a mixture of chloro(tetraphenylporphyrinato) iron(III) (34 mg, 0.05 mmol), imidazole (6.7 mg, 0.1 mmol) and α-guaiene 91% (1 g, 4.5 mmol) in polyethylenglycol (20 mL) under stirring, light irradiation with a 300 W Osram Ultra Vitalux lamp and at 45° C. After 38 hours, GCMS indicated a quantitative conversion to a mixture of rotundone 2 (27%), epoxy-guaiene 4 (6%), ketone 5 (11%), hydroxy-rotundone 6 (19%), corymbolon 7 (1%). The orange product mixture was extracted with tert-butyl methyl ether against water. The combined organic layers were dried over MgSO.sub.4, filtered and evaporated to 0.7 g of an orange oil, which was purified by flash chromatography over 30 g silica gel 15-40 μm using an hexane/ethyl acetate gradient 98:2-50:50. After evaporation of the solvents this gave 0.23 g of rotundone 2 with 52-59% GC-purity (13% corr) and 24 mg of Ketone 5 with a GC-purity of 71% (2% corr). Ketone 5 was further purified by preparative GC to a purity of >99.5% (containing less than 0.03% of rotundone 2) and its odor analyzed on blotter and by sniff-GC being woody, cedary, dry, isoraldeine-guaiac, smokey, fruity, spicy.

[0107] Analytical data of ketone 5 ((4S,7R)-4-methyl-7-(prop-1-en-2-yl)-3,4,5,6,7,8-hexahydroazulen-1(2H)-one): .sup.1H-NMR (benzene-D.sub.6, 400 MHz): 4.85-4.9 (2s, 2H), 3.1-3.14 (2h), 2.5-1.4 (10H), 1.75 (s, 3H), 0.9 (d, 3H) ppm; .sup.13C-NMR (benzene-D.sub.6, 100 MHz): 206.5 (s), 176.6 (s), 150.3 (s), 139.0 (s), 108.9 (t), 45.2 (d), 36.4 (d), 33.8 (t), 32.1 (t), 30.9 (t), 29.5 (t), 28.1 (t), 20.4 (q), 16.5 (q) ppm; .sup.13C-NMR (CDCl.sub.3, 100 MHz): 209.3 (s), 179.9 (s), 150.6 (s), 139.2 (s), 108.9 (t), 45.3 (d), 36.85 (d), 34.3 (t), 32.3 (t), 31.0 (t), 30.15 (t), 27.2 (t), 20.6 (q), 17.0 (q) ppm; IR (cm.sup.−1): 2661 (w), 2922 (m), 2854 (w), 1697 (s), 1642 (m), 1452 (w), 1438 (w), 1375 (w), 1304 (w), 1286 (w), 1260 (w), 1236 (w), 1173 (w), 1154 (w), 1071 (w), 1042 (w), 1023 (w), 992 (w), 886 (m), 532 (w); GCMS (EI, m/z): 204 (2%, [M].sup.+), 189 (11%, [M−15].sup.+), 161 (12%), 148 (51%), 147 (48%), 134 (10%), 133 (100%), 121 (18%), 119 (28%), 107 (19%), 106 (11%), 105 (43%), 93 (25%), 91 (18%), 91 (39%), 81 (34%), 81 (17%), 79 (27%), 77 (22%); [α].sub.D.sup.22=−11.4 (c 0.35, CHCl.sub.3); HRMS (ESI): Calculated for C.sub.14H.sub.21O [M+H].sup.+: 205.1587; Found: 205.1586.