PROCESS FOR CONVERTING ATRANOL AND ITS DERIVATIVES INTO HYDROSOLUBLE COMPOUNDS

Abstract

The present disclosure relates to a process for converting atranol and/or its derivatives into hydrosoluble compound(s). In particular, the present disclosure relates to a process for converting atranol and/or its derivative(s), said process comprising a step of mixing a composition comprising atranol and/or its derivative(s) with an enzyme belonging to the peroxidase family and a peroxide. The present disclosure also relates to a process for producing an oakmoss extract having less than 100 ppm of atranol and/or its derivative(s).

Claims

1. Process for converting atranol and/or its derivative(s) into hydrosoluble compound(s), said process comprising a step of mixing a composition comprising atranol and/or its derivative(s) with an enzyme belonging to the peroxidase family and a peroxide.

2. Process according to claim 1, wherein the composition comprising atranol and/or its derivative(s) is a moss extract.

3. Process according to claim 1, wherein the composition comprising atranol and/or its derivative(s) is an oakmoss concrete.

4. Process according to claim 1, wherein the composition comprising atranol and/or its derivative(s) is an oakmoss absolute.

5. Process according to claim 1, wherein the enzyme is horseradish peroxidase (HRP).

6. Process according to claim 1, wherein the peroxide is H.sub.2O.sub.2.

7. Process according to claim 6, wherein the amount of peroxide is at least 0.5 molar equivalent compared to atranol and/or its derivatives.

8. Process according to claim 1, wherein the amount of enzyme belonging to the peroxidase family is at least 0.1% by weight compared to the weight of atranol and/or its derivatives.

9. Process according to claim 1, wherein the mixing step lasts at least 0.5 hour.

10. Process according to claim 1, wherein the process is carried out in an aqueous buffer solution.

11. Process according to claim 1, wherein the process is carried out at a pH comprised between 8 and 10.

12. Process according to claim 1, wherein atranol is converted into a hydrosoluble dimer of formula (I) ##STR00006##

13. Process for producing an oakmoss extract having less than 100 ppm of atranol and/or its derivative(s), said process comprising the following steps: a) mixing an oakmoss extract comprising atranol and/or its derivative(s) with an enzyme belonging to the peroxidase family and a peroxide, b) incubating the mixture, to convert atranol and/or its derivative(s) into hydrosoluble compound(s), c) eliminating the hydrosoluble compound(s) using liquid/liquid extraction.

14. (canceled)

15. Process according to claim 7, wherein the amount of peroxide is at least 1 molar equivalent

16. Process according to claim 7, wherein the amount of peroxide is at least 2 molar equivalents.

17. Process according to claim 8, wherein the amount of enzyme belonging to the peroxidase family is at least 0.5% compared to the weight of atranol and/or its derivatives.

18. Process according to claim 8, wherein the amount of enzyme belonging to the peroxidase family is at least 1% compared to the weight of atranol and/or its derivatives.

19. Process according to claim 1, wherein the mixing step lasts at least 0.5 hour.

20. Process according to claim 1, wherein the mixing step lasts at least 1 hour.

21. Process according to claim 1, wherein the mixing step lasts at least 2 hours.

Description

FIGURES LEGENDS

[0068] FIG. 1 shows the HPLC-PDA chromatograms of an oakmoss absolute before (top) and after (bottom) treatment according to example 4 (2 equivalents of H.sub.2O.sub.2 and 2 hours reaction time). Insets are zoom of the area of the chromatogram where atranol and chloroatranol are eluting (respectively at 16.9 and 20.8 min).

EXAMPLES

Example 1: Conversion of Pure Atranol

[0069] Atranol (0.66 mmol) was dissolved in pH9 carbonate buffer 20 mM at room temperature to reach a concentration of 2 g/L. HRP was then introduced at a 1% wt ratio and the reaction was started with the slow addition of 2 equiv. of H.sub.2O.sub.2 (30% w/w aqueous solution) at a 0.1 mL/h flow rate. After 4 hours, the aqueous phase was extracted with AcOEt, and both the organic and aqueous phases were evaporated and analyzed. The same protocol was used to perform control experiments. The different conditions and results are summarized in table 1.

TABLE-US-00001 TABLE 1 Conversion of pure atranol Product in Product in the organic the aqueous Entry H.sub.2O.sub.2 HRP phase (yield) phase (yield) 1 Atranol (95%).sup.a 2 1% (w/w) Atranol (92%).sup.a 3 2 eq. 5-methylpyrogallol (84%) 4 2 eq. 1% (w/w) 5-methylpyrogallol (2%) Dimer (75%) .sup.aRecovery of starting material.

[0070] In the absence of H.sub.2O.sub.2, atranol was recovered unchanged upon extraction with AcOEt either with or without HRP used in 1% w/w (table 1, entries 1 and 2). In the presence of H.sub.2O.sub.2 but without HRP, 5-methylpyrogallol was obtained in 84% yield upon extraction with AcOEt (table 1, entry 3). Surprisingly, in the presence of HRP (1% w/w) and H.sub.2O.sub.2 (2 equiv.), only 2% of 5-methylpyrogallol were isolated by extraction with AcOEt. Upon evaporation of the aqueous phase, a dimer was obtained in 75% yield.

Example 2: HRP-Catalyzed Oxidation of Pure Atranol

[0071] HRP (124 U/mg, 4 mg) was dissolved in 65 mL of pH9 carbonate buffer (20 mM) containing atranol (200 mg, 1.32 mmol). The reaction flask was covered with an aluminum foil to avoid peroxide decomposition. Reaction was initiated by the slow addition of a 30% aqueous H.sub.2O.sub.2 solution at 0.1 mL/h to ensure the final addition of 2 equivalents of hydrogen peroxide (0.264 mL) with respect to atranol. After 6 hours at room temperature, an aqueous HCl solution (0.1 M) was added until pH4 is reached. This aqueous layer was extracted with ethyl acetate (370 mL). The aqueous phases were concentrated by rotary evaporation to give the dimer as a white powder (193 mg, 44%). ESI-MS: m/z=310. .sup.1H RMN (D.sub.2O, 400 MHz): ppm 6.37 (t, 1H), 3.97 (d, 1H), 3.37 (s, H), 2.68 (s, 2H), 2.62 (s, H), 2.14 (s, 3H), 1.79 (s, 3H) .sup.13C RMN: (D.sub.2O, 100 MHz) ppm 197.5 (C), 178.1 (C), 172.3 (C), 164.3 (C), 127.1 (CH), 88.9 (C), 87.1 (C), 85.7 (C), 77.7 (C), 60.2 (CH), 54.9 (CH), 52.9 (CH), 25.7 (CH.sub.3), 23.7 (CH.sub.3).

Example 3: Acetylation of the Dimer

[0072] Since the dimer could not be analyzed directly by GC-MS, its acetylated derivative was prepared. Dimer (0,100 g, 0.32 mmol) was dissolved in distilled CH.sub.2Cl.sub.2 (2 ml). Triethylamine was then added (0.28 ml, 2.1 mmol) followed by Ac.sub.2O (0.2 ml; 2.1 mmol) under an inert atmosphere. After completion of the reaction, the crude reaction mixture was evaporated in vacuo. The obtained solid was dissolved in CH.sub.2Cl.sub.2 and washed with water. After drying of the organic layer with MgSO.sub.4 and evaporation, the triacetylated dimer was obtained as an oil (0.12 g, 90%). HRMS: C.sub.20H.sub.19O.sub.10 for [MH.sup.+], calc. 419.0978; found 419.0950, =6.6 ppm.

Example 4: HRP-Catalyzed Removal of Atranol (4.3%) and Chloroatranol (2.3%) from Oakmoss Absolute at the Milligram Scale

[0073] Oakmoss absolute (250 mg) containing atranol (4.3%) and chloroatranol (2.3%) was dissolved in 270 mL of pH9 carbonate buffer (20 mM). After sonication of the reaction mixture during 2 hours, HRP (124 U/mg, 2 mg) was added. The reaction flask was covered with an aluminum foil, and 1, 2, 3 or 4 equivalents of 30% aqueous H.sub.2O.sub.2 solution were added. The agitation was maintained for 2 or 4 hours at room temperature. Extraction by ethyl acetate allowed the recovery of the modified absolute (231 mg, 92%, with 2 equivalents of H.sub.2O.sub.2 and 4 hours reaction time) after drying over magnesium sulfate, filtration and solvent removal by rotary evaporation. HPLC-UV-MS and GC-MS analysis of the modified oakmoss absolute confirmed the disappearance of atranol and chloroatranol. HPLC with UV detection was not suitable to quantify trace amounts of atranol and in this regard, MS detection was used with an external calibration method (S. C. Rastogi, R. Bossi, J. D. Johansen, T. Menn, G. Bernard, E. Gimnez-Arnau and J.-P. Lepoittevin, Content of oak moss allergens atranol and chloroatranol in perfumes and similar products Contact Dermatitis 2004 50, p. 367-370, R. Bossi, S. C. Rastogi, G. Bernard, E. Gimenez-Arnau, J. D. Johansen, J.-P. Lepoittevin and T. Menn A liquid chromatography-mass spectrometric method for the determination of oak moss allergens atranol and chloroatranol in perfumes Journal of Separation Science 2004 27, p. 537-540). The results are presented in table 2.

[0074] To assess the overall effect on the entire oakmoss absolute, HPLC-UV was used. The chromatograms of the starting oakmoss absolute and the obtained modified oakmoss absolute are shown in FIG. 1. They show that chemical composition of the absolute remained broadly the same.

TABLE-US-00002 TABLE 2 Atranol content of the modified oakmoss absolute after enzymatic reaction Atranol title Entry H.sub.2O.sub.2 Reaction time (ppm) 1 1 equiv. 2 h 141 2 2 equiv. 2 h 65 3 3 equiv. 2 h 32 4 4 equiv. 2 h 12 5 1 equiv. 4 h 130 6 2 equiv. 4 h 62 7 3 equiv. 4 h 28 8 4 equiv. 4 h 7

[0075] Residual values of atranol below 100 ppm were obtained when more than 2 equivalents of H.sub.2O.sub.2 were used. With 4 equivalents of H.sub.2O.sub.2 and upon 4 hours of reaction, a residual title as low as 7 ppm of atranol was observed.

Example 5: HRP-Catalyzed Removal of Atranol (4.3%) and Chloroatranol (2.3%) from Oakmoss Absolute at the Gram Scale

[0076] The oakmoss absolute (1.2 g) was dissolved in 1.3 L of pH9 carbonate buffer (20 mM). After sonication of the reaction mixture during 2 hours, HRP (124 U/mg, 10 mg) was added. The reaction flask was covered with an aluminum foil, and 2 equivalents of 30% aqueous H.sub.2O.sub.2 solution (97 l) were added. The agitation was maintained 2 hours at room temperature. Extraction by ethyl acetate allowed the recovery of the modified absolute (1.1 g, 91%) after drying over magnesium sulfate, filtration and solvent removal by rotary evaporation. The modified oakmoss absolute contained 60 ppm of atranol and chloroatranol was not detected (HPLC-MS, SIM mode). HPLC-UV-MS and GC-MS analysis confirmed this result.

Example 6: Sensory Analysis

[0077] The conserved olfactory quality of the modified oakmoss absolute was assessed by sensory analysis following the triangular testing methodology. Three identical vials containing 2 samples of oakmoss absolute and 1 sample of modified oakmoss absolute, as solution in EtOH (0.5% w/w), were submitted to a panel of 56 persons taken separately which were asked to identify the modified sample. The following formulae were used, with n.sub.1-3 being the value one should exceed to be sure that the result is not statistical distribution for a given level of confidence and N the number of panelists:

[00001]$\mathrm{For}.Math..Math.95.Math.\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{confidence}.Math.\text{:}.Math..Math.n.Math.1=0.77.Math.\sqrt{N}+\frac{2.Math.N+3}{6}+0.5$$\mathrm{For}.Math..Math.98.Math.\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{confidence}.Math.\text{:}.Math..Math.n.Math..Math.2=1.10.Math.\sqrt{N}+\frac{2.Math.N+3}{6}+0.6$$\mathrm{For}.Math..Math.9.99.Math.\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{confidence}.Math.\text{:}.Math..Math.n.Math..Math.3=1.46.Math.\sqrt{N}+\frac{2.Math.N+3}{6}+0.8$

[0078] For a panel of 56 persons, n.sub.1=25.9, n.sub.2=28.0, and n.sub.3=30.9.

[0079] As a result, the scores obtained were 18, 16 and 22, respectively. A score of 22 means that, even at the lowest level of confidence, the panel was far from being able to distinguish the modified sample.