Production of the Pepper Aroma Compound,(−)-Rotundone, by Aerial

Article pubs.acs.org/JAFC

Production of the Pepper Aroma Compound, (−)-Rotundone, by Aerial Oxidation of α‑Guaiene An-Cheng Huang, Stacey Burrett, Mark A. Sefton, and Dennis K. Taylor* Department of Wine Science, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia ABSTRACT: The aroma link between pepper and wine has recently been elucidated to be due to the important aroma compound rotundone. To date, rotundone is the only known impact odorant with a peppery aroma. Although the concentration found in products of natural origin is small, the odor detection threshold is among the lowest of any natural product yet discovered. We report herein the identification of the first known precursor to rotundone, namely, α-guaiene, and that one mechanism of transformation is simple aerial oxidation. KEYWORDS: (−)-rotundone, α-guaiene, aerial oxidation, pepper, wine

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INTRODUCTION In 2008, a breakthrough by Australian wine chemists identified the obscure sesquiterpene ketone (−)-rotundone, 1, to be responsible for the peppery aroma found in some wine varieties.1 It was also observed, for the first time, in a large range of common herbs and spices (viz., marjoram, oregano, geranium, nut grass, rosemary, saltbush, basil, and thyme) and was found at highest concentration in extracts of black and white peppercorns, the processing of which leads to the most widely utilized spice condiment in the world and aptly crowned as the king of spices (Piper nigrum).2 Hitherto, the spicy “black pepper” aroma of this historically important spice had been thought to result from a complex interaction of multiple odorants, none of which had a pepper-like odor in isolation.3,4 To date, (−)-rotundone, 1, is the only known impact odorant with a peppery aroma. Although the concentration found in products of natural origin is small (from ng/L amounts in red wines up to 2 mg/kg in peppercorns), the odor threshold of rotundone (8 ng/L in water, 16 ng/L in red wine) is among the lowest of any natural product yet discovered.1 Thus, a trace of rotundone imparts strong pepper notes to foods and beverages. In terms of grapes and wines, the accumulation of rotundone in two strongly peppery Italian grape varieties, one red and one white, has been described along with a convenient method for analysis.5 It has also recently been found that rotundone accumulates during grape berry ripening and is concentrated in the berry skins (exocarp), suggesting that skin contact during winemaking may be important for modulating the peppery character of wines.6 Another study has shown that the presence of additional grape leaves and stems during fermentation can also considerably enhance the concentration of rotundone in a finished wine, with the presence of stems being more important.7 Furthermore, it has recently been found that berry rotundone concentrations within a vineyard of Shiraz varietal grapes are markedly spatially variable and are associated with variations in vineyard soil properties and topography.8 Finally, the sesquiterpene rotundone was detected in an above threshold concentration only in a cooler climate Shiraz when compared to a warmer climate premium Australian Shiraz, © 2014 American Chemical Society

suggesting that its formation and stability may be higher in cooler environments.9 However, nothing is known of the mechanism of formation of rotundone, 1, during grape ripening or throughout the wine making process or, indeed, in other food products such as black and white pepper. Sesquiterpenes may be formed in plants by highly controlled enzymatic pathways or may also be formed by simple downstream chemical manipulations of abundant common sesquiterpene feeding stocks from the chiral pool by processes such as oxidation. Processes such as the latter are commonly induced as a response to environmental/external stimuli such as attack by pathogens or excessive heat/light exposure on plant material, or indeed induced by the plant itself and used as communication mechanisms for the recruitment of protective carnivores resulting from herbivore plant attack: the so-called plants’ “cry for help”.10,11 A common and abundant sesquiterpene produced by numerous plants is α-guaiene, 2, which is structurally related to rotundone, 1, in that oxidation at C3 has yet to occur (Figure 1). α-Guaiene has been identified in the essential oil of Cyperus rotundus L.,12 in agarwood oil extract,13−15 in black pepper (roasted),16 in patchouli oil,17,18 in nagarmotha oil produced from Cyperus scariosus R. Br.,19,20 and also in various wine grapes.21,22 At the same time, rotundone has also been identified in the majority of these plant extracts and various wines.1,7−9,12−15,19,23 Given the close structural similarity of rotundone, 1, and α-guaiene, 2, coupled with what appears to be a coincidental occurrence in various plants, we therefore speculated that rotundone in processed plant products, including dried/roasted herbs and spices and perhaps also wine must, could be formed as a result of air contact of the plant matter or foodstuff which is coated with a certain concentration of α-guaiene, 2, rather than, or in addition to, being a product of a specific enzymatically controlled step on Received: Revised: Accepted: Published: 10809

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MHz spectrometer (Agilent Technologies, USA). All compounds for NMR analysis were dissolved in C6D6. All 1H and 13C NMR spectra were calibrated with residual deuterated solvent signals set at 7.16 and 128.06 ppm. HRMS (ESI-TOF) analysis was performed with a triple TOF 5600 mass spectrometer from AB Sciex Instruments. Whatman grade 1 cellulose filter paper was used to mimic the oxidation of αguaiene on plant cell wall cellulose.27 Silica column chromatography was performed using either LC60A 40−63 μm silica (Grace Davison) or silica gel 60 (0.015−0.040 mm) from Merck. TLC was conducted with TLC silica gel 60 F254 plates (Merck KGaA) using standard vanillin stain for visualization. Guaiac wood essential oil (steamdistilled from Bulnesia sarmientoi, Paraguay) was purchased from the Australian Botanical Products Company as viscous oil. This oil contains ca. 40% (−)-guaiol as determined by GC−MS analysis. Preparation of Standards. α-Guaiene, 2, was prepared as follows. Recrystallization of guaiac wood essential oil from warm acetonitrile several times afforded (−)-guaiol as white needles in an overall yield of approximately 14% by weight in a similar fashion to that described previously.1 Treatment with acetic anhydride under reflux conditions afforded the guaiyl acetate in a good yield of 71.9% after workup. Elimination of acetic acid from the guaiyl acetate through pyrolysis was achieved under nitrogen at 210 °C in a similar fashion to that reported previously.28 Workup and purification by flash chromatography afforded α-guaiene, 2, in a very good yield of 84%. Analysis by GC−MS indicated that the purity of the α-guaiene, 2, was >98% with traces of β-guaiene and δ-guaiene also present. Rotundone, 1, was synthesized as follows. Pyridinium dichlorochromate (37.6 g, 0.1 mol) was added to CH2Cl2 (400 mL) and the resulting mixture cooled in an ice-bath before introduction dropwise of a solution of tert-butyl hydroperoxide (5−6 M in decane, 20 mL) in CH2Cl2 (80 mL) with stirring for 20 min. To this mixture was added dropwise a solution of α-guaiene, 2 (20.4 g, 0.1 mol), in CH2Cl2 (80 mL), and the resulting mixture was stirred at 0 °C under N2 for 4.5 h. The reaction was terminated by filtering through a plug of silica (ca. 80 g). The residue in the flask was further extracted with CH2Cl2 (2 × 100 mL), which was filtered through the same silica plug. The combined CH2Cl2 extracts were concentrated under vacuum at 40 °C and the concentrate purified by silica column chromatography (n-hexane:Et2O, gradient elution from 100:0 to 85:15) to yield rotundone, 1 (1.39 g, 27% based on 25% conversion), as a pale yellow oil with the recovery of unreacted α-guaiene, 2 (15.28 g, 75% recovery). Spectroscopic data of 1 including NMR and MS were in agreement with those reported.1 Aerial Oxidation of α-Guaiene, 2, and GC−MS Analysis. αGuaiene, 2 (1.0 g, 4.9 mmol), was dissolved in CH2Cl2 (200 mL) at ambient temperature. The mixture was stirred while being purged slowly with a stream of oxygen (ca. 1 mL/min) through a glass frit. Small aliquots (0.2 mL) were removed daily, diluted with CH2Cl2, and analyzed by GC−MS using an Agilent 6890 gas chromatograph coupled with a 5973N mass spectrometer detector (MSD) and a Gerstel multipurposed autosampler (Gerstel, Germany) and equipped with a HP-wax bonded polyethylene glycol capillary column (DB-Wax 30 m × 250 μm i.d. × 0.25 μm film thickness, Agilent Technologies). Samples were prepared in dry dichloromethane and analyzed in split mode with a split ratio of 50.1:1. The inlet and transfer line temperatures were set at 250 °C. Injection volume was 1 μL. The column flow was set at 1.9 mL/min with a pressure of 15.96 psi. Helium was used as carrier gas. The oven temperature was initially set at 50 °C and held for 0.5 min and ramped up to 240 °C at 10 °C/min and held for a further 6 min. The MSD was operated in full scan mode scanning m/z ions from 50 to 550 amu with EI energy set at 70 eV. MS source and quadrupole temperatures were set at 230 and 150 °C, respectively. The total and selected ion response ratios of α-guaiene and its oxidation products were used as indicators for the extent of autoxidation of α-guaiene under the different conditions. At the same concentrations and under the same GC−MS conditions, the total ion (TI) response ratio of α-guaiene (2):epoxy guaienes (5a/b):rotundone (1) was ca. 36:33:31 and the response ratio of α-guaiene (204) and rotundone (218) was found to be 1.0:1.11 (Figure 3). Preparation of Epoxides 5a/b. To a stirred solution of αguaiene, 2 (103 mg, 0.50 mmol), in CH2Cl2 (3 mL) at −78 °C was

Figure 1. Hypothetical aerial oxidation of α-guaiene, 2, to form rotundone, 1. IUPAC chemical names: (−)-(3S,5R,8S)-3,8-dimethyl5-(prop-1-en-2-yl)-3,4,5,6,7,8-hexahydroazulene-1(2H)-one 1 and (−)-(1S,4S,7R)-1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene, 2.

some precursor substrate. Mechanistically, any proposed aerial oxidation of α-guaiene, 2, is likely to begin with a traditional free radical initiation step, namely, hydrogen atom abstraction from C3 to produce the resonance stabilized allylic radical, 3 (Figure 1). This initiation could be caused by dioxygen (O2) itself or by other initiators such as reactive oxygen species (ROS) or the breakdown of H2O2 produced due to plant− pathogen interactions. Indeed, H2O2 has recently been identified in distinct cellular compartments of the skins of grapes during grape berry ripening, and as mentioned above, both rotundone and α-guaiene have clearly been identified on the skins of grapes.24 The allylic radical, 3, could then enter a propagation sequence by first reacting with dioxygen to form an allylic hydroperoxy radical, which in turn abstracts another hydrogen atom from either another guaiene molecule or some other abstractable hydrogen atom source to produce the guaiene hydroperoxide, 4. Exposure of this hydroperoxide to trace metals (e.g., copper sprays in vineyards) or nucleophiles (e.g., basic amines of plant proteins or pathogens) would allow for decomposition to the ketone rotundone, 1, via well established chemical mechanisms (Figure 1).25 Given this background, the current study has been carried out to evaluate whether α-guaiene could be a natural precursor to rotundone via the process of simple aerial oxidation. Autoxidation was carried out both in organic solvents and also on filter paper derived from cellulose to mimic more closely biological matrices of some plant products.

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MATERIALS AND METHODS

General Experimental Procedures. Acetic anhydride (≥98% ACS reagent grade), pyridinium dichlorochromate, tert-butyl hydroperoxide (5−6 M in decane), and m-chloroperbenzoic acid (max 77%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). d6Benzene was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). All reagents were used directly unless otherwise stated. Solvents for synthesis were dried according to known procedures where necessary.26 Solvents for general chromatography were AR grade except that those used for GC−MS and HRMS analysis were HPLC grade. NMR data were recorded with a Varian-Inova 600 10810

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Figure 2. GC−MS traces of the aerial oxidation of α-guaiene, 2, after (A) 4 days and (B) 29 days. added dropwise a solution of m-CPBA (128 mg, 0.57 mmol) in CH2Cl2 (3 mL). After stirring at −78 °C for 30 min, the reaction was quenched with KI (10 mg), saturated aqueous Na2S2O3 (1 mL) and aqueous NaHCO3 (3 mL). The resulting mixture was extracted with Et2O (3 × 10 mL). The combined ether layers were washed with brine (10 mL), dried over anhydrous MgSO4, and filtered. The filtrate was concentrated in vacuo and purified on neutral alumina to furnish the

pair of epoxy diastereomers, 5a:5b (ca. 1:2, 103 mg, 93%), as a colorless oil. 5a: 1H NMR (600 MHz, C6D6) δ 4.77 (m, 1H, H12a), 4.71 (dq, J = 1.8, 1.5 Hz, 1H, H12b), 2.15 (ddd, J = 12.0, 10.8, 6.0 Hz, 1H, H7), 1.94 (br d, J = 14.4 Hz, 1H, H6a), 1.88 (dd, J = 13.8, 8.4 Hz, 1H, H3a), 1.84−1.78 (m, 2H, H6b and 10), 1.72 (ddq J = 9.6, 7.2, 7.2 Hz, 1H, H4), 1.65 (m, 1H, H9a), 1.58 (br s, 3H, H13), 1.55 (m, 1H, H8a), 1.47 (ddd, J = 14.4, 10.8, 4.8 Hz, 1H, H8b), 1.42 (dd, J = 12.6, 8.4 Hz, 10811

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Figure 3. Structures of epoxides 5a and 5b and diketone 6.

Figure 4. Autoxidation of α-guaiene, 2, and formation of rotundone, 1, on cellulose filter paper vs none at various temperatures. 1H, H2a), 1.28 (ddd, J = 13.8, 10.8, 8.4 Hz, 1H, H3b), 1.16 (m, 1H, H9b), 1.09 (ddd, J = 12.6, 10.8, 8.4 Hz, 1H, H2b), 1.06 (d, J = 7.2 Hz, 3H, H15), 1.02 (d, J = 7.2 Hz, 3H, H14); 13C NMR (150 MHz, C6D6) δ 150.6 (C11), 109.1(C12), 72.6 (C5), 72.5(C1), 43.5 (C7), 38.5 (C4), 34.5 (C10), 32.2 (C8), 31.8 (C6), 31.4 (C3), 28.6 (C9), 28.4 (C2), 20.0 (C13), 18.5 (C15), 13.9 (C14); EI-MS m/z (rel intensity) 220 (8), 205 (32), 162 (87), 147 (46), 107 (95), 81 (89), 67 (100). 5b: 1H NMR (600 MHz, C6D6) δ 4.78 (m, 1H, H12a), 4.73 (dq, J = 1.8, 1.5 Hz, 1H, H12b), 2.47 (dddd, J = 12.6, 12.0, 2.4, 1.8 Hz, 1H, H7), 2.24 (m, 1H, H10), 2.05 (dd, J = 14.4, 2.4 Hz, 1H, H6a), 2.02 (br q, J = 7.2 Hz, 1H, H4), 1.98 (dddd, J = 13.8, 12.6, 3.0, 1.8 Hz, 1H, H8a), 1.86 (dd, J = 14.4, 12.0 Hz, 1H, H6b), 1.83−1.76 (m, 2H, H2a, 3a), 1.64 (s, 3H, H13), 1.58 (m, 1H, H9a), 1.52−1.48 (m, 1H, H9b), 1.34 (dddd, J = 13.8, 6.6, 4.2, 1.8 Hz, 1H, H8b), 1.24 (dddd, J = 14.4, 13.2, 12.6, 1.8 Hz, 1H, H3b), 0.94 (m, 1H, H2b), 0.89 (d, J = 7.2 Hz, 3H, H15), 0.70 (d, J = 7.2 Hz, 3H, H14); 13C NMR (150 MHz, C6D6) δ 151.8 (C11), 108.9 (C12), 73.0 (C5), 71.9 (C1), 43.8 (C7), 40.6 (C4), 34.2 (C6), 32.7 (C10), 31.3 (C8), 29.77 (C3), 29.74 (C9), 27.7 (C2), 21.0 (C13), 17.0 (C14), 16.0 (C15); EI-MS m/z (rel intensity) 220 (7), 205 (22), 162 (63), 145 (52), 107 (94), 81 (85), 67 (100). These NMR data were consistent with low field data reported previously.29 Identification of (2S,3S,6R)-3-Methyl-2-(3-oxobutyl)-6-(prop1-en-2-yl)cycloheptanone; 7-epi-Chabrolidione A, 6. A portion of the autoxidation mixture was subjected to silica (40−60 μm, ca. 100 g) column chromatography using a gradient elution of Et2O:hexanes (200 mL, 2:98, 8:92, 12:88, 15:85, 17:83, 20:80, 23:77, 26:74, 50:50). Fractions from gradient 20:80 Et2O:hexanes (ca. 100 mg) containing diketone 6 were further purified using ultrafine silica (15−40 μm, 10 g, Et2O:hexanes, 15:85, 200 mL) column chromatography repeatedly to furnish 6 (2 mg) as a colorless oil: Rf = 0.1 (30% Et2O:hexanes); 1H NMR (600 MHz, C6D6) δ 4.66 (br s, 1H, H12a), 4.63 (appr quint, J = 1.5 Hz, 1H, H12b), 2.65 (ddd, J = 9.6, 4.2, 2.4 Hz, 1H, H1), 2.37 (td, J = 10.2, 1.2 Hz, 1H, H6a), 2.35 (d, J = 10.2 Hz, 1H, H6b), 2.30 (m, 1H, H7), 2.23 (dddd, J = 13.8, 9.6, 8.4, 6.0 Hz, 1H, H2a), 2.13 (ddd, J = 16.8, 8.4, 6.0 Hz, 1H, H3a), 1.88 (ddd, J = 16.8, 8.4, 6.6 Hz, 1H, H3b), 1.65 (s, 3H, H14), 1.67−1.62 (m, 1H, H10), 1.53−1.46 (m, 2H, H9a,b),

1.48 (s, 3H, H13), 1.45−1.38 (m, 2H, H2b and 8a), 1.29−1.25 (m, 1H, H8b), 0.67 (d, J = 7.2 Hz, 3H, H15); 13C NMR (150 MHz, C6D6) δ 210.7 (C5), 206.5 (C4), 149.8 (C3), 109.6 (C12), 54.2 (C1), 50.2 (C6), 43.4 (C7), 41.7 (C3), 37.1 (C9), 35.0 (C10), 29.9 (C8), 29.3 (C14), 24.8 (C2), 19.9 (C13), 14.1 (C15); EI-MS m/z (rel intensity): 236 (9), 221 (15), 203 (6), 179 (27), 161 (21), 150 (19), 135 (25), 123 (53), 109 (70), 95 (100), 67 (87); HRMS (ESI-TOF) m/z [M + H]+ calcd for C15H25O3 237.1855, found 237.1847. Aerial Oxidation of α-Guaiene, 2, on Filter Paper. Autoxidation of α-guaiene, 2, on filter paper was carried out on 50 mg quantities in duplicate at ambient temperature, 40 °C, and 50 °C. Filter paper (10 cm2) was finely cut into approximately 2 mm2 shreds, and these were placed into 20 mL, 75 mm × 25 mm glass vials. Six vials (3 treatments in duplicate) contained approximately 50 filter paper shreds while another six vials contained no filter paper shreds and were used as controls. α-Guaiene, 2 (neat, 50 mg), was added to each of the vials followed by the addition of CH2Cl2 (0.5 mL), and the mixtures were gently shaken for several minutes. The solvent was then removed under a gentle stream of N2 at 10 °C, which ensured an even coating of guaiene onto the filter paper. The control experiments were identical except for the fact that they did not contain filter paper and were also conducted at ambient temperature, 40 °C, and 50 °C at the same time. The process of autoxidation was monitored by GC−MS by sampling a small fraction of the substrate from each control experiment and by removing one piece of filter paper shred from each filter paper treatment at various time points. A portion of the substrate from the control experiments or the filter paper sheds were placed in dry CH2Cl2 (1.5 mL) for GC−MS analysis. The selected ion response ratio of rotundone to α-guaiene at their corresponding retention times in the GC chromatogram was used as an indicator for the extent of the aerial oxidation of α-guaiene. The ratio of the areas of rotundone (m/z 218) to those of α-guaiene (m/z 204) under the different autoxidation conditions were plotted against time (Figure 4). Note that the total ion (TI) or other selected ion response ratios of rotundone and α-guaiene can also be used as indicators for the reaction rates of autoxidation of α-guaiene under the different 10812

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conditions without altering the trend of the curves shown in Figure 4. At the same concentrations and under the same GC−MS conditions, the total ion (TI) response ratio of α-guaiene (2):epoxy guaienes (5a/ b):rotundone (1) is ca. 36:33:31 and the response ratio of α-guaiene (m/z 204) and rotundone (m/z 218) was found to be 1.0:1.11. GC− MS analysis was performed on the same instrument (6890 GC-5973N MSD) and with the same capillary column (DB-Wax 30 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent Technologies). Acquisition parameters were as follows: initial oven temperature set at 100 °C, ramped up to 200 °C at 8 °C/min and then to 240 °C at 20 °C/min and held at 240 °C for 3 min. Injection was in a splitless mode with a purge flow of 6 mL/min in 0.05 min. The inlet temperature was set at 240 °C with a pressure of 12.92 psi. Injection volume was 2 μL and gas flow rate set at 1.2 mL/min. Helium was the carrier gas. MSD was in full scan mode (50−550 Da). Gain factor was 1.0 and EI 70 eV. Transfer line temperature was set at 240 °C, MS source at 230 °C, and MS quadrupole at 150 °C.

Table 1. Percentages Determined by GC−MS of Major Products Formed and α-Guaiene Remaining on Aerial Oxidation over Time time (days)

α-guaiene (2)

β-cis5a

α-trans5b

rotundone (1)

diketone 6

total (%)

0 4 7 9 17 29

100.0 49.9 25.0 15.8 2.4 0.2

0.0 2.5 3.9 4.5 5.2 5.0

0.0 11.3 18.6 20.2 24.6 23.6

0.0 9.5 13.6 15.1 17.3 16.7

0.0 6.0 10.0 12.2 14.9 15.1

100.0 79.2 71.2 67.8 64.3 60.6

(days 0, 4, 7, 9, 17, and 29) as a percentage of the peak area of α-guaiene plus total products observed by GC-MS. As can be seen from Table 1, the concentration of α-guaiene, 2, steadily decreased over time, However, the relative proportions of the oxidation products, rotundone, 1, monoepoxides 5a and 5b, and the diketone, 6, remain approximately constant over the time period, suggesting that they are all being produced from a common intermediate such as the hydroperoxide 4, as shown in Figure 1, formed first along the reaction coordinate. Removal of the proton α to the peroxide linkage by trace amounts of external nucleophiles would result in the formation of rotundone, 1, and the loss of H2O.25 Furthermore, the formation of the monoepoxides (5a and 5b) is highly consistent with the initial formation of hydroperoxide 4. Once 4 is formed in solution it would be expected to act as an epoxidizing agent, reacting with the more electron rich internally bridged CC of α-guaiene, 2, to produce either the cis or trans epoxides (5a and 5b) in a similar fashion to the action of m-CPBA on α-guaiene, 2, reported herein. The hydroperoxide 4 could act as an epoxidizing agent toward any substrate containing CC bonds, a situation which may well lead to some of the minor oxidized products seen in the later stages of this autoxidation sequence. A matrix or scaffold that may well mimic the cellulose biolayers of some processed plant materials is filter paper. Indeed, filter paper disks which are made from compacted cellulose fibers have been used to monitor the disruption of hydrogen bonding between plant cell wall polymers by proteins that induce wall extension and in assays for the measurement of lipid biosynthesis.27,31 With this scenario in mind, we coated αguaiene onto cellulose filter paper shreds and allowed it to undergo autoxidation at various temperatures. The selected ion response ratio of rotundone to α-guaiene at their corresponding retention times in the GC chromatogram was used as an indicator for the extent of guaiene degradation (Figure 4). Again the same major products were formed over time, and it was observed that the extent of aerial oxidation of α-guaiene in general rose significantly with increasing temperature. Oxidation of α-guaiene on filter paper was more advanced than that without filter paper at the same temperature. These observations are consistent with the fact that coating α-guaiene onto the surface of filter paper would greatly enlarge the overall contact area between molecular oxygen and the substrate αguaiene, thus leading to a substantial increase in oxidation rate. Sesquiterpenes such as α-guaiene are mainly biosynthesized via the mevalonate acid (MVA) pathway in the cytosol of plant cells32 though the newly discovered plastidial 2-C-methyl-Derythritol-4-phosphate (MEP) pathway also generate sesquiterpenes and takes place in plastids.33,34 A recent labeling study has suggested that the precursors supplied for the biosynthesis

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RESULTS AND DISCUSSION With pure α-guaiene, 2, in hand we placed 1 g in dichloromethane and bubbled oxygen through the solution over a number of weeks and followed the autoxidation by GC− MS on a daily basis. Figure 2A shows the oxidation at day 4 while Figure 2B displays the progress of oxidation of α-guaiene after 29 days. We indeed found that the aerial oxidation of αguaiene, 2 (tR 8.84 min), after 4 days resulted in the formation of rotundone, 1 (tR 15.87 min), as one of the major products (Figure 2A). Identification was confirmed by comparison with an authentic sample via MS, by symmetrical peak enhancement on coinjection with synthetic rotundone utilizing several column types, and through isolation by column chromatography and comparison by 1H NMR. In addition, two reaction products at retention times of 11.69 and 12.19 min displayed molecular masses of m/z 220 (C15H24O) indicating that they had incorporated one oxygen atom when compared to α-guaiene (m/z 204, C15H24) itself. These two compounds were identified as the monoepoxides (β(cis)-5a, tR 11.69, and α-(trans)-5b, tR 12.19) by comparison with synthetic samples. One other major product at retention time 18.15 min was clearly observable after 4 days of oxidation (Figure 2A). It displayed a molecular mass of m/z 236 indicating that it was likely to be an oxidized product (C15H24O2) with two oxygen atoms incorporated. After careful column chromatography of the autoxidation mixture, this product was isolated and identified as the diketone, 6, by 1H, 13 C, and 2D NMR analysis. This oxidation product presumably results from the oxidative cleavage of the C1−C8a carbon bond within α-guaiene itself. Indeed, such a cleavage has been proposed for the formation of several related epimers of this diketone isolated from various soft coral extracts in nature.30 We followed the aerial oxidation for 29 days, at which point complete consumption of α-guaiene, 2, was observed (Figure 2B). Again, formation of rotundone, 1, the two epoxides (5a and 5b), and the diketone, 6, were clearly visible. In addition, numerous other minor oxidation products were now present, many of which displayed molecular masses of 220 or 236 indicating the incorporation of one or two oxygen atoms when compared to α-guaiene (m/z 204). Conducting the aerial oxidation in a range of other organic solvents (e.g., ether, chloroform or acetonitrile) also resulted in the same four major compounds being formed in very similar time frames. Table 1 displays the relative GC peak area of the four major products formed at various stages of the autoxidation process 10813

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of sesquiterpenes in the grape berry exocarp are mainly transported from plastids to the cytosol in cells of grape berry skin.35 Dried grape skins consist of ca. 15% of cell wall materials which are made up of ca. 30−35% of neutral polysaccharides and ca. 20% of acidic pectic substances with 62% moderately methyl-esterified.36 The only barrier between the cytosol and the plant cell wall is the cell membrane which is permeable to small hydrophobic molecules such as sesquiterpenes according to the well-established solubility−diffusion model (Overton’s rule).37−40 While enzymic oxidation of terpenes by plant or fungal oxidases is well-known,41 it is possible that hydrophobic sesquiterpenes formed in the cytosol of plant cell matrices such as grapes could also be secreted onto their cell wall matrices and accumulate thereafter, and in the case for α-guaiene might be further oxidized into sesquiterpenoids such as rotundone by ROS or atmospheric oxygen. Overall our studies have shown that simple aerial oxidation of α-guaiene, 2, results in significant formation of the peppery aroma compound rotundone, 1. Therefore, the mode of formation of rotundone in processed plant products, including dried herbs and spices, may simply be a result of air contact rather than rotundone being a product of enzymatically controlled processes. Furthermore, this autoxidation sequence is enhanced at higher temperatures as indicated by our findings when exploiting cellulose filter paper to mimic nature’s biological scaffolds and may have significance when considering the amount of rotundone that may be formed during the cooking, heating, or drying of foodstuffs.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61-8-8313-7239. Fax: +61-8-8313-4711. E-mail: [email protected]. Funding

This project was supported by the School of Agriculture, Food and Wine, the University of Adelaide, as well as by Australia’s grape growers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. A.-C.H is indebted to the University of Adelaide and the China Scholarship Council for a joint Ph.D. scholarship. S.B. thanks the Faculty of Science for a Ph.D. scholarship. Notes

The authors declare no competing financial interest.

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REFERENCES

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