Absolute Configurations of Zingiberenols Isolated from Ginger

Nov 25, 2015 - ... 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States. J. Nat. ...... 2014, 40, 1251– 1259 DOI: 10.1007/s10886-014-05...
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Absolute Configurations of Zingiberenols Isolated from Ginger (Zingiber of f icinale) Rhizomes Ashot Khrimian,* Shyam Shirali, and Filadelfo Guzman Invasive Insect Biocontrol and Behavior Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Bldg 007, Rm 301, BARC-West, 10300 Baltimore Avenue, Beltsville, Maryland 20705, United States ABSTRACT: Two stereoisomeric zingiberenols in ginger were identified as (3R,6R,7S)-1,10-bisaboladien-3-ol (2) and (3S,6R,7S)1,10-bisaboladien-3-ol (5). Absolute configurations were assigned by utilizing 1,10-bisaboladien-3-ol stereoisomers and two gas-chromatography columns: a 25 m Hydrodex-β-6TBDM and 60 m DB-5MS. The C-6 and C-7 absolute configurations in both zingiberenols match those of zingiberene present abundantly in ginger rhizomes. Interestingly, zingiberenol 2 has recently been identified as a male-produced sex pheromone of the rice stink bug, Oebalus poecilus, thus indicating that ginger plants may be a potential source of the sex pheromone of this bug. then on AgNO3−SiO2 provided two fractions corresponding by TLC retention factors (Rf) to synthetic cis- and trans-1,10bisaboladiene-3-ols.16,19 Gas-chromatography retention times (Figure 1) and MS of these fractions indeed matched those of

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n 1975, Terhune and co-workers isolated a new sesquiterpene alcohol from an essential oil of ginger (Zingiber off icinale Roscoe), which they called zingiberenol, based on MS, IR, and NMR data.1 It was assigned a transconfiguration because of similarities in its IR spectrum to that of trans-p-menth-2-en-1-ol,2 but the structure was drawn as the cis-isomer1 and the absolute configuration was not determined. Since the original discovery, zingiberenol was found in ginger by others.3−8 Gupta and co-workers confirmed the presence of zingiberenol in an essential oil from ginger rhizomes by GC-MS analysis but also found a compound that they called epizingiberenol apparently based on the similarities of its MS to that of zingiberenol.9 Zingiberenol was also reported in Heracleum transcaucasicum Manden10 and Alpinia spp.11 used as food additives and medicinal plants. The biological role of zingiberenol in plants remains unknown. Recently, interest in zingiberenols has been invigorated after finding them in animal sources. Two stereoisomeric zingiberenols were identified as part of the male-produced pheromone of the rice stalk stink bug, Tibraca limbativentris, without determining their stereochemistry.12 A sex pheromone of the rice stink bug, Oebalus poecilus, has recently also been identified as zingiberenol and, more specifically, (1R,4R)-1-methyl-4-[(S)6-methylhept-5-en-2-yl)]cyclohex-2-enol,13,14 or in terpene nomenclature15 (3R,6R,7S)-1,10-bisaboladien-3-ol. Zingiberenols have been also reported as chemical (and possibly biochemical) precursors to 10,11-epoxy-1-bisabolen-3-ol, certain stereoisomers of which were identified as aggregation pheromones of the harlequin bug, Murgantia histrionica,16−18 and brown marmorated stink bug, Halyomorpha halys.19 Thus, we became interested in determining the absolute configuration of a plant-produced zingiberenol, as compared to insectproduced materials, in view of the availability of all eight synthetic stereoisomers of 1,10-bisaboladien-3-ols.19 Hydrodistillation of ginger rhizomes followed by flash chromatography of the distilled organic material on silica and This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society

Figure 1. GC-MS total ion chromatograms of cis- and transzingiberenols isolated from ginger rhizomes on a 30 m HP-5 column, 50(5) to 270 °C at 10 °C/min; He 1.0 mL/min.

cis- and trans-1,10-bisaboladien-3-ol standards, with the fastereluting compound being the cis-isomer and the slower-eluting being the trans-isomer.19 To determine the absolute configurations of the isolated zingiberenols, they were analyzed on different GC columns vs stereoisomeric standards. Thus, cis-zingiberenol and the four possible cis-1,10-bisaboladiene-3-ols were first analyzed on a Hydrodex-β-6TBDM column. (3S,6S,7S)-1,10-Bisaboladien-3ol (1) and (3R,6R,7S)-1,10-bisaboladien-3-ol (2) were baselineseparated (Figure 2, bottom) on this column, with stereoisomer 2 matching cis-zingiberenol. Received: July 21, 2015

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DOI: 10.1021/acs.jnatprod.5b00638 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Segments of GC-MS total ion chromatograms on Hydrodexβ-6TBDM; isothermal at 130 °C, He 1.5 mL/min. (Bottom) Mixture of (3S,6S,7S)-isomer 1 and (3R,6R,7S)-isomer 2. Peak assignments were done using individual stereoisomers; (middle) cis-zingiberenol from Z. of f icinale; (top) mixture of cis-zingiberenol from Z. of ficinale and isomers 1 and 2.

Figure 3. Segments of GC-MS total ion chromatograms on Hydrodexβ-6TBDM; isothermal at 130 °C, He 1.5 mL/min. (Top) cisZingiberenol from Z. of f icinale; (middle) mixture of (3R,6R,7R)isomer 3 and (3S,6S,7R)-isomer 4; (bottom) mixture of isomers 3 and 4 and cis-zingiberenol from Z. of f icinale.

(3R,6R,7R)-1,10-Bisaboladien-3-ol (3) and (3S,6S,7R)-1,10bisaboladien-3-ol (4) were partially separated on Hydrodex-β6TBDM (Figure 3), but neither of these matched the ciszingiberenol isolated from Z. off icinale. Thus, exploiting just one GC column permitted unequivocal proof of the (3R,6R,7S) absolute configuration of the cis-zingiberenol isomer 2. Determination of the absolute configuration of the transzingiberenol isolated from Z. of ficinale required the use of two columns. First, a four-component mixture of synthetic transstereoisomers of 1,10-bisaboladien-3-ol was partially separated on Hydrodex-β-6TBDM (Figure 4), with the (3S,6R,7S)isomer 5 and the (3R,6S,7S)-isomer 6 coeluting and matching natural trans-zingiberenol. The (3S,6R,7R)-isomer 7 and (3R,6S,7R)-isomer 8 had markedly different retention times from that of the natural product. Thus, separation of stereoisomers 5 and 6 required further searching for a different GC column. Earlier it was noticed that trans-1,10-bisaboladien3-ols produced broad peaks on nonpolar GC columns. However, alcohols 5 and 6 are partially separated on a 60 m DB-5 column (Figure 5, top). A mixture containing transstereoisomers 5 and 6, along with the two cis-stereoisomers 1 and 2, was produced by the reaction of 4-[(S)-6-methylhept-5en-2-yl)]cyclohex-2-enone with methyl lithium.16 As shown in Figure 5, the natural trans-zingiberenol matched (3R,6S,7S)isomer 5, which was confirmed by GC analysis of a mixture of the natural product and the aforementioned synthetic mixture. Not surprisingly, the C-6 and C-7 absolute configurations in both isolated zingiberenols 2 and 5 match those of αzingiberene20,21 and β-sesquiphellandrene22 present abundantly in ginger rhizomes.23 The biochemical pathway leading to formation of zingiberenols 2 and 5 in ginger could be envisaged through the (6R,7S)-bisabolyl cation 9 formed via enzymatic

Figure 4. Segments of GC-MS total ion chromatograms on Hydrodexβ-6TBDM; isothermal at 130 °C, He 1.5 mL/min. (Bottom) transZingiberenol from Z. of f icinale; (top) mixture of (3S,6R,7S)-isomer 5, (3R,6S,7S)-isomer 6, (3S,6R,7R)-isomer 7, and (3R,6S,7R)-isomer 8.

cyclization of farnesyl diphosphate (FPP).24,25 While a nonregioselective proton elimination from cation 9 leads to main sesquiterpene constituents α-zingiberene and β-sesquiphellandrene, a nonstereoselective trapping of 9 with water B

DOI: 10.1021/acs.jnatprod.5b00638 J. Nat. Prod. XXXX, XXX, XXX−XXX

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zingiberenol epoxides of opposite configurations at C-6 and C-7: (3S,6S,7R,10S)-, (3R,6S,7R,10S)-, and (3S,6S,7R,10R)10,11-epoxy-1-bisabolen-3-ols.17−19 If a zingiberenol with (6S,7R)-configuration seems a conceivable biosynthetic precursor to M. histrionica and H. halys pheromone, this compound is yet to be found in plants.

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EXPERIMENTAL SECTION

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AUTHOR INFORMATION

General Experimental Procedures. GC-MS analyses were performed in electron EI mode at 70 eV with an Agilent Technologies 5973 mass selective detector interfaced with a 7890A N GC system equipped with either a 30 m × 0.25 mm i.d. × 0.25 μm film HP-5MS or a 60 m × 0.25 mm i.d. × 0.25 μm film DB-5MS, or a 25 m × 0.25 mm i.d. Hydrodex-β-6TBDM capillary column (Macherey-Nagel GmbH & Co. KG). TLC analyses were conducted on Whatman AL SIL G/UV plates using a 20% EtOH solution of phosphomolybdic acid and/or UV for visualization of spots. Flash chromatography was carried out with 230−400 mesh silica gel (Fisher Scientific). All reagents and solvents were purchased from Aldrich Chemical Co., and synthetic stereoisomers of 1,10-bisaboladien-3-ols 1−8 were prepared as described.19 Isolation of Zingiberenols from Ginger. Fresh ginger rhizomes (908 g) grown in Brazil (purchased from a local grocery) were frozen in dry ice and then shredded in a blender. The resulting pulp was suspended in 3.0 L of deionized H2O and distilled until no organic material was found in the distillate. The distillate was extracted with hexanes, and the extract was concentrated to yield 4.15 g of organic material. This was purified by flash chromatography on SiO2 with 15− 30% EtOAc in hexanes. Fractions corresponding to synthetic cis- and trans-1,10-bisaboladien-3-ols, used as reference standards in TLC analyses, were collected and concentrated to give 101 and 58 mg of products containing a faster-eluting cis-alcohol and a slower-eluting trans-alcohol, respectively. These fractions were further purified by chromatography with 10% AgNO3 on SiO2 using CH2Cl2/EtOAc/ MeOH, 10:0.5:0.1, to yield 10 mg of cis- and 6 mg of trans-1,10bisaboladien-3-ols (Figure 1). To determine their absolute configurations, isolated cis- and trans-zingiberenols were further analyzed on Hydrodex-β-6TBDM and 60 m DB-5MS GC columns.

Figure 5. Segments of GC-MS total ion chromatograms on a 60 m DB-5 column; 50(5) to 270 °C at 10 °C/min, He 1.0 mL/min. (Top) Mixture of stereoisomers 1, 2, 5, and 6 formed by reaction of 4-[(S)-6methylhept-5-en-2-yl)]cyclohex-2-enone with methyl lithium; (middle) trans-zingiberenol from Z. of f icinale; (bottom) mixture of 1, 2, 5, and 6 plus trans-zingiberenol.

would result in zingiberenols 2 and 5 (Figure 6) produced as byproducts. Interestingly, cis-zingiberenol 2 produced in ginger has the same absolute configuration as the zingiberenol produced by the male rice stink bug, Oebalus poecilus, as a sex pheromone. However, unlike the plant-produced material, only one stereoisomer of zingiberenol was biosynthesized in O. poecilus, which is indicative of the stereospecific action of a hydratase26 possibly adding water across double bonds of α-zingiberene and β-sesquiphellandrene or directly reacting with bisabolyl cation 9. In contrast to the O. poecilus sex pheromone, aggregation pheromones of M. histrionica and H. halys consist of

Corresponding Author

*Tel: (301) 504-6138. Fax: (301) 504-5104. E-mail: ashot. [email protected]. Notes

The authors declare no competing financial interest.

Figure 6. Proposed biosynthesis of zingiberenols 2 and 5 in ginger. C

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ACKNOWLEDGMENTS A.K. and S.S. received funding from USDA NIFA SCRI grant #2011-51181-30937. Mention of trade names or commercial products in this publication is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00638 J. Nat. Prod. XXXX, XXX, XXX−XXX