Biosynthesis of the sesquiterpene patchoulol from farnesyl pyrophosphate in leaf extracts of Pogostemon cablin (patchouli): Mechanistic considerations

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 256, No. 1, July, pp. 56-68, 1987

Biosynthesis of the Sesquiterpene Patchoulol from Farnesyl Pyrophosphate in Leaf Extracts of Pogostemon cab/in (Patchouli): Mechanistic Considerations’ RODNEY

Institute

CROTEAU:

of Biological

SHARON L. MUNCK, CASIMIR C. AKOH, AND D. MICHAEL SATTERWHITE Chemistry,

Washington State University,

Pullman,

Received December 29,1986, and in revised form February

HENRY

J. FISK,

Washington 99164-6340 27,198’7

Several mechanistic alternatives have been proposed for the enzyme-catalyzed, electrophilic cyclization of farnesyl pyrophosphate to the tricyclic sesquiterpene alcohol patchoulol, which is the characteristic component of the essential oil of Pogostemon cablin (patchouli). These alternatives include schemes involving deprotonation-reprotonation steps and the intermediacy of the monocyclic and bicyclic olefins germacrene and bulnesene, respectively, and involving a 1,3-hydride shift with only tertiary cationic intermediates and without any deprotonation-reprotonation steps. Analytical studies, based on analyses of P. cablin leaf oil at different stages of plant development, and in viva time-course investigations, using 14C02 and [‘4C]sucrose, gave no indication that germacrene and bulnesene were intermediates in patchoulol biosynthesis. A soluble enzyme system from P. cablin leaves was prepared, which was capable of converting farnesyl pyrophosphate to patchoulol, and isotopic dilution experiments with both labeled and unlabeled olefins were carried out with this system to confirm that sesquiterpene olefins did not participate as free intermediates in the transformation of the acyclic precursor to patchoulol. Patchoulol derived biosynthetically from [12,13-14C;1-3H]farnesyl pyrophosphate was chemically degraded to establish the overall construction pattern of the product. Similar studies with [12,13-‘4C,6-3HSfarnesyl pyrophosphate as a precursor eliminated deprotonation steps to form bound olefinic intermediates in the biosynthesis of patchoulol, while providing supporting evidence for the hydride shift mechanism. 0 1987 Academic

Press, Inc.

Patchouli alcohol (patchoulol) (6) is the major component of the essential oil of patchouli (Pogostemon cablin Benth.; syn. P. heyneanus and P. patchcruli Pellet var. suavis Hook.) and is an important perfumery raw material because of its character-

istic woody fragrance and fixative properties (1, 2). In addition to patchoulol, the structure and absolute configuration of which were elucidated in the mid 1960s (35), the oil of patchouli contains the related sesquiterpene olefins a-patchoulene (7), ppatchoulene (8), and y-patchoulene (9), as well as a-guaiene (lo), a-bulnesene (ll), and caryophyllene (2, 3, 6-8). Patchoulol and the patchoulenes are also constituents of spikenard oil (from Nardostachys spp.) where they cooccur with aristolane and gurjunane sesquiterpenes, rather than with sesquiterpenes of the bulnesane/ guaiane type (9,10).

r This is Scientific Paper No. 7649, Project 0268 from the Research Center, College of Agriculture and Home Economics, Washington State University, Pullman, Washington 99164. This investigation was supported in part by National Science Foundation Grant DMB8507121 and by a grant from the Haarmann and Reimer Corporation. ’ To whom correspondence should be addressed. 0003-9861/87 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

56

PATCHOULOL

FROM

FARNESYL

PYROPHOSPHATE

The several thousand sesquiterpenes of plant and microbial origin comprise nearly 200 skeletal types, and all are considered to be derived from trans,trans-farnesyl pyrophosphate (1) (11,12). However, the biosynthesis of relatively few sesquiterpenes has been examined, either in vivo, or in cellfree enzyme systems where the details of the cyclization of farnesyl pyrophosphate can be most carefully explored (11-14). Based largely on the earlier mechanistic considerations of Hendrickson (15) and Parker and associates (16), and on “biomimetic” syntheses, the biogenesis of the patchoulane sesquiterpenes from farnesyl pyrophosphate is thought to involve protonation of the isopropenyl group of an (+)a-bulnesene intermediate (ll), followed by cyclization and a series of Wagner-Meerwein rearrangements (17-19) (11 * 4 + 5 + 6 of Scheme I). As pointed out by Coates (19), a-bulnesene is itself regarded as being formed by proton-induced cyclization of a germacratriene (e.g., (+)-germacrene A, (14)). Thus, the proposed overall conversion of farnesyl pyrophosphate to the patchoulanes would be unusual in proceeding via two deprotonated (olefinic)

IN Pogostemm

57

intermediates (via 1 + 2 + 14 + 15 -+ 11 + 4 + 5 + 6). A minor variant on this scheme replaces bulnesene with bulnesol as the key intermediate, but is otherwise identical in concept (20). Reexamination of the biogenetic possibilities revealed an alternate cyclization mode without deprotonation-reprotonation steps, but rather involving only tertiary cationic intermediates and a 1,3-hydride shift in the construction of cation (4) from which the patchoulenes (7, 8, 9) and patchoulol (6) could ultimately arise (i.e., 1 + 2 -+ 3 + 4). A stereoelectronically feasible variation on this carbocationic scheme might involve a pair of 1,2-hydride shifts, rather than the 1,3-shift, in conversion of (3) to (4). The cyclization of the germacryl cation (2) to the patchoulyl skeleton (12), which by hydride shift then capture of the resulting cation by Hz0 would yield patchoulol directly (1 + 2 + 12 + 13 + 6) while bypassing the patchoulenes (7, 8, 9), is less favorable on steric grounds because of the transient generation of the trans-cyclooctene system in the 2 + 12 conversion. Earlier investigations with P. cablin have not addressed the origin of the patchoulane

\fiigide

8

c&din

7

5 SCHEME I

9

Shift

58

CROTEAU

congeners (8, 21, 22), and no other information was available bearing on the proposed deprotonation-reprotonation steps or hydride shift alternatives for the biosynthesis of these complex tricyclic compounds. In this communication we report on in vivo studies with P. cab&, and describe the development of a cell-free enzyme system for the conversion of farnesyl pyrophosphate to the patchoulenes and patchoulol. The results obtained from both experimental approaches support the alternative 1,3-hydride shift mechanism for the cyclization.

ET AL.

6-methyl-5-hepten-2-iodide to [6-3Hjgeranyl THP3 ether trisnoraldehyde, and thus differed from an earlier synthesis of the 6-3H-labeled precursor (28) in minimizing the number of steps required following the introduction of tritium. The [6-aH]geranyl THP ether trisnoraldehyde was prepared from geranyl THP ether (29) by epoxidation with m-chloroperbenzoic acid (30) and cleavage of the 6,7-epoxide with periodic acid in dioxane (31). The crude trisnoraldehyde was purified by column chromatography on silica (gradient development with ether in hexane) and 0.53 mmol of the product was treated with NaBaH (25 mCi, 528 Ci/mol, New England Nuclear) in ethanol/Hz0 to afford the corresponding 6-aH-labeled alcohol (40% radiochemical yield, -132 Ci/mol) which was purified by TLC (silica gel G, with hexane:ether (l:l, v/v)). The alcohol was reoxidized to the aldehyde by treatment with pyriEXPERIMENTAL PROCEDURES dinium chlorochromate and sodium acetate in CHaCla Plant materials, substrates, and reagents. P. cab& (32), and the product was purified by TLC (silica gel plants were obtained from Longwood Gardens (KenG, with hexane:ether (l:l, v/v)) to afford pure [6nett Square, PA) and were propagated from cuttings 3H]geranyl THP trisnoraldehyde which was diluted in peat:pumice:sand (55:35:10) in a greenhouse with to a specific activity of 40 Ci/mol before use. supplemental lighting (21,000 lx minimum) for 16 h For the preparation of the alkylidenephosphorane, days, with a 90/7O”C (day/night) temperature cycle. 6-methyl-5-hepten-2-01, prepared by NaBH, reduction Plants were fertilized weekly with a complete fertilizer of 6-methyl-5-hepten-2-one, was converted to the pplus micronutrients. Plants were allowed to grow to toluene sulfonate and thence to the iodide (33) which 20 cm in height (at least six leaf pairs) before use, was purified by column chromatography on silica with and the shoot apex and first leaf pair (l-l.5 cm in hexane. A solution of the 6-methyl-5-hepten-2-iodide length) were used in in viva experiments with r4COz and a slight excess of triphenylphosphine in anhyand [‘*C]sucrose, and for the preparation of cell-free drous acetonitrile was refluxed under N2 for 48 h. The extracts. solvent was removed under vacuum, the remaining [U-‘4C]Sucrose (3.6 CVmol; radiochemical purity residue was dissolved in acetone, and ether was added. > 99%) was obtained from New England Nuclear The precipitate so formed was pulverized, collected, Corp., and 14C0, was generated by the addition of and recrystallized from CH&&:ether to give B-methylH,POI to aliquots of aqueous NaH14C03, a stock so- 5-hepten-2-(triphenyl)phosphonium iodide as a creamlution of the latter having been prepared from Bar4C03 colored solid (mp = 232-234°C; confirmed by ‘H NMR). (48 Ci/mol, New England Nuclear Corp.). tranqtransThe corresponding phosphorane was generated by the [l-3H]Farnesyl pyrophosphate (84.8 Ci/mol) was preaddition of 0.13 ml of 1.35 M n-butyl lithium to 0.18 pared and purified as described previously (23, 24), mmol of the phosphonium iodide in anhydrous THF and trans,trans-[12,13-‘4C]farnesyl pyrophosphate (2 at 0°C under N,, and the resulting deep red solution Ci/mol) was a gift from Professor D. E. Cane of Brown was stirred for 2.5 h while warming slowly to room University (25). [12,13-‘4C;1-3H]Farnesyl pyrophostemperature. The solution was cooled again to 0°C phate was prepared by admixture to a ‘H:r4C ratio of and 0.12 mmol[6-aH]geranyl THP trisnoraldehyde in about one, and the mixture repurified by ion-exchange 0.5 ml THF was added. After stirring at 0°C for 2 h, chromatography to a aH?C ratio of 0.93 f 0.02, as the solution was stirred overnight at room temperadetermined by enzymatic hydrolysis of a sample, TLC ture during which time it slowly changed color from purification of the resulting farnesol, and preparation red to pale yellow. The resulting slurry was poured of the crystalline pnitrobenzoate (mp = 30-31°C from into a solution of 20% methanol in half-saturated hexanes). The position of labeling of the product was NH&l and the mixture was extracted with ether. The also verified by MnOa oxidation (26) of farnesol to farcombined organic extracts were washed with satunesal (3H:‘4C = 0.47) and oxidation of the aldehyde to rated NaCl, dried over MgSO*, and the truns,trarzsfarnesoic acid (3H:14C = ~0.01) with Tollen’s reagent isomer was purified from the mixture by repeated (27). [12,13-‘4C;6-3H]Farnesyl pyrophosphate was also prepared by admixture of the corresponding r4C-labeled and 3H-labeled pyrophosphate esters. The latter 3 Abbreviations used: THP, tetrahydropyranyl; was prepared by a technique involving the addition THF, tetrahydrofuran; Mes, 4-morpholineethanesulof the alkylidenetriphenylphosphorane prepared from fonic acid.

PATCHOULOL

FROM

FARNESYL

PYROPHOSPHATE

preparative argentation TLC (silica gel G containing 15% AgN03 with hexane:ether (2:1, v/v)). The trans,trans-[6-3Hlfarnesyl THP ether was treated with pyridinium ptoluene sulfonate in ethanol at 55°C for 3 h to provide trun.s,truns-[6-3H]farnesol after evaporation of solvent, addition of ether, and passage through a short column of silica. The radiochemical purity and specific activity of this product (98%, 40 Ci/mol, 8% radiochemical yield) were verified by combination of radio-GLC and capillary-GLC, and the material was pyrophosphorylated by published procedures and purified by passage through Dowex 5OWX8 and CF-11 cellulose (23). The [6-3H]farnesyl pyrophosphate was then mixed with the 12,13-‘4C-labeled product to a ‘H?C ratio of about one and repurified by ion-exchange chromatography on DEAE-cellulose, whereupon a major ‘H-bearing contaminant, eluting after farnesyl pyrophosphate, was noted. This product of unknown origin was of a polarity consistent with a tetraphosphate. The [12,13-‘4C,6-3H]farnesyl pyrophosphate so isolated was again repurified by ion-exchange chromatography using a flatter gradient to ensure removal of the unidentified contaminant. A sample of this material was enzymatically hydrolyzed to farnesol, which after addition of carrier, TLC purification, and preparation of the crystalline pnitrobenzoate (mp = 30-31”C), confirmed that the ‘H?C ratio had dropped to 0.48 f 0.02. Authentic standards of nerolidol (85% truns, 15% ~1’s)and tran.s,truns-farnesol were obtained from K & K Laboratories and Aldrich Chemical Co., respectively, while oil of patchouli was provided through the generosity of Dr. R. Hopp, Haarmann and Reimer GmbH, Holzminden, West Germany. The sesquiterpene olefins of patchouli and pure patchoulol were obtained from patchouli oil by dilution in hexane and passage through a silicic acid column to obtain the olefin fraction. Patchoulol was then obtained by gradient elution with hexane:ether (9:l to 8:2), the fractions containing the patchoulol being combined and concentrated and the product recrystallized from hexane. Verification of identity was by mp (55-56”C, lit. mp = 55-56°C (34)), GC-MS, ir, and ‘H NMR (5). The purity of the product was confirmed by capillary GLC to be in excess of 98%.The sesquiterpene triene fraction from immature Douglas fir needle oil (containing germacrenes A, B, and D) was obtained by hexane extraction of the plant tissue, partitioning of the hydrocarbons on silicic acid, and preparative argentation TLC (12% AgNO3, with hexanes:benzene: ether (50:50:1, v/v/v), Rf= 0.2-0.4). This material was shown by GLC-MS to consist of germacrene D (-30%) (.35), germacrene A (-8%) (36), and germacrene B (-5%)(37). In viva experiments. For experiments with [U“C]sucrose, matched cuttings (shoot tip and first leaf pair) were placed in vials and administered an aqueous solution containing 1.39 pm01 of [“Clsucrose (5.0 pCi

IN Pogostemon

w&in

59

in 0.1 ml) through the cut stems. Incubation was at 30°C in the light (21,000 lx). After the uptake of labeled material (l-2 h), the vials were kept filled with distilled water and, after periods of 3, 6, 9, 12, and 18 h, two cuttings were transferred to a Ten-Broeck homogenizer and thoroughly extracted with 15 ml diethyl ether. Authentic patchouli oil (20 pl) was added to the extract, which was concentrated to 5.0 ml and subjected to exhaustive microscale steam distillation (38). The aqueous phase of the distillate was saturated with NaCl and the volatile organic products were recovered by ether extraction. Aliquots were taken for determination of radioactivity and for radio-GLC analysis. Experiments with 14C02 were similarly performed, except in this instance 12 cuttings were exposed to 50 &Ci ‘*CO2 in air in a 250-ml chamber for 2 h in the light. After flushing the chamber with air, a pair of cuttings was taken at 3, 6, 9, 12, and 18 h intervals, and the labeled products were isolated and analyzed as before. Experiments with 14C02 and [‘4C]sucrose were run in duplicate. Enzyme extraction and partial pur$cation Enzyme extracts were prepared by a general technique described previously for the isolation of monoterpene and sesquiterpene cyclases from sage leaves (13). Thus, 5-10 g of immature leaves were homogenized in a TenBroeck homogenizer with 25% tissue weight of insoluble polyvinylpolypyrrolidone (Polyclar AT (39)) in cold 75 mM Mes-5 mM phosphate buffer (pH 6.0,2 ml/ g tissue), containing 20% glycerol, 5 mM dithiothreitol, 10 mM Na&05, 10 mM sodium ascorbate, and 15 mM MgCl*, and the homogenate was then slurried with an equal tissue weight of polystyrene resin (XAD-4 (40)). Following the removal of the polymeric adsorbents and cell debris by filtration through cheesecloth and centrifugation at 3OOOg,the extract could be assayed. In most instances, the preparation was sequentially centrifuged at 27,OOOgand 145,OOOgto obtain the soluble supernatant in which the bulk of the cyclase activity resided. Partial purification was achieved by concentration of the extract (by ultrafiltration through an Amicon PM-30 membrane) and passage through a 2.5 X 120 cm column of Sephacryl S-200 equilibrated and run with 10 mM Mes-5 mM phosphate buffer, pH 6.0, containing 10% glycerol, 1 mM dithiothreitol, and 5 mM sodium ascorbate. Assay procedure. In a typical assay, a l-ml aliquot of the preparation (50-100 pg protein) was incubated in a screw-capped vial in the presence of 30 mM MgCl* and 10 jaM [1-‘Hlfarnesyl pyrophosphate. Following incubation at 30°C for 1 h, the tube was chilled in ice, and the radioactive pentane-soluble products were extracted (2 X 1.5 ml) and partitioned on a silicic acid column (13) into a sesquiterpene hydrocarbon fraction eluted by pentane (typically 5% conversion of substrate) and a polar, oxygenated sesquiterpene fraction eluted with ether (typically 20% conversion of sub-

60

CROTEAU

strate). After counting an aliquot of each fraction, appropriate carrier standards were added (20 pl of the olefin fraction isolated from patchouli oil, and a mixture containing 5 mg each of patchoulol, nerolidol, and farnesol) prior to radio-GLC analysis. For assays in which it was necessary to isolate individual products, the two fractions were concentrated under vacuum and separated by TLC (sesquiterpene olefins on silica gel G containing 15% AgNOa using hexane:benzene:ether (50:50:1, v/v/v), and oxygenated sesquiterpenes on silica gel G using hexane:ether (7:3, v/v)). When only patchoulol was of interest, the ether eluant was treated with excess 090, in pyridine to convert unsaturated compounds to the corresponding polyols (13, 41), thereby facilitating TLC isolation of the product. To search for possible phosphorylated intermediates in the reaction, the aqueous phase of the incubation mixture remaining after pentane extraction was treated with a mixture of 3 units acid phosphatase and 2 units apyrase (13,41) and the terpenols so liberated were examined by radio-GLC and radio-TLC as before. Preparative incubations were carried out at 5- to lo-fold the normal assay scale for 3 h in the presence of 30 mM Mg*+ and 20 pM substrate, and the extracts from several such incubations were pooled before product separation. Product degradation and preparation of derivatives. To locate the tritium in radioactive patchoulol derived enzymatically from [12,13-14C;1-SH]- and [12,13-i4C,63H]-labeled farnesyl pyrophosphates, a degradative scheme employed by Btichi and co-workers (42) for the structure elucidation of patchoulol was modified. Thus, the biosynthetic patchoulol(6) isolated by TLC, was diluted with -2.5 mmol authentic carrier and crystallized to constant specific radioactivity, followed by treatment with 0.15 ml of 50% aqueous HzSO* in 7 ml diethyl ether at reflux for 20 min to effect the conversion to fl-patchoulene (8) (see Scheme III). The reaction mixture was poured into water and the ether extract was washed with dilute NaHC03 and water, then dried over NazS04 and concentrated to an oily residue. The residue was dissolved in pentane containing 2% ether and the solution was passed through a short column of silicic acid to provide crude @patchoulene. The column was then rinsed with ether to obtain the unreacted patchoulol which was recycled through the HaSO treatment. The P-patchoulene was purified by preparative argentation TLC on silica gel G containing 15% AgNOa with hexane:benzene:ether (50:50:1, v/v/v) (combined average yield 55%). The purity of the product was verified by capillary GLC (>98%)and aliquots of the sample were weighed and counted. The /3-patchoulene (8,1 mmol) and NaI04 (10 mmol) were added to 49 ml of a solution of CH.J&, acetonitrile, and water (223, v/v/v) to which RuC&. Hz0

ET AL. (2 mmol) was added and the golden-brown mixture was stirred vigorously at room temperature for 2 h (43). Additional CHzClz was added and the phases were separated. The aqueous phase was washed with CHzClz and the combined organic extracts were washed with saturated NaCl and dried over NasSO, before evaporation of solvent. The residue was dissolved in ether and, following filtration through celite (43) and concentration, this material was separated by preparative TLC (silica gel G, with hexane:ethyl acetate (7~3, v/ v)) to afford crystalline patchoulidione (16, Scheme III) (mp = 95’C from hexane; Lit. mp = 95-96°C (42); 53% average yield), portions of which were weighed and counted. To a solution of patchoulidione (0.5 mmol) in a minimum amount of ether was added 3 ml of BFs-etherate which was stirred at 30°C for 48 h (42). The reaction mixture was cooled on ice and poured into water, and the aqueous layer was extracted with ether. The combined ether extract was washed with water and dried over Na,SO, before evaporation of solvent. The residue was then dissolved in hexane and the solution was passed over neutral alumina (Type I) with 5% ethyl acetate in hexane (42) to provide the substituted cyclohexenone (17) (0.3 mmol) plus an unidentified diketone (0.15 mmol) similar in MS to, but not identical by GLC retention with, the starting material. Since the enone (17) and diketone were only separable by GLC, the mixture was hydrogenated (HZ over 10% Pd/C) in hexane at room temperature for 2 h, after which the suspension was filtered through celite and the solvent evaporated. The residue was dissolved in the minimum quantity of ether and the unchanged diketone removed by preparative TLC (silica gel G, with hexane:ethyl acetate (4:1, v/v)) to give the pure saturated ketone (18) (0.27 mmol average yield) of which portions were weighed and counted. The hydrogenation reaction was also evaluated using 2H2 over 10% Pd/C and the product was purified as before and examined by GLC-MS. The deuterated ketone was then subjected to exhaustive base-catalyzed exchange of the a-hydrogens by treatment with excess 5% sodium methoxide in methanol on a steam bath for 1 h. The product, reisolated by pouring the reaction mixture into water and ether extraction, was examined for deuterium content by GLC-MS. Verification of the identity of each degradation product of patchoulol was by mp (where appropriate), MS, ir, and ‘H NMR, and purity was determined by capillary GLC. Terpenyl pnitrobenzoates and terpenone oximes were prepared by standard techniques (44). Analytical procedures. The general procedures for radio-TLC and radio-GLC have been described (13). TLC developing solvents are indicated in the text, and the radio-GLC columns were 10 ft X 0.125 in. o.d., stainless steel, packed with either 15% FFAP, 15% Carbowax 20M, or 15% SE-30 on Chromosorb WHP

PATCHOULOL

FROM

FARNESYL

PYROPHOSPHATE

IN Pogostemm cablin

61

cis-, lO cm in length, 0.8% fresh wt yield) contained 20% (Yguaiene, 16% a-bulnesene, 10% a-patchoulene, 5% seychellene, 3% B-patchoulene, minor amounts of y-patchoulene, /3-eleRESULTS AND DISCUSSION mene, and a-gurgenene, and -35% paPatchouli leaf oil analysis. As with other tchoulol, with less than 4% caryophyllene members of the Lamiaceae, the sesquiter- and 3% nerolidol. In both yield and compenoids of P. cablin are produced and ac- position, the oil of mature patchouli leaves cumulated in glandular trichomes (8), the resembled the commercial oil (6-8). Aloily contents of which are readily isolated though the composition of patchouli oil for analysis by combination of solvent ex- changed significantly during development traction-steam distillation (45). The oil with regard to caryophyllene and nerolidol produced by patchouli leaves was isolated content, the changes in relative proportions from representative samples at various of the remaining constituents were ministages of development ranging from emer- mal and gave no indication of possible pregence to full expansion. Each crude oil cursor-product relationships. sample was initially analyzed by capillary In viva studies. To further examine GLC, followed by partitioning of the sam- possible precursor-product relationships ple on silica into a hydrocarbon fraction among the sesquiterpenes of patchouli oil, and oxygenated terpenoid fraction (13) pulse-chase experiments with 14C02 and from which individual subfractions were [U-14C]sucrose were carried out with isolated by TLC and argentation TLC, The patchouli shoot tip cuttings. Following identity of each sesquiterpenoid component pulse labeling of a set of matched cuttings, was confirmed by comparison of retention two cuttings were removed at 3-h intervals, index and mass spectrum, and where nec- the oil was extracted with ether, and the essary ir and ‘H NMR spectra, to those of extract was subjected to microscale steam the authentic compound (2, 6, 46-48). distillation (38). An aliquot of the distillate Monoterpenes (largely CX-and fl-pinene) was taken for determination of 14Ccontent, comprised 15-30% of the oil and were not and the remainder was concentrated and examined in detail. separated by radio-GLC to determine the The oil from emerging leaves (co.5 cm distribution of labeled components. The in length, 0.6% fresh wt yield) was shown levels of incorporation of label into volatile to contain caryophyllene (16%),a-guaiene sesquiterpenes were typical for experi(17%), cY-bulnesene (13%), a-patchoulene ments of this type (49) (maximum label(G%),P-patchoulene (1.6%),and minor lev- ing of 0.3% with 14C02 was observed at els of ,&elemene, humulene, seychellene, y- 12 h; maximum labeling of 0.9% with patchoulene, and gurgenene, and the oxy- [14C]sucrosewas observed at 9 h). With the genated compounds nerolidol (tram-, 13%; exception of caryophyllene and nerolidol,

(operated at 150°C for 10 min and programmed at 4’C/min to 220°C or operated isothermally at 1’70°C if only olefins were examined, with a He flow rate of 60 ml/min). Analytical GLC and GLC-MS (at 70 eV, Hewlett-Packard 5840-5985) were performed on 25m fused silica capillary columns (Superox or RSL150). Infrared and ‘H-NMR spectra were obtained on a Beckman IR-20 spectrophotometer and JEOL FX-90 spectrometer, respectively. Radioactivity in liquid and solid samples was determined in a counting solution (15 ml) consisting of 03% (w/v) Omnifluor (New England Nuclear) dissolved in 30% ethanol in toluene. The counting efficiency for aH was 27% and for 14C was 82%, and all assays were counted to a standard error of less than 1%. Quench correction in double-label experiments was accomplished by internal standardization using ‘H- and i4C-labeled toluene.

62

CROTEAU ET AL.

which acquired label at a somewhat faster rate than all other sesquiterpenes, and exhibited detectable turnover of 15-20% at the last time period examined, no differential labeling of the other sesquiterpenoid components of patchouli was observed. Thus, all olefins but caryophyllene acquired label at a relative rate correspondingly less than that of patchoulol, all reached a labeling maximum at 12 and 9 h for COz and sucrose, respectively, and all exhibited no apparent change throughout the remainder of the time course. The distribution of label from both 14C02and [‘4C]sucrose resembled closely the relative composition of the leaf oil at this stage of maturity (i.e., caryophyllene (13-17%),aguaiene (15-18%),abulnesene (lo-13%),a-patchoulene (-5%), nerolidol (lo-14%), and patchoulol (2833%)), and, with the exceptions noted above, all sesquiterpene olefins and patchoulol exhibited a similar smooth increase in specific activity to maximum for the duration of the time course. Thus, neither the analytical studies nor the in viva pulse-chase experiments gave evidence to support any precursor-product relationships between sesquiterpene olefins and patchoulol. To address this point in any greater detail required a cell-free biosynthetic system. Patchoulol synthesis in a cell-free system. Patchouli leaf homogenates were prepared using a general method previously developed for the isolation and assay of monoterpene and sesquiterpene cyclases (13), and this crude extract was examined for the ability to convert [l-3H]farnesyl pyrophosphate (10 pM) to patchoulol in the presence of 1.5 mM Mnzf and 15 mM Mg’+. Pentane extraction of the reaction mixture and partitioning of the extract on silica gave an olefin fraction (5% conversion) and, by ether elution of the column, an oxygenated sesquiterpene fraction (20% conversion). Radio-GLC analysis of the olefin fraction revealed this material to contain five major labeled components chromatographically coincident with authentic standards of caryophyllene (31% ), a-bulnesene (23.5%), cu-guaiene (20.5%), apatchoulene (14.9%), and P-patchoulene

(7%). Radio-GLC analysis of the oxygenated sesquiterpenoids revealed the presence of nerolidol(‘7.5%),patchoulol(17.5%), and farnesol (75%). Boiled controls produced negligible levels of olefins or patchoulol and very low levels of solvolytitally generated nerolidol and farnesol, indicating that the activities observed were enzymatic. The oxygenated sesquiterpene fraction pooled from several incubations was treated with excess Os04 in pyridine to convert farnesol and nerolidol to the corresponding polyols, and 0.5 mmol of authentic patchoulol was added to the 3H-labeled patchoulol and this material was purified from the polyols by TLC. Crystallization to constant specific activity from hexane (0.182 mCi/mol; mp = 55-56”C, Lit. mp = 55-56°C (34)) confirmed the identity of the biosynthetic patchoulol. The identities of the labeled nerolidol and farnesol were similarly verified (in samples not treated with 0~0~) by dilution with authentic (*)-nerolidol and trans,trana-farnesol, TLC purification, and crystallization of the nitrobenzoates. Identities of the biosynthetic sesquiterpene olefins were confirmed by dilution of the olefin fraction with authentic olefins (from patchouli oil), separation of components by argentation TLC, and radio-GLC coincidence on two columns of widely differing polarity. The component identified as a-guaiene on initial radio-GLC was shown by subsequent argentation TLC to be comprised of CXguaiene and an unidentified sesquiterpene olefin likely to be a triene. Differential centrifugation of the homogenate indicated that the ability to convert farnesyl pyrophosphate to patchoulol, nerolidol, and sesquiterpene olefins resided exclusively in the 145,000~supernatant, as did the bulk of the phosphatase activity. Particulate fractions pelleting at 27,OOOg and 145,000gcontained negligible farnesyl pyrophosphate cyclase activity, although residual phosphatase activity was present. Recombination of the supernatant with the particulate fractions provided cyclase activities no greater than that of the supernatant alone.

PATCHOULOL

FROM

FARNESYL

PYROPHOSPHATE

A preliminary attempt to fractionate the soluble enzyme preparation was made by concentration of the extract (by ultrafiltration) and passage through a 2.5 X 120cm Sephacryl S-200 column. Assay for all relevant activities indicated the activities for the conversion of farnesyl pyrophosphate to caryophyllene and nerolidol to elute very slightly ahead of patchoulol cyclase, which was coincident with the cyclase activities for the other sesquiterpene olefins. Phosphatase activity, as evidenced by the formation of farnesol, was prominent and eluted in a broad region encompassing all the other activities. Based on the elution profile on the gel permeation column, the molecular weight of the patchoulol cyclase was estimated to be about 70,000. Although the partially purified preparation contained relatively high levels of phosphatase activity (responsible for up to 20% loss of the substrate), an attempt was made to examine the reaction products for the presence of potential phosphorylated intermediates, such as nerolidyl and patchoulyl pyrophosphate, by enzymatic hydrolysis (apyrase plus acid phosphatase) of products remaining in the aqueous incubation mixture after solvent extraction. Evidence for the presence of only residual substrate was obtained (i.e., only farnesol was released by the hydrolysis procedure). Removal of divalent cations from the partially purified preparation by treatment with 5 mM EDTA and exhaustive dialysis reduced both sesquiterpene olefin cyclase and patchoulol cyclase activity (as well as the ability to produce nerolidol) to negligible levels, with only minor influence on phosphatase activity. Readdition of 15 mM MgClz restored activity nearly completely, with maximum cyclase activity observed at about 30 mM. MnClz was an inefficient substitute for MgClz, restoring olefin and patchoulol cyclase activity to about 30% of maximum at the optimum concentration of 3 mM. Other divalent metal ions tested ( co2+, Ni2+ Fe2+, Ca*+) were no more effective than Mn’+. Significantly, the product distributions generated in the presence of the different cations differed markedly, indicating the presence of several distinct

IN Pogostemon

cd&n

63

enzymatic activities. Thus, in the presence of Mg2+ (20 mM) substrate was converted to patchoulol at about twice the rate as to nerolidol, and the olefin fraction was comprised primarily of 40% caryophyllene, 18% cu-patchoulene, 17% a-bulnesene, and 21% cu-guaiene plus an unidentified sesquiterpene (coincident with a-guaiene on radioGLC but more like a germacrene on argentation TLC), whereas in the presence of Mn*+ (2 mM) the overall proportion of patchoulol decreased (patchoulol and nerolidol were formed in comparable amounts) and the olefin fraction was comprised largely of a-bulnesene (42% ), caryophyllene (33%),and a-patchoulene (20%), with very little a-guaiene and unidentified olefin (-1%). Combinations of Mn2+ and Mg2+ resulted in lower levels of cyclase activity than Mg 2+ alone, and so Mg2+ alone (30 mM) was used in subsequent studies. Product distribution was also shown to vary with pH in the range 5.3 to 7.4 (adjusted by dialysis); the pH optimum for patchoulol formation was near 6.0. Similarly, the distribution of oxygenated products and olefins varied somewhat from preparation to preparation (e.g., from 3 to 8% conversion to patchoulol, 5 to 10% conversion to olefins) and with time of storage of the preparation. The partially purified preparation lost 50-60% of cyclization activity within 2 days, and was essentially inactive after 5 days storage at 0-4°C (phosphatase activity, on the other hand, was quite stable). The only products found to vary consistently with patchoulol formation were the closely related olefins LYand P-patchoulene (y-patchoulene was always a minor component, the variation in production of which was difficult to estimate accurately). Since the activities for the cyclization of farnesyl pyrophosphate to patchoulol and structurally related olefins were essentially coincident on gel permeation chromatography, it was not possible to determine if the olefins were coproducts or intermediates of patchoulol biosynthesis, or the products of distinct cyclases. As an alternate approach to the question of olefinic intermediates in patchoulol biosynthesis,

64

CROTEAU

isotopic dilution experiments were carried out in which the conversion of [l3H]farnesyl pyrophosphate (10 FM) to patchoulol was examined in the presence of 0.5 mM of patchouli olefins (a mixture containing 29% a-bulnesene, 23% (Yguaiene, 17% a-patchoulene, 7% P-patchoulene, 6% caryophyllene, and less than 4% each of other olefins), both with and without Tween 20 at 50 pg/pmol total olefin. The presence of the unlabeled olefins, either added directly in 5 ~1 hexane or in aqueous solution containing Tween, had no discernible effect on the conversion of farnesyl pyrophosphate to patchoulol when compared to the appropriate controls. Similarly, the sesquiterpene olefin fraction isolated from immature Douglas fir needles (containing germacrenes A, B, and D), when incubated with [l-3H]farnesyl pyrophosphate and the enzyme under the same conditions, had no detectable effect on patchoulol biosynthesis. In an additional approach to addressing the question of olefinic intermediates in patchoulol biosynthesis, the labeled sesquiterpenes were isolated from preparative incubations of the enzyme with [l3H]farnesyl pyrophosphate. This material (0.91 &i, containing 8% @patchoulene, 24% caryophyllene, 20% a-patchoulene, 30% a-bulnesene, and 18% a-guaiene plus unidentified olefin) was divided into portions, 0.3 &i of which (in 5 ~1 hexane) was incubated with a fresh enzyme preparation containing Mg2+ but no [l-3H]farnesyl pyrophosphate. An identical 0.3 &i portion of the labeled olefins was suspended in 0.25 ml buffer containing 20 pg Tween before incubation with the enzyme. Following incubation, authentic carrier was added and the patchoulol was isolated by OsOl treatment and TLC; this product was not detectably labeled. To ensure that the patchoulol cyclase had not been inactivated under these incubation conditions, the experiment was repeated, but in this instance the 3H-labeled olefins were coincubated with 5 pM [12,13-‘4C]farnesyl pyrophosphate. The patchoulol so isolated was labeled with 14C(2.7% conversion), but once again no 3H-labeled product was detected.

ET AL.

Although it is possible that the exogenous sesquiterpene olefins may not be as accessible to the enzyme as those generated endogenously from the acyclic precursor, farnesyl pyrophosphate, the results would seem to argue against the intermediacy of free germacrene and a-bulnesene (or the other sesquiterpene olefins) in the biosynthesis of patchoulol, but leave open the possibility (by the coincidence of activities on gel permeation chromatography) that patchoulol and related olefins may be coproducts of the same cyclase enzyme. Labeling studies with [l-‘HIand [6‘H] farnesyl pgrophosphate. The summation of results from oil analysis, in viva studies, and isotopic dilution experiments provided no support for the intermediacy of sesquiterpene olefins in patchoulol biosynthesis, nor was any direct evidence provided bearing on the possibility of the hydride shift mechanism in the cyclization to patchoulol. To probe the latter question, the conversion of [12,13-‘4C;6-3Hlfarnesyl pyrophosphate to patchoulol was examined, since cyclization with retention of 3H would confirm the hydride shift (Scheme II), while appropriate degradation of the product would reveal the nature of the shift. Conversely, loss of 3H in the cyclization would be indicative of the deprotonation step to an olefin (germacrene + bulnesene) as an intermediate process in patchoulol biosynthesis (see Scheme I). Before undertaking studies with [12,13“C;6-3H]farnesyl pyrophosphate, the overall pattern of the cyclization was first con-

“o@ - @$L&L Ts TB 6

5

SCHEME II

4

PATCHOLJLOL

FROM

FARNESYL

PYROPHOSPHATE

firmed by employing [12,13-14C;1-3H]farnesyl pyrophosphate as the precursor. Thus, preparative scale incubation of this precursor (3H:‘4C = 0.93) with the partially purified enzyme preparation afforded 0.36 &i-3H (-3% yield) of patchoulol (presumably labeled as in Scheme II) which was diluted with 2.5 mmol of carrier and crystallized (mp = 56°C) to constant specific activity of 0.15 mCi-3H/mol with unchanged 3H:14Cratio of 0.91 rfr 0.02. Treatment of [‘4C;3H]patchoulol with H,S04 afforded /3-patchoulene (8) (purified by argentation TLC, 52% yield, 3H:‘4C = 0.91) which was oxidized with Ru04 to the crystalline patchoulidione (16) of unchanged isotope ratio (purified by TLC, 58% yield, 3H.‘4C = 0.92, mp = 94-95°C) (see Scheme III). Treatment of the diketone (16) with BF3-etherate to effect the conversion to the cyclohexenone (17) gave, by radio-GLC, the expected product (17) (56% yield by mass, but of diminished radioactivity) and an unidentified dione, similar but not identical to the starting material (31% yield by mass, with specific activity similar to patchoulidione) from which the cyclohexenone derivative was essentially inseparable by TLC. The mixture therefore was hydrogenated, and the unchanged dione then removed by TLC to yield the pure cyclohexanone derivative (18), which was analyzed as an oil (3H:‘4C = 0.01) and as the corresponding oxime (3H:‘4C = ~0.01). The experiment was repeated in toto with another preparation of patchoulol (0.154 mCi-3H/ mol; 3H:14C= 0.91) and again gave the 3H-

+&

@+3

14

CA

T6

I

0

Tl

1

)-,-&-&9 0

0

18

Ts

17 SCHEME III

T,

16

IN Pogostemcm coldin

65

depleted cyclohexanone derivative (18) in overall yield of 13% with 3H:14C < 0.01. These results indicate that 3H of the product was lost on cyclization of patchoulidione (16) to the cyclohexenone derivative (17), a finding fully consistent with the labeling pattern predicted for patchoulidione (16) and thus for patchoulol derived from [12,13-‘4C;1-3H]farnesyl pyrophosphate (Scheme II). Labeling studies with [12,13-‘4C;6-3H]farnesyl pyrophosphate (3H:14C = 0.48 f 0.02) were next undertaken; preparativescale incubations with the enzyme preparation affording 0.107 &i-3H (-3% yield) of patchoulol. The product was diluted with 2.5 mmol carrier as before and crystallized to constant specific activity of 0.045 mCi3H/mol (mp = 56°C) with 3H:‘4C = 0.51 + 0.02, thus indicating that 3H originally at C6 of the farnesyl substrate had undergone migration in the transformation to patchoulol (i.e., C6 of patchoulol bears no hydrogens). Conversion of [3H:‘4C]patchoulol to P-patchoulene (8) as before, gave the olefin in 53% yield, and subsequent oxidation provided patchoulidione (16) in 49% yield (mp = 94°C); both derivatives bearing essentially identical, and unchanged, 3H:14Cratios of 0.48 + 0.03. The sample was subjected to BF3-etherate treatment, to effect conversion of patchoulidione to the cyclohexenone (17), and to the routine hydrogenation procedure, to permit separation of the derived cyclohexanone (18) from the contaminating dione. Surprisingly, the isolated cyclohexanone (18) (obtained in 11% overall yield from patchoulol) bore very little 3H (3H:14C - 0.01). A duplicate sample of patchoulidione (3H:‘4C = 0.49) which had been run in parallel confirmed the result, providing the cyclohexanone nearly devoid of 3H (3H: 14C= 0.02 -+ 0.01). Unfortunately, the specific activities of the products derived from [12,13-‘4C;6-3H]farnesyl pyrophosphate were too low to permit radio-GLC analysis of the cyclohexenone (17) following BF3etherate treatment, so it was not possible to determine whether 3H had been lost on conversion of patchoulidione (16) to the cyclohexenone (17) or during the hydro-

66

CROTEAU

genation to the cyclohexanone derivative (18) (Scheme III). Since BFs-etherate treatment of patchoulidione seemed unlikely to result in loss of hydrogen from other than the Cl position of the acyclic precursor (as shown above), the catalytic hydrogenation step was examined in detail using an unlabeled sample of the cyclohexenone and ‘Hz. The reduction product was examined by GLC-MS, and by analysis of the [P(220) + n]’ ions was shown to be comprised of species bearing l(ll% ), 2(53%),3(31%),and 4(5%)‘H atoms. Furthermore, vigorous treatment of the deuterated product with NaOMe/MeOH to exchange the a-hydrogens, followed by GLCMS analysis of the two major diastereomers (of four) in the resulting mixture, indicated these products to be comprised of species bearing 1(74%),2(21%),and 3(5%) 2H atoms. Thus, ‘H introduced in hydrogenation was shown to be present at both exchangeable positions, as well as at carbons other than C6. It therefore seemed probable that the 3H present in the biosynthetically derived cyclohexenone (17) had been lost on hydrogenation due, at least in part, to double-bond migration throughout the methyl-substituted cyclohexene ring. An attempt to circumvent the entire problem by base-catalyzed exchange of labeled patchoulidione (16) failed due to the generation of unidentified oligomers and polar substances even under mild conditions. The results of the degradation of patchoulol derived from [12,13-14C;63H]farnesyl py ro p hosphate did not unequivocally establish the position of 3H in the product; yet, a reasonable argument can be made that patchoulol was labeled at the methyl-substituted carbon (C3 of Schemes II and III) as a result of the proposed 1,3-hydride shift during the course of the cyclization. Thus, patchoulidione (16) of identical 3H:‘4C ratio as the acyclic precursor was obtained, a result which in itself confirmed that the 3H at C6 of the farnesyl chain had undergone migration (i.e., C6 could bear no 3H), and ruled out the possibility of coupled 1,2:1,2-hydride shifts (C2 -+ C3:C6 -P C2) since C2 could bear no

ET AL.

3H. By a sim’11ar line of reasoning, a single 1,2-hydride shift (C2 + C3) with deprotonation to ,&patchoulene and reprotonation to (4) could be eliminated as intervening steps in patchoulol formation. The earlier results with [12,13- “C;1-3H]-farnesyl pyrophosphate eliminated Cl of the product as the migration terminus. The appended trimethylcyclopentane fragment of patchoulidione (16) (carbons 7-13 and 15) also seemed an unlikely site for the migration since the only feasible mechanism for this is an internal deprotonation (at C6)reprotonation (at C12/13) in the conversion of germacrene A (14) to a-bulnesene (11) and thence to intermediate (4). This process would leave 3H at a location (Cl2 or C13) from which it would not have been lost under the conditions of hydrogenation. Of the remaining carbons (C3, C4, C5), C4 and C5 are methylenes which undergo no change in bonding in the course of the reaction, leaving C3 as the most probable site of labeling in the derived [14C: 3H]patchoulol. The argument for labeling at C3 is not solely by default, in that the proposed 1,3-hydride shift is both stereochemically consistent with the absolute configuration of patchoulol and favored by the near parallel alignment of the interacting orbitals in the ,&patchoulyl intermediate (3) (Scheme II). Additionally, similar 1,3-hydride shifts have been demonstrated in the construction of other sesquiterpene types (50). Although reprotonation of the olefinic intermediate humulene has been reported to occur in the biosynthesis of pentalenene (12,51), and similar steps are presumed to occur in the construction of several other sesquiterpene skeletal types (11, 12), the present results based on analytical studies and on both in vivo and in vitro investigations argue against the involvement of olefins (germacrene, bulnesene, patchoulenes, and other CG-deprotonated isomers (52)) in the biosynthesis of patchoulol. Rather, the results of studies using 12,1314C;1-3H- and 12,13-‘4C;6-3H-labeled farnesyl pyrophosphate support a mechanism involving only tertiary cationic intermediates in the construction of patchoulol

PATCHOULOL

FROM

FARNESYL

PYROPHOSPHATE

with a concomitant 1,3-hydride shift, a step for which there is also ample precedent in sesquiterpene biosynthesis (50). Although the summation of the evidence weighs heavily against a role for olefins as intermediates in patchoulol biosynthesis, the possibility that olefins, such as the patchoulenes, may be coproducts of the patchoulol cyclization remains uncertain. The cyclization to patchoulol need not, for stereochemical reasons, involve nerolidyl pyrophosphate as a mandatory intermediate (11-14); however, the coproduction of nerolidol by the relatively crude cell-free system (presumably by combination of farnesyl pyrophosphate isomerase and pyrophosphatase activities) suggests the possibility of an isomerization-cyclization sequence. To adequately address these questions will require further purification of the patchoulol cyclase and a means of stabilizing this activity. ACKNOWLEDGMENTS W’e thank D. E. Cane for the sample of [12,13‘%]iarnesyl pyrophosphate, R. Hopp for providing authentic P. cab&n oil, T. Michnick for assistance in obtaining NMR spectra, G. Wichelns for raising the plants, and B. Frazier for typing the manuscript. REFERENCES 1. HEROUT, V. (1982) in Fragrance Chemistry (Theimer, E. T., Ed.), pp. 222-265, Academic Press, New York. 2. MOOKHERJEE, B. D., LIGHT, K. K., AND HILL, I. D. (1981) in Essential Oils (Mookherjee, B. D., and Mussinan, C. J., Eds.), pp. 247-272, Allured Pub. Corp., Wheaton, IL. 3. WOLFF, G., AND OURISSON, G. (1969) Tetrahedron 25,4903-4914. 4. DANITSCHEVSKY, S., AND DUMAS, D. (1968) J. Chem Sot. Chem. Commun., 1287-1288. 5. DOBLER, M., DUNITZ, J. D., GULBERG, B., WEBER, H. P., BUCHI, G., AND PADILLA 0, J. (1963) Proc. Chem. Sot. 383. 6. TSUBAKI, N., NISHIMURA, K., AND HIROSE, Y. (1967) Bull. Chem. Sot. Japan 40,597-600. 7. LAWRENCE, B. M. (1981) Perfum. Flavor. G(Aug/ Sept), 73-76. 8. HENDERSON, W., HART, J. W., How, P., AND JUDGE, J. (1970) Phytochemistry 9,1219-1228. 9. ROCKER, G., TALITGES, J., MAHESHWARI, M. L., AND SAXENA, D. M. (1976) Phytochemistry 15,224.

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10. LAWRENCE, B. M. (1986) Perfum. Flavor. lO(Dec/ Jan), 32-36. 11. CANE, D. E. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., Eds.), Vol. 1, pp. 283-374, Wiley, New York. 12. CANE, D. E. (1985) Act. Chem Res. 18,220-226. 13. CROTEAU, R., AND CANE, D. E. (1985) in, Methods in Enzymology (Law, J. H., and Rilling, H. C., Eds.), Vol. 110, Part A, pp. 383-405, Academic Press, Orlando. 14. CROTEAU, R. (1986) in Herbs, Spices and Medicinal Plants-Recent Advances in Botany, Horticulture and Pharmacology (Craker, L. E., and Simon, J. E., Eds.), Vol. 1, pp. 81-133, Oryx Press, Phoenix. 15. HENDRICKSON, J. B. (1959) Tetrahedron 7, 82-89. 16. PARKER, W., ROBERTS,J. S., AND RAMAGE, R. (1967) Q. Rev. Chem. Sot. 21,331-363. 17. HEROUT, V. (1971) in Aspects of Terpenoid Chemistry and Biochemistry (Goodwin, T. W., Ed.), pp. 53-94, Academic Press, London. 18. DEVON, T. K., AND SCOTT, A. I. (1972) Handbook of Naturally Occurring Compounds, Vol. II, Terpenes, pp. 56-57, Academic Press, New York. 19. COATES, R. M. (1976) in Progress in the Chemistry of Organic Natural Products (Herz, W., Grisebath, H., and Kirby, G. W., Eds.), Vol. 33, pp. 73-230, Springer-Verlag, Vienna. 20. BANTHORPE, D. V., AND CHARLWOOD, B. V. (1980) in. Encyclopedia of Plant Physiology (Bell, E. A., and Charlwood, B. V., Eds.), Vol. 8, Secondary Plant Products, pp. 185-220, SpringerVerlag, Berlin. 21. JONES, L. H., BARRETT, J. N., AND GOPAL, P. P. S. (1973) J. Exp. Bot. 24, 145-158. 22. FRANCIS, M. J. 0. (1972) Planta Med. 22,201-204. 23. DIXIT, V. M., LASKOVICS, F. M., NOALL, W. I., AND POULTER, C. D. (1981) J. Org. Chem. 46, 19671969. 24. CROTEAU, R., AND GUNDY, A. (1984) Arch. Biochem. Biophys. 233,838-841. 25. CANE, D. E., IYENGAR, R., AND SHIAO, M.-S. (1981) J. Amer. Chem. Sot. 103,914-931. 26. ATTENBURROW, J., CAMERON, A. F. B., CHAPMAN, J. H., EVANS, R. M., HENS, B. A., JANSEN, A. B. A., AND WALKER, T. (1952) J. Chem. Sot., 1094-1111. 27. BARUA, R. K., AND BARUA, A. B. (1964) Biochem. J 92,21-22c. 28. ARIGONI, D., CANE, D. E., M%LER, B., AND TAMM, C. (1973) Helv. Chem. Acta 56,2946-2949. 29. BALSEVICH, J. (1982) Canad J. Chem 61,1053-1059. 30. ANDERSON, W. K., AND VEYSOGLU, T. (1973) J. Org. Chem. 38,2267-2268. 31. MAERKER, G., AND HAEBERER, E. T. (1966) J. A?ner. Oil Chem. Sot. 43,97-100.

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32. COREY,E. J., AND SUGGS,J. W. (1975) Tetrahedron L&t., 2647-2650. 33. COREY, E. J., D’ALARCAO, M., AND K~ER, K. S.

(1985) Tetrahedron Letts, 3919-3922. 34. GLASBY,J. S. (1982) Encyclopedia of the Terpenoids, p. 1879, Wiley, New York. 35. KEPNER, R. E., ELLISON,B. O., AND MAARSE, H. 36.

37. 38. 39.

ET AL. SHARPLESS, B. K. (1981) J. Org Chem 46,39363938.

44. STERRE~, F. S. (1975) in The Essential Oils (reprinted) (Guenther, E., Ed.), Vol. 2, pp. 808810, Kreiger, Huntington, NY.

45. EL-KELTAWI, N. E., AND CROTEAU, R. (1986) Phgtochemistry 25,1285-1288. (1975) Agric Food Chem 23,343~344. 46. WENNINGER, J. A., YATES, R. L., AND DOLINSKY, M. (1966) Proc Sci. Sect, Tuilet Goods Assoc. 46, WEINHEIMER,A. J., YOUNGBLOOD, W. W., WASHECHECK, P. H., KARNS,T. K. B., AND CIERESZKO, 44-56. L. S. (1970) Tetrahedron Lett., 497-500. 47. TEISSEIRE, P., MAUPETIT, P., AND CORBIER,B. (1974) NISHIMURA, K. (1969) Tetrahedron Let& 3097-3100. Recherches 19,8-35. CROTEAU, R., AND KARP, F. (1976) Arch Biochem. 48. CROTEAU, R., AND RONALD, R. C. (1983) in ChroBiophys. 176,734-746. matography: Fundamentals and Applications LOOMIS, W. D. (1974) in Methods in Enzymology of Chromatographic and Electrophoretic

(Fleischer, S., and Packer, L., Eds.), Vol. 31, pp. 528-544, Academic Press, New York. 40. LOOMIS, W. D., LILE, J. D., SANDSTROM, R. P., AND BURBOTT, A. J. (1979) Phytodemist7y 18,1049-

1054. 41. CROTEAU, R., AND KARP, F. (1979) Arch. Biochem. Biophys. 198,512-522. 42. BUCHI, G., ERICKSON, R. E., AND WAKABAYASHI, N. (1961) J. Amer. Chem. Sot. 83,927-938. 43. CARLSEN,P. H. J., KATSUKI, T., MARTIN, V. S., AND

Methods (Heftmann, E., Ed.), Part B, pp. 147189, Elsevier, Amsterdam. 49. CROTEAU, R., BURBOTT, A. J., AND LOOMIS, W. D. (1972) Ph@chemi&y 11,2459-2467. 50. ARIGONI, D. (1975) Pure Appl Chem 41,219~245. 51. CANE, D. E., ABELL, C., AND TILLMAN, A. M. (1984) Bioorg. Chem. 12,312-328. 52. NES, W. R., AND MCKEAN, M. L. (1977) Biochem-

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