ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 277, No. 2, March, pp. 374-381,199O
Biosynthesis of Monoterpenes: Stereochemistry of the Coupled lsomerization and Cyclization of Geranyl Pyrophosphate to Camphane and lsocamphane Monoterpenes’ Rodney Institute
Croteau,2
Jonathan
of Biological Chemistry,
Gershenzon, Washington
Carl J. Wheeler,3
State University,
Received September 25,1989, and in revised form November
and D. Michael Washington
Satterwhite
99164-6340
17,1989
The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia oficinalis (sage) and Tanacetum v&are (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[ 1Z3H]linalyl pyrophosphate plus [ l-‘4C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3s)-[1Z-3H]linalyl pyrophosphate plus [ 1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:‘4C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-a-hydrogen of the derived ketone. The finding that the 1S3H of the linalyl precursor was positioned at the endo-a-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at Cl of the geranyl substrate in these transforma-
i This investigation was supported in part by National Health Grant GM 31354, and by Project 0268 from the State University Agricultural Research Center, Pullman, * To whom correspondence should be addressed. 3 Present address: Revlon Science Institute, San Diego,
Pullman,
Institutes of Washington WA 99164. CA 92111.
tions, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endoconformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids. 0 1990 Academic Press, Inc.
Bornyl pyrophosphate cyclase from common sage (Saluia oficinalk) catalyzes the divalent metal ion-dependent conversion of [ l-3H]geranyl pyrophosphate to (+)-(1R,4R)-[3-3H]bornyl pyrophosphate, whereas the bornyl pyrophosphate cyclase from tansy (Tanacetum vulgare) catalyzes the transformation of the same acyclic precursor to (-)-(lS,4S)-[3-3H]-bornyl pyrophosphate (1, 2) (Scheme I). These enantiomeric cyclizations, which ultimately lead to the formation of (+)- and (-)camphor, respectively, in these species, are accomplished without loss of hydrogen from Cl of the acyclic precursor and without formation of detectable free intermediates (l-3). Since the trans-geometry of the C2-C3 bond of the geranyl substrate prevents direct cyclization of this precursor, it is clear that the bornyl pyrophosphate cyclases are able to catalyze the isomerization of geranyl pyrophosphate to a bound intermediate capable of cyclizing, as well as the cyclization reaction itself. A stereochemical model for the coupled isomerization and cyclization of geranyl pyrophosphate to bornyl pyrophosphate (4) and related bicyclic monoterpenes (5-7) has been proposed (8). This reaction sequence (Scheme I) is initiated by ionization of geranyl pyrophosphate to the allylic cation, with suprafacial migration of the pyro-
374 All
0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
STEREOCHEMISTRY
OF CAMPHANE
phosphate counter ion of the resulting pair (1) to afford a bound linalyl pyrophosphate intermediate. Rotation about the newly generated C2-C3 single bond to the cisoid conformer overcomes the original stereochemical impediment to the direct cyclization of geranyl pyrophosphate, while subsequent ionization of this tertiary allylic isomer promotes Cl-C6 cyclization of the antiendo form (2). A second electrophilic cyclization involving the cyclohexenyl double bond of the cu-terpinyl intermediate (3) gives the bornyl (camphyl) cation (4), which is captured by the paired pyrophosphate anion. The biosynthetic transformations catalyzed by the (+)- and (-)bornyl pyrophosphate cyclases are thus described by mirror-image schemes involving antipodal linalyl pyrophosphate and a-terpinyl cation (3) intermediates. Closely related antipodal cyclizations are catalyzed by the (+)- and (-)-pinene cyclases from sage, which produce (+)-(lR,4S)and (-)-(lS,4R)-camphene, respectively, as minor products by Wagner-Meerwein rearrangement of the respective bornyl cations (4) to the related isocamphyl cations (5) and deprotonation to the corresponding olefins (9, 10) (Scheme I). [l-3H]Geranyl pyrophosphate thus leads to specific labeling of C7 (the methylene bridge hydrogens) of these bicyclo[2.2.l]olefins. A key feature of this general stereochemical model is the predicted intermediacy of (--)-(SE)-linalyl pyrophosphate in the construction of (+)-bornyl pyrophosphate and (+)-camphene, and the intermediacy of the antipodal (+)-(3S)-isomer in the corresponding enantiomeric cyclizations to (-)-bornyl pyrophosphate and (-)-camphene. These predictions were recently confirmed by direct testing of the optically pure linalyl pyrophosphates with the relevant cyclases (10,ll). Interestingly, the bornyl pyrophosphate and camphene (pinene) cyclases from sage are capable of cyclizing the unnatural linalyl pyrophosphate enantiomer to the antipodal products, but at a slower rate than that of the predicted intermediate (10,ll). The mechanistic basis of these anomalous reactions has been discussed (8, 10, 11). Although the linalyl pyrophosphates are the first explicitly chiral intermediates in the cyclization scheme, the eventual configuration is predetermined by the helical conformation of geranyl pyrophosphate achieved on initial binding to the cyclase; the left-handed helical form yielding (3R)-linalyl pyrophosphate, the right-handed helical counterpart affording the (3S)-enantiomer (8). As an essential consequence of this syn-isomerization-C2-C3-rotation-anti-cyclization scheme, the configuration at Cl of the geranyl substrate should be retained in the transformations to the bornyl pyrophosphates and camphenes (Scheme I). These predictions also were recently confirmed by stereoselective degradation of the products derived enzymatically from [ 1R-3H,‘4C]-labeled and [1S-3H,‘4C]-labeled geranyl pyrophosphate (4, 7, 10). Therefore, the results, thus far, are entirely consistent
AND
ISOCAMPHANE
375
BIOSYNTHESIS Geranyl
Pyrophosphate
(-)-3R-LPP OPP A+ 4 PPOcJ 1
I OOPP -2 /,
h
-
>1
2
2
4
I /
6PP
(+)-Bornyl Pyrophosphate
6 3 I
,
(+I-Camphene
(-bcamphene
SCHEME I. LPP indicates linalyl pyrophosphate. OPP indicates the pyrophosphate moiety. 1 is the geranyl cation, 2 is the linalyl cation, 3 is the a-terpinyl cation, 4 is the bornyl (camphyl) cation, and 5 is the isocamphyl cation.
with the proposed isomerization-cyclization model. However, although indirect evidence for the isomerization step is available (12, 13), the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate has
376
CROTEAU
never been explicitly demonstrated because, in the tightly coupled sequence, linalyl pyrophosphate is never free of the enzyme surface. There is ample precedent for syn-stereochemistry in this reaction type, based on examination of the origin of linalool itself (14) and of the corresponding transposition of trans,trans-farnesyl pyrophosphate to nerolidyl pyrophosphate in the biosynthesis of sesquiterpenoids (l&16). In this communication, we describe the stereochemical alteration at Cl of linalyl pyrophosphate in the transformations to bornyl pyrophosphate and camphene.
This
information,
in conjunction
with
the pre-
viously established retention of configuration at Cl of the geranyl precursor in the coupled reaction, provides strong supporting evidence for the proposed syn-isomerization-anti-endo-cyclization stereochemistry in the construction of the camphane (bornane) and isocamphane monoterpenes. EXPERIMENTAL
PROCEDURES
Plant materials, substrates, and reagents. Sage (S. oficinalis L.) and tansy (2’. u&are, L.) plants were grown from seed under greenhouse conditions described previously (1, 2). Young emerging leaves from 18 to Pl-day-old plants were used as the source of the monoterpene cyclases. The preparation of each of the following substrates has been previously described: [l-3H]geranyl pyrophosphate (80 Ci/mol) (l), [l“‘Clgeranyl pyrophosphate (25 Ci/mol) (6), (3R)-[1Z-3H]linalyl pyrophosphate (21.6 Ci/mol) (17), and (3S)-[lZ-3H]linalyl pyrophosphate (40 Ci/mol) (17). (f)-Camphene ([a]? + 6” (C = 10, EtOH)) was obtained from K & K Laboratories (Plainview, NY). rat-Borneo1 was from U.S. Biochemical Corp. (Cleveland, OH), and rat-camphor was from Aldrich Chemical Co. (Milwaukee, WI). All other standards, reagents, and biochemicals were purchased from Aldrich or Sigma Chemical Co. Enzyme preparation and assay. The preparation, assay, and general properties of (+)-pinene cyclase (cyclase I), (-)-pinene cyclase (cyclase II) (9, 18) and (+)-bornyl pyrophosphate cyclase (1, 3, 11) from S. oficinalis, and (-)-bornyl pyrophosphate cyclase (2, 4, 11) from T. uulgare, have been described. These procedures employ selective extraction of the leaf epidermal oil glands (19) and partial purification of the cyclase by gel permeation and/or ion-exchange chromatography to remove the bulk of the competing phosphatase activities. The standard assay involves conversion of radiolabeled substrate to chromatographically purified product. T. uulgare was used as the source of (-)-bornyl pyrophosphate cyclase for this work because S. oficinalis extracts contain very low levels of this activity (4, ll), and the preparative conversions were thus inefficient. Preparative enzymatic conversions and product isolation. For the enzymatic synthesis of (+)-camphene, 5-ml aliquots of the partially purified cyclase I preparation from sage (containing 80-100 yg of protein) in 20 mM Mes,4 5 mM potassium phosphate buffer, pH 6.5, containing 0.5 mM dithiothreitol, 15 mM MgClz and 10% (v/v) glycerol were employed. The same conditions were used for preparative conversions with (+)-bornyl pyrophosphate cyclase from sage and with (-)-bornyl pyrophosphate cyclase from tansy. For the enzymatic synthesis of (-)-camphene, similar preparations of cyclase II from sage
’ Abbreviations used: Mes, 4-morpholineethanesulfonic tris(hydroxymethyl)aminomethane; TLC, thin-layer phy; GLC, gas-liquid chromatography.
acid; Tris, chromatogra-
ET AL. (containing 50-70 pg of protein) were employed. However, the pH of the buffer system was adjusted to the optimum of 7.2. In each case, the reaction was initiated by the addition of a saturating concentration of the allylic pyrophosphate substrate (10 FM of [l-3H]geranyl pyrophosphate in the case of the standard assay), and the mixture in a screwcapped vial was incubated with gentle swirling for 3 h at 30°C. For camphene isolation, the solution was chilled in ice following incubation, and 2 ml of pentane added to extract olefinic products. Thorough mixing of the biphasic system was followed by centrifugation to facilitate separation of the phases, and the pentane layer was passed through a short column of silica gel (Mallinckrodt type 60A) overlaid with anhydrous MgSO,. Extraction and elution were repeated with an additional 2 ml of pentane to provide a combined eluate containing only hydrocarbons. Samples from replicate incubations were pooled, carrier standards were added (10 mg of (+)-camphene plus -10 ~1 of the olefin fraction from sage oil), and the camphene was isolated by argentation TLC (Silica Gel G containing 15% AgNOB, using 1% diethyl ether in benzene:pentane (1:2, v/v); R,, 0.55). The scraped gel from the appropriate region was eluted with pentane. An aliquot of this eluate was counted and another aliquot analyzed by radio-GLC (20, 21) (>97% radiochemical purity after repetition of argentation TLC using 2% n-hexene in pentane). A sufficient number of such preparative incubations were carried out to accumulate in excess of 3 nmol of camphene from the double-labeled precursor mixture. In order to isolate the bornyl pyrophosphate produced, the enzymatic reaction mixtures, following incubation, were first extracted with ether. These ether extracts contained negligible levels of borneol, but did give, by aliquot counting, a measure of substrate loss to competing phosphatases and solvolysis. The reaction mixtures were then purged with a stream of N2 to remove traces of ether, and 1 ml of 200 mM sodium acetate buffer, pH 5.0, containing 1 unit each of wheat
germ acid phosphatase and potato apyrase, was added. The mixture was incubated with gentle agitation for 75 min at 30°C. After cooling in ice, 2 ml of ether was added and the biphasic system was vigorously mixed and then centrifuged to separate phases. The organic layer was dried by passage through a short column of MgSO,, and the entire extraction repeated with an additional 1 ml portion of ether. All of the ether extracts from like incubations were combined to provide a sample of borne01 (in excess of 5 nmol), which represented all of the bornyl pyrophosphate produced. To facilitate the TLC separation of borne01 from unsaturated monoterpenes present in the combined extract (i.e., from hydrolysis of the substrate), excess OsOl (0.1 mg) in pyridine (10 ~1) was added, and the sample was stirred overnight. Next, 1 ml of saturated aqueous NaHSO, was added (followed by stirring for 3 h at room temperature) to decompose osmate esters, thereby completing the conversion of unsaturated compounds to the corresponding diols, and drastically reducing their mobility in the subsequent TLC step. The organic phase, and an additional 1 ml ether extract of the aqueous phase, were combined, washed twice with small portions of 0.1 N HCl, and dried over MgSO,. Each sample was diluted with 10 mg (k)-borneol carrier, and then concentrated under vacuum (Savant Speed Vat) and separated by TLC on Silica Gel G with hexanes:ether (2:1, v/v). The band corresponding to borne01 (Rf = 0.4) was scraped directly into a glass wool-plugged Pasteur pipet, and the product eluted with diethyl ether or dichloromethane. An aliquot of the sample was taken for determination of radioisotope content and another for radio GLC analysis (>97% radiochemical purity). Conversion of bornyl pyrophosphate to camphor. The biosynthetitally generated, labeled borne01 (>0.22 &i-3H) released by enzymatic hydrolysis of the pyrophosphate ester and isolated by TLC as above, was diluted with additional racemic carrier to a specific radioactivity of -0.3 PCi-sH/mmol, dissolved in CHzClz, and oxidized to camphor with a slight excess of pyridinium chlorochromate in CH&lz for 40 min at room temperature (22). The reaction mixture was poured through Florisil to remove chromate salts, and the product (-0.6 mmol) isolated from the eluate by concentration and TLC as before.
STEREOCHEMISTRY An aliquot of the derived camphor was converted oxime (4) for determination of isotope content.
OF CAMPHANE to the crystalline
Conversion of camphene to camphor. The biosynthetically generated, 3H:‘4C-labeled camphene (>0.14 &i-3H), isolated as above, was diluted with additional (+)-camphene to a specific radioactivity of -0.2 &i-3H/mmol, and subjected to acetolysis essentially as described by Erman (23). Thus, 0.6 mmol of camphene was diluted with 1.2 mmol of glacial acetic acid, to which -0.02 mmol BF,-etherate was added by syringe, and the mixture heated at 100°C in a sealed tube under Ar for 3 h. The contents of the cooled tube were partitioned between saturated aq NaCl and ether, and the ether-soluble products analyzed by GLC and shown to contain -50% isobornyl acetate and -10% bornyl acetate, along with numerous other products. The crude mixture was hydrolyzed in 50% ethanolic 0.1 N KOH, and the resulting alcohols were partitioned into ether, following saturation with NaCl. Drying of the extract over MgS04, and concentration of the solvent, afforded an oily mass that was dissolved in CHzClz and subjected to pyridinium chlorochromate oxidation, as before, for the conversion of isoborneol (and borneol) to camphor. Preparative TLC of this mixture yielded camphor (-50% yield from camphene) that was well-resolved from contaminating products of higher and lower R, value (>95% by GLC). Aliquots of the derived camphor were converted to the crystalline oxime, as before, for determination of isotope content. Several attempts at employing the chromic acid oxidation of camphene to produce camphor directly (24) gave the ketone in higher yields, but the method was accompanied by more extensive racemization of the product (see below). Base-catalyzed enchange procedure. The a-protons of 3H-labeled camphor derived from the enzymatically generated bornyl pyrophosphate and camphene were subjected to base-catalyzed exchange by a procedure described in detail elsewhere (4, 7). In brief, the derived camphor samples (at a total of 40 mg from camphene or 80 mg from bornyl pyrophosphate) were dissolved in 0.2 ml dioxane and added by syringe to 2.3 ml of dioxane:0.5 N aqueous NaOH (l:l, v/v), which was heated with constant stirring at 60°C. Aliquots comprising 20% of the total sample were removed at 4 min intervals and added to 0.5 ml of cold 0.25 N HN03, followed by NaCl saturation, recovery of the camphor by ether-extraction, purification by TLC, and determination of ‘H:14C content by liquid scintillation spectrometry. At the end of the exchange run (4 samples including the zero-time control), 2.5 ml of the dioxane:aqueous NaOH solution was added to the remaining sample (20% of the starting material), and the mixture heated on a steam bath for 2 h. Following this exhaustive exchange procedure, the residual camphor was recovered and purified as before, and the 3H:‘4C ratio of this material (as the oxime) was determined. This latter procedure was required in order to evaluate the extent of racemization that had occurred during the chemical transformations (i.e., racemization results in the placement of ‘H in a position inaccessible to exchange) and to allow correction of the rate curves which are based on 3H loss from the exchangeable total. Hydrolysis ojpyrophosphate esters and prenol analysis. To examine the possible interconversion of [Wlgeranyl pyrophosphate and [3H]linalyl pyrophosphate, the aqueous phases of the corresponding incubation mixtures remaining after extraction of (+)- or (-)-camphene were combined, centrifuged to remove denatured protein, and lyophilized. The resulting glass was dissolved in 5 ml 0.1 N Tris-HCl buffer, pH 8.0, containing 5 units of calf intestine alkaline phosphatase, and incubated for 3 h at 30°C in a sealed tube. Following chilling of the incubation mixture, the liberated prenols (75-80% recovery) were extracted into ether and diluted with 5 mg each of carrier geraniol and (+-)-linalool, and, following concentration of solvent, the products were separated by TLC (Silica Gel G using hexane:ether (l:l, v/v); R, geraniol, 0.45, R, linalool, 0.50). Each product was further purified by TLC on Silica Gel G containing 15% AgN03 with hexane:ether (2:3, v/ v) before determination of radioisotope content by liquid scintillation spectrometry. Incubation mixtures of the bornyl pyrophosphate cyclases with the geranyl and linalyl substrates were similarly treated;
AND
ISOCAMPHANE
BIOSYNTHESIS
however, the 0~0~ treatment necessarily omitted.
377
employed for the analysis of borne01 was
Other analytical procedures. Detailed procedures for liquid scintillation spectrometry, TLC, and radio-GLC of the monoterpenoids have been described (4,11,20,21).
RESULTS
AND
DISCUSSION
The proposed model for the cyclization of linalyl pyrophosphate to bornyl pyrophosphate predicts that the Zhydrogen at Cl of the acyclic precursor will become the en&-a-hydrogen of this product (Scheme II). Similarly, cyclization of [ 1Z3H]linalyl pyrophosphate to camphene should result in specific labeling of the exo-hydrogen of the methylene bridge of this olefin (Scheme II). Validation of this prediction would provide strong evidence for the anti,endo-cyclization of (3R)- and (3S)-linalyl pyrophosphate to the respective antipodal bornane and isocamphane monoterpenes. Additionally, since geranyl pyrophosphate is known to be transformed to the enantiomeric bicyclic products with retention of configuration at Cl (4), such a result would confirm the helical conformation of the geranyl precursor achieved at the outset of the coupled isomerization-cyclization sequence, as well as the syn-stereochemistry of the geranyl-to-linalyl isomerization (i.e., with suprafacial pyrophosphate migration, the l-proR-hydrogen of geranyl pyrophosphate will become the l-Z-hydrogen of 3Rlinalyl pyrophosphate, whereas the l-pros-hydrogen will become the l-Z-hydrogen of the 3S-enantiomer (Scheme II)). To approach this problem, it was necessary to devise a means of specifically locating the relevant hydrogens of the bicyclic products derived from Cl-labeled precursors. The conversion of bornyl pyrophosphate to camphor has been exploited for this purpose in the past (4, 7), since the C1-3H atom of the precursor resides on the C3-methylene (adjacent to the carbonyl) of the derived bicyclo[2.2.l]ketone, and the exo- and e&o-protons can be readily distinguished by differential base-catalyzed exchange (i.e., Kexo:Kend,- 2O:l (25-27)). A similar strategy was developed for conversion of camphene to camphor to permit location of the 3H tracer by exchange procedures. The loss of 3H via base-catalyzed exchange of the bicyclic ketone is most readily determined by using product derived from racemic Cl-labeled precursors as a reference, and by employing a 14C-labeled internal standard (4, 5, 7). Lacking both 1E,Z-3H-labeled and ‘*C-labeled linalyl pyrophosphate enantiomers for this purpose, [ 1R,S-3H]geranyl pyrophosphate and [ 1-14C]geranyl pyrophosphate were employed. Preliminary experiments were, therefore, directed toward confirming that geranyl pyrophosphate and linalyl pyrophosphate were converted to product at the same active site of each cyclase by employing the mixed substrate method of Dixon and Webb (28). Thus, all combinations of [ l-3H]geranyl py-
378
CROTEAU
GPP
ET AL.
’
Q (z)Q PPO:d--“;-1 (+I-Bornyl Pyrophosphate
(+)-Borneo1
4 0 (z)Q .‘Q 4
(-)-Bornyl Pyrophosphate
(-)-Borneo1
I 8 (z)Q d-
0 (-I-lS,4S-Camphor
(+I- 1 R,4R-Camphor
t
0 (2) -b (+I-Camphene
(+I-lsobornyl Acetate
(-klsobornyl Acetate
The circled R designates the l-proR SCHEME II. 2 designates the 1-Z hydrogen of linalyl pyrophosphate
hydrogen, (LPP).
and the circled
rophosphate and [ 1Z-3H]linalyl pyrophosphate (2-8 PM range) gave rise to total rates of product formation significantly less than the arithmetic sum calculated from each precursor when tested alone (Table I), confirming that both substrates competed for the same active site and, therefore, that both isomerization and cyclization steps occur at the same reaction center. This observation is entirely consistent with a tightly coupled isomerization-cyclization reaction in which no free intermediates are formed, and it substantiates identical results obtained with all other cyclases examined (6, 7,29). On the basis of the observed relative changes in rates of conversion of the geranyl and linalyl precursors alone and in combination, the appropriate (3R)- and (3S)-[ lZ3H]linalyl pyrophosphate enantiomers were mixed with [l-‘*C]geranyl pyrophosphate to give roughly a 10~1 ratio of 3H:14C in the bornyl pyrophosphate and camphene products. [1R,S-3H]Geranyl pyrophosphate and [l-14C]geranyl pyrophosphate were admixed at a 3H:14C ratio of 1O:l to provide the precursor needed to generate racemitally labeled product for use as a reference standard for base-catalyzed exchange.
S the l-pros
(-)-Camphene hydrogen,
of geranyl
pyrophosphate
(GPP).
Preparative-scale incubation of (+)-bornyl pyrophosphate cyclase (S. oficinalis) with the mixture of (3R)[ 1Z-3H]linalyl pyrophosphate and [ 1-l*C]geranyl pyrophosphate precursors gave 0.23 &i-3H of product (isolated as borneol) at a 3H:‘4C ratio of 7.6 + 0.4:1 (as the phenylurethane (4)). The reference standard of (+)-bornyl pyrophosphate (isolated as borneol) prepared from [1R,S-3H,1-14C]geranyl pyrophosphate was obtained at a level of 0.34 pCi-3H (3H:‘4C = 10.2 f 0.2:1). In the case of the (-)-bornyl pyrophosphate cyclase (7’. uulgure), the (3S)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate mixture gave 0.24 &i-3H of product at 3H:‘4C = 12.3 + 0.51, and the incubation with [lR,S3H,1-14C]geranyl pyrophosphate gave a reference standard of 0.31 &i-3H at a ratio of 9.9 k 0.2:1. Each borne01 sample was diluted with carrier to a specific radioactivity and oxidized with pyridinium of -0.3 &i-3H/mmol, chlorochromate to camphor (TLC purified) without significant change in the 3H:‘4C ratio (determined as the oxime). Each camphor sample was then subjected to base-catalyzed exchange in dioxane:aqueous NaOH at 6O”C, as described under Experimental Procedures, US-
STEREOCHEMISTRY OF CAMPHANE AND ISOCAMPHANE BIOSYNTHESIS
379
TABLE I Conversion of Geranyl Pyrophosphate and Linalyl Pyrophosphate Mixtures to Bornyl Pyrophosphate and Camphene Substrate” (3R)-LPP
GPP Product
PM
(+)-Bornyl pyrophosphate
2 4 2 4
(+)-Camphene
VGb 38.6 53.3 12.1 26.6
PM
VLb
Calculated sum tv,+ Vdb
Actual sum (observed)b
4 2 6 3
25.3 17.5 4.6 2.4
63.9 70.1 16.7 29.0
31.4 50.6 7.2 19.3
(3&s)-LPP
GPP PM (-)-Bornyl pyrophosphate (-)-Camphene
4 8 4 8
VGb 49.6 57.5 11.6 22.7
GM
VLb
(VG•t VI,)*
(observed)*
8 4 8 4
98.3 53.4 29.3 17.0
147.9 110.9 40.9 39.7
66.6 57.1 24.9 21.2
’ Abbreviations used:GPP, geranyl pyrophosphate;LPP, linalyl pyrophosphate. * Velocities are given in cpm X 10e3and are the averagesof duplicate runs differing by lessthan 11% of the mean. ing a technique in which samples were periodically removed, the reaction quenched in HNOB, and the camphor reisolated by TLC for radioisotope determination. To evaluate the extent of racemization that occurred during the chemical transformations leading to camphor, the remaining sample at the end of each exchange run (26% of total) was subjected to exhaustive exchange, and the camphor was reisolated as before. In all cases, essentially all of the tritium was lost (3H:14C < O.l:l as the oxime), as expected (1, 2, 4), indicating complete specificity in the positioning of 3H at C3 of the ketone, and no racemization leading to nonexchangeable 3H. Relative exchange rates of the a-hydrogens were then established by comparing 3H loss from the starting material in each case to that observed for the corresponding 3R,S-3H-labeled camphor sample exchanged under identical conditions. Since the exo:endo exchange rate ratio is 20 (25-27), the theoretical rate ratio for the exo-a-3H exchange, with reference to the racemically labeled standard, is 2, whereas that for the endo-a-‘H, with reference to the same standard, is 0.1. For (+)-camphor derived from (+)-bornyl pyrophosphate cyclase and the (3R)[ 1Z-3H]linalyl pyrophosphate precursor, the observed ratio calculated from the linear portion of the curve was 0.11 f 0.02. For (-)-camphor derived from (-)-bornyl pyrophosphate cyclase and the (3S)-[1Z-3H]linalyl pyrophosphate precursor, the observed ratio was 0.12 ? 0.02. Thus, for both cases, the 1Z-3H of the linalyl precursor was placed in the endo-a-position of the bornane product, in agreement with the proposed isomerizationcyclization model (Scheme II). To locate the 3H atom in the biosynthetically derived camphenes, the well-known (30,31) acid-catalyzed rear-
rangement of the isocamphane to the bornane (camphane) skeleton was exploited. Thus, acetolysis of camphene, under the conditions described (23), gave rise to roughly 50% isobornyl acetate (and 10% bornyl acetate), which was hydrolyzed and oxidized to camphor in about 50% overall yield (Scheme II), providing a minimum of 0.07 &i-3H of product (at a specific radioactivity of -0.2 &i/mmol) in each case. The transformation of camphene (measured as camphenilone oxime (10)) to camphor (measured as the oxime) was accomplished without significant change in the 3H:‘4C ratio, which in this set of products ranged from 6.7 to 9.3. The 3H:14Clabeled ketones were then subjected to base-catalyzed exchange, as before, to selectively remove the exo-a-protons. The transformation of the isocamphane to the bornane ring system is accompanied by partial racemization, largely as the result of e3co-3,2-methyl migration (in the isocamphyl cation (5)) and endo-6,2-hydride shift (in the bornyl cation (4)) (See Scheme 1) (32). In the present instance, methyl migration leads to camphor of the opposite configuration, in which the relative orientations of the exo- and endo-a-hydrogens are unaffected. The hydride shift, similarly, leads to the enantiomeric camphor, but in this instance, the 3H label is rendered inaccessible to exchange, since Cl of the precursor now resides at C5 of the dimethylene bridge of the resulting ketone. The latter process was readily corrected for by exhaustive base-treatment of the remaining camphor sample following an exchange run, and was shown to vary from 14 to 18% (as residual 3H label); the extent of racemization is known to be highly dependent on the precise reaction conditions (32). The corrected exchange rate for (+)-[3H]camphor derived from (+)-[3H]cam-
380
CROTEAU
phene generated from (3R)- [ 1Z-3H]linalyl pyrophosphate, with reference to the R,S-3H-labeled standard, was 0.11 f 0.03. The rate for (-)-[3H]camphor derived from (-)-[3H]camphene generated from (3S)-[lZ3H]linalyl pyrophosphate, with reference to the corresponding racemically labeled standard, was 0.12 f 0.03. Thus, as was the case with (+)- and (-)-bornyl pyrophosphate, the 1Z-3H of the (3R)- and (3S)-linalyl precursors was transformed to the en&-o-position of the respective (+)- and (-)-camphor products, as predicted. These observations serve to confirm the syn-stereochemistry in the isomerization of geranyl pyrophosphate to linalyl pyrophosphate when taken with the net retention of configuration at Cl of the geranyl precursor in the coupled transformation to these enantiomeric bicyclic products (4). The results presented here, in addition to confirming the syn-isomerization, rotation, anti-cyclization sequence (increasing evidence suggests a universal preference for anti-allylic displacement in related isoprenoid cyclizations (16,33-37)), establish the helical conformation of the reacting geranyl precursor and the anti-end0 conformation (38) of the cyclizing tertiary intermediate and rule out all other possible conformers for this coupled reaction. When taken together with the previously described configurational imperative for (3R)-linalyl pyrophosphate in the cyclization to (+)-bornyl pyrophosphate and (+)-camphene, and for (3S)-linalyl pyrophosphate in the cyclization to (-)-bornyl pyrophosphate and (-)-camphene (10, ll), the summation of these studies unequivocally establishes all of the absolute stereochemical elements in the isomerization and cyclization of geranyl pyrophosphate to these enantiomeric bornane and isocamphane products. Even the aberrant cyclizations of the unnatural linalyl antipodes by the sage enzymes are entirely consistent with this stereochemical picture (10, 11, 39). The present work completes studies on the stereochemical origin of all of the major classes of bicyclic monoterpenes (bornanes (camphanes), isocamphanes, thujanes, pinanes, fenchanes). All of the results with these diverse skeletal types are fully consistent (5-8, 10, 17), and lend very strong support to the general syn-isomerization-anti,endo-cyclization model for the transformation of the universal acyclic precursor geranyl pyrophosphate (8). An interesting ancillary observation arising from the present work was provided by analysis of the [14C]geranyl pyrophosphate and [3H]linalyl pyrophosphate remaining at the completion of the preparative enzyme conversions to bornyl pyrophosphate and camphene (most of the substrates were not consumed in this process). Thus, enzymatic hydrolysis of the residual pyrophosphate esters by estabhshed methods (17,20,40), followed by dilution with carrier and TLC separation of the alcohols, demonstrated the presence of only [14C]geranyl pyrophosphate (3H:14C < 0.03:1) and [3H]linalyl pyro-
ET AL.
phosphate (3H:14C > 98:l). The absence of observable cross contamination, in each case, confirms that [‘“Cllinalyl pyrophosphate, which arises by isomerization of [14C]geranyl pyrophosphate, is enzyme-bound and not free to equilibrate with [3H]linalyl pyrophosphate in solution, and confirms that isomerization is not reversible (i.e., from [3H]linalyl pyrophosphate to free [3H]geranyl pyrophosphate) in the time frame of the overall reaction. ACKNOWLEDGMENTS We thank Greg Wichelns for typing the manuscript.
for raising the plants and Nancy Madsen
REFERENCES 1. Croteau,
R., and Karp, F. (1979) Arch. Biochem. Biophys.
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512-522. 2. Croteau, R., and Shaskus, J. (1985) Arch. Biochem. Biophys. 236, 535-543. 3. Croteau, R., and Felton, N. M. (1981) Arch. B&hem. Biophys. 207,460-464. 4. Croteau, R., Felton, N. M., and Wheeler, C. J. (1985) J. Biol. Chem. 260,5956-5962.
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D. M., Wheeler, C. J., and Felton, N. M. (1988) J. Biol. Chem. 263, 15,449-15,453. Hallahan, T. W., and Croteau, R. (1989) Arch. Biochem. Biophys. 269,313-326. Croteau, R., Satterwhite, D. M., Wheeler, C. J., and Felton, N. M. (1989) J. Biol. Chem. 264,2075-2080. Croteau, R. (1987) C!zem. Reu. 87,929-954. Gambliel, H., and Croteau, R. (1984) J. Biol. Chem. 259,740-748. Croteau, R., Satterwhite, D. M., Cane, D. E., and Chang, C. C. (1988) J. Biol. Chem. 263, 10,063-10,071. Croteau, R., Satterwhite, D. M., Cane, D. E., and Chang, C. C. (1986)J. Biol. Chem. 261,13,438-13,445. Wheeler, C. J., and Croteau, R. (1986) Arch. Biochem. Biophys.
246,733-742. 13. Wheeler,
C. J., and Croteau, R. (1987) Proc. Natl. Acad. Sci. USA
84,4856-4859. 14. Gotfredsen, S. E. (1979) Ph.D. thesis, ETH Zurich, No. 6243, as cited in Refs. (15, 16). 15. Cane, D. E., Iyengar, R., and Shiao, M.-S. (1981) J. Amer. Chem.
sot. 103,914-931. 16. Cane, D. E. (1980) Tetrahedron 36,1109-1159. 17. Satterwhite, D. M., Wheeler, C. J., and Croteau, R. (1985) J. Biol. Chem. 260,13,901-13,908. 18. Gambliel, H., and Croteau, R. (1982) J. Biol. Chem. 257, 23355
2342. 19. Gershenzon, J., Duffy, M. A., Karp, Anal. Biochem. 163,159-164.
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20. Croteau, R., and Cane, D. E. (1985) in Methods
in Enzymology (Law, J. H., and Rilling, H. C., Eds.), Vol. 110, pp. 383-405, Academic Press, New York. 21. Satterwhite, D. M., and Croteau, R. (1988) J. Chromatogr. 452, 61-73. 22. Corey, E. J., and Suggs, J. W. (1975) Tetrahedron Lett., 2647-
2650. 23 Erman, W. F. (1964) J. Amer. Chem. Sot. 86,2887-2897. 24. Berlin, A. A., Davankov, A. B., and Kalliopin, L. E. (1945) J. Appl. Chem.(USSR) 18,217-220.[CA:40,37417].
STEREOCHEMISTRY 25. Thomas, A. F., Schneider, Chem. Sot. 89.6870. 26. Tidwell,
OF CAMPHANE
R. A., and Meinwald,
J. (1967) J. Amer.
T. T. (1970) J. Amer. Chem. Sot. 42,1448-1449.
27. Abad, G. A., Jindal, S. P., and Tidwell, Chem. Sot. 96,6326-6331.
T. T. (1973) J. Amer.
ISOCAMPHANE
BIOSYNTHESIS
381
32. Erman, W. F. (1985) in Studies in Organic Chemistry Vol. 11, Chemistry of the Monoterpenes (Gassman, P. G., Ed.), Part B, pp. 1151-1168, Dekker, New York. 33. Overton, K. H. (1979) Chem. Sot. Reu. 8.447-473. 34. Coates, R. M., and Cavender, P. L. (1980) J. Amer. Chem. Sot.
102,6358-6359.
28. Dixon, M., and Webb, E. C., Eds. (1979) in Enzymes, 3rd ed., pp. 72-75, Academic Press, New York. 29. Croteau, R., Miyazaki, J. H., and Wheeler, them. Biophys. 269,507-516.
AND
C. J. (1989) Arch. Bio-
30. Simonsen, J. L. (1949) in The Terpenes, Vol. II, 2nd ed., pp. 315322, Cambridge University Press, Cambridge. 31. Barton, D. H. R., and Harper, S. H. (1953) in Chemistry of Carbon Compounds (Rodd, E. H., Ed.), Vol. IIB, pp. 573-587, Elsevier, New York.
35. Drengler, K. A., and Coates, R. M. (1980) J. Chem. Sot. Chem. Commun., 856-857. 36. Cane. D. E.. Ha. H.-J.. Paraellis. C.. Waldmeier. F.. Swanson. S.. and Murthy, P. 6. N. (1985)Biodrg. Chem. 13,246:265. 37. Cane, D. E. (1985) Accts. Chem. Res. 18,220-226. 38. Gotfredsen, S., Obrecht, J. P., and Arigoni, D. (1977) Chimia, 31,
62-63. 39. Croteau, R., and Satterwhite. D. M. (1989) J. Biol. Chem. 264. i5,309-15,315. 40. Croteau, R., and Karp, F. (1979) Arch. Biochem. Biophys. 198,
523-532.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 277, No. 2, March, pp. 374-381,199O
Biosynthesis of Monoterpenes: Stereochemistry of the Coupled lsomerization and Cyclization of Geranyl Pyrophosphate to Camphane and lsocamphane Monoterpenes’ Rodney Institute
Croteau,2
Jonathan
of Biological Chemistry,
Gershenzon, Washington
Carl J. Wheeler,3
State University,
Received September 25,1989, and in revised form November
and D. Michael Washington
Satterwhite
99164-6340
17,1989
The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia oficinalis (sage) and Tanacetum v&are (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[ 1Z3H]linalyl pyrophosphate plus [ l-‘4C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3s)-[1Z-3H]linalyl pyrophosphate plus [ 1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:‘4C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-a-hydrogen of the derived ketone. The finding that the 1S3H of the linalyl precursor was positioned at the endo-a-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at Cl of the geranyl substrate in these transforma-
i This investigation was supported in part by National Health Grant GM 31354, and by Project 0268 from the State University Agricultural Research Center, Pullman, * To whom correspondence should be addressed. 3 Present address: Revlon Science Institute, San Diego,
Pullman,
Institutes of Washington WA 99164. CA 92111.
tions, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endoconformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids. 0 1990 Academic Press, Inc.
Bornyl pyrophosphate cyclase from common sage (Saluia oficinalk) catalyzes the divalent metal ion-dependent conversion of [ l-3H]geranyl pyrophosphate to (+)-(1R,4R)-[3-3H]bornyl pyrophosphate, whereas the bornyl pyrophosphate cyclase from tansy (Tanacetum vulgare) catalyzes the transformation of the same acyclic precursor to (-)-(lS,4S)-[3-3H]-bornyl pyrophosphate (1, 2) (Scheme I). These enantiomeric cyclizations, which ultimately lead to the formation of (+)- and (-)camphor, respectively, in these species, are accomplished without loss of hydrogen from Cl of the acyclic precursor and without formation of detectable free intermediates (l-3). Since the trans-geometry of the C2-C3 bond of the geranyl substrate prevents direct cyclization of this precursor, it is clear that the bornyl pyrophosphate cyclases are able to catalyze the isomerization of geranyl pyrophosphate to a bound intermediate capable of cyclizing, as well as the cyclization reaction itself. A stereochemical model for the coupled isomerization and cyclization of geranyl pyrophosphate to bornyl pyrophosphate (4) and related bicyclic monoterpenes (5-7) has been proposed (8). This reaction sequence (Scheme I) is initiated by ionization of geranyl pyrophosphate to the allylic cation, with suprafacial migration of the pyro-
374 All
0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
STEREOCHEMISTRY
OF CAMPHANE
phosphate counter ion of the resulting pair (1) to afford a bound linalyl pyrophosphate intermediate. Rotation about the newly generated C2-C3 single bond to the cisoid conformer overcomes the original stereochemical impediment to the direct cyclization of geranyl pyrophosphate, while subsequent ionization of this tertiary allylic isomer promotes Cl-C6 cyclization of the antiendo form (2). A second electrophilic cyclization involving the cyclohexenyl double bond of the cu-terpinyl intermediate (3) gives the bornyl (camphyl) cation (4), which is captured by the paired pyrophosphate anion. The biosynthetic transformations catalyzed by the (+)- and (-)bornyl pyrophosphate cyclases are thus described by mirror-image schemes involving antipodal linalyl pyrophosphate and a-terpinyl cation (3) intermediates. Closely related antipodal cyclizations are catalyzed by the (+)- and (-)-pinene cyclases from sage, which produce (+)-(lR,4S)and (-)-(lS,4R)-camphene, respectively, as minor products by Wagner-Meerwein rearrangement of the respective bornyl cations (4) to the related isocamphyl cations (5) and deprotonation to the corresponding olefins (9, 10) (Scheme I). [l-3H]Geranyl pyrophosphate thus leads to specific labeling of C7 (the methylene bridge hydrogens) of these bicyclo[2.2.l]olefins. A key feature of this general stereochemical model is the predicted intermediacy of (--)-(SE)-linalyl pyrophosphate in the construction of (+)-bornyl pyrophosphate and (+)-camphene, and the intermediacy of the antipodal (+)-(3S)-isomer in the corresponding enantiomeric cyclizations to (-)-bornyl pyrophosphate and (-)-camphene. These predictions were recently confirmed by direct testing of the optically pure linalyl pyrophosphates with the relevant cyclases (10,ll). Interestingly, the bornyl pyrophosphate and camphene (pinene) cyclases from sage are capable of cyclizing the unnatural linalyl pyrophosphate enantiomer to the antipodal products, but at a slower rate than that of the predicted intermediate (10,ll). The mechanistic basis of these anomalous reactions has been discussed (8, 10, 11). Although the linalyl pyrophosphates are the first explicitly chiral intermediates in the cyclization scheme, the eventual configuration is predetermined by the helical conformation of geranyl pyrophosphate achieved on initial binding to the cyclase; the left-handed helical form yielding (3R)-linalyl pyrophosphate, the right-handed helical counterpart affording the (3S)-enantiomer (8). As an essential consequence of this syn-isomerization-C2-C3-rotation-anti-cyclization scheme, the configuration at Cl of the geranyl substrate should be retained in the transformations to the bornyl pyrophosphates and camphenes (Scheme I). These predictions also were recently confirmed by stereoselective degradation of the products derived enzymatically from [ 1R-3H,‘4C]-labeled and [1S-3H,‘4C]-labeled geranyl pyrophosphate (4, 7, 10). Therefore, the results, thus far, are entirely consistent
AND
ISOCAMPHANE
375
BIOSYNTHESIS Geranyl
Pyrophosphate
(-)-3R-LPP OPP A+ 4 PPOcJ 1
I OOPP -2 /,
h
-
>1
2
2
4
I /
6PP
(+)-Bornyl Pyrophosphate
6 3 I
,
(+I-Camphene
(-bcamphene
SCHEME I. LPP indicates linalyl pyrophosphate. OPP indicates the pyrophosphate moiety. 1 is the geranyl cation, 2 is the linalyl cation, 3 is the a-terpinyl cation, 4 is the bornyl (camphyl) cation, and 5 is the isocamphyl cation.
with the proposed isomerization-cyclization model. However, although indirect evidence for the isomerization step is available (12, 13), the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate has
376
CROTEAU
never been explicitly demonstrated because, in the tightly coupled sequence, linalyl pyrophosphate is never free of the enzyme surface. There is ample precedent for syn-stereochemistry in this reaction type, based on examination of the origin of linalool itself (14) and of the corresponding transposition of trans,trans-farnesyl pyrophosphate to nerolidyl pyrophosphate in the biosynthesis of sesquiterpenoids (l&16). In this communication, we describe the stereochemical alteration at Cl of linalyl pyrophosphate in the transformations to bornyl pyrophosphate and camphene.
This
information,
in conjunction
with
the pre-
viously established retention of configuration at Cl of the geranyl precursor in the coupled reaction, provides strong supporting evidence for the proposed syn-isomerization-anti-endo-cyclization stereochemistry in the construction of the camphane (bornane) and isocamphane monoterpenes. EXPERIMENTAL
PROCEDURES
Plant materials, substrates, and reagents. Sage (S. oficinalis L.) and tansy (2’. u&are, L.) plants were grown from seed under greenhouse conditions described previously (1, 2). Young emerging leaves from 18 to Pl-day-old plants were used as the source of the monoterpene cyclases. The preparation of each of the following substrates has been previously described: [l-3H]geranyl pyrophosphate (80 Ci/mol) (l), [l“‘Clgeranyl pyrophosphate (25 Ci/mol) (6), (3R)-[1Z-3H]linalyl pyrophosphate (21.6 Ci/mol) (17), and (3S)-[lZ-3H]linalyl pyrophosphate (40 Ci/mol) (17). (f)-Camphene ([a]? + 6” (C = 10, EtOH)) was obtained from K & K Laboratories (Plainview, NY). rat-Borneo1 was from U.S. Biochemical Corp. (Cleveland, OH), and rat-camphor was from Aldrich Chemical Co. (Milwaukee, WI). All other standards, reagents, and biochemicals were purchased from Aldrich or Sigma Chemical Co. Enzyme preparation and assay. The preparation, assay, and general properties of (+)-pinene cyclase (cyclase I), (-)-pinene cyclase (cyclase II) (9, 18) and (+)-bornyl pyrophosphate cyclase (1, 3, 11) from S. oficinalis, and (-)-bornyl pyrophosphate cyclase (2, 4, 11) from T. uulgare, have been described. These procedures employ selective extraction of the leaf epidermal oil glands (19) and partial purification of the cyclase by gel permeation and/or ion-exchange chromatography to remove the bulk of the competing phosphatase activities. The standard assay involves conversion of radiolabeled substrate to chromatographically purified product. T. uulgare was used as the source of (-)-bornyl pyrophosphate cyclase for this work because S. oficinalis extracts contain very low levels of this activity (4, ll), and the preparative conversions were thus inefficient. Preparative enzymatic conversions and product isolation. For the enzymatic synthesis of (+)-camphene, 5-ml aliquots of the partially purified cyclase I preparation from sage (containing 80-100 yg of protein) in 20 mM Mes,4 5 mM potassium phosphate buffer, pH 6.5, containing 0.5 mM dithiothreitol, 15 mM MgClz and 10% (v/v) glycerol were employed. The same conditions were used for preparative conversions with (+)-bornyl pyrophosphate cyclase from sage and with (-)-bornyl pyrophosphate cyclase from tansy. For the enzymatic synthesis of (-)-camphene, similar preparations of cyclase II from sage
’ Abbreviations used: Mes, 4-morpholineethanesulfonic tris(hydroxymethyl)aminomethane; TLC, thin-layer phy; GLC, gas-liquid chromatography.
acid; Tris, chromatogra-
ET AL. (containing 50-70 pg of protein) were employed. However, the pH of the buffer system was adjusted to the optimum of 7.2. In each case, the reaction was initiated by the addition of a saturating concentration of the allylic pyrophosphate substrate (10 FM of [l-3H]geranyl pyrophosphate in the case of the standard assay), and the mixture in a screwcapped vial was incubated with gentle swirling for 3 h at 30°C. For camphene isolation, the solution was chilled in ice following incubation, and 2 ml of pentane added to extract olefinic products. Thorough mixing of the biphasic system was followed by centrifugation to facilitate separation of the phases, and the pentane layer was passed through a short column of silica gel (Mallinckrodt type 60A) overlaid with anhydrous MgSO,. Extraction and elution were repeated with an additional 2 ml of pentane to provide a combined eluate containing only hydrocarbons. Samples from replicate incubations were pooled, carrier standards were added (10 mg of (+)-camphene plus -10 ~1 of the olefin fraction from sage oil), and the camphene was isolated by argentation TLC (Silica Gel G containing 15% AgNOB, using 1% diethyl ether in benzene:pentane (1:2, v/v); R,, 0.55). The scraped gel from the appropriate region was eluted with pentane. An aliquot of this eluate was counted and another aliquot analyzed by radio-GLC (20, 21) (>97% radiochemical purity after repetition of argentation TLC using 2% n-hexene in pentane). A sufficient number of such preparative incubations were carried out to accumulate in excess of 3 nmol of camphene from the double-labeled precursor mixture. In order to isolate the bornyl pyrophosphate produced, the enzymatic reaction mixtures, following incubation, were first extracted with ether. These ether extracts contained negligible levels of borneol, but did give, by aliquot counting, a measure of substrate loss to competing phosphatases and solvolysis. The reaction mixtures were then purged with a stream of N2 to remove traces of ether, and 1 ml of 200 mM sodium acetate buffer, pH 5.0, containing 1 unit each of wheat
germ acid phosphatase and potato apyrase, was added. The mixture was incubated with gentle agitation for 75 min at 30°C. After cooling in ice, 2 ml of ether was added and the biphasic system was vigorously mixed and then centrifuged to separate phases. The organic layer was dried by passage through a short column of MgSO,, and the entire extraction repeated with an additional 1 ml portion of ether. All of the ether extracts from like incubations were combined to provide a sample of borne01 (in excess of 5 nmol), which represented all of the bornyl pyrophosphate produced. To facilitate the TLC separation of borne01 from unsaturated monoterpenes present in the combined extract (i.e., from hydrolysis of the substrate), excess OsOl (0.1 mg) in pyridine (10 ~1) was added, and the sample was stirred overnight. Next, 1 ml of saturated aqueous NaHSO, was added (followed by stirring for 3 h at room temperature) to decompose osmate esters, thereby completing the conversion of unsaturated compounds to the corresponding diols, and drastically reducing their mobility in the subsequent TLC step. The organic phase, and an additional 1 ml ether extract of the aqueous phase, were combined, washed twice with small portions of 0.1 N HCl, and dried over MgSO,. Each sample was diluted with 10 mg (k)-borneol carrier, and then concentrated under vacuum (Savant Speed Vat) and separated by TLC on Silica Gel G with hexanes:ether (2:1, v/v). The band corresponding to borne01 (Rf = 0.4) was scraped directly into a glass wool-plugged Pasteur pipet, and the product eluted with diethyl ether or dichloromethane. An aliquot of the sample was taken for determination of radioisotope content and another for radio GLC analysis (>97% radiochemical purity). Conversion of bornyl pyrophosphate to camphor. The biosynthetitally generated, labeled borne01 (>0.22 &i-3H) released by enzymatic hydrolysis of the pyrophosphate ester and isolated by TLC as above, was diluted with additional racemic carrier to a specific radioactivity of -0.3 PCi-sH/mmol, dissolved in CHzClz, and oxidized to camphor with a slight excess of pyridinium chlorochromate in CH&lz for 40 min at room temperature (22). The reaction mixture was poured through Florisil to remove chromate salts, and the product (-0.6 mmol) isolated from the eluate by concentration and TLC as before.
STEREOCHEMISTRY An aliquot of the derived camphor was converted oxime (4) for determination of isotope content.
OF CAMPHANE to the crystalline
Conversion of camphene to camphor. The biosynthetically generated, 3H:‘4C-labeled camphene (>0.14 &i-3H), isolated as above, was diluted with additional (+)-camphene to a specific radioactivity of -0.2 &i-3H/mmol, and subjected to acetolysis essentially as described by Erman (23). Thus, 0.6 mmol of camphene was diluted with 1.2 mmol of glacial acetic acid, to which -0.02 mmol BF,-etherate was added by syringe, and the mixture heated at 100°C in a sealed tube under Ar for 3 h. The contents of the cooled tube were partitioned between saturated aq NaCl and ether, and the ether-soluble products analyzed by GLC and shown to contain -50% isobornyl acetate and -10% bornyl acetate, along with numerous other products. The crude mixture was hydrolyzed in 50% ethanolic 0.1 N KOH, and the resulting alcohols were partitioned into ether, following saturation with NaCl. Drying of the extract over MgS04, and concentration of the solvent, afforded an oily mass that was dissolved in CHzClz and subjected to pyridinium chlorochromate oxidation, as before, for the conversion of isoborneol (and borneol) to camphor. Preparative TLC of this mixture yielded camphor (-50% yield from camphene) that was well-resolved from contaminating products of higher and lower R, value (>95% by GLC). Aliquots of the derived camphor were converted to the crystalline oxime, as before, for determination of isotope content. Several attempts at employing the chromic acid oxidation of camphene to produce camphor directly (24) gave the ketone in higher yields, but the method was accompanied by more extensive racemization of the product (see below). Base-catalyzed enchange procedure. The a-protons of 3H-labeled camphor derived from the enzymatically generated bornyl pyrophosphate and camphene were subjected to base-catalyzed exchange by a procedure described in detail elsewhere (4, 7). In brief, the derived camphor samples (at a total of 40 mg from camphene or 80 mg from bornyl pyrophosphate) were dissolved in 0.2 ml dioxane and added by syringe to 2.3 ml of dioxane:0.5 N aqueous NaOH (l:l, v/v), which was heated with constant stirring at 60°C. Aliquots comprising 20% of the total sample were removed at 4 min intervals and added to 0.5 ml of cold 0.25 N HN03, followed by NaCl saturation, recovery of the camphor by ether-extraction, purification by TLC, and determination of ‘H:14C content by liquid scintillation spectrometry. At the end of the exchange run (4 samples including the zero-time control), 2.5 ml of the dioxane:aqueous NaOH solution was added to the remaining sample (20% of the starting material), and the mixture heated on a steam bath for 2 h. Following this exhaustive exchange procedure, the residual camphor was recovered and purified as before, and the 3H:‘4C ratio of this material (as the oxime) was determined. This latter procedure was required in order to evaluate the extent of racemization that had occurred during the chemical transformations (i.e., racemization results in the placement of ‘H in a position inaccessible to exchange) and to allow correction of the rate curves which are based on 3H loss from the exchangeable total. Hydrolysis ojpyrophosphate esters and prenol analysis. To examine the possible interconversion of [Wlgeranyl pyrophosphate and [3H]linalyl pyrophosphate, the aqueous phases of the corresponding incubation mixtures remaining after extraction of (+)- or (-)-camphene were combined, centrifuged to remove denatured protein, and lyophilized. The resulting glass was dissolved in 5 ml 0.1 N Tris-HCl buffer, pH 8.0, containing 5 units of calf intestine alkaline phosphatase, and incubated for 3 h at 30°C in a sealed tube. Following chilling of the incubation mixture, the liberated prenols (75-80% recovery) were extracted into ether and diluted with 5 mg each of carrier geraniol and (+-)-linalool, and, following concentration of solvent, the products were separated by TLC (Silica Gel G using hexane:ether (l:l, v/v); R, geraniol, 0.45, R, linalool, 0.50). Each product was further purified by TLC on Silica Gel G containing 15% AgN03 with hexane:ether (2:3, v/ v) before determination of radioisotope content by liquid scintillation spectrometry. Incubation mixtures of the bornyl pyrophosphate cyclases with the geranyl and linalyl substrates were similarly treated;
AND
ISOCAMPHANE
BIOSYNTHESIS
however, the 0~0~ treatment necessarily omitted.
377
employed for the analysis of borne01 was
Other analytical procedures. Detailed procedures for liquid scintillation spectrometry, TLC, and radio-GLC of the monoterpenoids have been described (4,11,20,21).
RESULTS
AND
DISCUSSION
The proposed model for the cyclization of linalyl pyrophosphate to bornyl pyrophosphate predicts that the Zhydrogen at Cl of the acyclic precursor will become the en&-a-hydrogen of this product (Scheme II). Similarly, cyclization of [ 1Z3H]linalyl pyrophosphate to camphene should result in specific labeling of the exo-hydrogen of the methylene bridge of this olefin (Scheme II). Validation of this prediction would provide strong evidence for the anti,endo-cyclization of (3R)- and (3S)-linalyl pyrophosphate to the respective antipodal bornane and isocamphane monoterpenes. Additionally, since geranyl pyrophosphate is known to be transformed to the enantiomeric bicyclic products with retention of configuration at Cl (4), such a result would confirm the helical conformation of the geranyl precursor achieved at the outset of the coupled isomerization-cyclization sequence, as well as the syn-stereochemistry of the geranyl-to-linalyl isomerization (i.e., with suprafacial pyrophosphate migration, the l-proR-hydrogen of geranyl pyrophosphate will become the l-Z-hydrogen of 3Rlinalyl pyrophosphate, whereas the l-pros-hydrogen will become the l-Z-hydrogen of the 3S-enantiomer (Scheme II)). To approach this problem, it was necessary to devise a means of specifically locating the relevant hydrogens of the bicyclic products derived from Cl-labeled precursors. The conversion of bornyl pyrophosphate to camphor has been exploited for this purpose in the past (4, 7), since the C1-3H atom of the precursor resides on the C3-methylene (adjacent to the carbonyl) of the derived bicyclo[2.2.l]ketone, and the exo- and e&o-protons can be readily distinguished by differential base-catalyzed exchange (i.e., Kexo:Kend,- 2O:l (25-27)). A similar strategy was developed for conversion of camphene to camphor to permit location of the 3H tracer by exchange procedures. The loss of 3H via base-catalyzed exchange of the bicyclic ketone is most readily determined by using product derived from racemic Cl-labeled precursors as a reference, and by employing a 14C-labeled internal standard (4, 5, 7). Lacking both 1E,Z-3H-labeled and ‘*C-labeled linalyl pyrophosphate enantiomers for this purpose, [ 1R,S-3H]geranyl pyrophosphate and [ 1-14C]geranyl pyrophosphate were employed. Preliminary experiments were, therefore, directed toward confirming that geranyl pyrophosphate and linalyl pyrophosphate were converted to product at the same active site of each cyclase by employing the mixed substrate method of Dixon and Webb (28). Thus, all combinations of [ l-3H]geranyl py-
378
CROTEAU
GPP
ET AL.
’
Q (z)Q PPO:d--“;-1 (+I-Bornyl Pyrophosphate
(+)-Borneo1
4 0 (z)Q .‘Q 4
(-)-Bornyl Pyrophosphate
(-)-Borneo1
I 8 (z)Q d-
0 (-I-lS,4S-Camphor
(+I- 1 R,4R-Camphor
t
0 (2) -b (+I-Camphene
(+I-lsobornyl Acetate
(-klsobornyl Acetate
The circled R designates the l-proR SCHEME II. 2 designates the 1-Z hydrogen of linalyl pyrophosphate
hydrogen, (LPP).
and the circled
rophosphate and [ 1Z-3H]linalyl pyrophosphate (2-8 PM range) gave rise to total rates of product formation significantly less than the arithmetic sum calculated from each precursor when tested alone (Table I), confirming that both substrates competed for the same active site and, therefore, that both isomerization and cyclization steps occur at the same reaction center. This observation is entirely consistent with a tightly coupled isomerization-cyclization reaction in which no free intermediates are formed, and it substantiates identical results obtained with all other cyclases examined (6, 7,29). On the basis of the observed relative changes in rates of conversion of the geranyl and linalyl precursors alone and in combination, the appropriate (3R)- and (3S)-[ lZ3H]linalyl pyrophosphate enantiomers were mixed with [l-‘*C]geranyl pyrophosphate to give roughly a 10~1 ratio of 3H:14C in the bornyl pyrophosphate and camphene products. [1R,S-3H]Geranyl pyrophosphate and [l-14C]geranyl pyrophosphate were admixed at a 3H:14C ratio of 1O:l to provide the precursor needed to generate racemitally labeled product for use as a reference standard for base-catalyzed exchange.
S the l-pros
(-)-Camphene hydrogen,
of geranyl
pyrophosphate
(GPP).
Preparative-scale incubation of (+)-bornyl pyrophosphate cyclase (S. oficinalis) with the mixture of (3R)[ 1Z-3H]linalyl pyrophosphate and [ 1-l*C]geranyl pyrophosphate precursors gave 0.23 &i-3H of product (isolated as borneol) at a 3H:‘4C ratio of 7.6 + 0.4:1 (as the phenylurethane (4)). The reference standard of (+)-bornyl pyrophosphate (isolated as borneol) prepared from [1R,S-3H,1-14C]geranyl pyrophosphate was obtained at a level of 0.34 pCi-3H (3H:‘4C = 10.2 f 0.2:1). In the case of the (-)-bornyl pyrophosphate cyclase (7’. uulgure), the (3S)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate mixture gave 0.24 &i-3H of product at 3H:‘4C = 12.3 + 0.51, and the incubation with [lR,S3H,1-14C]geranyl pyrophosphate gave a reference standard of 0.31 &i-3H at a ratio of 9.9 k 0.2:1. Each borne01 sample was diluted with carrier to a specific radioactivity and oxidized with pyridinium of -0.3 &i-3H/mmol, chlorochromate to camphor (TLC purified) without significant change in the 3H:‘4C ratio (determined as the oxime). Each camphor sample was then subjected to base-catalyzed exchange in dioxane:aqueous NaOH at 6O”C, as described under Experimental Procedures, US-
STEREOCHEMISTRY OF CAMPHANE AND ISOCAMPHANE BIOSYNTHESIS
379
TABLE I Conversion of Geranyl Pyrophosphate and Linalyl Pyrophosphate Mixtures to Bornyl Pyrophosphate and Camphene Substrate” (3R)-LPP
GPP Product
PM
(+)-Bornyl pyrophosphate
2 4 2 4
(+)-Camphene
VGb 38.6 53.3 12.1 26.6
PM
VLb
Calculated sum tv,+ Vdb
Actual sum (observed)b
4 2 6 3
25.3 17.5 4.6 2.4
63.9 70.1 16.7 29.0
31.4 50.6 7.2 19.3
(3&s)-LPP
GPP PM (-)-Bornyl pyrophosphate (-)-Camphene
4 8 4 8
VGb 49.6 57.5 11.6 22.7
GM
VLb
(VG•t VI,)*
(observed)*
8 4 8 4
98.3 53.4 29.3 17.0
147.9 110.9 40.9 39.7
66.6 57.1 24.9 21.2
’ Abbreviations used:GPP, geranyl pyrophosphate;LPP, linalyl pyrophosphate. * Velocities are given in cpm X 10e3and are the averagesof duplicate runs differing by lessthan 11% of the mean. ing a technique in which samples were periodically removed, the reaction quenched in HNOB, and the camphor reisolated by TLC for radioisotope determination. To evaluate the extent of racemization that occurred during the chemical transformations leading to camphor, the remaining sample at the end of each exchange run (26% of total) was subjected to exhaustive exchange, and the camphor was reisolated as before. In all cases, essentially all of the tritium was lost (3H:14C < O.l:l as the oxime), as expected (1, 2, 4), indicating complete specificity in the positioning of 3H at C3 of the ketone, and no racemization leading to nonexchangeable 3H. Relative exchange rates of the a-hydrogens were then established by comparing 3H loss from the starting material in each case to that observed for the corresponding 3R,S-3H-labeled camphor sample exchanged under identical conditions. Since the exo:endo exchange rate ratio is 20 (25-27), the theoretical rate ratio for the exo-a-3H exchange, with reference to the racemically labeled standard, is 2, whereas that for the endo-a-‘H, with reference to the same standard, is 0.1. For (+)-camphor derived from (+)-bornyl pyrophosphate cyclase and the (3R)[ 1Z-3H]linalyl pyrophosphate precursor, the observed ratio calculated from the linear portion of the curve was 0.11 f 0.02. For (-)-camphor derived from (-)-bornyl pyrophosphate cyclase and the (3S)-[1Z-3H]linalyl pyrophosphate precursor, the observed ratio was 0.12 ? 0.02. Thus, for both cases, the 1Z-3H of the linalyl precursor was placed in the endo-a-position of the bornane product, in agreement with the proposed isomerizationcyclization model (Scheme II). To locate the 3H atom in the biosynthetically derived camphenes, the well-known (30,31) acid-catalyzed rear-
rangement of the isocamphane to the bornane (camphane) skeleton was exploited. Thus, acetolysis of camphene, under the conditions described (23), gave rise to roughly 50% isobornyl acetate (and 10% bornyl acetate), which was hydrolyzed and oxidized to camphor in about 50% overall yield (Scheme II), providing a minimum of 0.07 &i-3H of product (at a specific radioactivity of -0.2 &i/mmol) in each case. The transformation of camphene (measured as camphenilone oxime (10)) to camphor (measured as the oxime) was accomplished without significant change in the 3H:‘4C ratio, which in this set of products ranged from 6.7 to 9.3. The 3H:14Clabeled ketones were then subjected to base-catalyzed exchange, as before, to selectively remove the exo-a-protons. The transformation of the isocamphane to the bornane ring system is accompanied by partial racemization, largely as the result of e3co-3,2-methyl migration (in the isocamphyl cation (5)) and endo-6,2-hydride shift (in the bornyl cation (4)) (See Scheme 1) (32). In the present instance, methyl migration leads to camphor of the opposite configuration, in which the relative orientations of the exo- and endo-a-hydrogens are unaffected. The hydride shift, similarly, leads to the enantiomeric camphor, but in this instance, the 3H label is rendered inaccessible to exchange, since Cl of the precursor now resides at C5 of the dimethylene bridge of the resulting ketone. The latter process was readily corrected for by exhaustive base-treatment of the remaining camphor sample following an exchange run, and was shown to vary from 14 to 18% (as residual 3H label); the extent of racemization is known to be highly dependent on the precise reaction conditions (32). The corrected exchange rate for (+)-[3H]camphor derived from (+)-[3H]cam-
380
CROTEAU
phene generated from (3R)- [ 1Z-3H]linalyl pyrophosphate, with reference to the R,S-3H-labeled standard, was 0.11 f 0.03. The rate for (-)-[3H]camphor derived from (-)-[3H]camphene generated from (3S)-[lZ3H]linalyl pyrophosphate, with reference to the corresponding racemically labeled standard, was 0.12 f 0.03. Thus, as was the case with (+)- and (-)-bornyl pyrophosphate, the 1Z-3H of the (3R)- and (3S)-linalyl precursors was transformed to the en&-o-position of the respective (+)- and (-)-camphor products, as predicted. These observations serve to confirm the syn-stereochemistry in the isomerization of geranyl pyrophosphate to linalyl pyrophosphate when taken with the net retention of configuration at Cl of the geranyl precursor in the coupled transformation to these enantiomeric bicyclic products (4). The results presented here, in addition to confirming the syn-isomerization, rotation, anti-cyclization sequence (increasing evidence suggests a universal preference for anti-allylic displacement in related isoprenoid cyclizations (16,33-37)), establish the helical conformation of the reacting geranyl precursor and the anti-end0 conformation (38) of the cyclizing tertiary intermediate and rule out all other possible conformers for this coupled reaction. When taken together with the previously described configurational imperative for (3R)-linalyl pyrophosphate in the cyclization to (+)-bornyl pyrophosphate and (+)-camphene, and for (3S)-linalyl pyrophosphate in the cyclization to (-)-bornyl pyrophosphate and (-)-camphene (10, ll), the summation of these studies unequivocally establishes all of the absolute stereochemical elements in the isomerization and cyclization of geranyl pyrophosphate to these enantiomeric bornane and isocamphane products. Even the aberrant cyclizations of the unnatural linalyl antipodes by the sage enzymes are entirely consistent with this stereochemical picture (10, 11, 39). The present work completes studies on the stereochemical origin of all of the major classes of bicyclic monoterpenes (bornanes (camphanes), isocamphanes, thujanes, pinanes, fenchanes). All of the results with these diverse skeletal types are fully consistent (5-8, 10, 17), and lend very strong support to the general syn-isomerization-anti,endo-cyclization model for the transformation of the universal acyclic precursor geranyl pyrophosphate (8). An interesting ancillary observation arising from the present work was provided by analysis of the [14C]geranyl pyrophosphate and [3H]linalyl pyrophosphate remaining at the completion of the preparative enzyme conversions to bornyl pyrophosphate and camphene (most of the substrates were not consumed in this process). Thus, enzymatic hydrolysis of the residual pyrophosphate esters by estabhshed methods (17,20,40), followed by dilution with carrier and TLC separation of the alcohols, demonstrated the presence of only [14C]geranyl pyrophosphate (3H:14C < 0.03:1) and [3H]linalyl pyro-
ET AL.
phosphate (3H:14C > 98:l). The absence of observable cross contamination, in each case, confirms that [‘“Cllinalyl pyrophosphate, which arises by isomerization of [14C]geranyl pyrophosphate, is enzyme-bound and not free to equilibrate with [3H]linalyl pyrophosphate in solution, and confirms that isomerization is not reversible (i.e., from [3H]linalyl pyrophosphate to free [3H]geranyl pyrophosphate) in the time frame of the overall reaction. ACKNOWLEDGMENTS We thank Greg Wichelns for typing the manuscript.
for raising the plants and Nancy Madsen
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