Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (-)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis

Article abstract of DOI:10.1021/ol991230p

(equation presented) Syntheses and enzymatic cyclizations of 8α,hydroxy-17-nor copalyl diphosphate (8a), (15R)-[15-²H₁] 8b, and (15R,17E)-[15-³H₁,17-²H₁] copalyl diphosphate ([²H,³H] 2) catalyzed by recombinant abietadiene synthase (rAS) gave 17-nor manoyl oxide (9a), (16E)-[16-²H₁] 9b, and (15S,16R)-[16-²H₁,16-³H₁] abietadiene ([²H₁,³H₁] 4), respectively. These and other results indicate that conversion of CPP (2) to abietadiene (4) occurs by anti S_N′ cyclization to a sandaracopimar-15-en-8-yl carbocation intermediate (13⁺, 13β-methyl)' followed by hydrogen transfer and methyl migration suprafacially on the si face of the vinyl group.

Full text of DOI:10.1021/ol991230p

ORGANIC
LETTERS
2000
Vol. 2, No. 5
573-576
Stereochemistry of the
Cyclization-Rearrangement of
(+)-Copalyl Diphosphate to
(−)-Abietadiene Catalyzed by
Recombinant Abietadiene Synthase from
Abies grandis
,†
Matthew M. Ravn,† Robert M. Coates,* Janice E. Flory,‡ Reuben J. Peters,‡ and
Rodney Croteau‡
Department of Chemistry, UniVersity of Illinois, 600 S. Mathews AVenue,
Urbana, Illinois 61801, and Institute of Biological Chemistry,
Washington State UniVersity, Pullman, Washington 99164
r-coates@uiuc.edu
Received November 6, 1999
ABSTRACT
Syntheses and enzymatic cyclizations of 8r-hydroxy-17-nor copalyl diphosphate (8a), (15R)-[15-2H ] 8b, and (15R,17E)-[15-3H ,17-2H ] copalyl
1
1
1
diphosphate ([2H,3H] 2) catalyzed by recombinant abietadiene synthase (rAS) gave 17-nor manoyl oxide (9a), (16E)-[16-2H ] 9b, and (15S,16R)-
1
[16-2H ,16-3H ] abietadiene ([2H ,3H ] 4), respectively. These and other results indicate that conversion of CPP (2) to abietadiene (4) occurs by
1
1
1
1
anti SN′ cyclization to a sandaracopimar-15-en-8-yl carbocation intermediate (13+, 13â-methyl) followed by hydrogen transfer and methyl
migration suprafacially on the si face of the vinyl group.
The abietanes comprise a large family of perhydrophenath-
rene-type diterpenes characterized by an isopropyl group,
or its functionalized equivalent, at C13.1 Familiar examples
are abietic acid (5) and the isomeric levopimaric, neoabietic,
and palustric acids, which are major diterpene constituents
of conifer oleoresin.2 This defensive secretion is produced
by pines, firs, spruces, and other conifers as a primary
response to wounds caused by physical injuries, insects, large
herbivores, and microbial diseases.3 The derivation of the
abietane carbon skeleton from the predicted head-to-tail
connectivity of isoprene units led L. Ruz´ıcka to propose that
the aberrant isopropyl group might arise by proton-induced
rearrangement of a regular isoprenoid precusor, such as
pimaric acid.4 The acid-induced conversion of pimaric and
isopimaric acids to abietic acid affords chemical precedent
for this biogenetic relationship.5
The native enzymes responsible for the cyclization of
(E,E,E)-geranylgeranyl diphosphate (1) to (-)-abietadiene
(4) (Scheme 1) were isolated in partially purified form from
the stems of both wound-induced grand fir (Abies grandis)
* Tel: (217) 333-4280. FAX: (217) 244-8068.
† University of Illinois.
‡ Washington State University.
(1) CRC Handbook of Terpenoids: Diterpenoids Vol I-IV; Dev, S.,
Misra, R., Eds.; CRC Press: Boca Raton, FL, 1985-1986.
(2) Soltes, J.; Zinkel, D. F. In NaVal Stores: Production, Chemistry and
Utilization; Zinkel, D. F., Russel, J., Eds.; Pulp Chemicals Association:
New York, 1989; Chapter 9.
(3) Phillips, M. A.; Croteau, R. Trends Plant Sci. 1999, 4, 184.
(4) Ruz´ıcka, L. Experentia 1953, IX, 357.
(5) Wenkert, E.; Chamberlin, J. W. J. Am. Chem. Soc. 1959, 81, 688.
10.1021/ol991230p CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/15/2000

The coupled cyclization-rearrangement of 2 was inter-
cepted by use of an oxa analogue (8a) bearing an 8R-
hydroxyl function in place of the exocyclic methylene group
(Scheme 2). 8-Oxo-17-nor copalol (6a), obtained in three
Scheme 1
Scheme 2
and lodgepole pine (Pinus contorta) saplings.6 Cytochrome
P450 mixed function oxygenases and a dehydrogenase from
the same sources, which carry out the sequential oxidation
of the C18 methyl group of 4 to produce abietic acid, have
also been characterized.7 The gene encoding abietadiene
synthase (AS) from grand fir has been cloned, and the cDNA
was functionally expressed as a single polypeptide bearing
a putative N-terminal plastidial targeting sequence.8 Recom-
binant AS (rAS) was heterologously expressed in E. coli as
a pseudomature enzyme without the N-terminal 84 amino
acids9 and used in crude bacterial extracts after lysis and
clarification (at 0.1 g wet cell weight/mL buffer). Although
the resulting recombinant AS catalyzed the divalent metal
ion-dependent cyclizations of both 1 and the predicted
bicyclic intermediate (+)-copalyl diphosphate (2) to 4,9,10
none of the four plausible pimaradiene intermediates was
converted into the rearranged diterpene to an appreciable
extent under the same assay conditions.10 Recent deuterium
labeling experiments revealed that the cyclization-rearrange-
ment of (+)-CPP to abietadiene occurs with stereospecific
“intramolecular” transfer of a hydrogen atom from the 17-
pro-E position of 2 to the terminal carbon of the side chain,
which becomes the pro-S methyl of the isopropyl group.9,10
In this communication, we present evidence indicating a 13â-
methyl group in the presumed pimaradiene (3b) or pimarenyl
carbocation intermediate and a suprafacial relationship
between the hydrogen reincorporated at C16 and the
subsequent methyl migration to C15.11
steps from methyl copalate,12 underwent reduction with Li/
NH3 (EtOH, ether, -33 °C) to 8R-hydroxy-17-nor copalol
(7a, 83%).13,14 Conversion to the corresponding allylic
diphosphate 8a was accomplished in two steps by phos-
phorylation [(EtO)2P(O)Cl, py, CH2Cl2, 0 °C, 3 h],15
displacement with pyrophosphate anion [(Bu4N)3HP2O7, CH3-
CN, rt, 3.7 d], and purification according to the Poulter
methods (66%).14,16 Incubation of 8a with rAS8,10,17 effected
clean cyclization to 17-nor manoyl oxide 9a (yield, ca. 150
µg, 46% by 1H NMR estimate), the 13â-methyl configuration
of which was established by NOE measurements.14 Acid-
catalyzed dehydration (pTsOH‚H2O, ether, rt, 1.5 h) of 7a
afforded a 1.5:1 mixture of 9a and its 13R-methyl isomer
(76%). The formation of 17-nor manoyl oxide in the
enzymatic cyclization implicates a â-methyl group at C13
(12) (a) catalytic OsO4, NMO, acetone; 3:1 mixture of 8,17- and 13,-
14-diols (78%); (b) LiAlH4 (or LiAlD4 for 6b), ether; (c) NaIO4, aqueous
acetone (60% for two steps).
(13) All new compounds were characterized at high purity by appropriate
1H NMR, 13C NMR, and IR spectra. Purity g 95% was established by
satisfactory combustion analysis and/or inspection of 1H and 13C NMR
spectra. Detailed experimental procedures and characterization data are
provided in the Supporting Information. Key data are presented in the text
or in footnote 14.
(6) LaFever, R. E.; Vogel, B. S.; Croteau, R. Arch. Biochem. Biophys.
1994, 313, 139.
(7) Funk, C.; Croteau, R. Arch. Biochem. Biophys. 1994, 308, 258.
(8) Vogel, B. S.; Wildung, M. R.; Vogel, G.; Croteau, R. J. Biol. Chem.
1996, 271, 23262.
(9) Ravn, M. M.; Coates, R. M.; Jetter, R.; Croteau, R. Chem. Commun.
1998, 21.
(10) Peters, R. J.; Flory, J.; Jetter, R.; Croteau, R., manuscript in
preparation.
(11) The occurrence of an analogous proton transfer to form the pro-S
methyl group of a presumptive abietadiene intermediate was inferred from
the labeling pattern of ginkgolide A biosynthesized from [6,6,6-2H3]
mevalonate in Ginkgo biloba cell cultures: (a) Schwarz, M.; Arigoni, D.
In Isoprenoids Including Carotenoids and Steroids; Cane, D. E., Ed.; Vol.
2 in ComprehensiVe Natural Products Chemistry; Barton, D., Nakanishi,
K., Meth-Cohn, O., Eds.; Elsevier: Oxford, 1999; Chapter 14. (b) Schwarz,
M. K. Dissertation 10951, ETH Zu¨rich, Switzerland, 1994.
(14) Key spectral and physical data for selected compounds: 7a, mp
1
85-86 °C, H NMR δ 3.41 (td, J ) 10.6, 4.9 Hz, 1H, H8); 8a, 31P NMR
δ -7.02, -9.72 (2d, J ) 22 Hz); 9a, NOE irrad at 3.52 obs 0.74 (C20
CH3, 7.3%), 1.21 (C17 CH3, 6.7%); 9b, 1H NMR δ 5.37 and 6.04 (2d, J )
17.4 Hz, 1H each, CHdCHD).
(15) Ravn, M. M.; Jin, A. Q.; Coates, R. M. Eur. J. Org. Chem., in
press.
(16) For leading references, see: (a) Davisson, V. J.; Woodside, A. B.;
Neal, T. R.; Stremler, K. E.; Muehlbacher, M.; Poulter, C. D. J. Org. Chem.
1986, 51, 4768. (b) Woodside, A. B.; Huang, Z.; Poulter, C. D. Org. Synth.,
Coll. Vol. VIII, 1993, 616.
(17) Incubation conditions:8,10 30 mM HEPES, pH 7.2, 5.0 mM dithio-
threitol, 7.5 mM MgCl2, 20 µM MnCl2, 5% (v/v) glycerol, 80 µM 8a (0.40
µmol total), 3 × 1 mL rAS cell lysate; 31 °C, 16 h. Purification by pipet
chromatography over MgSO4 and silica gel; yield, ca. 150 µg (46%) by 1H
NMR estimate.
574
Org. Lett., Vol. 2, No. 5, 2000

of the tricyclic intermediate corresponding to sandaracopi-
maradiene (3b) which in fact is a consistent, albeit very
minor, byproduct (ca 2%) that accompanies abietadiene in
the extractable products from incubations of 1 with rAS.8,10
The stereochemistry of the enzyme-catalyzed SN′ cycliza-
tion was determined by deuterium labeling at C15 (Scheme
2). Allylic oxidation (MnO2, hexane, rt) of 6b followed by
re-selective hydride reduction with (R)-Alpine borane18
(THF, H2O2, NaOH)19 and Li/NH3 reduction gave 7b. The
15S configuration and stereohomogeneity (98% de) of the
Scheme 4
1
intermediate keto alcohol 6c were confirmed by H NMR
analysis of the corresponding camphanate ester.20 Conversion
to 8b by diphosphate displacement as described above for
unlabeled 7a was assumed to proceed with complete inver-
sion of configuration, as established for (S)-[1-2H1] ge-
raniol.15,20 rAS-catalyzed cyclization of 8b gave rise to 9b,
the 16E stereochemistry of which was established by the
large coupling (J ) 17 Hz) between the two vinyl protons.14
Hence, the stereochemistry of this enzyme-catalyzed SN′
cyclization, and by inference that of 2, is anti, in agreement
with similar SN′ cyclizations that occur in diterpene biosyn-
thesis.21
hydride reduction and dehydrobromination and afforded
(17E)-17-bromocopalol (11). The corresponding tetrahydro-
pyranyl ether was lithiated, deuterated, and hydrolyzed to
(17E)-[17-2H1] copalol (12). A four-step reaction sequence
consisting of MnO2 oxidation, NaBH3T reduction, MnO2
oxidation, and (R)-Alpine borane reduction gave (15S,17E)-
[15-2H1,17-3H1] copalol ([2H,3H] 12).18,19 Conversion to the
diethyl phosphate followed by displacement with (Bu4N)3-
P2O7H in CH3CN with inversion of configuration15 provided
(15R,17E)-[17-3H1,15-2H1] 2 (0.13 mCi, 40% radiochemical
yield, 21.8 mCi/mmol).
The stereochemistry of the hydrogen reincorporation at
C16 during the rearrangement that generates the isopropyl
group was elucidated by double isotope labeling (Schemes
3 and 4). (15R,17E)-[15-3H1,17-3H1] 2 was synthesized from
Scheme 3
Incubation with rAS17 and addition of carrier afforded [16-
2H1,16-3H1] abietadiene (4) (1.8 mg, 1.12 µCi/µmol, 1.57 ×
108 dpm), which was subjected to Kuhn-Roth oxidation (aq
H2CrO4, reflux, 16 h)22 (Scheme 4). The resulting [2H,3H]
acetic acid (3.1 × 107 dpm, 20% radiochemical yield, 90%
radiochemical purity) was isolated by steam distillation and
analyzed by enzymatic chirality assay with malate synthase
and fumarase.23 The F values observed, 74.1 ( 2.1 and 74.7
( 1.6, establish R stereochemistry for the chiral acetate,
formation of (15S,16R)-[16-2H1,16-3H1] abietadiene (4) in
the enzymatic reaction, and overall retention of configuration
in the replacement of diphosphate of [2H,3H] 2 with
deuterium.
Because an anti SN′ cyclization of doubly labeled 2 would
generate a vinyl group bearing tritium in the 16E position
of the pimarenyl intermediate (e.g., 13+), the deuterium atom
from C17 and the methyl group from C13 both migrate to
the si,si face of the vinyl group of 3b, i.e., a formal
suprafacial relationship.24 This stereochemical finding is at
variance with either a concerted pericyclic mechanism
methyl copalate as shown in Scheme 3. Bromination of the
exocyclic double bond followed by diisobutylaluminum
(18) (a) Midland, M. M. Chem. ReV. 1989, 89, 1553. (b) Midland, M.
M.; Greer, S.; Tramantano, A.; Zderic, S. A. J. Am. Chem. Soc. 1979, 101,
2352. (c) Prabhakaran, P. C.; Gould, S. J.; Orr, G. R.; Coward, J. K. J. Am.
Chem. Soc. 1988, 110, 5779.
(19) (a) Midland, M. M.; Graham, R. S. Org. Synth., Coll. Vol. VIII,
1990, 402. (b) Krishnamurthy, S.; Brown, H. C. J. Org. Chem. 1977, 42,
1197.
(20) (a) Gerlach, H.; Zagalak, B. J. Chem. Soc., Chem. Commun. 1973,
274. (b) Gerlach, H.; Kappes, D.; Boeckman, R. K.; Maw, G. N. Org. Synth.,
Coll. Vol. IX, 1998, 151.
(21) (a) MacMillan, J.; Beale, M. H. In Isoprenoids Including Carotenoids
and Steroids; Cane, D. E., Ed.; Vol. 2 in ComprehensiVe Natural Products
Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Elsevier:
Oxford, 1999; Chapter 8 (b) Cane, D. E. Tetrahedron 1980, 36, 1109.
(22) Phillips, G. T.; Clifford, K. H. Eur. J. Biochem. 1976, 61, 271.
(23) (a) Floss, H. G.; Tsai, M. D. AdV. Enzymol. 1982, 50, 243. (b)
Unpublished protocols and calibration standards of (R)- and (S)-[2H,3H]
acetate were kindly provided by H. G. Floss and S. Lee, University of
Washington, Seattle, WA. The F values obtained for the chiral acetate
standards at the UI were as follows: R, 78.0 ( 3.6; S, 28.1 ( 3.6.
Org. Lett., Vol. 2, No. 5, 2000
575

predicted to be antarafacial on the basis of orbital symmetry
considerations11b or a synchronous proton addition to C16
and antiperiplanar methyl migration to C15. Consequently
a secondary sandaracopimaren-15-yl carbocation (14+)/
diphosphate anion pair, or alternatively a covalently bonded
pimarenyl-enzyme adduct,25,26 is implicated prior to the
methyl group rearrangement and proton elimination steps
leading to abietadiene.
The occurrence of bona fide secondary carbocation
intermediates in terpene synthase-catalyzed cyclizations and
rearrangements is rare, reflecting the higher energy of these
species. Examples are found in mechanistic schemes leading
to monoterpenes (bornyl diphosphate and fenchol synthases),27a
sesquiterpenes (pentalenene and trichodiene synthases),27b
tetracyclic diterpenes (e.g., ent-kaurene synthase),21a and
polycyclic triterpenes (squalene and oxido-squalene
synthases).27c,d However, in all of these cases the 15 kcal/
mol thermodynamic energy barrier between the secondary
ion28 and a preceding tertiary carbocation would be offset
by the substantial enthalpic gains associated with cyclizations
into CdC double bonds (∆HBE ≈ -20 kcal/mol) or with
relief of ring strain (bicyclo[3.1.1]heptane/bicyclo[2.2.1]-
heptane, ∆∆HSE ) -19 kcal/mol),29 together with the
stabilization arising from charge delocalization (bridged ions)
and concerted bond-forming reactions.
The thermodynamically uphill isomerization of the tertiary
pimarenyl ion 13+ to the apparently localized secondary ion
14+ in the absence of an exothermic counterbalance or the
possibility of a lower energy concerted mechanism is
unprecedented. Although the 15 kcal/mol thermodynamic
barrier between tertiary and secondary carbenium ions28
should be surmountable at room temperature, it nevertheless
seems likely that AS participates overtly in catalyzing the
process and lowering the thermodynamic deficit. Some
important factors to consider in the enzyme-induced desta-
bilization of 13+/OPP- and/or stabilization of 14+/OPP-
carbocation-diphosphate anions are ion pair distances and
forces,30 homoallyl interaction in 14+, π-complexation with
the aromatic rings of amino acid side chains at the active
site,31 interactions with proximal peptide carbonyl dipoles
or nucleophilic heteratoms,32 and electrostatic field gradients.
Interestingly, binding of a sandaracopimarenyl amine, mim-
icing the secondary carbocation intermediate, is greatly
enhanced (∼1000-fold) by the addition of inorganic diphos-
phate,24b suggesting that AS utilizes the diphosphate anion
to stabilize the secondary carbocation intermediate. The
X-ray crystallographic structure of rAS and its complexes
with pimarenyl ion mimic inhibitors may reveal what specific
interactions and mechanisms are involved in the remarkable
pimarenyl+-abietadienyl+ rearrangement.
(24) (a) The relatively efficient enzymatic cyclization of 8a and the
unproductive binding of its 8â-OH isomer (Ki ) 0.18 µM),24b as well as
precedent,21 suggest that the SN′ cyclization probably takes place on the
re,re(R) face of C17 to generate 13+ with a 14R deuterium substituent. (b)
Ravn, M. M.; Coates, R. M.; Peters, R. J.; Croteau, R., unpublished results.
(25) Cornforth, J. W. Angew. Chem., Int. Ed. Engl. 1968, 7, 903.
(26) Incubations of (13S,15S)-isopimar-8(14)-en-15-yl diphosphate and
a mixture of (13S,15S) and (13S,15R) isomers with rAS did not produce
detectable quantities of abietadiene. Thus, internal return of OPP- to form
a freely exchangeable covalent diphosphate intermediate appears to be
excluded.
(27) (a) Wise, M. L.; Croteau, R. In Isoprenoids Including Carotenoids
and Steroids; Cane, D. E., Ed.; Vol. 2 in ComprehensiVe Natural Products
Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Elsevier:
Oxford, 1999; Chapter 5. (b) Cane, D. E. In Isoprenoids Including
Carotenoids and Steroids; Cane, D. E., Ed.; Vol. 2 in ComprehensiVe
Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O.,
Eds.; Elsevier: Oxford, 1999; Chapter 6. (c) Abe, I.; Prestwich, G. D. In
Isoprenoids Including Carotenoids and Steroids; Cane, D. E., Ed.; Vol. 2
in ComprehensiVe Natural Products Chemistry; Barton, D., Nakanishi, K.,
Meth-Cohn, O., Eds.; Elsevier: Oxford, 1999; Chapter 10. [d] Poralla, K.
In Isoprenoids Including Carotenoids and Steroids; Cane, D. E., Ed.; Vol.
2 in ComprehensiVe Natural Products Chemistry; Barton, D., Nakanishi,
K., Meth-Cohn, O., Eds.; Elsevier: Oxford, 1999; Chapter 11.
(28) (a) Bittner, E. W.; Arnett, E. M.; Saunders, M. J. Am. Chem. Soc.
1976, 98, 2724. (b) Arnett, E. M.; Petro, C. J. Am. Chem. Soc. 1978, 100,
5408.
Acknowledgment. We thank the National Institutes of
Health for research grants GM 13956 (R.M.C.) and GM
31354 (R.B.C.) and Drs. H.G. Floss and S. Lee for authentic
standards of (R)- and (S)-[2H1,3H1] acetates and assistance
with the enzymatic chirality assays. R.M.C. thanks D.
Arigoni and F.-G. Kla¨rner for helpful discussions. R.J.P. is
a fellow of the Jane Coffin Childs Memorial Fund for
Medical Research, and this investigation has been supported
in part by the Jane Coffin Childs Memorial Fund for Medical
Research.
Supporting Information Available: Experimental pro-
cedures and full characterization data. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL991230P
(30) (a) Poulter, C. D. Acc. Chem. Res. 1990, 23, 70. (b) Poulter, C. D.;
Capson, T. L.; Thompson, M. D.; Bard, R. S. J. Am. Chem. Soc. 1989,
111, 3734.
(31) Dougherty, D. A. Science 1996, 271, 163.
(29) Liebman, J. F.; Greenberg, A. Chem. ReV. 1976, 76, 320.
(32) Jenson, C.; Jorgensen, W. L. J. Am. Chem. Soc. 1997, 119, 10846.
576
Org. Lett., Vol. 2, No. 5, 2000