Stereochemistry of the macrocyclization and elimination steps in taxadiene biosynthesis through deuterium labeling

Article abstract of DOI:10.1021/jo0502091

Taxadiene synthase catalyzes the cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) to taxa-4(5),11(12)-diene (Scheme 1, 5 → 2) as the first committed step of Taxol biosynthesis. Deuterated GGPPs labeled stereospecifically at C-1, C-4, and C-16 were

Full text of DOI:10.1021/jo0502091

Stereochemistry of the Macrocyclization and Elimination Steps in
Taxadiene Biosynthesis through Deuterium Labeling
Qingwu Jin,† David C. Williams,‡ Mehri Hezari,‡ Rodney Croteau,‡ and Robert M. Coates*,†
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, and
The Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
coates@scs.uiuc.edu
Received February 1, 2005
Taxadiene synthase catalyzes the cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) to taxa-
4(5),11(12)-diene (Scheme 1, 5 f 2) as the first committed step of Taxol biosynthesis. Deuterated
GGPPs labeled stereospecifically at C-1, C-4, and C-16 were synthesized and incubated with
recombinant taxadiene synthase from Taxus brevifolia to elucidate the stereochemistry of the
cyclization reaction at these positions. The deuterium-labeled taxadienes obtained from (R)-[1-
2H1]-, (S)-[1-2H1]-, and [16,16,16-2H3]GGPPs (9, 10, and 23b) were established to have deuterium
1
in the 2R and 2â CH2 and 16CH3 positions, respectively, by high-field H NMR spectroscopy (eqs
1-3). Incubation of (R)-[4-2H1]GGPP (17) with the recombinant enzyme gave a 10:10:80 mixture of
[5â-2H1]taxa-3(4),11(12)-diene, [5â-2H1]taxa-4(20),11(12)-diene, and unlabeled taxa-4(5),11(12)-diene
according to GC/MS analyses of the products (eq 4). It follows that C-1 of GGPP underwent inversion
of configuration, that the A ring cyclization occurs on the si face of C15, and that the terminating
proton abstraction removes H5â from the final taxenyl carbocation intermediate. Thus, the
C1-C14 and C15-C10 bonds are formed on the opposite faces of the 14,15 double bond of the
substrate, i.e., overall anti electrophilic addition. The implications of these findings for the
mechanism of the cyclization and rearrangement are discussed.
Introduction
tion developed by Bristol-Myers-Squibb involves harvest-
ing 10-deacetylbaccatin III and attaching the side chain
chemically.7a Since 2002, production by BMS has switched
Taxol (1) is an established anticancer drug with annual
sales exceeding one billion US dollars. The use of this
diterpene alkaloid for treatment of ovarian and breast
cancer was approved by the FDA in the early 1990s.1
Despite considerable effort in the synthetic community
and completion of total syntheses by several labora-
tories,2-5 the commercial production by purely chemi-
cal means is not viable owing to the length and low
overall yields of these schemes.6 Semisynthetic produc-
(3) (a) Holton, R. A.; Somoza, C.; Kim, H.-B.; Liang, F.; Biediger, R.
J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.;
Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile,
L. N.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1597. (b) Holton, R. A.;
Kim, H.-B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boatman, P. D.;
Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.;
Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. N.; Liu, J. H. J.
Am. Chem. Soc. 1994, 116, 1599.
(4) (a) Danishefsky, S. J.; Masters, J. J.; Young, W. B.; Link, J. T.;
Snyder, L. B.; Magee, T. V.; Jung, D. K.; Issacs, R. C. A.; Bornmann,
W. G.; Alaimo, C. A.; Coburn, C. A.; Grandi, M. J. D. J. Am. Chem.
Soc. 1996, 118, 2843. (b) Magee, T. V.; Bornmann, W. G.; Isaacs, R. C.
A.; Danishefsky, S. J. J. Org. Chem. 1992, 57, 3274.
* To whom correspondence should be addressed. Tel: (217) 333-
4280. Fax (217) 244-8024.
† University of Illinois.
‡ Washington State University.
(5) (a) Wender, P. A.; Badham, N. F.; Conway, S. P.; Floreancig, P.
E.; Glass, T. E.; Gra¨nicher, C.; Houze, J. B.; Ja¨nichen, J.; Lee, D.;
Marquess, D. G.; McGrane, P. L.; Meng, W.; Mucciaro, T. P.; Mu¨hle-
bach, M.; Natchus, M. G.; Paulsen, H.; Rawlins, D. B.; Satkofsky, J.;
Shuker, A. J.; Sutton, J. C.; Taylor, R. E.; Tomooka, K. J. Am. Chem.
Soc. 1997, 119, 2755-2756. (b) Wender, P. A.; Badham, N. F.; Conway,
S. P.; Floreancig, P. E.; Glass, T. E.; Houze, J. B.; Krauss, N. E.; Lee,
D.; Marquess, D. G.; McGrane, P. L.; Meng, W.; Natchus, M. G.;
Shuker, A. J.; Sutton, J. C.; Taylor, R. E. J. Am. Chem. Soc. 1997,
119, 2757-2758.
(1) (a) Holms, F. A.; Kudelka, A. P.; Kavanogh, J. J.; Huber, M. H.;
Ajani, J. A.; Valero, V. In Taxane Anticancer Agents: Basic Science
and Current Status; George, G. I., Chen, T. T., Ojima, I., Vyas, D. M.,
Eds.; American Chemical Society: Washington DC, 1995; pp 31-57.
(b) Arbuck, S. G.; Blaylock, B. A. In Taxol: Science and Applications;
Suffness, M., Ed.; CRC Press: Boca Raton, FL, 1995; pp 379-415. (c)
Pezzuto, J. Nat. Biotechnol. 1996, 14, 1083.
(2) Nicolau, K. C.; Yang. Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.;
Guy, R. K.; Clairborne, C. F.; Renaud, J.; Coluadourous, E. A.;
Paulvannan, K.; Sorensen, E. J. Nature 1994, 367, 630.
(6) Borman, S. Chem. Eng. News 1994, 72, 32.
10.1021/jo0502091 CCC: $30.25 © 2005 American Chemical Society
Published on Web 05/12/2005
J. Org. Chem. 2005, 70, 4667-4675
4667

Jin et al.
to a plant cell culture-based method, but most generic
Taxol suppliers still rely on semisynthetic methods.7b
a dependence upon a divalent metal ion for activity. The
gene for this novel cyclase has been cloned and overex-
pressed in E. coli.14,15 Taxadiene synthase shares more
gene sequence homology with abietadiene synthase from
grand fir16,17 than it does with casbene synthase from
castor beans,18 despite the very different polyene cycliza-
tion effected by the former and the similar macrocycliza-
tion catalyzed by the latter, indicating that phylogeny is
of greater significance than mechanistic similarity.19
The mechanism proposed for the cyclization is shown
in Scheme 1.16,20,21 GC/MS analysis of [2H1]taxadiene
arising from incubation of [10-2H1]GGPP with taxadiene
synthase established that the deuterium is transferred
from C11 to the C ring of the product.8 NMR data
demonstrated that this deuterium is located at C-7 on
the R-face, and a mechanism involving an intramolecular
11Rf7R proton transfer was advanced.21,22 Incubations
of the related macrocyclic diterpenes, cembrene A (3) and
verticillene (4), with taxadiene synthase did not produce
taxa-4(5),11(12)-diene.8 However, it remains a possibility
that partially cyclized intermediates are tightly bound
in the active site and have no opportunity to exchange
with exogenous additives during the catalytic cycle. An
alternative mechanism in which the C-ring is formed first
was ruled out by the lack of incorporation of deuterium
label from 2,7-cyclo-GGPP-d2.15,23 Recent labeling results
support a similar macrocyclization via the same verti-
cillen-12-yl intermediate (6) in the biosynthesis of the
diterpene precursor to phomactin.24
Considerable progress has been made in characterizing
the initial and final stages of Taxol biosynthesis. The first
and possibly rate-limiting step is the cyclization of
(E,E,E)-geranylgeranyl diphosphate (GGPP, 5) to the
parent tricyclic diterpene, taxa-4(5),11(12)-diene (2,
Scheme 1).8 This complex reaction is catalyzed by the
SCHEME 1
Several stereochemical issues are presented by the
mechanism shown in Scheme 1, i.e., inversion or reten-
tion of configuration at C1 of GGPP, re vs si face attack
at C15 of GGPP, H11R vs H11â stereochemistry of the
verticillen-12-yl ion intermediate 6, and HR vs Hâ
elimination in the formation of the 4,5 double bond. Both
inversion and retention have been observed at the
diphosphate-bearing carbon in the cyclizations catalyzed
by monoterpene and sesquiterpene synthases.25 Retentive
stereochemistry is attributed to prior allylic rearrange-
ment to transient tertiary diphosphate intermediates
that undergo anti SN′ cyclizations. This paper reports the
elucidation of three of these stereochemical unknowns
by means of deuterium labeled substrates. A separate
enzyme taxadiene synthase, the mechanism of which will
be discussed in this and a closely related paper submitted
separately.9 The structure of the cyclization product was
confirmed by total synthesis of (()-taxadiene.10 Microso-
mal preparations from Taxus brevifolia catalyze the
conversion of taxadiene to taxa-4(20),11(12)-dien-5R-ol
with migration of the C-ring double bond into the
exocyclic position, a structural characteristic of many
taxane diterpenes.11
(14) Wildung, M. R.; Croteau, R. J. Biol. Chem. 1996, 271, 9201.
(15) Williams, D. C.; Wildung, M. R.; Jin, Q.; Dalal, D.; Oliver, J.
S.; Coates, R. M.; Croteau, R. Arch. Biochem. Biophys. 2000, 379, 137.
(16) MacMillan, J. A. In Comprehensive Natural Products Chemis-
try; Barton, D., Nakanishi, K., Eds.; Elsevier: Amsterdam, 1999; Vol.
2 (Cane, D. E., Ed.), Chapter 8.
Taxadiene synthase isolated from T. brevifolia12,13 has
a molecular weight of 79 000, an optimal pH of 8.5, and
(17) (a) Ravn, M. M.; Coates, R. M.; Jetter, R.; Croteau, R. B. Chem.
Commun. 1998, 21. (b) Ravn, M. M.; Coates, R. M.; Flory, J. E.; Peters,
R. J.; Croteau, R. B. Org. Lett. 2000, 2, 573. (c) Peters, R. J.; Ravn, M.
M.; Coates, R. M.; Croteau, R. B. J. Am. Chem. Soc. 2001, 123, 8974.
(18) (a) Robinson, D. R.; West, C. A. Biochemistry 1970, 9, 80. (b)
Guilford, W. J.; Coates, R. M. J. Am. Chem. Soc. 1982, 104, 3506.
(19) Trapp, S. C.; Croteau, R. Genetics 2001, 158, 811.
(20) (a) Harrison, J. W.; Scrowton, R. M.; Lythgoe, B. J. Chem. Soc.
C 1966, 1933. (b) Gueritte-Voegelein, F.; Guenard, D.; Potier, P. J.
Nat. Prod. 1987, 50, 9.
(7) (a) Commercon, A.; Bourzat, J. D.; Didier, E.; Lavelle, F. In
Taxane Anticancer Agents: Basic Science and Current Status; George
G. I., Chen, T. T., Ojima, I., Vyas, D. M., Eds.; American Chemical
Society: Washington DC, 1995; pp 233-246. (b) Ritter, S. K. Chem.
Eng. News 2004, 82, 28.
(8) Lin, X.; Hezari, M.; Koepp, A. E.; Floss, H. G.; Croteau, R.
Biochemistry 1996, 35, 2968.
(9) The stereochemistry of the transient verticillen-12-yl ion inter-
mediate was inferred to have the bridgehead hydrogens in a trans 1âH/
11RH relationship by TS-catalyzed cyclization of 6-fluoroGGPP to
7-fluoroverticilla-3(4),7(8),11(12)-triene. See: Jin, Y.; Williams, D. C.;
Croteau, R.; Coates, R. M. J. Am. Chem. Soc. 2005, 127, 7834.
(10) Rubenstein, S. M.; Williams, R. M. J. Org. Chem. 1995, 60,
7215.
(11) Hefner, J.; Rubenstein, S. M.; Ketchum, R. E. B.; Gibson, D.
M.; Williams, R. M.; Croteau, R. Chem. Biol. 1996, 3, 479.
(12) Hezari, M.; Lewis, N. G.; Croteau, R. Arch. Biochem. Biophys.
1995, 322, 437.
(13) LaFever, R. E.; Stofer Vogel, B.; Croteau, R. Arch. Biochem.
Biophys. 1994, 313, 139.
(21) Williams, D. C.; Carroll, B.; Jin, Q.; Rithner, C.; Lenger, S. R.;
Floss, H.; Coates, R. M.; Williams, R. M.; Croteau, R. Chem. Biol. 2000,
7, 969.
(22) Intramolecular proton transfers have been observed in cycliza-
tions brought about by abietadiene and pentalenene synthases. See
refs 17 and: Cane, D. E.; Weiner, S. E. Can. J. Chem. 1994, 72, 188.
(23) Coates, R. M.; Jin, A. Q. J. Org. Chem. 1997, 62, 7475.
(24) Tokiwano, T.; Fukushi, E.; Endo, T.; Oikawa, H. Chem. Com-
mun. 2004, 1324.
(25) (a) Croteau, R. Chem. Rev. 1987, 87, 929. (b) Cane, D. E. Chem.
Rev. 1990, 90, 1089.
4668 J. Org. Chem., Vol. 70, No. 12, 2005

Stereochemistry of Taxadiene Biosynthesis
SCHEME 2
alpine borane (THF, 25 °C) gave the chiral alcohol (1R)-
[1-2H1]-4-benzyloxy-2-methyl-2E-buten-1-ol (13a, 96%).
The enantiomeric purity was determined to be 95R/5S
1
by means of H NMR analysis of the camphanate ester
derivative.27,31 Reaction of the labeled alcohol with meth-
anesulfonyl chloride (Et3N, CH2Cl2, 0 °C) provided me-
sylate 13b (97%) for the alkylation reaction.
The key coupling reaction was carried out by lithiation
of (E,E)-farnesyl phenyl sulfone (14)32 (1 equiv of nBuLi,
3:1 THF-HMPA, -78 and -23 °C, 10 and 60 min)
followed by alkylation of the sulfonyl-stabilized carbanion
with mesylate 13b (1 equiv, -78 and -23 °C, 1 and 2 h).
The resulting sulfone ether (15, 50%) was reduced with
Li/EtNH2 (4:3 ether-EtNH2, -78 °C) to cleave both the
allylic C-S bond and the benzyl ether. (R)-[4-2H1]-
Geranylgeraniol (16, 29%) was purified by chromatog-
raphy on 15% AgNO3-silica gel to remove a small
amount of the 5,6 double-bond isomer (∼7:1 6,7-5,6
isomer ratio) formed in the reaction. The enantiomeric
purity of (R)-[4-2H1]geranylgeraniol was determined by
asymmetric epoxidation of the 2,3-double bond (Ti(OiPr)4,
D-(-)-diethyl tartrate, tBuOOH, CH2Cl2, -20 °C).33 2H
NMR analysis (77 MHz, C6H6) of the resulting (2R,3R,4R)-
[1-2H1] epoxide (18) showed an 8:1 ratio for the two
diastereomers (δD 1.35 major and 1.51 minor) which
corresponds to 91:9 ratio of 4R and 4S enantiomers after
allowance for the expected 95:5 enantioselectivity of the
asymmetric epoxidation. The labeled alcohol 16 was
converted to (R)-[4-2H1]GGPP (17) by activation as the
chloride (MsCl, LiCl, collidine, DMF, 0 °C)34 and allylic
displacement (Bu4NHOPP, CH3CN, 25 °C, 30%) followed
by ion exchange to the NH4 salt and cellulose chroma-
tography.35
GGPP labeled with deuterium in the trans terminal
methyl group (23b) was accessed by regioselective cleav-
age of the 14,15 double bond of (E,E,E)-geranylgeranyl
benzyl ether36,37 and E-selective Wittig olefination. The
polyene ether was converted to the trisnoraldehyde (19)
in four steps (38% overall yield): (a) terminal hypobro-
mination38 with NBS in aqueous THF (4:7 H2O/THF, 0
°C, 2.5 h, 62%), (b) epoxide formation (KOH pellets, ether,
25 °C, 78%),39 (c) hydrolysis to the 14,15 diol (HClO4, 2:1
THF/H2O, 25 °C, 82%), and (d) periodate cleavage (NaIO4,
1:1 THF/H2O, 25 °C, 96%).38 Wittig reaction of the
aldehyde with ethyl 2-(triphenylphosphoranylidene)-
propionate (1.5 equiv, CH2Cl2, 25 °C, 98%) gave the R,â-
unsaturated ester 20 with an E/Z selectivity of 97:3
publication deals with the C11 stereochemistry of the
verticillenyl intermediate in the cyclization.9
Synthesis of Deuterium-Labeled GGPPs. Sub-
strates labeled with deuterium in the 1HR/1HS, 4HR/4HS,
and 16(E-CH3)/17(Z-CH3) positions were required to
elucidate the cryptic stereochemistry occurring at C1, C4,
and C15 in the cyclization mechanism. Both (R)- and (S)-
[1-2H1]GGPPs (9 and 10, see eqs 1 and 2) were prepared
by reduction of the 1-deuterio aldehyde precursor, (E,E,E)-
geranylgeranial, with (R)- and (S)-alpine boranes, con-
version to diethyl phosphates, and SN2 displacements
with tris(tetrabutylammonium) diphosphate.26 The enan-
tiopurities were assumed to be the same as those of (R)-
and (S)-[1-2H1]geranyl PP (98-99%) prepared in exactly
the same way from deuterium-labeled geraniols. Thus,
enzymatic hydrolysis of (R)-[1-2H1]geranyl PP with bacte-
rial alkaline phosphatase, derivatization of (R)-[1-2H1]-
geraniol with (1S)-camphanoyl chloride, and 1H NMR
analysis indicated the diasteromeric purity to be
99:1.26, 27
(R)-[4-2H1]Geranylgeraniol (16) was synthesized by
means of a C15 + C5 Biellmann coupling approach
outlined in Scheme 2.11,28 Reduction of ethyl 4-benzyloxy-
2-methyl-2E-butenoate (11)29 with LiAlD4 (ether, 0 °C,
77%) followed by Swern oxidation (DMSO, (COCl)2, Et3N,
CH2Cl2, -78 °C)30 afforded the corresponding deuterio
aldehyde (12, 92%). Enantioselective reduction with (S)-
(30) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651.
(31) Lampe, D.; Mills, S. J.; Potter, B. V. L. J. Chem. Soc., Perkin
Trans. 1 1992, 2899.
(32) (a) Eren, D.; Keinan, E. J. Am. Chem. Soc. 1988, 110, 4356. (b)
Guertin, K. R.; Kende, A. S. Tetrahedron Lett. 1993, 34, 5369. (c) Torii,
S.; Uneyama, K.; Matsunami, S. J. Org. Chem. 1980, 45, 16.
(33) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974.
(34) Collington, E. W.; Meyers, A. I. J. Org. Chem. 1971, 36, 3044.
(35) Woodside, A. B.; Huang, Z.; Poulter, C. D. Organic Syntheses;
Wiley: New York, 1993; Collect. Vol. VIII, p 616.
(26) Ravn, M. M.; Jin, Q.; Coates, R. M. Eur. J. Org. Chem. 2000,
1401.
(27) (a) Gerlach, H.; Zagalak, B. J. Chem. Soc. Chem. Commun.
1973, 274. (b) Gerlach, H.; Kappes, D.; Boeckman, R. K., Jr.; Maw, G.
N. Org. Synth. 1998, Coll. Vol. 9, 151.
(36) Coates, R. M.; Ley, D. A.; Cavender, P. L. J. Org. Chem. 1978,
43, 4915.
(28) (S)-[4-2H1]Geraniol has been prepared by an asymmetric epox-
idation-deuteride reduction approach and (S)-[4-2H1]farnesyl PP
was obtained by prenyl transferase coupling of (E)-[4-2H1]isopentenyl
and geranyl PPs: (a) Pyun, H.-J.; Waschal, K. C.; Jung, D.-I.; Coates,
R. M.; Croteau, R. Arch. Biochem. Biophys. 1994, 308, 488. (b) Cane,
D. E.; McIlwaine, D. B.; Oliver, J. S. J. Am. Chem. Soc. 1990, 112,
1285.
(37) Yanagisawa, A.; Hibino, H.; Habaue, S.; Hisada, Y.; Yasue, K.;
Yamamoto, H. Bull. Chem. Soc. Jpn. 1995, 68, 1263.
(38) (a) Van Tamelen, E. E.; Curphey, T. J. Tetrahedron Lett. 1962,
3, 121. (b) Van Tamelen, E. E.; Nadeau, R. G. Bioorg. Chem. 1982, 11,
197. (c) Grigoreva, N. Y.; Avrutov, I. M.; Semenovskii, A. V.; Odinokov,
V. N.; Akhunova, V. R.; Tolstikov, G. A. Akad. Nauk. Sci. Bull. (Engl.)
1979, 28, 353.
(29) Marshall, J. A.; Trometer, J. D.; Blough, B. E.; Crute, T. D. J.
Org. Chem. 1988, 53, 4274.
(39) Clarke, H. T.; Hartman, W. W. Organic Syntheses, 2nd ed.;
Wiley: New York, 1964; Collect. Vol. I, p 233.
J. Org. Chem, Vol. 70, No. 12, 2005 4669

Jin et al.
SCHEME 3
assignments in C6D6 (Table 1) correspond well with those
previously reported41 in CDCl3, the maximum deviations
1
being 0.15 ppm for H NMR and 0.7 ppm for 13C NMR.
HMBC, HMQC, TOCSY, and NOE spectral correla-
tions were critical in securing unambiguous NMR as-
signments for taxadiene. The HMBC long-range corre-
lation grid shows cross-peaks between both methyl
resonances at δH1.09 and 1.32 ppm and the vinylic carbon
at δC137.5 ppm. This indicates that the 13C peak at δC
137.5 can be assigned to C11. These methyl resonances
were also correlated with peaks at δC 38.9 and 44.2 ppm
on the HMBC plot. According to the DEPT spectra, the
signal at 44.2 ppm is a methine and the peak at 38.9 ppm
is a quaternary carbon. Thus, these two peaks in the 13
C
NMR spectrum were assigned to C1 and C15, respec-
tively, and the signals at δH 1.09 and 1.32 belong to the
geminal methyl groups (C16 and C17).
The NOE data in Table 2 allowed stereochemical
assignments to be made. The CH3 group at δH 0.93 (C19)
showed a small positive NOE (0.7%) upon irradiation at
1.32 ppm. This allows assignment of the 1.32 and 1.09
ppm peaks to the C17 and C16 geminal methyl groups,
respectively. These assignments are supported by NOEs
between the C16 CH3 (δH 1.09) and both H13â (δH 2.24,
2.1%) and H14â (δH 2.02, 1.2%) on the A ring. The C-5
vinyl proton clearly belongs to the peak at δH 5.38 ppm,
and by HMQC correlation, its 13C resonance was assigned
to 121.3 ppm. The C5 carbon resonance is also correlated
by HMBC to the C-20 methyl group at δH 1.73 ppm. The
peaks at 137.5 and 129.3 ppm were related to the C-18
vinyl methyl peak at 1.60 ppm. In the COSY spectrum,
the vicinal protons at δH 1.94 and 2.11 ppm for C-6
showed cross-peaks with the vinyl proton at 5.38 ppm.
The C5 proton is correlated on the HMBC map with the
C7 carbon peak at 38.5 ppm, which in turn is matched
with vicinal proton peaks at δH 1.19 and 1.74 ppm in the
HMQC plot. Irradiation of the C19 methyl peak resulted
in a positive NOE (1.1%) at 1.19 ppm, identifying the 7â
proton.
Rigorous assignments for H2R and H2â were compli-
cated by the proximity of their signals (δH 1.62 and 1.72)
to each other and to that for H1 (δH 1.66) in the proton
spectrum and the extensive overlap in this region.
However, NOEs observed at δH 1.72 (1.8 and 2.1%) after
irradiation at the C17 and C19 methyl signals enabled
attribution of the higher field resonance to H2â.
An examination of Dreiding models shows the proxim-
ity of the C16 and C19 methyl groups, and the resulting
steric repulsion is a serious limiting factor for the
conformations available to the molecule. According to the
coupling constants between the H1, H2R, H2â, and H3
protons (Table 1), the following dihedral angles can be
deduced:43 1/2R, 36°; 1/2â, 58°; 3/2R, 112°; 3/2â, 132°. The
7R proton shows two additional couplings of 5.9 Hz
besides the geminal coupling, and the 7â proton shows
one coupling of 6.2 Hz. The dihedral angels between these
protons and the C6 protons can be deduced from the
coupling constants:43 6R/7â, 90°; 6â/7â, 28°; 6R/7R, 32°;
6â/7R, 143°. The C ring needs to adopt a pseudo-chair
conformation to fulfill these dihedral angles.
(Scheme 3). The identification of the trans and cis isomers
was based on the H NMR chemical shifts for the C14
1
vinyl protons (δH 6.74 E isomer, 5.90 Z isomer) and
comparisons with literature data for similar compounds.36
Reduction of ester 20 with LiAlD4 (Et2O, 25 °C, 2 h)
afforded the dideuterated alcohol (21, 82%). The trans
CD2OH group was converted to CD3 by the mesylation
(MeSO2Cl, Et3N, CH2Cl2, -20 °C, 30 min), and reductive
displacement with LiBEt3D (1.0 M in THF, 20 equiv, -20
°C, 45 min, 82%) in the same solution afforded labeled
ether 22.40 Reductive cleavage of the benzyl protecting
group by lithium in NH3-THF (-78 °C, 30 min) gave
[16,16,16-2H3]geranylgeraniol (23a, 96%).36 The localiza-
tion of the deuterium predominantly in the trans termi-
nal methyl group was verified by the absence of the
corresponding signals in the 1H and 13C NMR spectra (δH
1.72 and δC 25.6 absent). The allylic alcohol was con-
verted to [16,16,16-2H3]GGPP (23b) following known
procedures.34,35
1H NMR Assignments for Taxadiene in C6D6 1H
and 13C NMR assignments for taxadiene in CDCl3 were
previously reported for the product formed enzymati-
cally41 and for synthetic (()-taxadiene.10 The proton
resonances arising from the C-ring hydrogens, specifically
H5, H6R and H6â, H7R and H7â, and the H19 methyl
protons, were assigned by Floss and Rithner using 1D
DPFGSC-TOCSY and NOESY NMR techniques.21
Halsall reported the 13C NMR spectra for a series of
4(20),11-taxadiene derivatives.42
In the present work, a typical amount of taxadiene
obtained from an enzyme incubation was 50-100 µg. At
this scale, the presence of a small amount of HCl from
air oxidation of CDCl3 might jeopardize the integrity of
the sample and the quality of the spectra. Accordingly,
we decided to obtain independent assignments in C6D6,
in which solvent the microgram samples of taxadiene
were expected to be more stable. 1H and 13C NMR spectra
were obtained with synthetic (()-taxa-4(5),11(12)-diene
kindly provided by R. M. Williams.10 The data and
The 13â, 14R, and 14â protons show vicinal coupling
(40) Andres, H.; Morimoto, H.; Williams, P. G. J. Chem. Soc., Chem.
Commun. 1990, 627.
(41) Koepp, A. E.; Hezari, M.; Zajicek, J.; Vogel, B. S.; LaFever, R.
E.; Lewis, N. G.; Croteau, R. J. Biol. Chem. 1995, 270, 8686.
(42) Halsall, T. G. Org. Magn. Reson. 1983, 21, 524.
constants greater than 10 Hz. Thus, the conformation of
(43) Karplus, M. J. Chem. Phys. 1959, 30, 11.
4670 J. Org. Chem., Vol. 70, No. 12, 2005

Stereochemistry of Taxadiene Biosynthesis
TABLE 1. 600 MHz 1H and 150 MHz 13C NMR Data and Assignments for (()-Taxa-4(5),11(12)-diene (2) in C6D6
C no.
δC
δH
m
J (Hz)
7.4, 6.8
C no.
δC
δH
m
J (Hz)
1
2R
44.2
1.66
1.62
dd
ddd
11
12
137.5
129.3
15.7, 5.2, 2.1
28.4
2â
1.72
2.62
ddd
15.5, 6.4, 1.9
13R
1.79
29.6
3
4
39.8
138.1
13â
14R
2.24
1.58
br t
ddd
14.7
14.7, 10.6, 4.3
23.3
38.9
5
121.3
22.7
5.38
1.94
m
14â
2.02
dddd
14.9, 12.1, 8.8, 3.1
6R
br d
17.4
15
6â
7R
2.11
1.76
m
16
17
30.6
26.1
1.09
1.32
s
s
38.5
37.3
41.4
7â
8
1.19
1.39
18
19
20
21.2
21.5
23.9
1.60
0.93
1.73
s
s
s
9R
dt
14.9, 5.9
9â
10R
1.81
2.15
ddd
15.0, 10.0, 5.1
24.5
10â
2.63
ddd
15.0, 10.4, 5.4
TABLE 2. NOE Enhancements in the 600 MHz 1H NMR Spectrum of (()-Taxa-4(5),11(12)-diene (2) in C6D6 after
Irradiations at 0.93, 1.09, and 1.32 ppm
irradiation at 16 CH3 (1.09 ppm)
irradiation at 17 CH3 (1.32 ppm)
irradiation at 19 CH3 (0.93 ppm)
proton
δH
NOE (%)
proton
δH
NOE (%)
proton
δH
NOE (%)
17CH3
1CH
14â
1.32
1.66
2.02
2.24
2.62
1.6
1.6
1.2
2.1
0.5
19CH3
16CH3
1CH
2â
0.93
1.09
1.66
1.72
1.81
2.15
2.62
0.7
1.7
1.1
1.8
1.9
1.5
0.1
7â
1.19
1.32
1.39
1.72
1.81
2.11
2.62
1.1
0.7
0.7
2.1
1.2
2.2
0.5
17CH3
9R
13â
2â
3 CH
9â
9â
10R
3CH
6â
3CH
the A ring is likely a twisted pseudo-boat form to match
these coupling constants. These conclusions about the
conformation of taxa-4(5),11(12)-diene are in agreement
with the X-ray crystal structure and data published by
Williams and Rubenstein for the related synthesis of
20-nor-4,5-dihydrotaxen-20-one.10
Incubations of Labeled GGPPs with Taxadiene
Synthase. Because the full-length taxadiene synthase
cDNA is translated as a catalytically impaired prepro-
tein,14 a series of N-terminally truncated enzymes was
generated by expression in E. coli of the corresponding
5′-truncated cDNAs from a suitable vector.15 A pseudoma-
ture recombinant form of taxadiene synthase having 60
amino acids deleted from the preprotein was found to be
most efficient, with kinetics comparable to those of the
mature native enzyme.15 Incubations were performed
under established assay conditions in the presence of 1
mM MgCl2, the labeled GGPPs, and a small amount of
[1-3H]GGPP. The resulting nonpolar products were ex-
tracted into pentane, and the solvent was removed with
a nitrogen stream before GC and GC-MS analyses. Typi-
cal yields were ∼6%. Enzymatic cyclization with unla-
beled subtrate afforded a 5:95 mixture of exocyclic and
endocyclic taxadiene isomers according to GC analyses.
The location and configuration of the deuterium in the
[2-2H1]taxadiene epimers formed from (R)- and (S)-[1-2H1]-
GGPPs (9 and 10) were elucidated by 1D TOCSY 1H
NMR spectra (Figure 1 and the Supporting Information).
The presence of one deuterium in both [2-2H1]taxadiene
samples is apparent from their nearly identical GC-MS
spectra (Supporting Information) that show M+ at m/z
273 and the most intense fragment ion at m/z 123, both
J. Org. Chem, Vol. 70, No. 12, 2005 4671

Jin et al.
FIGURE 2. 1H NMR spectra (600 MHz, C6D6, 15°C) of
unlabeled (()-taxadiene (2) (a) and [2H3]taxadiene biosynthe-
sized from [16,16,16-2H3]GGPP (23b) (b). The positional
numbers are shown above the five methyl signals in panel a.
The signal at 1.09 (16 CH3) is absent in panel b. The extra
peak at 1.36 in the lower spectrum is from an impurity.
occurs mainly by means of the geminal H2â-H2R coupling
interaction, owing to the small magnitude of the direct
vicinal coupling interaction, i.e., J3-2R. ) 2.1 Hz. The
presence of deuterium at H2â would block the H2â-H2R
spin transmission path. The presence of hydrogen in the
2R position of the sample was confirmed by the observa-
tion of an NOE at 1.59 upon irradiation at 1.73 (C20
CH3). The puckered conformation of the B ring in
taxadiene brings the C-ring vinyl methyl (C20) into close
proximity with H2R. We conclude that enzyme-catalyzed
cyclization of (S)-[1-2H1]GGPP gave rise to [2â-2H1]-
taxadiene (eq 2).
FIGURE 1. 1D TOCSY NMR spectra (750 MHz, C6D6, 15 °C)
of taxadienes generated by irradiations at 2.62 ppm: (a)
unlabeled taxadiene; (b) [2-2H1]taxadiene from (R)-[1-2H1]-
GGPP (9); (c) [2-2H1]taxadiene from (S)-[1-2H1]GGPP (10).
increased by 1 amu compared to those in the GC-MS of
unlabeled 2. The TOCSY pulse sequence was employed
to eliminate peak overlap in the region of interest, i.e.,
1.62 (H2R) and 1.72 (H2â), which coincided with, or
overlapped, peaks for H1 (1.66), H20 CH3 (1.73), H7R
(1.76), and H9â (1.81). The TOCSY spectrum of unlabeled
taxadiene following irradiation at 2.62 (H3 and H10â)
led to five peaks at 1.39 (H9R), 1.62 (H2R), 1.72 (H2â),
1.81 (H9â), and 2.15 (H10R) (Figure 1a). The TOCSY
spectrum (2.62 irradiation) of the labeled product from
(R)-[1-2H1]GGPP (9) displays a strong peak at 1.69 and
no detectable peak in the vicinity of 1.62 (Figure 1b). The
small upfield displacement (∆δ -0.03 ppm) is typical for
a geminal deuterium isotope shift.44 This spectrum
clearly locates the deuterium in the 2R position (eq 1).
1
The H NMR spectrum of the taxadiene product from
incubation of [16,16,16-2H3]GGPP (23b) showed a neg-
ligible peak at 1.09 ppm and a normal sized peak at 1.32
ppm (Figure 2). This clear-cut result proves that the C16
methyl group of the [2H3]taxadiene product is deuterated
(eq 3).
In contrast, the TOCSY spectrum of the [2-2H1]-
taxadiene from (S)-[1-2H1]GGPP (10) generated by the
same method showed no peaks in the same region. The
absence of a TOCSY signal for H2R can be explained by
the assumption that the spin propagation from H3 to H2R
GC-MS analysis of the incubation products from (R)-
[4-2H1]GGPP (17) showed three peaks in a 10:10:80 area
ratio with retention times of 10.29, 10.49, and 10.70 min
(MS, M+ 273, 273, and 272, respectively). The second
and third peaks in retention time order were identi-
(44) Bovey, F. A. Nuclear Magnetic Resonance Spectroscopy, 2nd ed.;
Academic Press: San Diego, 1988; p 141.
4672 J. Org. Chem., Vol. 70, No. 12, 2005

Stereochemistry of Taxadiene Biosynthesis
SCHEME 4
SCHEME 5
fied as [5-2H1]taxa-4(20),11(12)-diene (25) and unlabeled
4(5),11(12)-taxadiene (2) by GC and MS comparisons with
synthetic samples (eq 4).10 The first component eluted
was assigned tentatively as [5-2H1]taxa-3(4),11(12)-diene
(24), from its molecular ion peak in the MS and the
absence of the usual C-ring fragment ion.41 This product
was barely detectable (∼1%) in the mixture from unla-
beled substrate while the proportion of the exocyclic
isomer 25 was typically 4-5%. Presumably, the primary
kinetic isotope associated with D5â elimination from the
taxen-4-yl ion to form the 4,5 double-bond isomer magni-
fies the extent of competition of the minor pathways (H3R
and H20 eliminations) that gives rise to the minor
taxadiene isomers.15 These results confirm that proton
abstraction occurs predominantly on the â-face of C5 of
taxen-4-yl ion.15,21
literature with trans (1H-11H) hydrogens on the six-
membered A ring. In the case of verticillol,45 the trans
bridging was firmly established by means of an X-ray
crystal structure determination.45c,47 The same holds for
phomactatriene, the rearranged verticillane diterpene
derived from the 1â,11R-trans-verticillen-12-yl ion (6A)
by vicinal 11RH f 12RH and 15âMe f 11âMe rear-
rangements.24 Recent results from incorporation of la-
beled acetate into phomacatriene point to an initiating
macrocyclization similar to that shown in Scheme 4. The
parallel finding in this laboratory that taxadiene syn-
thase-catalyzed cyclization of 6-fluoroGGPP to predomi-
nantly 7-fluoroverticilla-3,7,12(18)-triene with trans
H1â/H11R bridgehead configurations affords compelling
evidence in favor of the trans-bridged verticillen-12-yl+
ion intermediate 6A.9
The six-membered A ring of the initially formed trans-
bridged verticillen-12-yl positive ion 6A must undergo
conformational inversion to enable formation of the C
ring (Scheme 5). The intramolecular transfer of the C11
bridgehead proton to C7 shown has been verified by
deuterium labeling.8,21 Molecular modeling indicates that
a direct intramolecular 11R f 7R proton transfer is
sterically and energetically favorable.21 Alternatively an
indirect process mediated by an active-site residue would
appear feasible. Similar nonexchanging proton translo-
cations take place in cyclization mechanisms producing
the sesquiterpene pentalenene22 and the diterpene abi-
etadiene.17 In the case of pentalenene biosynthesis, it was
postulated that a histidine residue was responsible for
carrying out a vicinal deprotonation-reprotonation.48
However, site-directed mutagenesis showed that this
enzyme base is not essential for catalytic activity.49
Discussion
The known 1âH stereochemistry of taxadiene and the
new finding that the trans terminal CH3 of GGPP
becomes the C16 methyl in the tricyclic product (eq 3)
require that the C1-C14 and C15-C10 bonds are formed
on the opposite faces of the 14,15 double bond (14 re, 15
si) of GGPP, corresponding to an anti addition. The
observed inversion of configuration at CH2OPP (eqs 1 and
2) indicates the likelihood of a stereoelectronically favor-
able antiperiplanar macrocyclization and renders un-
likely the possibility of allylic rearrangement followed by
SN′ cyclization. A boat or chair folding of the terminal
diene of GGPP would allow antiperiplanar alignment of
C1 with the 14,15 and 10,11 double bonds (Scheme 4,
5A vs 5B). Addition to the C11-C12 double bond
might occur on the re-re or si-si face to generate trans
(H1â-H11R) or cis (H1â-H11â) bridged verticillen-12-
yl ion intermediates (6A vs 6B).
(45) (a) Kaneko, C.; Hayashi, S.; Ishhikawa, M. Chem. Pharm. Bull.
1964, 12, 1510. (b) Erdtman, H.; Norin, T.; Sumimoto, M.; Morrison,
A. Tetrahedron Lett. 1964, 51, 3879. (c) Karlsson, B.; Pilotti, A.-M.;
So¨derholm, A.-C.; Norin, T.; Sundin, S.; Sumimoto, M. Tetrahedron
1978, 34, 2349.
(46) (a) Harrison, L. J.; Tori, M.; Taira, Z.; Asakawa, Y. The 28th
Symposium on the Chemistry of Terpenes, Essential Oils, and Aromat-
ics; Kanasawa, Japan, 1984; p 285. (b) Nagashima, F.; Toyota, M.;
Asakawa, Y. Phytochemistry 1990, 29, 2169. (c) Asakawa, Y. In
Progress in the Chemistry of Natural Products; Herz, W., Kirby, G.
W., Moore, R. E., Steglich, W., Tamm, Ch., Eds); Springer: Vienna,
1995; Vol. 65, p 1. (d) Nagashima, F.; Tamada, A.; Fujii, N.; Asakawa,
Y. Phytochemistry 1997, 46, 1203. (e) Basar, S.; Koch, A.; Konig, W.
A. Flavour Frag. J. 2001, 16, 315. (f) Duh, C.-Y.; El-Gamal, Ali Ali H.;
Wang, S.-K.; Dai, C.-F. J. Nat. Prod. 2002, 65, 1429.
All known natural products belonging to the bio-
genetically related verticillane group of diterpenes45,46
and retaining both bridgehead protons are shown in the
(47) The absolute configuration of (+)-verticillol has been revised
as H1â/H11R (1S,11S) through anomalous dispersion analysis in the
X-ray crystallographic determination of its p-iodobenzoate derivative.9
J. Org. Chem, Vol. 70, No. 12, 2005 4673

Jin et al.
+
SCHEME 6
11 f 7R H transfer, cyclization onto the 3,4 double
bond, and terminating elimination of the 5â hydrogen
from a twist-boat conformation of a taxa-11-en-4-yl ion
intermediate complete the complex tricyclization mech-
anism.
Experimental Section
Representative preparative procedures and characterization
data for (R)-[4-2H1]GGPP (17) and [16,16,16-2H3]GGPP (23b),
and for the enzymatic incubation experiments, are given below.
General experimental aspects, procedures, and characteriza-
tion data for the other compounds and NMR spectra and
GC-MS traces of taxadiene and deuterium-labeled taxadienes
are available in the Supporting Information.
(4S,E,E,E)-[4-2H1]5-Benzenesulfonyl-1-benzyloxy-3,7,-
11,15-tetramethyl-2,6,10,14-hexadecatetraene (15). The
procedure of Yee and Coates for a different compound was
followed.50 A patent by Kuraray Co. mentioned the preparation
of the same compound in unlabeled form in their synthesis of
all-trans-polyprenols.51 A solution of sulfone 14 (412 mg, 1.19
mmol) in HMPA (2 mL) and THF (6 mL) was stirred and cooled
at -78 °C as n-BuLi in hexane (0.76 mL, 1.56 M, 1.19 mmol)
was added dropwise. After 10 min at - 78 °C and 1 h at - 23
°C, the solution was cooled back to -78 °C. A solution of
mesylate 13b (322 mg, 1.19 mmol) in THF (3 mL) was added
dropwise, stirring was continued for 1 h at -78 °C and 2 h at
-23 °C, and MeOH (10 drops) was added. The yellow solution
was allowed to warm to 25 °C, and H2O (30 mL) and 1 M HCl
(2 mL) were added. The product was extracted with hexane
(4 × 30 mL), and the combined organic extracts were washed
with H2O (3 × 50 mL), dried (Na2SO4), and evaporated to give
a yellow oil (760 mg). Purification by flash chromatography
(15% EtOAc in hexane) provided 312 mg (50%) of sulfone 15:
Rf 0.44 (30% EtOAc in hexane); 1H NMR (CDCl3, 400 MHz) δ
1.55 (s, 3 H, CH3), 1.56 (d, 3 H, J ) 1.0 Hz, CH3), 1.57 (s, 3 H,
CH3), 1.59 (s, 3 H, CH3), 1.67 (d, 3 H, J ) 1.0 Hz, CH3), 1.89-
2.09 (m, 9 H, CH2), 3.91 (m, 1 H, CH), 3.96 (t, 2 H, J ) 6.5 Hz,
CH2), 4.43 (s, 2 H, CH2), 4.93 (d, 1 H, J ) 10.5 Hz, vinyl H),
5.02 (m, 1 H, vinyl H), 5.07 (t of quintets, 1 H, J ) 6.8, 1.5 Hz,
vinyl H), 5.39 (t, 1 H, J ) 6.7 Hz, vinyl H), 7.25-7.88 (m, 10
H, ArH); 13C NMR (CDCl3, 101 MHz) δ 15.8, 16.3, 17.6, 25.6,
26.2, 26.6, 39.56, 39.60, 63.03, 63.05, 66.0, 71.6, 116.81, 116.84,
123.2, 124.1, 124.66, 124.70, 127.43, 127.56, 128.2, 128.6,
129.1, 133.3, 135.5, 137.6, 138.2, 145.4.
Inversion of the cyclohexene ring to a half-boat form and
rotations of the nonenyl chain (7A f 7B) are required
in order to bring the carbocation site into position for
cyclization onto the re-re face of the 3(4) double bond to
generate the taxen-4-yl carbocation, 7B f 8A.
The loss of deuterium from (R)-[4-2H1]GGPP in the
final step of the mechanism indicates that proton elimi-
nation forming taxa-4(5),11(12)-diene occurs exclusively
on the â-face (Scheme 6). The C ring of the tax-11-en-4-
yl+ ion presumably adopts a twist-boat conformation
(8Af 8B) in order for a stereoelectronically favorable
elimination of a ψ-axial 5â proton to occur. It is not clear
why the ostensibly higher energy elimination of the 5â
proton predominates, other than enforced proximity to
an active site base. The elimination of H3R in the
formation of the tetrasubstituted isomer 24-d1 indicates
access of a proton acceptor on the opposite face. The
increased proportions of the 3(4) and 4(20) double bond
isomers (24 and 25) of taxadiene reflect the operation of
a substantial primary kinetic isotope effect.15, 21
(4R,E,E,E)-[4-2H1]-3,7,11,15-Tetramethyl-2,6,10,14-hexa-
decatetraen-1-ol (16). The procedure of Yee and Coates for
a different compound was followed.50 To a solution of sulfone
15 (266 mg, 0.51 mmol) in Et2O (6 mL) at -78 °C was added
EtNH2 (8 mL) through Tygon tubing by evaporation from the
metal container (40 °C) and condensation in the reaction flask
(-78 °C). Lithium pieces (141 mg, 20.4 mmol) were added, and
the suspension was stirred at -78 °C until the blue color
persisted. 1-Hexyne (1 mL) was added, the Li pieces were
removed, and the yellow color was quenched by addition of
MeOH (1 mL). The solution was allowed to warm to 25 °C,
and the solvents were evaporated. The residue was partitioned
between H2O (20 mL) and ether (20 mL), and the aqueous
layer was extracted with ether (3 × 25 mL). The combined
organic extracts were washed with 1% HCl (1 × 40 mL), satd
NaCl (1 × 40 mL), satd NaHCO3 (1 × 40 mL), and satd NaCl
(1 × 40 mL). The ethereal solution was dried (Na2SO4) and
evaporated to give a yellow oil (106 mg). Purification by flash
chromatography (15% AgNO3 in SiO2, 10% EtOAc in hexane
to 50% EtOAc in hexane) gave (4R,E,E,E)-[4-2H1]GGOH (16,
43 mg, 29%). (The AgNO3-impregnated silica gel was prepared
as follows: To 85 g of SiO2 and 15 g of AgNO3 was added 50
mL of CH3CN. After the mixture was stirred for 30 min at 25
°C, the solvent was removed under reduced pressure until the
Conclusions
The labeling results reported herein indicate that the
initiating macrocyclization step in taxadiene biosynthesis
occurs by a stereoelectronically favorable alignment of
CH2OPP with the 14,15 and 10,11 double bonds of the
GGPP substrate. The potentially concerted alkylation/
π-cyclization proceeds directly to a verticillen-12-yl car-
bocation intermediate having either a trans (1â,11R) or
cis (1â,11â) stereochemical relationship of the bridgehead
hydrogens (6A vs 6B). The taxadiene synthase-catalyzed
conversion of 6-fluoroGGPP to 7-fluoroverticilla-3,7,12-
(18)-triene with trans bridgehead stereochemistry and
other evidence strongly favor 1,11-trans verticillenyl ion
6A.9 Inversion of the A ring followed by direct or indirect
(48) (a) Cane, D. E.; Tillman, A. M. J. Am. Chem. Soc. 1983, 105,
122. (b) Cane, D. E.; Abell, C.; Tillman, A. M. Bioorg. Chem. 1984, 12,
312. (c) Lesburg, C. A.; Zhai, G.; Cane, D. E.; Christianson, D. W.
Science 1997, 277, 1820.
(49) Seemann, M.; Zhai, G.; Umezawa, K.; Cane, D. E. J. Am. Chem.
Soc. 1999, 121, 591.
(50) Yee, N. K. N.; Coates, R. M. J. Org. Chem. 1992, 57, 4598.
4674 J. Org. Chem., Vol. 70, No. 12, 2005

Stereochemistry of Taxadiene Biosynthesis
weight was constant.) The 1H NMR spectrum of 16 was
identical with that of unlabeled GGOH except that the CH2
peaks at 1.94-2.13 ppm showed one less proton upon integra-
tion: 1H NMR (CDCl3, 400 MHz) δ 1.13 (s, 1 H, OH), 1.60 (s,
9 H, CH3), 1.68 (s, 6 H, CH3), 1.94-2.13 (m, 11 H, CH2), 4.15
(dd, 2 H, J ) 5.9, 3.2 Hz, CH2), 5.11 (m, 3 H, vinyl H), 5.42 (t
of quintets, 1 H, J ) 6.8, 1.2 Hz, vinyl H).
5.42 (t of sextets, 1 H, J ) 6.6, 1.2 Hz, vinyl H), 7.26-7.38 (m,
5 H, aryl H); 13C NMR (CDCl3, 101 MHz) δ 15.87, 15.90, 16.4,
17.5, 26.2, 26.5, 26.6, 39.50, 39.57, 39.59, 66.4, 71.8, 120.7,
123.7, 124.1, 124.3, 127.4, 127.7, 128.2, 131.0, 134.8, 135.2,
138.5, 140.3.
(E,E,E,E)-[16,16,16-2H3]-3,7,11,15-Tetramethyl-2,6,10,14-
hexadecatetraen-1-ol (23a). The deprotection was carried
out according to Coates’ procedure.50 To a solution of 22 (82
mg, 0.21 mmol) in THF (6 mL) and NH3 (9 mL) at - 78 °C
was added Li (30 mg, 0.43 mmol). After 30 min at - 78 °C,
3-hexyne (1 mL) was added, and after 5 min, satd NH4Cl (5
mL) was added slowly. The white suspension was allowed to
warm to rt, and satd NH4Cl (15 mL) was added. The aqueous
layer was extracted with hexane (4 × 20 mL), and the
combined organic extracts were washed with satd NaCl
(1 × 25 mL). Drying (Na2SO4) and evaporation gave a light
yellow oil (67 mg), which was purified by flash chromatogra-
phy (20% EtOAc in hexane) to give (E,E,E,E)-[16,16,16-2H3]-
geranylgeraniol (60 mg, 96%) as a yellow oil. The 1H NMR
spectrum was identical to that of unlabeled GGOH except that
the integration of the singlet at 1.68 ppm changed from 6 to 3
H: 1H NMR (CDCl3, 500 MHz) δ 1.60 (s, 9 H, CH3), 1.68 (s, 3
H, CH3), 1.95-2.14 (m, 12 H, CH2), 4.15 (d, 2 H, J ) 7.0 Hz,
CH2), 5.11 (m, 3 H, vinyl H), 5.42 (td, 1 H, J ) 7.0, 1.1 Hz,
vinyl H).
(4R,E,E,E)-[4-2H1]-3,7,11,15-Tetramethyl-2,6,10,14-hexa-
decatetraen-1-yl Diphosphate, Triammonium Salt (17).
The diphosphorylation was carried out according to the
procedure reported by Poulter.35 To a solution of LiCl (22 mg,
0.52 mmol), collidine (112 mg, 0.93 mmol), and alcohol 16 (30
mg, 0.10 mmol) in DMF (4 mL) at 0 °C was added CH3SO2Cl
(71 mg, 0.62 mmol). After 1 h at 0 °C, ice-water (20 mL) was
added, and the product was extracted with cold pentane (3 ×
20 mL). The combined organic extracts were washed with satd
Cu(NO3)2 (5 × 20 mL), satd NaCl (2 × 20 mL), and satd
NaHCO3 (1 × 20 mL). Drying (Na2SO4) and evaporation gave
the chloride as a yellow oil (32 mg, quantitative). The chloride
(32 mg, 0.10 mmol) was dissolved in CH3CN (1 mL), and
powdered 3 Å molecular sieves (200 mg) and HOPP (NBu4)3
(136 mg, 0.14 mmol) were added. After 16 h at 25 °C, CH3CN
(30 mL) was added, and the solids were filtered. The filtrate
was washed with hexane (4 × 10 mL), and the acetonitrile
layer was evaporated to give a brown oil (186 mg). The oil was
dissolved in ion exchange buffer (2 mL) and loaded onto an
ion-exchange column (6 mL ion-exchange resin). Elution with
ion-exchange buffer (12 mL) and lyophilization gave a yellow
solid, which was purified by cellulose chromatography (10 mL
cellulose, cellulose buffer) to give 15.3 mg (30%) of the labeled
diphosphate 17 as a white solid: Rf 0.35 (cellulose TLC, 2:1:1
(E,E,E,E)-[16,16,16-2H3]-3,7,11,15-Tetramethyl-2,6,10,14-
hexadecatetraen-1-yl Diphosphate, Triammonium Salt
(23b). Diphosphorylation was carried out as described above
for 17. Alcohol 23a (26 mg, 0.088 mmol) gave 8.7 mg (20%) of
23b, Rf 0.34 (cellulose TLC, cellulose buffer). The 1H NMR
spectrum was identical to that of unlabeled GGPP except that
the CH3 resonance at 1.48 ppm was absent: 1H NMR (D2O,
400 MHz) δ 1.39 (s, 3 H, CH3), 1.42 (s, 3 H, CH3), 1.52 (s, 3 H,
CH3), 1.75-1.95 (m, 12 H, CH2), 4.26 (t, 2 H, J ) 6.1 Hz,
CH2O), 4.89-5.00 (m, 3 H, vinyl H), 5.25 (t, 1 H, J ) 6.8 Hz,
vinyl H); 31P NMR (D2O, 162 MHz) δ -9.5 (d, J ) 20.8 Hz),
-5.7 (d, J ) 20.8 Hz).
Preparative Incubations with Recombinant Taxadi-
ene Synthase. The truncated (M60) version of recombinant
taxadiene synthase, from which the plastidial transit peptide
had been deleted, was overexpressed in E. coli and purified
(>96%) as previously described.15 Preparative incubations were
carried out in 3 mL of the standard assay buffer (25 mM
Hepes, pH 8.0, containing 10% (v/v) glycerol) in the presence
of 1 mM MgCl2 and saturating levels of deuterated GGPP plus
a trace amount of [1-3H]GGPP (106 dpm) in order to monitor
the conversion easily. Up to 1 mg of protein was employed in
each incubation for 8-12 h at 31 °C, which afforded pentane-
soluble product yields in the 5-10% range.
1
2-propanol/acetonitrile/0.1 M NH4HCO3). The H NMR spec-
trum was the same as the unlabeled GGPP except that
integration for the CH2 peaks at 1.76-1.98 ppm changed from
12 to 11 H: 1H NMR (D2O, 500 MHz) δ 1.41 (s, 6 H, CH3),
1.43 (s, 3 H, CH3), 1.48 (s, 3 H, CH3), 1.53 (s, 3 H, CH3), 1.76-
1.98 (m, 11 H, CH2), 4.28 (t, 2 H, J ) 6.2 Hz, CH2), 4.97 (m, 3
H, vinyl H), 5.26 (t, 1 H, J ) 6.7 Hz, vinyl H); 31P NMR (D2O,
162 MHz) δ -9.54 (d, J ) 20.8 Hz), -5.72 (d, J ) 20.8 Hz).
(E,E,E,E)-[16,16,16-2H3]-1-Benzyloxy-3,7,11,15-tetra-
methyl-2,6,10,14-hexadecatetraene (22). The mesylation
followed Woggon’s procedure,52 and the reduction followed
Williams’ procedure.40 To a solution of alcohol 21 (104 mg, 0.26
mmol) and Et3N (106 mg, 1.04 mmol) in CH2Cl2 (6 mL) at -
20 °C was added CH3SO2Cl (39 mg, 0.34 mmol). After 30 min
at - 20 °C, LiBEt3D (5.2 mL, 1.0 M in THF, 5.2 mmol) was
added dropwise. After 45 min at - 20 °C, the reaction was
quenched by slow addition of H2O (20 mL). The aqueous layer
was extracted with hexane (3 × 20 mL), and the combined
organic extracts were washed with 1% HCl (2 × 30 mL), satd
NaHCO3 (1 × 30 mL), and satd NaCl (1 × 30 mL). Drying
(Na2SO4) and evaporation gave a colorless oil (160 mg), which
was purified by flash chromatography (30% CH2Cl2 in pentane)
Acknowledgment. This work was supported by
National Institutes of Health Grants GM 13956 to
R.M.C. and GM 31354 to R.C. We would like to thank
Dr. Feng Lin of the University of Illinois for assistance
with acquiring the NMR data and Yinghua Jin for
proofreading the manuscript drafts. We are grateful to
Prof. Robert M. Williams of Colorado State University
for providing us with synthetic (()-taxadiene.
1
to give 22 (82 mg, 82%) as a pale yellow oil. The H and 13C
NMR spectra were identical to the unlabeled compound except
that the peak at 1.72 ppm (d, 3 H, J ) 1.0 Hz, CH3) was absent
1
in the H NMR spectrum and the 25.6 ppm peak was absent
in the 13C NMR spectrum. Data for 22: Rf 0.40 (35% CH2Cl2
1
in pentane); H NMR (CDCl3, 400 MHz) δ 1.61 (s, 9 H, CH3),
1.66 (s, 3 H, CH3), 1.95-2.17 (m, 12 H, CH2), 4.04 (d, 2 H, J )
6.6 Hz, CH2), 4.51 (s, 2 H, CH2Ph), 5.12 (m, 3 H, vinyl H),
Supporting Information Available: General experimen-
tal aspects, preparative procedures, and chracterization data
for compounds not given in the Experimental Section and other
NMR figures and spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
(51) Asanuma, G.; Tamai, Y.; Kanehira, K. Eur. Pat. Appl. EP
771778, 1997.
(52) Fretz, H.; Woggon, W. D.; Voges, R. Helv. Chim. Acta 1989,
72, 391.
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