From the
The biosynthesis of taxol (paclitaxel) and related taxoids in
Pacific yew ( Taxus brevifolia) is thought to involve the
cyclization of geranylgeranyl diphosphate to a taxadiene followed by
extensive oxygenation of this diterpene olefin intermediate. A
cell-free preparation from sapling yew stems catalyzed the conversion
of [1-
The highly functionalized diterpenoid taxol
(paclitaxel)
The biosynthesis of taxol 1 (Fig. S1) is presumed to involve cyclization of
geranylgeranyl diphosphate 3, via (1 S)-verticillene 4, to taxa-4
(20) ,11
(12) -diene 5 (20, 21, 22) since taxoids bearing the
4
(20) - and 11
(12) -pair of double bonds are very common
(23) . Such a cyclization to establish the taxane skeleton could
then be followed by oxidative elaboration of this diterpene olefin
intermediate
(22, 24) . Previous investigations on taxol
biosynthesis have involved feeding early precursors, such as labeled
acetate, to various yew tissue preparations
(25, 26) ,
or have focused on late stage modifications to the taxane nucleus, such
as the origin of appended acyl moieties
(27, 28) . No
experimental work on the enzymology of taxol biosynthesis has been
reported, and the first dedicated step of the pathway and the identity
of the presumed olefinic precursor of taxol have remained uncertain. In
this communication, we describe the enzymatic cyclization of
geranylgeranyl diphosphate to taxa-4
(5) ,11
(12) -diene as
the first committed reaction, and a very slow step, of taxol
biosynthesis.
Aliquots of the taxoid fraction were analyzed by HPLC on a 250 mm
NMR spectra were recorded at 499.8 MHz
(
Radio-GLC was performed on a Gow-Mac 550P
gas chromatograph coupled to a Packard 894 gas proportional counter
(37) using a 30 m
Pacific yew saplings were chosen as an experimental system
because the taxoid content of immature tissue is relatively high
(9) , sapling stems contain a high proportion of phloem
parenchyma cells in which taxol is thought to be produced
(25, 38, 39) , and saplings can be maintained in
a greenhouse to minimize the effects of environmental variation
(10, 40) . A soluble enzyme extract was prepared from
T. brevifolia stem sections by methods previously developed
for the isolation of other terpenoid cyclases from gymnosperm stem that
minimize the deleterious effects of co-extracted phenolic materials
(31) . Incubation of this preparation with 10 µ
M
[1-
To determine if this
presumptive diterpene olefin could serve as a precursor of taxol, the
purified biosynthetic product (5.36 µCi) was suspended in buffer
and vacuum-infiltrated into two batches of T. brevifolia stem
discs. Following incubation for 8 days, the labeled products were
extracted and separated into a taxoid fraction containing metabolites
more polar than (+)-taxusin (the tetraacetate of
taxa-4
(20) ,11
(12) -dien-5,9,10,13-tetraol). This
fraction represented about 30% incorporation of radioactivity from the
olefin and, when separated by reversed-phase radio-HPLC, revealed the
presence of at least a dozen labeled compounds of polarity between
10-deacetylbaccatin III (10-deacetyl 2,
R
To confirm
the identity of the labeled taxoids, 10-deacetylbaccatin III,
cephalomannine, and taxol (for which substantial amounts of authentic
standards were available) were isolated by TLC, diluted with the
corresponding unlabeled carrier, and crystallized to constant specific
activity and melting point, thereby verifying the incorporation of the
tritium-labeled olefin into 10-deacetylbaccatin III (4.1%),
cephalomannine (0.5%), and taxol (1.1%). (The incorporation percentages
should be regarded as minimum levels, as some tritium label (depending
on the kinetic isotope effect) will be lost on oxidation at C-2
(Fig. S1) of the taxane skeleton of these advanced metabolites.)
It is not uncommon for the level of 10-deacetylbaccatin III to exceed
the levels of taxol and cephalomannine in T. brevifolia stem
tissue
(40) . These results indicated that the diterpene olefin
product of the cell-free system did serve as a precursor of taxol and
other very closely related taxoids of yew stem, thus suggesting that
the cyclization product of geranylgeranyl diphosphate was a taxadiene.
To identify the biosynthetic diterpene olefin product, it was
necessary to acquire sufficient material for spectroscopic analysis. To
this end, the hydrocarbon fraction from an extract of 750 kg of dried
T. brevifolia bark powder was diluted with 0.5 µCi of the
enzymatically derived, tritium-labeled olefin, and the corresponding
product (
The structure of the diterpene olefin was
determined to be taxa-4
(5) ,11
(12) -diene 7 by
analysis of one- and two-dimensional
The
The
The resonances of the
C-3 and C-7 carbons showed a cross-peak with the proton singlet at
Following the interpretation of the TOCSY and DQF-COSY
spectra, the methylene CH
The
gem-dimethyl groups (CH
To confirm that the
biosynthetic product generated in yew stem extracts and the olefin
isolated from bark were the same, large scale enzyme incubations with
geranylgeranyl diphosphate were carried out and the olefin fraction was
analyzed by combined GLC-MS. The retention time and mass spectrum of
the enzymatic product (>99% pure) were identical to those of
taxa-4
(5) ,11
(12) -diene isolated from the bark extract
(Fig. 1, b and c). (1 S)-Verticillene 4, a possible enzyme-bound intermediate in the cyclization, was
not detectable as a product of the cell-free extract.
The next step of the pathway to taxol is presumed to
involve oxygenation of taxadiene
(22, 24) . The
observations that no oxygenated taxoids bearing the 4
(5) -double
bond have yet been reported, whereas taxoids with the exo-methylene at
the 4
(20) -position and that also bear an oxygen function at C-5
are exceedingly common
(23) , therefore suggest that
hydroxylation at C-5 of taxa-4
(5) ,11
(12) -diene, with
migration of the double bond, must occur as an early, if not the first,
oxygenation step of the pathway (Scheme 2). The transformation of
taxa-4
(5) ,11
(12) -diene 7 to
taxa-4
(20) ,11
(12) -diene-5-ol 8 would also set
the stage for the later elaboration of the unusual oxetane moiety 11, which is considered
(22, 24) to arise by
conversion of the 5-hydroxy-4
(20) -methylene 9 to the
corresponding epoxide function 10 followed by ring expansion
(Fig. S2).
We thank David Bailey (Hauser Chemical Research Inc.,
Boulder, CO) for the bark extract and authentic taxoid standards,
Nicholas Wheeler (Weyerhaeuser Research Center, Centralia, WA) for the
T. brevifolia saplings, and Gerald Pattenden (University of
Nottingham, Nottingham, United Kingdom) for the mass spectrum of
verticillene.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]geranylgeranyl diphosphate to a cyclic
diterpene olefin that, when incubated with stem sections, was converted
in good radiochemical yield to several highly functionalized taxanes,
including 10-deacetyl baccatin III and taxol itself. Addition of the
labeled olefin to a yew bark extract, followed by radiochemically
guided fractionation, provided sufficient product to establish the
structure as taxa-4
(5) ,11
(12) -diene by two-dimensional
NMR spectroscopic methods. Therefore, the first dedicated step in taxol
biosynthesis is the conversion of the universal diterpenoid precursor
geranylgeranyl diphosphate to taxa-4
(5) ,11
(12) -diene,
rather than to the 4
(20) ,11
(12) -diene isomer previously
suggested on the basis of the abundance of taxoids with double bonds in
these positions. The very common occurrence of taxane derivatives
bearing the 4
(20) -ene-5-oxy functional grouping, and the lack
of oxygenated derivatives bearing a 4
(5) -double bond, suggest
that hydroxylation at C-5 of taxadiene with allylic rearrangement of
the double bond is an early step in the conversion of this olefin
intermediate to taxol.
(
)1 (1) is of
interest because of its efficacy in the treatment of a range of cancers
(2) . A principal limitation to the wider therapeutic use of
taxol was the limited supply of the drug isolated from the bark of
Pacific yew ( Taxus brevifolia Nutt.:Taxaceae); the yields are
low (0.01-0.1% dry weight), the species is very slow growing and
sparsely distributed, and the harvest is destructive
(3, 4) . Alternate approaches for obtaining taxol
include isolation from the renewable foliage and other tissues of
plantation-grown Taxus species
(5, 6, 7, 8, 9, 10) ,
production in tissue culture systems
(11, 12) , and
semisynthesis of the drug and its analogs from baccatin III 2 and related late stage taxane diterpenoid (taxoid) metabolites that
are more readily available
(13, 14, 15, 16) . Total synthesis of
taxol, at present, is not commercially viable
(17) , and
efficient and economical supply of the drug must rely on biological
production systems for the foreseeable future
(18) . Therefore,
it is essential to understand the origin of taxol and related taxoids
in yew species from the universal precursor of plant diterpenoids,
geranylgeranyl diphosphate 3 (19) . To this end, we have
undertaken a systematic study of taxol biosynthesis at the enzyme
level, with the goal of targeting the slow steps of the pathway for
ultimate manipulation.
Figure S1:
Proposed cyclization of geranylgeranyl
diphosphate to taxadiene, and conversion of this intermediate to highly
functionalized taxoids. OPP represents the diphosphate
moiety.
Plants, Substrates, and Standards
Two-year-old
T. brevifolia saplings in active growth were obtained from the
Weyerhaeuser Research Center, Centralia, WA.
[1-H]Geranylgeranyl diphosphate (118 Ci/mol) was
prepared as described previously
(29) . Bark extracts and taxoid
standards (taxol, cephalomannine, and baccatin III, and their
10-deacetyl derivatives) were obtained from Hauser Chemical Research
Inc., Boulder, CO. (+)-Taxusin was isolated from the wood of
T. brevifolia (30) . Other diterpenoid standards were
from our own collection.
Cell-free Extracts
Methods previously developed
for the isolation and assay of terpenoid cyclases from conifer stem
tissue were employed
(29, 31) . Yew sapling stem
sections were frozen in liquid Nand pulverized in a hammer
mill to separate the bark and adhering tissue from the woody core. The
resulting ``bark'' powder was extracted with a buffer
containing vinylpyrrolidone polymers and polystyrene resin to adsorb
phenolics, and the 27,000
g supernatant was partially
purified by ion-exchange chromatography on DEAE-cellulose
(31) .
The preparation was then dialyzed to assay conditions (30 m
M
HEPES (pH 8.0), 5 m
M dithiothreitol, 10% glycerol) and
adjusted to
50 µg of protein/ml before incubation in the
presence of 10 µ
M geranylgeranyl diphosphate and 1
m
M MgCl
(29) . Following incubation
(1-3 h), the pentane-soluble products of the reaction mixture
were extracted and the extract passed through a short column of silica
gel to provide the hydrocarbon fraction. Quantification was by aliquot
counting, and the products were analyzed by radio-GLC.
(
)
To obtain sufficient amounts of the biosynthetic olefin for
use in in vivo studies and for GLC-MS analysis, the enzyme
preparation was scaled up by a factor of 10.
In Vivo Incorporation Experiments
The biosynthetic
olefin (5.36 µCi of H) that had been purified by CC
(silica gel; pentane) and argentation TLC (silica gel; 10%
AgNO
:pentane) was suspended by sonication in a buffer (1.5
ml) consisting of 5 m
M KH
PO
(pH 5.5),
10 m
M sucrose, 2 m
M KCl, 2 m
M phenylalanine,
1 m
M dithiothreitol, 1 m
M MgCl
, 1
m
M sodium acetate, 1 m
M sodium benzoate, 0.2
m
M EDTA, 0.1 m
M
Na
H
P
O
, and 0.05% Tween
20. Following filter sterilization, the suspension was split and
vacuum-infiltrated (six times) into two 1.75-g batches of yew stem
discs (1-2 mm thick) prepared from sapling stem sections that had
been surface-sterilized in 70% ethanol (1 s), followed by 3% sodium
hypochlorite, 0.5% Tween 20 (1 min) and washing, and then sectioned
using sterile technique. The tissue was incubated for 8 days at room
temperature, in air, in the light with slow shaking in a glass vial and
then thoroughly extracted with hexane (2
10 ml), which was
passed through a silica gel column that was rinsed with diethyl
ether:pentane (1:1) to remove residual substrate and labeled
metabolites less polar than (+)-taxusin. The column was then
rinsed with 10 ml of chloroform:acetonitrile:methanol (6:3:1), and this
material was used to extract the tissue, as before. The process was
repeated with three additional portions of
chloroform:acetonitrile:methanol, at which point a methanol extraction
afforded no additional extracted radioactivity. This taxoid fraction
was decolorized, without loss of tritium, by elution through Davisil
(Alltech C
on silica) with a gradient from 55% water, 25%
methanol, 20% acetonitrile to 100% acetonitrile. Approximately 90% of
the applied tracer was recovered in organic solvent-soluble products.
4.6-mm Econosil C8 column (5 µm, Alltech) by gradient
elution (30 ml) from solvent A (55% water, 25% methanol, 20% acetonite)
to 75% solvent A plus 25% solvent B (acetonitrile), while collecting
1-ml fractions for liquid scintillation counting. Retention times of
the standards were as follows: 10-deacetylbaccatin III (3.08 min),
baccatin III (5.24 min), 10-deacetylcephalomannine (20.48 min),
10-deacetyltaxol (22.99 min), cephalomannine (26.21 min), and taxol
(27.82 min). The taxoid fraction was subsequently diluted with 2 mg
each of 10-deacetylbaccatin III, cephalomannine, and taxol and
subjected to preparative TLC on silica gel G with chloroform:methanol
(20:1). The products (10-deacetylbaccatin III,
R
0.17; cephalomannine plus taxol,
R
0.47-0.51) were eluted with
methanol, diluted with an additional 200 mg of authentic carrier, and
crystallized from aqueous methanol to constant specific activity and
m.p. Because cephalomannine and taxol were not separable, the eluted
mixture was split in half; one-half was diluted with cephalomannine,
the other with taxol. Repeated crystallization gave 10-deacetylbaccatin
III, 150 nCi/mmol, m.p. 243-245 °C decomposes cephalomannine,
14.0 nCi/mmol, m.p. 183-186 °C decomposes taxol, 31.7
nCi/mmol, m.p. 215-218 °C decomposes. All m.p. values are in
agreement with the literature
(23) .
Product Isolation and Analysis
An extract from 750
kg of T. brevifolia bark powder, that had been enriched in
non-polar materials by partitioning from methanol into heptane, was
batch processed through silica gel with hexane to afford a hydrocarbon
fraction (3.1 g). This material was diluted with 0.5 µCi of the
enzymatically prepared olefin and chromatographed repeatedly on a
silica gel column with hexane, while monitoring fractions by liquid
scintillation counting and GLC-MS, to yield a fraction (230 mg; >90%
recovery of label) eluting between sandaracopimaradiene and
abietatriene. This material, a very complex mixture of sesquiterpene
and diterpene hydrocarbons, was next purified by argentation column
chromatography (10% AgNO-silica gel with a 0 to 10%
gradient of Et
O in pentane) to afford 72 mg of an oil.
Another passage through silica gel (with pentane) followed by
reversed-phase column chromatography on Davisil (Alltech C
on silica, 2.5-20% CCl
in acetonitrile) and
argentation TLC (10% AgNO
-silica gel with 5% ether in
pentane) yielded about 1 mg of 85% taxadiene ( R
0.8-0.9, with 2% mixed sesquiterpene olefins and 11%
abietatriene).
H, DQF-COSY, TOCSY) and at 125.7 MHz
(
C(
H), DEPT, HETCOR) on a Varian VXR-500S
instrument, and at 300.1 MHz (HMQC, HMBC) using a Bruker AMX-300
instrument. All experiments were run at ambient temperature (21 °C)
with a 5 µ
M solution in CDCl
. Chemical shifts
are reported in
(ppm) using tetramethylsilane as an internal
standard. The DQF-COSY
(32) and TOCSY
(33) experiments
(mixing time 60 ms) were run in the phase sensitive mode. The 512
t
increments of 96 scans each were sampled in 2000
data points for each of the 512 t
increments. Zero-filling
the F1 domain to 2000 and a Gaussian weighting function were applied in
both F
and F
domains prior to double Fourier
transformation. The HETCOR
(34) spectrum was obtained using
2000 data points in the F
domain and 256 t
increments (800 scans each), which were zero-filled to 1000 in
the F
domain. A sine-bell squared weighting function phase
shifted by
/2 was applied in both domains prior to double Fourier
transformation. The HMQC spectrum was measured employing the pulse
sequence of Bax et al. (35) . Delay
was set to
3.571 ms corresponding to the average one bond carbon-proton coupling
constant, 145 Hz. In this experiment, 256 t
increments were sampled in 2000 data points using 464 scans for
each of the t
increments. Data in the F
domain
were zero-filled to 1000, and the sine-bell squared weighting function
phase shifted by
/2 was applied in both F
and F
domains prior to double Fourier transformation. Analogous
parameters were adopted for the HMBC spectrum that was obtained using
the pulse sequence of Bax and Summers
(36) and involved
low-pass J-filtering to suppress correlations due to one-bond
couplings. Delay
was set to 62.5 ms corresponding to
the average long range (through two or three bonds) carbon-proton
coupling constant, 8 Hz.
0.53-mm diameter fused silica column
coated with a 1.2-µm film of Superox FA (Alltech); 8 p.s.i. He,
isothermal at 200 °C with injector and detector at 220 °C.
GLC-MS analysis was performed on a Hewlett-Packard 5840A/5985B system
using a 30 m
0.25-mm diameter fused silica column with
0.25-µm film of Superox FA (Alltech) operated at 10 p.s.i. H
and programmed from 100 °C (5-min hold) at 10 °C/min to
220 °C. Electron impact spectra were recorded at 70 eV.
H]geranylgeranyl diphosphate, in the presence
of 1 m
M MgCl
, yielded radiolabeled,
pentane-soluble products that were purified by column chromatography on
silica gel to afford the hydrocarbon fraction (1 nmol; 5% conversion in
1 h). The production of the hydrocarbon material from geranylgeranyl
diphosphate by the enzyme preparation was absolutely dependent on the
presence of the divalent metal ion, as expected for a terpenoid cyclase
(41) , and negligible activity was observed in thermally
inactivated control incubations. Radio-GLC analysis of the hydrocarbon
fraction revealed the presence of a single major component that eluted
between the tricyclic diterpene olefin standards sandaracopimaradiene
and abietatriene (Fig. 1 a).
3.08 min) and taxol 1 ( R
27.82 min), including the common
bark taxoids baccatin III 2, cephalomannine 6, and taxol,
and their 10-deacetyl derivatives
(40) (Scheme 1). Negligible
incorporation of olefin label into this taxoid fraction was observed in
control experiments with thermally inactivated tissue.
1 mg) was isolated by radiochemically guided
chromatographic fractionation. The mass spectrum of the product gave
principal ions at m/z 122 (100%), 107 (26%), 121 (25%), 123
(20%), and 105 (10%), and a parent ion at m/z 272 (1.5%)
consistent with a cyclic diterpene olefin of molecular formula
C
H
. Radio-GLC analysis of the isolated
product demonstrated coincidental elution of radioactivity and the
principal mass peak, as expected. Based on recovery, it was estimated
that the level of this olefin in bark tissue was in the 5-10
µg/kg range.
H and
C
NMR spectra. Proton connectivities were determined by DQF-COSY and
TOCSY experiments, and the signals of all carbons with directly
attached protons were assigned using HETCOR and HMQC spectra. Finally,
the HMBC spectrum was used to assign quaternary carbons and to check
the correctness of the connectivities established by the interpretation
of the other spectra.
C DEPT experiment revealed a
total of 20 carbon resonances (Table I), of which five corresponded to
methyl carbons, seven to methylene carbons, three to methine carbons,
and five to non-proton bearing carbons. One of the methine resonances
and three non-proton bearing carbon resonances occurred in the
downfield region: 120.0-140.0 ppm. All remaining carbon
resonances were in the high field region: 20.0-45.0 ppm.
H NMR spectrum () showed a one-proton multiplet
(
J = 11.1 Hz) at
5.28. All other signals
were in the region from 0.65 to 2.63 ppm. In the TOCSY spectrum, the
olefinic CH-5 (
5.28) proton multiplet exhibited cross-peaks to
four different sets of protons corresponding to the methylene
CH
-6 (
1.88, 2.01) and CH
-7 (
1.18,
1.67) protons, the methine CH-3 (
2.51) proton, and the methyl
CH
-20 (
1.68) protons. By analyzing the HETCOR
spectrum, the carbon signals at
24.0, 38.4, 39.7, and 121.1 were
assigned to C-20, C-7, C-3, and C-5, respectively; they correlated with
the corresponding proton signals (see above).
0.82 in the HMBC spectrum. Therefore, the proton signal was
assigned to the methyl CH
-19 protons. Additionally, this
proton resonance (
0.82) correlated with another two carbon
signals at
37.2 and 41.4, corresponding to the quaternary C-8 and
methylene CH
-9 carbons, respectively. That the signal at
37.2 belonged to a quaternary carbon, and the signal at
41.4 to a methylene carbon, was further supported by the DEPT data.
Analysis of the HMBC spectrum also allowed the carbon signal at
138.5 to be assigned to the olefinic C-4 carbon, as it exhibited a
cross-peak with the methyl CH
-20 proton signal (
1.68)
due to a two-bond linkage. The methylene CH
-9 proton
signals at
1.42 and 1.82 were identified based upon their
correlation with the C-9 carbon signal at
41.4 in the HETCOR
spectrum.
-2 and CH
-10 proton
signals were assigned. The CH-3 proton signal (
2.51) displayed
two cross-peaks at
1.57 and 1.66, corresponding to the methylene
CH
-2 protons. One of the CH
-9 proton signals
(
1.42) correlated with the signal at
2.15, and the other
signal (
1.82) showed a cross-peak at
2.61. Therefore, both
signals at
2.15 and 2.61 were assigned to the methylene
CH
-10 protons. Moreover, they exhibited a mutual cross-peak
due to geminal coupling. In addition, both correlated with the carbon
signal at
24.5 in the HETCOR spectrum. Consequently, the latter
was assigned to the C-10 carbon. The HETCOR analysis also allowed the
carbon signal at
28.4 to be assigned to the C-2 carbon.
-16, CH
-17)
attached to the quaternary C-15 carbon of the taxadiene A-ring were
easily identified in the HMBC spectrum. Both methyl proton resonances
at
1.01 (CH
-16) and 1.33 (CH
-17)
displayed cross-peaks with the same carbon signals at
44.2,
137.7, and 39.0, corresponding to the carbons C-1, C-11, and C-15,
respectively. Furthermore, the CH
-16 proton resonance
correlated with the carbon CH
-17 signal at
26.3
(assigned using HETCOR analysis) and, due to the same three-bond
coupling, the proton signal of the methyl CH
-17 correlated
with the carbon CH
-16 resonance at
30.7 (assignment
based on HETCOR analysis). HMBC analysis also revealed the correlation
of the carbon C-11 resonance with the proton singlet at
1.66. At
the same time, this proton resonance exhibited cross-peaks with the
carbon signals at
29.8 and 129.5. These facts allowed assignment
of the proton resonance at
1.66 to the methyl CH
-18
protons; the carbon signals were assigned as the methylene
CH
-13 carbon (
29.8) and the olefinic carbon C-12
(
129.5). Having established the carbon resonances of the methine
CH-1 and methylene CH
-13, the HETCOR analysis permitted
assignment of the proton signals of the groups at
1.74 (CH-1),
and at
1.88 and 2.08 (CH
-13). Both the methylene
CH
-13 proton resonances, as well as the methine CH-1 proton
resonance, exhibited cross-peaks at
1.59 and 2.08 in the DQF-COSY
spectrum. At the same time, these two proton signals (
1.59, 2.08)
showed a mutual cross-peak. Therefore, they were assigned to the
methylene CH
-14 protons. Only one of the CH
-14
proton resonances displayed a cross-peak with the carbon signal at
22.6 in the HETCOR spectrum. However, this signal (
22.6)
corresponded to a methylene carbon based upon the DEPT data and was
assigned to C-14. The remaining unassigned methylene carbon resonance
at
23.2 was then assigned to the methylene CH
-6, even
though no cross-peak between protons and carbon of the CH
-6
group could be identified in the HETCOR and HMQC spectra. The
assignment of each carbon and hydrogen of the C
H
olefin left little doubt that the product was
taxa-4
(5) ,11
(20) -diene 7.
Figure 1:
Chromatographic analysis of taxadiene.
a, radio-GLC analysis of the diterpene olefin fraction
generated from [1-H]geranylgeranyl diphosphate by
the cell-free enzyme preparation from sapling yew stems. The arrows indicate the positions of the retention markers
sandaracopimaradiene (10.8 min) and abietatriene (21.0 min).
b, capillary GLC-MS analysis of an olefin fraction from yew
bark enriched in taxa-4(5),11(12)-diene (*), and of the diterpene
olefin fraction ( c) generated from geranylgeranyl diphosphate
by the cell-free enzyme preparation from sapling yew stems. The
biosynthetic olefin appears to be >99% pure, and its retention time
(26.800 ± 0.005 min) and mass spectrum are identical to those of
taxa-4(5),11(12)-diene isolated from yew
bark.
The
cyclization of geranylgeranyl diphosphate to
taxa-4
(5) ,11
(12) -diene, as the first dedicated step in
the biosynthesis of taxol and related metabolites, is consistent with
earlier suggestions that the pathway involves preliminary formation of
a parent taxane olefin followed by oxidative modification
(20, 21, 22) . However, the identification of
taxa-4
(5) ,11
(12) -diene 7 as the olefinic
intermediate, rather than taxa-4
(20) ,11
(12) -diene 5 as originally proposed on the basis of metabolite co-occurrence
(22) , was unexpected. Consequently, the previously proposed
cyclization to 5 (20, 21) (Fig. S1) can be
reformulated as involving ionization of the geranylgeranyl diphosphate
ester with closure of the first ring and deprotonation to afford
(1 S)-verticillene. Protonation at C-7 can then initiate
transannular cyclization to generate the taxenyl cation, which upon
deprotonation at C-5 yields the endocyclic double bond of the taxadiene
product.
Figure S2:
Proposed conversion of
taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5-ol, and
transformation of the 4(20)-ene-5-oxy functional grouping to the
corresponding oxirane and oxetane groups.
Although the enzyme that catalyzes the
transformation of geranylgeranyl diphosphate to taxadiene has not yet
been characterized in any detail, the low cyclization activity in
T. brevifolia stem extracts relative to that of other
diterpene cyclases of gymnosperm stem tissue
(29) , coupled to
the very low levels of this key olefin intermediate in the bark,
suggest that the cyclization step to generate the taxane skeleton is
very slow (relative to subsequent oxygenations) and, thus, is an
important target for manipulation. With the initial step of taxol
biosynthesis in Pacific yew now defined, it would be of interest to
determine if the taxol-producing fungus Taxomyces andreanae (42) employs a similar cyclization reaction.
Table: H and
C NMR
chemical shifts
(ppm) for taxa-4(5),11(12)-diene measured in
CDCl
solution at 293 K with tetramethylsilane as internal
standard
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.