Studies on taxadiene synthase: interception of the cyclization cascade at the verticillene stage and rearrangement to phomactatriene

Article abstract of DOI:10.1016/j.tet.2007.03.029

The cyclization of geranylgeranyl diphosphate (GGDP) to taxadiene catalyzed by taxadiene synthase has been suggested to proceed in stages, involving a transient bicyclic verticillyl carbocation intermediate, which also has been proposed in the biosyntheti

Full text of DOI:10.1016/j.tet.2007.03.029

Tetrahedron 63 (2007) 6204–6209
Studies on taxadiene synthase: interception of the cyclization
cascade at the verticillene stage and rearrangement to
phomactatriene
Siew Yin Chow,y Howard J. Williams, James D. Pennington, Samik Nanda,z
*
Joseph H. Reibenspies and A. Ian Scott
Department of Chemistry, Texas A&M University, College Station, TX 77843-3255, United States
Received 11 January 2007; revised 28 February 2007; accepted 2 March 2007
Available online 12 March 2007
Abstract—The cyclization of geranylgeranyl diphosphate (GGDP) to taxadiene catalyzed by taxadiene synthase has been suggested to pro-
ceed in stages, involving a transient bicyclic verticillyl carbocation intermediate, which also has been proposed in the biosynthetic pathway
leading to phomactatriene by marine fungi of Phoma sp. On incubation with des-7-methylGGDP, which would be expected to decrease the
stability of a carbocation produced by hydride migration, taxadiene synthase produced phomactatrienes as major products. This indicates that
the verticillyl carbocation was indeed formed but underwent further skeletal rearrangements, diverging from the usual pathway taken by
GGDP en route to taxadiene. Products were identified using GC–MS, one- and two-dimensional NMR, and X-ray crystallography.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
but extraction of this compound from bark of the tree gave
only low yields. Paclitaxel became a leading target for total
synthesis in the 1990s,3–8 but the lengthy sequence of steps
and the low overall yields associated with total synthesis
served as a drawback for large-scale commercial production.
The supply problem has been solved, and paclitaxel is now
prepared on a commercial scale either by semisynthesis
from 10-desacetyl baccatin III (2), which is a renewable ad-
vanced intermediate from the needles of Taxus baccata,9 or
by fermentation of the Chinese yew Taxus chinensis.10
Isoprenoids are found in virtually all forms of life, and as of
1997, as many as 23,000 isoprenoid compounds are found in
the literature.1 An important member of the diterpenoid fam-
ily is paclitaxel (1, Fig. 1), which has been widely used in the
treatment of breast and ovarian cancers since 1992.2 Pacli-
taxel is a natural product made by the yew tree (Taxus),
O
HO
AcO
O
O
OH
OH
Ph
NH
O
The phomactins are another important class of terpenoids
some of which act as platelet-activating factor (PAF) anta-
gonists.11 To date, 11 distinct phomactins have been isolated
from the marine fungus Phoma sp.11 and are all attractive
synthetic targets, with total synthesis already accomplished
for phomactins A, D, and G (3, 4, and 5, Fig. 1).12
HO
Ph
O
OH
O
O
H
OBz
2
H
OBz
OH
OH
OAc
OAc
1
O
CHO O
H
O
OH
OH
OH
H
H
The biosynthesis of paclitaxel begins with the cyclization
of geranylgeranyl diphosphate (GGDP, 6) to taxa-
4(5),11(12)diene (7), and followed by a series of monooxy-
genations on the parent compound and esterification of some
of the resulting hydroxyl groups to give paclitaxel. To date,
taxadiene synthase, a few monooxygenases, and all five
acyl/benzoyl transferases have been found and cloned into
common laboratory microorganisms.13–15 Although the ex-
act sequence of the transformations is not known, a statistical
survey of the oxygenation pattern of the entire taxoid family
suggests that oxygenation of taxadiene should occur in the
order C5, C10, C2, C9, C1, and C13 en route to paclitaxel.
O
O
O
H
3
4
5
Figure 1. Structures of taxane and phomactin diterpenes.
* Corresponding author. Tel.: +1 979 845 3243; fax: +1 979 845 5992;
e-mail: scott@mail.chem.tamu.edu
Present address: BASF Corporation, Charlotte Technical Center, 11501
Steele Creek Road, Charlotte, NC 28273, United States.
Present address: Biotechnology Research Center, Toyama Prefectural
University, 5180 Kurokawa Kosugi, Toyama, 939-0398, Japan.
y
z
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.03.029

S. Y. Chow et al. / Tetrahedron 63 (2007) 6204–6209
6205
P2O7
H
H
10
14
6
P2O7
P2O7
H
H
Taxus.
Phoma.
H
H
H
H
H
H
12
13
11
H
H
7
8
9
Figure 2. Proposed biosynthetic pathways leading to taxadiene and phomactatrienes.
Similarly, esterifications should occur in the order C5, C2,
C10, and C13.16
strategy has been to use substrate analogs that block later en-
zymatic steps to terminate the process at an intermediate
stage.20 Using this strategy, we have recently demonstrated
that cyclization of 6 to 3 could be intercepted at the mono-
cyclic isocembrene stage with suitably-designed GGDP
analogs.21
The biosynthesis of the phomactins, like paclitaxel and other
isoprenoids in general, should involve an initial cyclization
of GGDP to a parent hydrocarbon, followed by oxidative
modification of the carbon backbone. Although the biosyn-
thesis of the phomactins has not been demonstrated in vitro,
isolation of phomacta-1(14),3(4),7(8)-triene (8) and pho-
macta-1(15),3(4),7(8)-triene (9) from Phoma sp. supported
this mechanism.17,18
2. Results and discussion
In this paper, we extend the mechanistic study of TS by at-
tempting to intercept the cyclization cascade at the verticil-
lene stage with analog 14 whose synthesis is shown in
Scheme 1.
The biosyntheses of taxadiene 7 and phomactatrienes 8 and
9 are phylogenetically unrelated, with taxadiene following
the methyl erythritol phosphate (MEP) pathway,19a while
the phomactatrienes follow the mevalonate pathway,19b but
share certain similarities.17,18 The cyclization of GGDP 6
to taxadiene 3 was proposed to proceed with multiple carbo-
cationic intermediary stages such as 10–12, shown in
Figure 2. Formation of intermediate 11 followed by a series
of hydride- and methyl migrations would also lead to the
phomactatrienes.17,18
Compared to the natural substrate, this analog lacks the
methyl group at C7, which would lead to a less stable carbo-
cation if the trans-annular proton transfer (11/12) took
place. While this work was in progress, Coates et al. pub-
lished a related work on the taxadiene synthase-catalyzed
formation of verticillene-like products.22
2.1. Synthesis of substrates
Synthesis of 7-desmethylGGDP 14, shown in Scheme 1, em-
ployed aldehyde 15, prepared readily from geranyl benzyl
ether.23 A Wittig reaction gave unsaturated ester 16, having
Under normal circumstances, these carbocation intermedi-
ates are buried in the enzyme active site, with short lifetimes
that prohibit isolation and direct observation, and a common
Ph3P=CHCO2Et
O
OBn
X
OBn
15
16 X = CO2Et
17 X = CH2OH
18 X = CH2Br
DiBAL-H
CH3SO2Cl,
LiBr
1) BuLi
2) 18
SO2Tol
20
SO2Tol
OBn
19
Li/NH3
X
21 X = OH
22 X = Br
14 X = P2O7
PBr3
(Bu4N)3HP2O7
Scheme 1. Preparation of substrate for biotransformation using taxadiene synthase.

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S. Y. Chow et al. / Tetrahedron 63 (2007) 6204–6209
Table 1. NMR data for compound 23
the desired trans geometry at the newly created double bond.
Reduction of 16 with DiBAL-H gave allylic alcohol 17 in
75% yield. Conversion of alcohol 17 to bromide 18 was
accomplished by treatment with methanesulfonyl chloride
followed by lithium bromide. Coupling of bromide 18 with
geranyl sulfone 19, followed by reduction with lithium/
ammonia, furnished 7-desmethylGGOH 21 in 28% overall
yield from 17.24 Alcohol 21 was converted to 7-des-
methylGGDP 14 in 24% yield by activation as the bromide
22 (PBr3, Et2O, 0 ꢀC), followed by treatment with (Bu4N)3-
HOPP in anhydrous acetonitrile using a modified literature
procedure.25
Position
dC (mult.)
dH (mult., J)
1
2
3
4
5
6
7
8
133.1 (s)
34.1 (t)
125.1 (d)
130.3 (s)
37.8 (t)
31.9 (t)
129.1 (d)
131.8 (d)
28.1 (t)
34.0 (t)
40.8 (s)
33.2 (d)
27.5 (t)
32.6 (t)
132.7 (s)
21.4 (q)
15.3 (q)
16.3 (q)
16.4 (q)
3.12 (dd, J¼12.1, 12.1 Hz), 2.01 (m)
5.01 (m)
2.00 (m)
2.13 (m)
5.04 (m)
5.16 (m)
2.09 (m), 1.97 (m)
1.62 (m), 1.42 (m)
9
10
11
12
13
14
15
16
17
18
19
1.78 (m)
1.45 (m)
2.32 (m), 1.86 (m)
2.2. Enzymatic cyclization of 7-desmethylGGDP
0.78 (s)
1.49 (s)
Small-scale incubation of analog 14 with recombinant taxa-
diene synthase13 gave a mixture of hydrocarbon products, as
indicated by GC–MS analysis (Scheme 2). The molecular
weight of the all the hydrocarbon products was shown to
be 258, consistent with the loss of a proton and the diphos-
phate group. Large-scale incubation of analog 14 with the
enzyme (from 90 L of recombinant Escherichia coli) af-
forded 66 mg of hydrocarbon products, which were further
purified, or partially enriched by preparative GC to obtain
enough sample of the two major products for rigorous
NMR study. The yield of the cyclization reaction of 14
was about 22%, which was comparable to the yield of taxa-
diene formation. In the control experiment (not shown), in-
cubation of analog 14 with lysate of E. coli strain BL21/
DE3/plysS (which does not encode taxadiene synthase)
failed to give any hydrocarbon products on TLC.
0.83 (d, J¼6.8 Hz)
1.68 (s)
Table 2. NMR data for compound 24
Position
dC
dH (mult., J)
1
2
3
4
5
6
7
8
138.6 (s)
35.4 (t)
127.4 (d)
134.1 (s)
39.5 (t)
30.5 (t)
126.9 (d)
134.3 (d)
26.1 (t)
36.5 (t)
38.4 (s)
40.6 (d)
31.2 (t)
121.0 (d)
37.0 (d)
14.2 (q)
22.9 (q)
17.2 (q)
15.7 (q)
2.70 (d, J¼7.6 Hz)
5.04 (m)
2.13 (m), 2.03 (m)
2.13 (m)
5.18 (m)
5.20 (m)
2.03 (m), 2.01 (m)
1.73 (m), 1.21 (m)
9
10
11
12
13
14
15
16
17
18
19
Individual products were separated by preparative thin layer,
liquid, and gas chromatographies and analyzed by NMR and
mass spectroscopies. The two major components were iden-
tified as phomactatriene analogs 23 and 24 by NMR spectros-
copy, with details given in Tables 1 and 2 below. In general,
1H, 13C, and DEPT experiments combined with HSQC data
allowed chemical shift measurement and association of indi-
vidual protons to their attached carbons. HMBC and COSY
data were then used to determine connectivity.
1.25 (m)
2.41 (m), 1.62 (m)
5.26 (m)
2.21 (m)
1.15 (d, J¼8.0 Hz)
0.92 (s)
0.84 (d, J¼6.8 Hz)
1.53 (s)
H
TS
H
H
H
H
H
P2O7
taxadiene
6
H
17
18
12
7
10
9
11
8
3
TS
6
5
15 16
2
13
14
1
4
19
H
P2O7
23
14
+
17
18
12
13
H
7
10
9
11
8
3
6
5
TS
15 16
2
14
1
4
H
19
H
H
24
P2O7
14
+
minor
products
+
25
Scheme 2. Products derived from des-7-methylGGDP (14) incubation with taxadiene synthase.

S. Y. Chow et al. / Tetrahedron 63 (2007) 6204–6209
6207
25 mM aq NH4HCO3], loaded onto a column containing
190 mL of Sigma Dowex 50WX8-200 resin (pretreated with
concd aq NH4OH,26 washed with excess distilled water,
then pre-equilibrated with 380 mL of solvent A) and eluted
with 380 mL solvent A. Typically, the product would elute
immediately, and fractions containing the product were
cloudy and/or yellow colored, as followed by a silica gel
TLC with anisaldehyde visualization. The desired fractions
were pooled, concentrated in vacuo using a rotary evaporator
(ꢁ40 ꢀC), followed by lyophilization to dryness to give
either a gum or a thick liquid.
Solid–liquid extraction of the crude product was performed
by following the literature procedure.25b The lyophilized
product was dissolved in a minimal amount of 0.1 M aq
NH4HCO3, and treated with solvent B [1:1 (v/v) isopropa-
nol/acetonitrile]. The mixture was centrifuged at 11,000g
for 10 min, and the clear supernatant was collected and
saved. After two identical treatments, the supernatants were
combined, and concentrated in vacuo using a rotary evapora-
tor (bath temperature ꢁ40 ꢀC) to give a thick yellow liquid,
which was either stored frozen at ꢂ20 ꢀC, or used directly in
Figure 3. 50% Thermal ellipsoid plot (50% probability) of compound 23.
Sample 23 crystallized on standing in chloroform and the as-
signed structure was confirmed by X-ray crystallography,
which also was used to confirm stereochemical assignments
(Fig. 3).
1
the next chromatography step. H and 13C NMR in D2O at
this stage showed significant presence of the Bu4N group,
indicating that the initial cation-exchange step was not
completely successful. As such, cellulose chromatography
was not attempted, and the product was purified by silica
chromatography instead.27
Acyclic compound 25 found in lower concentration may be
an artifact, but none was formed when enzyme was not pres-
ent, the major product in that case being the alcohol pro-
duced on phosphate ester hydrolysis. Smaller amounts of
several other compounds were formed, but due to difficulties
in separation (and in some cases instability) were not identi-
fied.
The thick yellow liquid from the solid–liquid extraction was
dissolved in a minimal amount of 0.1 M NH4HCO3, and
loaded onto the silica column, saving a small amount for
TLC. Typically, approximately 250 mL of silica gel [equili-
brated with solvent C (2:1 v/v isopropanol/concd NH4OH)]
was used for every 3 mL of sample solution. The column
was eluted with solvent. ATLC analysis (in solvent C, anis-
aldehyde stain) of the crude mixture showed the desired
product at the baseline Rf 0.0–0.1, along with impurities
with Rfw0.5–1.0. When all the impurities have eluted from
the column, solvent D [6:3:10 (v/v/v) isopropanol/concd
NH4OH/doubly distilled H2O (ddH2O)] was used to elute
the desired product from the column. The fractions contain-
ing the product were pooled, and concentrated in vacuo
using a rotary evaporator (bath temperature ꢁ40 ꢀC) to
remove bulk solvent. Rotary evaporation was discontinued
when the product solution started to bubble, and the cloudy
product solution was lyophilized to give a colorless gum.
The product was taken up in ddH2O, and centrifuged at
11,000g for 10 min. The supernatant was lyophilized to give
7-desmethylGGDP 14 [1.27 g, 1.1 mmol, 24%, (Bu4N)3 salt
3. Experimental
3.1. General
NMR spectroscopy was performed on a Bruker ARX-500 in-
strument using 3 mm H-BB or BB-H probes, CDCl3 solvent
at 25 ꢀC. Details of X-ray procedures are available in Sup-
plementary Data. X-ray results have been submitted to the
Cambridge Database as CCDC 638433.
3.1.1. 7-Desmethylgeranylgeranyl bromide, 22. To a solu-
tion of 21 (1.21 g, 4.4 mmol) in Et2O (25 mL) at 0 ꢀC, was
added PBr3 (2.2 mmol, 200 mL) using a plastic delivery
pipette. The mixture was warmed to room temperature and
stirred for 30 min, and additional (200 mL) PBr3 was added,
if TLC showed any unreacted 21. The reaction was diluted
with Et2O (25 mL), and quenched with satd aq NaCl (50 mL).
The organic layer was separated, dried with MgSO4,
filtered, and transferred into a cold dry flask, and concen-
trated in vacuo to give 7-desmethylGGBr 22 (1.46 g,
4.3 mmol, 97%), which was used immediately in the next
step.
1
by H NMR]. The product was kept as a frozen aqueous
solution (25 mg/mL) at ꢂ20 ꢀC in plastic centrifuge tubes.
1
Solvent suppressed H NMR (D2O, 500 MHz) d 5.4 (m,
3H), 5.10 (t, J¼6.8 Hz, 1H), 5.05 (t, J¼6.8 Hz, 1H), 4.4
(br, 2H), 3.14 (t-like, J¼8.8 Hz, 24H), 2.0 (m, 12H), 1.6
(m, 36H), 1.3 (sextet, J¼7.4 Hz, 24H), 0.90 (t, J¼7.4 Hz,
36H). 13C NMR (D2O) d 143.1, 137.3, 133.7, 133.0,
3.1.2. 7-Desmethylgeranylgeranyl diphosphate, 14. To
bromide 22 (1.46 g, 4.3 mmol), was added anhydrous
CH3CN (25 mL), followed by (Bu4N)3HOPP (7.76 g,
8.6 mmol). The reaction was stirred at room temperature for
2 h, and concentrated using a rotary evaporator (bath tem-
perature ꢁ40 ꢀC). The clear syrup obtained was dissolved
in minimal amount of solvent A [1:49 (v/v) isopropanol/
3
132.1, 126.7, 126.6, 123.2 (d, JP,C¼9.8 Hz), 64.8 (d,
2JP,C¼5.4 Hz), 60.6 (Bu4N), 42.02, 41.99, 35.3, 33.4, 30.5,
29.0, 27.8, 25.7 (Bu4N), 21.7 (Bu4N), 19.7, 18.5, 18.2,
15.4 (Bu4N). 31P NMR (D2O) d ꢂ8.0 (d-like, 1P), ꢂ10.7
(d-like, 1P). ESIHRMS calcd for C19H33O7P2 435.1702,
found 435.1677.

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S. Y. Chow et al. / Tetrahedron 63 (2007) 6204–6209
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3.1.3. Incubation of 7-desmethylGGDP with taxadiene
synthase. Cell pellets from an 8 L culture of E. coli strain
BL21(DE3)plysS/pTS79H,13e were resuspended and lysed
in 200 mL buffer consisting of 30 mM NaHEPES, 5 mM
sodium metabisulfite, 2.5 mM L-ascorbic acid, 10 mM KF,
2 mM dithiothreitol, 2 mM b-cyclodextrin hydrate, and
1 mM MgCl2 at pH 8.4. Substrate analog 14 (100 mg,
86 mmol) was added, and the mixture was shaken gently
for 16–24 h at room temperature. The reaction was extracted
with 2ꢃ600 mL hexane (HPLC grade); the organic layer
was dried with MgSO4, and evaporated. The residue was re-
dissolved in 1 mL HPLC hexane, and was loaded onto
a 10 mL silica column. The column was eluted with
20 mL HPLC hexane to give 4.9 mg (19 mmol, 22%) crude
product after evaporation of solvent. The major products
were isolated by preparative GC, see Tables 1 and 2 for 1H
and 13C NMR. GC–EIMS: m/z 258.
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Acknowledgements
We thank the National Institute of Health, MERIT Award
DK32034, the Robert A. Welch Foundation, and the Texas
Advanced Technology and Research Program (TATRP) for
financial support. We also thank Dr. Charles Roessner for
advice on gene expression.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2007.03.029.
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