Accessing low-oxidation state taxanes: Is taxadiene-4(5)-epoxide on the taxol biosynthetic pathway?

Article abstract of DOI:10.1039/c5sc03463a

We have shown for the first time that taxadiene (3) can be epoxidised in a regio- and diastereoselective manner to provide taxadiene-4(5)-epoxide (12) as a single diastereoisomer, and that this epoxide can be rearranged to give taxa-4(20),11(12)-dien-5α-ol (4). Furthermore, the epoxide 12 rearranges under acidic conditions to give taxa-4(20),11(12)-dien-5α-ol (4), the known bridged ether OCT (5) and the new oxacyclotaxane (OCT2) 15. Contrary to previous speculation, taxadiene-4(5)-epoxide (12) is susceptible to rearrangement when exposed to an iron^III porphyrin, and these observations justify consideration of epoxide 12 as a chemically competent intermediate on the taxol biosynthetic pathway.

Full text of DOI:10.1039/c5sc03463a

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Accessing low-oxidation state taxanes: is
taxadiene-4(5)-epoxide on the taxol biosynthetic
Cite this: DOI: 10.1039/c5sc03463a
pathway?†
Naomi A. Barton,a Benjamin J. Marsh,a William Lewis,a Nathalie Narraidoo,b
b
b
a
*
Graham B. Seymour, Rupert Fray and Christopher J. Hayes
We have shown for the first time that taxadiene (3) can be epoxidised in a regio- and diastereoselective
manner to provide taxadiene-4(5)-epoxide (12) as a single diastereoisomer, and that this epoxide can be
rearranged to give taxa-4(20),11(12)-dien-5a-ol (4). Furthermore, the epoxide 12 rearranges under acidic
conditions to give taxa-4(20),11(12)-dien-5a-ol (4), the known bridged ether OCT (5) and the new
oxacyclotaxane (OCT2) 15. Contrary to previous speculation, taxadiene-4(5)-epoxide (12) is susceptible
to rearrangement when exposed to an ironIII porphyrin, and these observations justify consideration of
epoxide 12 as a chemically competent intermediate on the taxol biosynthetic pathway.
Received 14th September 2015
Accepted 26th January 2016
DOI: 10.1039/c5sc03463a
www.rsc.org/chemicalscience
(tobacco,7 E. coli8a) has also been described. In 2008 Rontein
showed that overexpression of both taxadiene synthase and
Introduction
taxa-4(5),11(12)-diene 5-hydroxylase (CYP725A4) in tobacco
(Nicotiana sylvestris) did not produce taxa-4(20),11(12)-dien-5a-
ol (4) as expected, but instead led to the production of 5(12)-oxa-
3(11)-cyclotaxane (OCT) 5 (Scheme 2).7
Since its isolation from the pacic yew (Taxus brevifolia), and
subsequent FDA approval in 1992, taxol and its close derivatives
continue to be used as frontline drugs for the treatment of
cancer.1 Its effectiveness in the clinic, coupled with an
intriguing tricyclic structure, has ensured that taxol has
endured as a molecule of interest to scientists for nearly 50
years.2 In this paper we show that a combination of metabolic
engineering and synthetic chemistry can be used to give ready
access to low oxidation state taxanes, giving new insight into the
early stages of the ‘oxidase-phase’ of the taxol biosynthetic
pathway.3
The rst committed step in the taxol biosynthetic pathway
(Scheme 1) is the taxadiene synthase-catalysed cyclisation of
geranylgeranyl pyrophosphate 1 to produce taxa-4(5),11(12)-
diene (3).4 The remaining biosynthetic steps involve a series of
oxidation, and functional group interconversion processes,
the rst of which is the taxadiene-5a-hydroxylase-mediated
oxidation of 3 into taxa-4(20),11(12)-dien-5a-ol (4).5
In 2010 Stephanopoulos reported a signicant improvement
in this area using E. coli as the chassis organism.8a Under their
optimised conditions, taxa-4(20),11(12)-dien-5a-ol (4) could be
produced, but unfortunately the desired product 4 was obtained
as a 1 : 1 mixture with OCT (5), thus severely limiting the amount
of 4 being produced. These two studies clearly demonstrate that
the presence of both taxadiene synthase and taxadiene-5a-
hydroxylase in a metabolically engineered chassis organism does
not guarantee satisfactory production of taxadien-5-ol 4, and
the catalytic promiscuity and multispecicity of taxadiene-5a-
hydroxylase has attracted recent attention.10
A number of research groups have reported the over-
production of taxa-4(5),11(12)-diene (3) in a variety of chassis
organisms (yeast,6 tobacco,7 E. coli,8 tomato9), and the incor-
poration of both taxadiene synthase and its 5a-hydroxylase
aSchool of Chemistry, University of Nottingham, University Park, NG7 2RD,
Nottingham, UK
bDivision of Plant and Crop Sciences, School of Biosciences, University of Nottingham,
Sutton Bonnington, LE12 5RD, Loughborough, UK. E-mail: chris.hayes@nottingham.
ac.uk; Fax: +44 (0)115 951 3564; Tel: +44 (0)115 951 3045
† Electronic supplementary information (ESI) available: Full experimental
procedures and copies of 1H and 13C NMR spectra. CCDC 1030909. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c5sc03463a
Scheme 1 Biosynthesis of Taxol® from geranylgeranyl-pyrophos-
phate, via taxadiene.
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Scheme
4
Taxadiene hydroxylase mediated oxidation of taxa-
Scheme 2 Production of oxidised taxanes in metabolically engineered
tobacco and E. coli containing both taxadiene synthase and taxadiene
hydroxylase.
4(5),11(12)-diene (3) to taxa-4(20),11(12)-dien-5a-ol (4).
supports the epoxide/rearrangement route for the conversion of
3 to 4, as small inverse secondary isotope effects are observed in
epoxidation reactions,11 but no further experiments have been
reported to examine this possibility.
The production of OCT 5, along with additional oxidation
products, in engineered taxadiene synthase/taxadiene hydroxylase-
containing organisms7,8a lead us to question whether epoxide 12
could be an intermediate in the taxadiene hydroxylase mechanism
as we could envisage viable pathways for the production of both 4
and 5 from epoxide 12. Therefore, we decided to synthesise 12 and
study it's chemistry in the context of the early stages of the taxol
biosynthetic pathway.
Our current understanding of the taxadiene-5a-hydroxylase
oxidation mechanism is derived from experiments performed
by Williams and Croteau (Scheme 3).5 The observation that
taxadiene-containing microsomes could convert both the 4(5)-3
(Scheme 1) and the 4(20)-6 alkene isomers of taxadiene to
taxadien-5-ol 4 with equal efficiency (Scheme 3, eqn (1)), lead
Williams and Croteau to suggest an H-atom abstraction/oxygen
rebound mechanism, via the allylic radical 10, as being the most
likely (path A, Scheme 4).
An alternative pathway involving epoxidation of 3 to give 12,
followed by rearrangement to give 4 (path B, Scheme 4) was also
considered, but was eventually discounted by the fact that the
4(20)-alkene isomer 6 is also converted to 4 by taxadiene
hydroxylase (via a process unlikely to involve 12).5 This
conclusion was further supported by the fact that the epoxide 12
has not been observed as an oxidation product of 3 in any
studies reported thus far. In order to provide further evidence
for the H-atom abstraction/oxygen rebound mechanism (path A,
Scheme 4), Williams et al. prepared deuterium-labelled
[C20–2H3]-taxadiene (7) and subjected this to taxadiene
hydroxylase. However, under these conditions, the expected
kinetic isotope effect was not observed for the transformation of
7 to 8 (Scheme 3, eqn (2)),5 which is at odds with the proposed
H-atom abstraction process. Furthermore, Williams et al. report
that their experiment ‘unexpectedly revealed that the deuter-
ated substrate yielded slightly more taxa-4(20),11(12)-dien-5a-ol
than did the unlabeled substrate’,5b thus indicating a small
inverse isotope effect. This experimental observation actually
Results and discussion
Epoxidation of taxadiene
Our studies began by isolating taxadiene from our previously
described taxadiene synthase-containing tomatoes,9 using
a slightly modied protocol that allows extraction directly from
fresh fruit (see ESI† for details). This procedure afforded taxa-
4(5),11(12)-diene (3) and taxa-4(20),11(12)-diene (6) as an insepa-
rable 17 : 1 (3 : 6) mixture. With ready access to taxadiene we next
turned our attention to epoxidation of 3, with DMDO being
selected as the oxidant due to its ease of use.12 As we were con-
cerned with the potential over-epoxidation of taxadiene, we per-
formed the reactions with substoichiometric quantities of oxidant.
Pleasingly, when taxa-4(5),11(12)-diene (3) was treated with 0.7
equivalents of DMDO, the desired epoxide 12 was obtained as the
1
major new product (95% purity as judged by H NMR; see ESI†)
and unreacted taxadiene was recovered (Scheme 5).
Whilst the epoxide derived from 6 was not observed, the
recovered taxadiene (20%) was signicantly enriched in 6 (1 : 2;
3 : 6) compared to the starting material (17 : 1; 3 : 6), thus indi-
cating that 3 is much more reactive towards epoxidation than 6.
Care had to be taken during chromatography on silica gel as the
epoxide 12 was acid sensitive (vide infra). Treatment of taxadiene
(3) with excess DMDO (2 equivalents), produced the bis-epoxide
13 in 75% yield, and this epoxide was found to be much more
stable than 12 to chromatography on silica gel (Scheme 5).
Synthesis of taxa-4(20),11(12)-dien-5a-ol (4)
With a reliable route to the key epoxide 12 secured, we
Scheme
3 Elucidating the taxadiene hydroxylase mechanism
(Williams and Croteau).
next wanted to assess its ability to act as a precursor to
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Fig. 1 X-ray crystal structure of the taxadiene derived epoxyalcohol
14.†
Scheme 5 DMDO epoxidation of taxa-4(5),11(12)-diene.
explored the behaviour of 12 under conditions of more rele-
vance to the biosynthesis. We speculated that if taxadiene
hydroxylase acts as a monooxygenase and epoxidises taxadiene
3 to produce 12, then this would initially leave a mild Lewis
acidic iron centre in close proximity to the epoxide, which could
catalyse subsequent rearrangement reactions. Therefore, we
decided to examine the behaviour of 12 under a range of acidic
conditions.
taxa-4(20),11(12)-dien-5a-ol (4). Before examining conditions of
relevance to the biosynthesis, we rst reacted 12 with Yama-
moto's aluminium amide reagent (TMPAlEt2) to produce 4 as
a reference sample (Scheme 6).13 As the epoxide 12 was prone to
decomposition during column chromatography (vide infra), we
used the epoxide in crude form directly from the DMDO
oxidation. Thus, treatment of unpuried 12 with freshly
prepared Yamamoto's reagent (BuLi, TMP, ClAlEt2, 0 ꢀC, PhMe)
gave taxa-4(20),11(12)-dien-5a-ol (4) in 60% isolated yield over
the two steps from taxadiene (3). The spectroscopic data for 4
matched that reported by Williams for the 5a-stereoisomer,5
and this enabled us to conrm that epoxidation (3 / 12) must
have occurred on the a-face of taxadiene. Having prepared bis-
epoxide 13, we next examined its behaviour under the same
rearrangement conditions. Thus, treatment of 13 with Yama-
moto's reagent provided epoxy-alcohol 14 as the major isolable
product (50%). It is interesting to note that the 11(12)-epoxide
moiety is also observed in natural taxanes such as taxinine A
11(12)-epoxide.14 Fortunately, 14 was obtained as a crystalline
solid and we were able to determine an X-ray crystal structure
(Fig. 1) to conrm the stereochemistry of the 11(12)-epoxide,
and also show that the C5-hydroxyl was on the a-face.
In order to simulate the acid-mediated decomposition
encountered during silica gel chromatography, the epoxide 12
ꢀ
was treated with silica gel in C6D6 at 70 C. Reaction progress
1
was monitored by H NMR (see ESI†), and we determined that
12 converts into OCT (5), the molecule that had previously been
produced in metabolically engineered tobacco by Rontein
(Scheme 7),7 and the new isomeric oxacyclotaxane 15 (OCT2).
Complete conversion of epoxide 12 was observed, as judged by
the loss of the C19 methyl 1H NMR signal at 0.58 ppm, and the
isomeric bridged ethers 5 and 15 were produced in an approx-
imately 3 : 2 ratio (1H NMR). Chromatographic separation gave
isolated samples of 5 (19%) and 15 (19%), which were then fully
characterised.
Treatment of epoxide 12 with a stronger acid (pTSA, C6D6)
gave taxa-4(20),11(12)-dien-5a-ol (4) as the major new product,
with OCT (5) and OCT2 (15) being produced as minor products
(isolated yields: 4 (17%); 5 (7%); 15 (7%)). The formation of
4(20),11(12)-dien-5a-ol (4) from the epoxide 12 under these
Rearrangements of taxadine-4(5)-epoxide 12
Encouraged by the successful conversion of epoxide 12 to taxa-
4(20),11(12)-dien-5a-ol (4) using Yamamoto's reagent, we next
Scheme
6
Synthesis of taxa-4(20),11(12)-dien-5a-ol (4) via rear- Scheme
7
Rearrangement of taxadiene-4(5)-epoxide (12) under
rangement of epoxide 12.
acidic conditions.
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strongly acidic conditions is readily explained by invoking
protonation of the epoxide 12 to produce 16 (Scheme 8). Ring-
opening then affords the cation 17, and loss of a proton from
the C20 methyl group installs the exo-methylene group in 4
(Scheme 8). The formation of OCT (5) also implicates the
cation 17 as an intermediate. A 1,2-hydride shi rst produces
the new tertiary cation 18, which next undergoes trans-
annulation with the C11(12)-alkene leading to the cation 19.
Etherication, involving trapping the cation 19 with the
secondary hydroxyl, then gives OCT (5). Similarly, the forma-
tion of OCT2 (15) can be rationalised by invoking a 1,2-alkyl
shi of the tertiary cation 19, leading to the new tertiary cation
20, which is then trapped as the ether 15 by reacting with the
C5-hydroxyl (Scheme 8).
As the biological oxidant (taxadiene hydroxylase5) acting
upon taxadiene is a cytochrome P450, it is tempting to speculate
that the reduced ironIII porphyrin (11, Scheme 4) is capable of
facilitating a Lewis acid-catalysed rearrangement of the epoxide
in vivo. Rontein, however, discounted this proposal17 on the
basis that previous work on very different chemical systems has
shown that ironIII porphyrins are poor catalysts for the rear-
rangement of epoxides.15 As we had access to the epoxide 12, we
could test this hypothesis experimentally, and we decided to
treat 12 with an ironIII porphyrin.
Contrary to the literature hypothesis, we were pleased to nd
that treatment of 12 with FeIII(TPP)Cl (2 equiv.) in C6D6 at 25 ꢀC
for 72 hours, lead to epoxide rearrangement, with the produc-
tion of OCT (5) and OCT2 (15) as the main new products in
a 1 : 1 ratio (1H NMR). Formation of taxa-4(20),11(12)-dien-5a-ol
(4) was not observed under these Lewis acidic conditions
(Scheme 9). As a control experiment, we exposed the similarly-
substituted cyclogeraniol-derived epoxide 2216 to the same
Fe(TPP)Cl rearrangement conditions,17 and as expected from
previous reports,15 no rearrangement was observed, thus high-
lighting the propensity of 12 to rearrange.
Scheme 9 IronIII porphyrin mediated rearrangement of taxadiene-
4(5)-epoxide (12).
Having shown that the two step epoxidation/FeIII induced
rearrangement mimics that seen in vivo (tobacco) mediated by
taxa-4(5),11(12)-diene 5-hydroxylase (CYP725A4), we wondered
if the initial oxidation of taxadiene could also be achieved using
the FeIII(TPP)Cl catalyst and a suitable stoichiometric oxidant
(Scheme 10). Thus, treatment of taxadiene (3) with FeIII(TPP)Cl
(10 mol%) and hydrogen peroxide (1 equiv.)18 lead to complete
consumption of starting material (as judged by t.l.c. and 1H
NMR), and the subsequent production of oxidation products.
Although the isolated yields were low, 1H NMR of the crude
reaction mixture showed that the two major products were OCT
(5) and the OCT2 (15). The production of taxa-4(20),11(12)-dien-
5a-ol (4) was not observed under these conditions (Scheme 10).
Implications for the taxol biosynthetic pathway
As discussed in the introduction (Scheme 2), the current
proposal for the biosynthesis of 4 from taxadiene 3 is that
taxadiene hydroxylase performs an H-atom abstraction from the
C20 methyl group of the 4(5)-alkene isomer of 3 to form the allyl
radical 10, and involvement of the epoxide 12 was rejected.
Further support for the involvement of a common allyl radical
10 came from the fact that the 4(20)-alkene isomer 6 was also
converted to taxa-4(20),11(12)-dien-5a-ol (4) by taxadiene
hydroxylase. However, our experiments, coupled with the
previously published kinetic isotope effect data,5 demonstrate
that the epoxide 12 cannot be discounted as an intermediate on
Scheme
8 Proposed mechanisms for the formation of taxa-
4(20),11(12)-dien-5a-ol (4), OCT (5) and OCT2 (15) from taxadiene- Scheme 10 IronIII porphyrin mediated oxidation of taxa-4(5),11(12)-
4(5)-epoxide (12).
diene (3).
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Scheme 11 Proposal for the role of epoxide 12 in the biosynthesis of
taxa-4(20),11(12)-dien-5a-ol (4).
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Conclusions
In this study, we have shown that taxa-4(5),11(12)-diene (3) can
be isolated from the fruit of metabolically engineered tomatoes
using our new optimised procedure. Furthermore, we have
shown that taxadiene (3) can be epoxidised in a regio- and
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(12), and that this epoxide can be rearranged to give taxa-
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We have shown that the epoxide 12 is sensitive to acids, and that
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19 Whilst this manuscript was under review, a complementary
study by Stephanopoulos et al. has been reported that
also proposes taxadiene epoxidation by taxadiene-5a-
hydroxylase as being a step on the taxol biosynthetic
pathway. Please see: S. Edgar, K. Zhou, K. Qiao, J. R. King,
J. H. Simpson and G. Stephanopoulos, ACS Chem. Biol.,
2016, DOI: 10.1021/acschembio.5b00767.
Chem. Sci.
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