6- and 14-Fluoro farnesyl diphosphate: Mechanistic probes for the reaction catalysed by aristolochene synthase

Article abstract of DOI:10.1039/b817194g

The catalytic mechanism of the enzyme aristolochene synthase from Penicillium roqueforti (PR-AS) has been probed with the farnesyl diphosphate analogues 6- and 14-fluoro farnesyl diphosphate (1b and 1c). Incubation of these analogues with PR-AS followed by analysis of the reaction products by GC-MS and NMR spectroscopy indicated that these synthetic FPP analogues were converted to the fluorinated germacrene A analogues 3b and 3c, respectively. In both cases the position of the fluorine atom prevented the formation of the eudesmane cation analogues 4b and 4c. These results highlight that germacrene A is an on-path reaction intermediate during PR-AS catalysis and shed light on the mechanism by which germacrene A is converted to eudesmane cation. They support the proposal that the role of PR-AS in the cyclisation is essentially passive in that it harnesses the inherent chemical reactivity present in the substrate by promoting the initial ionisation of farnesyl diphosphate and by acting as a productive template to steer the reaction through an effective series of cyclisations and rearrangements to (+)-aristolochene (7a).

Full text of DOI:10.1039/b817194g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
6- and 14-Fluoro farnesyl diphosphate: mechanistic probes for the reaction
catalysed by aristolochene synthase†
David J. Miller,a Fanglei Yu,b David W. Knighta and Rudolf K. Allemann*a
Received 1st October 2008, Accepted 28th November 2008
First published as an Advance Article on the web 20th January 2009
DOI: 10.1039/b817194g
The catalytic mechanism of the enzyme aristolochene synthase from Penicillium roqueforti (PR-AS) has
been probed with the farnesyl diphosphate analogues 6- and 14-fluoro farnesyl diphosphate (1b and
1c). Incubation of these analogues with PR-AS followed by analysis of the reaction products by
GC-MS and NMR spectroscopy indicated that these synthetic FPP analogues were converted to the
fluorinated germacrene A analogues 3b and 3c, respectively. In both cases the position of the fluorine
atom prevented the formation of the eudesmane cation analogues 4b and 4c. These results highlight
that germacrene A is an on-path reaction intermediate during PR-AS catalysis and shed light on the
mechanism by which germacrene A is converted to eudesmane cation. They support the proposal that
the role of PR-AS in the cyclisation is essentially passive in that it harnesses the inherent chemical
reactivity present in the substrate by promoting the initial ionisation of farnesyl diphosphate and by
acting as a productive template to steer the reaction through an effective series of cyclisations and
rearrangements to (+)-aristolochene (7a).
aristolochene synthases from Penicillium roqueforti (PR-AS) and
Introduction
Aspergillus terreus (AT-AS).6–10
All sesquiterpenes derive from farnesyl diphosphate (FPP 1a).1
The first steps in their diversification to this family of natural
PR-AS catalyses the magnesium dependent turnover of FPP
(1a) into the sesquiterpene (+)-aristolochene (7a) (Scheme 1).3
products are the transformations catalysed by sesquiterpene
The enzyme acts as a high precision template that directs
synthases.2,3 These enzymes, which rely on the mainly a-helical
class I terpene fold for catalysis,2 serve as high fidelity templates
that subtly channel conformation and stereochemistry during the
cyclisation reactions that are essential to the generation of the
wide diversity in structure and stereochemistry found in these
compounds. They bind their respective substrates together with
the obligatory Mg2+-cofactor, catalyse the loss of the diphosphate
group and chaperone the reaction intermediates along complex
reaction pathways often with exquisite specificity.2,3 Sesquiterpene
synthases catalyse highly regio- and stereospecific cyclisations,
rearrangements and eliminations while at the same time excluding
water from the active site to prevent premature quenching of
the highly reactive cationic reaction intermediates. The existence
of these carbocationic intermediates makes fluorinated FPP
analogues useful tools for their study. The highly electronegative
fluorine atoms can strongly influence the stability of carbocationic
species but have only a relatively modest effect on the size and
shape of these species.4,5 Appropriately positioned fluorine atoms
can therefore bias such carbocationic cyclisations and shed light
on the intricate details of the reaction mechanism. The success of
this approach has been evident in a series of recent studies of the
mechanisms of action of several terpene synthases including the
FPP into a reactive conformation.2,11,12 Structural analyses of
aSchool of Chemistry, Cardiff University, Main Building, Park Place, Cardiff,
CF10 3AT, UK. E-mail: allemannrk@cardiff.ac.uk; Fax: +44 (0)29 2087
4030
bNewChem Technologies Ltd., Bedson Building, Kings Road, Newcastle upon
Tyne, NE1 7RU, UK; Tel: +44 (0) 191 2226803
1
† Electronic supplementary information (ESI) available: H NMR spec-
trum of 31c and mass spectra of all major compounds eluted by gas
chromatography in this study. See DOI: 10.1039/b817194g
Scheme 1
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co-crystals of AT-AS and 12,13-difluorofarnesyl and 2-
fluorofarnesyl diphosphate suggested a possible metal ion binding
sequence for the generation of the reactive Michaelis complex7
from which the reaction cascade is initiated through the attack of
C1 by the C10,C11-p-bond of FPP and loss of the diphosphate
group leading to germacryl cation (2a).6 Incubation of PR-AS with
12,13-difluorofarnesyl diphosphate showed that this compound
was a competitive inhibitor of the enzyme and indicated that this
reaction did not go through farnesyl cation; hence ring closure
and loss of diphosphate occur in a concerted fashion.6
Several lines of evidence including the production of 2-
fluorogermacrene A (3d) from incubations of 2-fluorofarnesyl
diphosphate (1d) with PR-AS,8 support the proposal that (-)-
germacrene A (3a) is then formed by loss of a proton from C12
of germacryl cation (2a).8,13–15 Cyclisation of germacrene A takes
place by protonation of the C6,C7 double bond by an unidentified
active site acid13,16,17 followed by ring closure through electron flow
from the C2,C3-p-bond and formation of a C2,C7 s-bond giving
the eudesmane cation (5a). Formation of (+)-aristolochene (7a)
is then achieved by a [1,2]-hydride shift from C2 to C3, a [1,2]-
methyl shift from C7 to C2 and elimination of the si-proton from
C8.14 Here we report the synthesis of 6-fluoro-farnesyl diphosphate
(6F-FPP, 1b) and 14-fluoro-farnesyl disphosphate (14F-FPP, 1c)
and their application to probe the PR-AS reaction in an effort to
decipher mechanistic aspects of the protonation of germacrene A
(3a) to generate cation 4a.
one (8) underwent a Horner–Wadsworth–Emmons reaction with
triethyl 2-fluoro-2-phosphonoacetate using NaH as the base. The
resulting Z/E ester mixture was reduced with DIBAL-H to give
a mixture of Z and E vinylic fluorides (9 and 10) (Z/E =
approx. 1 : 1) that were separated chromatographically. The desired
Z isomer 9 was then transformed into an allylic bromide and
treated with the double enolate of ethyl acetoacetate using the
methodology of Weiler and Sum to give 11 in excellent yield.9,18
Stereoselective conversion of this ketone into the enolic phosphate
12 followed by treatment with lithium dimethylcuprate gave ester
13, again in good yield.9 Reduction of 13 using DIBAL-H and
then diphosphorylation of the resulting 6-fluorofarnesol analogue
14 by a modification of the method of Davisson et al.19 gave 6F-
FPP (1b).
Synthesis of 14F-FPP
14F-FPP (1c) was prepared using a modified version of the method
used by Dolence and Poulter for the synthesis of 15-fluorofarnesyl
diphosphate and is shown in Scheme 3.20 The key reaction in this
synthesis was a Wittig reaction to couple the fluorinated ketone
22 to phosphonium salt 28. Ethyl 4-chloroacetoacetate (15) was
treated with benzyl alcohol and sodium hydride and the resulting
benzylic ether 16 was subjected to hydrogenolysis using 10% Pd-C
and H2 to remove the benzyl group. The resulting alcohol was then
protected with a THP group under standard conditions to give 18
in excellent overall yield. As observed previously, this compound
was not easily purified and so was used directly in the next step
in approx. 80% purity after flash chromatography.20 The THP
ether 18 was alkylated with dimethylallyl bromide using NaH as
the base to give the b-keto ester 19. Saponification was followed
by decarboxylation using KOH to give ketone 20 in moderate
yield. The fluorine substituent was introduced by acid catalysed
Results and discussion
Synthesis of 6F-FPP
The synthesis of 6F-FPP (1b) followed a protocol similar to that
published by Faraldos et al. (Scheme 2).10 6-Methyl-5-hepten-2-
Scheme 2 Reagents and conditions: (i) Triethyl 2-fluoro phosphonoacetate, NaH, THF, 0 ◦C then DIBAL-H, THF, -78 ◦C, 51%; (ii) NEt3, MsCl, LiBr,
THF, -45 ◦C; (iii) ethyl acetoacetate, NaH, BuLi, 0 ◦C then intermediate bromide, 98%; (iv) NaH, diethylchlorophosphate, 0 ◦C, 97%; (v) Me2CuLi,
Et2O, -78 ◦C, 95%; (vi) DIBAL-H, THF, -78 ◦C, 91%; (vii) NEt3, MsCl, LiBr, THF, -45 ◦C; (viii) (Bu4N)3HP2O7, CH3CN, then DOWEX 40 W (NH4
+
form) cation exchange 48%.
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Scheme
3
Reagents and conditions: (i) BnOH, NaH, toluene, 82%; (ii) H2, 10% Pd-C, EtOH, 95%; (iii) 3,4-DHP, p-TsOH, CH2Cl2;
(iv) 1-bromo-3-methyl-but-2-ene, NaH, THF, 0 ◦C, 53%, 2 steps; (v) KOH, H2O, EtOH, 52%; (vi) PPTS, EtOH, 50 ◦C, 96%; (vii) triflic anhydride,
2,6-lutidine, CH2Cl2, 0 ◦C then Bu4NF, THF, -78 ◦C; (viii) HBr, H2SO4, toluene, 70 ◦C, 88%; (ix) triethyl 2-phosphonoacetate, NaH, THF, 0 ◦C, 68%;
(x) DIBAL-H, THF, -78 ◦C, 97%; (xi) 3,4-DHP, p-TsOH, CH2Cl2, 86%; (xii) PPh3, CH3CN, D; (xiii) LiHMDS, THF, -78 ◦C, 30%; (xiv) PPTS, EtOH,
50 ◦C, 94%; (xv) MsCl, NEt3, LiBr, THF -45 ◦C; (xvi) (Bu4N)3HP2O7, CH3CN then DOWEX 50 W (NH4 form) cation exchange, 45%.
+
removal of the THP group in ethanol followed by transformation
of the resulting b-keto alcohol 21 into a triflate ester and then
substitution with fluoride using TBAF in THF at -78 ◦C. The
resulting b-fluoroketone 22 was found to be highly volatile and was
easily lost during solvent concentration under reduced pressure. It
was therefore not isolated and was used directly as a solution in
hexane in the following Wittig reaction.
tractable products from each incubation indicated the production
of monofluorinated sesquiterpenoids characterised by molecular
ion peaks of m/z = 222 (Fig. 1).
The preparation of 28 was achieved from a-acetyl butyrolactone
(23) which was ring opened and decarboxylated by treatment with
HBr and H2SO4 in toluene at 70 ◦C to give g-bromoketone 24
in 88% yield.21,22 Horner–Wadsworth–Emmons reaction of this
ketone with triethyl 2-phosphonoacetate and NaH in THF yielded
25 with good stereoselectivity (E : Z = 11 : 1).23 Reduction of
bromoester 25 with DIBAL-H was followed by protection of
the resulting allylic alcohol with a THP group. Treatment of
the resulting THP protected bromide 27 with PPh3 in refluxing
acetonitrile gave 28.
Prior to Wittig reaction of 28 with fluoro ketone 22, the
phosphonium salt was purified by trituration with ether to remove
excess PPh3. The two compounds were then mixed and treated with
LiHMDS in THF at -78 ◦C. The desired fluorofarnesol derivative
29 (stereoselectivity E : Z 1 : 7) was isolated in 30% yield from 21.
Transformation to the target compound was achieved by standard
methodology: the THP group was removed using PPTS in ethanol
at 50 ◦C and then diphosphorylation was performed in the usual
way9,19 with 1c being isolated in 45% yield in the final step.
Fig. 1 (a) Total ion chromatogram (TIC) of the pentane extractable
products isolated from incubation of 1b with PR-AS; 3b elutes at 27.4 min
(m/z = 222), minor compounds elute at 28.0 min (m/z = 222) and 29.4 min
(m/z = 240). (b) TIC of the pentane extractable products isolated from
incubation of 1c with PR-AS; 3c elutes at 29.4 min (m/z = 222).
Enzymatic conversion of 1b and 1c to fluorinated sesquiterpenes
PR-AS was overproduced in E. coli, extracted from inclusion
bodies and purified according to established protocols.24 Each
of the two monofluorinated FPP analogues was then incubated
on a preparative scale (~10 mg) with PR-AS as previously
described.8,10 GC-mass spectroscopic analysis of the pentane ex-
In the case of 1c (Fig. 1b) a single sharp peak was observed
accompanied by the broad signal characteristically observed for
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germacrene A-like compounds as a consequence of their thermal
instability.25 Hence the gas chromatogram suggested that the
product was a germacrene A derivative, presumably 3c.
Incubation of 6F-FPP (1b) with PR-AS resulted in a more
complex product mixture with 3 major peaks at 27.4, 28.0 and
29.4 min (Fig. 1a). The two compounds corresponding to the
two peaks with shorter retention times showed molecular ion
peaks m/z = 222 whereas the compound eluting at 29.4 min
gave a molecular ion peak of m/z = 240 (see ESI for all mass
spectra). The mass spectrum of this latter compound showed
major fragmentation peaks at m/z = 222, 207 and 202, consistent
with loss of H2O from the parent compound followed by loss of
CH3 or HF respectively from the dehydrated ion. These results
suggested that this compound was a fluorinated sesquiterpene
alcohol but since it was the smallest of the three peaks in the
chromatogram, it was difficult to isolate substantial amounts of
this side product and its structure was not investigated further.
For the two other products it seemed likely that at least one of
these would be the germacrene A analogue 3b. The (+)-isomer of
3b had been reported previously as the sole product resulting from
incubations of 1b with the plant sesquiterpene synthase tobacco
5-epi-aristolochene synthase (TEAS).10
Fig. 2 TIC of the pentane extractable products isolated from incubation
of (a) 1b with PRAS. 31b elutes at 23.6 min (m/z = 222), minor product
elute at 23.5 min (m/z = 222), 27.4 min (m/z = 222) and 29.4 min (m/z =
240). (b) 1c with PR-AS, 31c elutes at 24.8 min (m/z = 222), a minor
product elutes at 24.5 min (m/z = 222). (c) Expansion of the TIC for
the products derived from 1b between tR = 22 and 30 min. All GC were
performed in these instances with an injection port temperature of 300 ◦C
to induce Cope rearrangement of the enzyme produced products.
Germacrene A is well known to undergo a Cope rearrangement
even upon mild heating13,25 and so each of the product mixtures
isolated above were analysed by GC-M◦S with increasing injection
◦
port temperatures between 50 C-300 C but otherwise identical
conditions. Again the product mixture was more complex for
the products derived from 6F-FPP (1b) than from 14F-FPP (1c),
which gave a single major new product with a retention time of
24.8 min and a small amount of a side product with a retention
time of 24.5 min (Fig. 2b). For products derived from 1b (Fig. 2a
and c) there were two major new products with retention times
of 23.5 and 23.6 min with the alcohol product at 29.4 min being
retained. The compound with retention time 28.0 min was fully
◦
reacted whereas that at 27.4 min was still evident with a 300 C
injection temperature, albeit with much reduced intensity relative
to the alcohol product.
The lowest energy conformation of (-)-germacrene A (3a) is
the so-called down-down (DD) conformation (Scheme 4a) which
upon Cope rearrangement produces (+)-b-elemene (31a).26–28
Therefore, the major products from the thermally induced rear-
rangements of 3b and 3c are most likely the fluoro-b-elemene
analogues 31b-c (Scheme 4), which elute at 23.6 min (pane1 a,
Fig. 2) and 24.8 min (Fig. 2b), respectively. The minor product
observed from rearrangement of 3c may result from Cope rear-
rangement from the more disfavoured up-down (UD) or down-up
(DU) conformers of 3c shown (Scheme 4b and c). Rearrangement
of either of these conformers leads to a different diastereoisomer
of the fluoro-b-elemene analogue than that formed from the DD
conformer, namely 32c. It is reasonable to speculate that this is the
compound that elutes with a retention time of 24.5 min (Fig. 2b).
The situation is more complex for the product isolated from 1b
since the two compounds with retention times of 27.4 and 28.0 min
(Fig. 1a) appear to undergo thermal rearrangement resulting
in two rearrangement products. The minor product eluting at
23.5 min (Fig. 2a) is likely to be the rearrangement product of that
compound eluting at 28.0 min when the injector is set at 50 ◦C
(Fig. 1a). Again, it may be speculated that this compound arises
from the formation of a (+)-germacrene A derivative in this case
Scheme 4 Cope rearrangements of (a) down-down, (b) up-down and (c)
down-up conformational isomers of germacrene A and related fluorinated
derivatives.
as a minor by-product in the incubation of 1b with PR-AS since
this compound does undergo an observed thermal rearrangement
although there is no further evidence of this.
NMR Spectroscopic analysis of the incubation products derived
from 1b and 1c
The products from the preparative incubations discussed above
were analysed by NMR spectroscopy. The product mixture
isolated from incubation of 6F-FPP (1b) and PR-AS gave a
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Fig. 3 1H NMR spectrum of 3b isolated from incubation of 6F-FPP and PR-AS. Inset, expansion of the vinylic region of the spectrum, arrows indicate
peaks associated with the major compound present 3b.10
1H NMR spectrum characteristic of a germacrene based com-
pound as it displayed broad peaks suggestive of a compound
slowly exchanging between two or more conformational isomers.8
Coates and co-workers recently published the 1H NMR spectrum
of a fluoro-germacrene A derivative resulting from incubation of
1b and TEAS, the compound they isolated being the opposite
enantiomer of the major product 3b proposed in this study.10 The
1H NMR spectrum of 3b should therefore be identical to that
measured by Coates and co-workers. Comparison of the 1H NMR
spectrum of the product mixture isolated in this study with that
published previously indeed showed that the expected compound
was present as the major component (Fig. 3). As shown in Fig. 3
and as discussed above, the compound was not isolated as the only
component and at least one other compound is present, as can be
seen from lack of resolution in the alkyl region of the spectrum
between dH = 1.00 and 2.70 ppm. In addition and as highlighted
in Fig. 3, the vinylic region of the 1H NMR spectrum (dH = 4.30–
5.50 ppm in this case) shows the expected peaks for 6F-germacrene
A 3b at dH = 4.50, 4.60, 4.81 and 5.05‡ but also additional peaks at
dH = 4.56 and 5.40 ppm. Comparison with the published spectra
for 1a-fluoro-b-selinene and 1a-fluoro-a-selinene (produced by
acid catalysed rearrangement of the respective fluoro-germacrene
A derivatives)10 indicated that this minor product was not either
of these compounds and hence remains an unknown by-product.
The 1H NMR spectrum of the product isolated from incubation
of 14F-FPP (1c) and PR-AS was also rather poorly resolved at
room temperature as would be expected for conformationally
flexible germacrene A derivatives. As there was no literature
spectrum to compare this to, interpretation of the data was
more difficult. 19F NMR spectrometry showed three resonances
at dF = -210.6, -216.2 and -218.2 ppm implying that there were
three interconverting conformational isomers at this temperature
since GC-MS analysis clearly showed that only one product had
been formed (presumably the down-down, up-down and down-up
conformers).27,28 In an attempt to resolve and hence fully interpret
these spectra, variable temperature (VT) 19F and 1H NMR , COSY
and HSQC spectra were recorded for 3c in d8-toluene in the
range -60 to 105 ◦C (Fig. 4). A similar VT NMR experiment
had been successfully used previously for the interpretation of the
NMR spectra of the germacrene A derivative produced by PR-AS
from 2-fluorofarnesyl diphosphate.8 At low temperatures, some
sharpening and resolution of resonances was observed but the
1H NMR spectrum remained too complex to interpret the spectra
for individual conformational isomers. Furthermore, despite some
apparent coalescence of signals at high temperatures (>80 ◦C),
the appearance of new sharp signals most obviously in the region
of the spectrum where vinyl protons resonate (dH ~4.5–6.5 ppm)
suggested that at this elevated temperature a new stable compound
formed, which did not disappear upon cooling. A new triplet signal
also appeared at dF = -231 ppm in the 19F NMR spectrum of this
compounds at elevated temperatures, which again was retained on
cooling.
Since it appeared that the Cope rearrangement of 3c was occur-
ring at high temperature in the NMR solvent, the tube containing
the solution was heated at 90 ◦C for 2 h in order to complete the
rearrangement of this compound. GC-MS analysis of the solution
at this stage showed that Cope rearrangement was complete
(the chromatogram being identical to that shown in Fig. 2b).
Consequently, this rearranged fluoro-sesquiterpenoid product was
purified by preparative scale thin layer chromatography and then
analysed by 1H and 19F NMR spectroscopy. The spectra were now
clearly resolved and fully consistent with structure 31c (Scheme 4,
see ESI for 1H NMR spectrum).
These results strongly support the proposal that germacrene A
is an on-path reaction intermediate of FPP cyclisation by PR-
AS in agreement with several previous reports.8,13,15,16 They also
shed some light onto the mechanism by which germacrene A
is converted to eudesmane cation and two possible mechanistic
pathways can be envisaged (Scheme 5). In pathway a, protonation
‡ Note that 3b is observed as two slowly-exchanging conformers by 1H
NMR spectroscopy at room temperature.10
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Fig. 4 1H NMR spectrum of 3c at room temperature in d8-toluene. The inset shows an expansion of the spectrum both at -60 ◦C (lower inset) and at
105 ◦C (upper inset) and indicates that a new compound is formed, most obviously from the double doublet that has appeared at 5.7–5.8 ppm.
to the C6-C7 p-system. The fluorine substituents on C6 or C14
may simply reduce the electron density of the C6-C7 double bond
thereby preventing the formation the s-bond between C2 and C7.
Hence PR-AS acts as a template that orientates the cyclodecadiene
system of germacrene A in a high energy conformation poised to
be converted to eudesmane cation even through catalysis by the
rather weak general acids normally found in enzyme active sites.
In an attempt to distinguish these two pathways 2-fluorofarnesyl
diphosphate (1d),8 and PR-AS were incubated in deuterated buffer.
Should deuterated germacrene A cation 33d be formed then
the reaction cannot proceed any further, as cation 4d would be
destabilised by the b-fluorine atom and 3d would be regenerated
by elimination of a proton/deuteron from C6 of 33d. If this
Scheme 5
elimination process is not stereospecific or there is sufficient
of the C6,C7-double bond is facilitated by the orientation of the
two endocyclic double bonds of the germacrene A ring system
leading to a reaction in which attack of the C2,C3-p-bond at C7
and protonation occur in a concerted reaction. In the alternative
stepwise formation of eudesmane cation (pathway b) protonation
of the C6,C7-double bond leads to formation of the discrete
cationic intermediate 33a that is subsequently transformed into
4a by movement of the C6,C7-p-electrons. While the results
described here do not allow a clear distinction between these two
mechanisms, together with previously published computational
studies,16,17 they are in good agreement with the concerted
pathway a.
time for the general acid/base to exchange with the solvent, C6
deuterated 3d should be formed. GC-MS analysis of the reaction
products from the turnover of 2F-FPP and PR-AS in D2O, showed
a single hexane extractable product (3d) with a molecular ion peak
of m/z = 222. While the absence of deuterium incorporation
in these experiments is in agreement with pathway a, it does
not completely rule out the stepwise mechanism since 33d may
indeed be formed under these conditions but revert to 3d via a
stereospecific elimination reaction.
Conclusions
While in a stepwise mechanism (pathway b), the fluorine
substituents in 3b and 3c should sufficiently destabilise the carbo-
cations on C7 to prevent their formation and to lead to the accumu-
lation of the fluorinated germacrene A compounds (3b and 3c), cal-
culations using density functional theory, MP2 and semi-empirical
methodologies found no evidence for protonated germacrene A
as an intermediate before cyclisation to a eudesmane cation.17
This makes it rather unlikely that protonation of the C6-C7
double bond occurs in a discrete step prior to cyclisation,
especially since a strong acid would be needed to activate the
olefinic substrate. However, the results presented here are also in
agreement with a concerted reaction mechanism for the generation
of eudesmane cation (Scheme 5, pathway a), in that PR-AS will
stabilise a conformation of germacrene A that provides optimal
orbital overlap for electron flow from the C2-C3 double bond
6F-FPP (1b) and 14F-FPP (1c) have been prepared and tested
as substrates of PR-AS. Both compounds were turned over by
PR-AS to fluorinated germacrene thereby highlighting again
that this monocyclic sesquiterpenoid is most likely an on-path
reaction intermediate during PR-AS catalysis. In addition, the
results support the proposal that the substrate itself provides
the chemical functionality and reactivity required for efficient
cyclisation. Thus the main function of terpene synthases appears
to be to act as productive templates to steer the reactivities
inherent in their substrates. This proposal is similar to the findings
with non-enzymatic cyclisations of squalene and oxido-squalene,
where the formation of tetra- and pentacyclic triterpenes has
never been achieved efficiently in the absence of enzymes and
the tight control of substrate conformation within an enzyme
active site is a prerequisite for highly selective formation of
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products such as hopene or lanesterol.29 However, in contrast
to triterpene formation where the hydrophobic effect leads to
some pre-organisation of the substrates, sesquiterpene cyclases are
required to convert FPP from an extended conformation, which
FPP predominately adopts in aqueous solution,30 into the reactive
quasicyclic form. Hence it is fascinating to note that the enzymes
that catalyse one of the most complex reaction mechanisms found
in nature, appear to play only a rather ‘passive’ role in promoting
these transformations.
Expression and purification of aristolochene synthase
Enzyme was produced in E. coli BL21(DE3) cells harbouring a
cDNA for AS under the control of◦the T7 promoter as previously
described.24 Cells were grown at 37 C in LB medium with 0.3 mM
ampicillin until they reached an A600 of 0.5. They were induced with
0.5 mM isopropyl-b-D-1-thiogalactopyranoside, incubated for a
further 3 h and harvested by centrifugation at 8000g for 10 min.
Protein was then extracted from the inclusion bodies and purified
following our established protocols.13,24,32 The enzyme was pure as
judged by SDS-gel electrophoresis.
Experimental
(Z)-3,7-Dimethyl-2-fluoroocta-2,6-dien-1-ol (9)
All chemicals were purchased from Sigma-Aldrich unless other-
wise stated. Tetrahydrofuran (THF) and diethyl ether were dis-
tilled from sodium/benzophenone ketyl under nitrogen. Acetoni-
trile, dichloromethane, toluene and triethylamine were distilled
from calcium hydride under nitrogen. All other chemicals were
of analar quality or better and used as received unless otherwise
stated. Reactions were stirred at room temperature in air unless
otherwise stated. All glassware was clean and dry before use.
1H NMR spectra were measured on a Bruker Avance 500 NMR
spectrometer or a Bruker Avance DPX400 NMR spectrometer
and are reported as chemical shifts in parts per million downfield
from tetramethylsilane, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet), coupling constant
(to the nearest 0.5 Hz) and assignment, respectively. 13C and
31P NMR spectra were measured on a Bruker Avance 500
NMR spectrometer and are reported as chemical shift downfield
from tetramethylsilane, coupling constant where appropriate and
assignment. Assignments are made to the limitations of COSY,
DEPT 90/135, gradient HSQC and gradient HMBC spectra.
19F and 31P NMR spectra were recorded on a Jeol Eclipse +300
NMR spectrometer and are reported in chemical shift downfield
from CFCl3 and 85% H3PO4 respectively followed by multiplicity
and coupling constant (to the nearest 0.5 Hz) if appropriate.
C2HCl3 was filtered through basic alumina prior to use in NMR
spectroscopy. IR spectra were recorded on a Perkin-Elmer 1600
series FTIR spectrometer and samples were prepared as thin films
of neat liquid on sodium chloride discs for oils and as KBr disks
for solids. EI+ mass spectra were measured on a Micromass LCT
premiere XE mass spectrometer ES- mass spectra were provided by
the UK EPSRC mass spectrometry service, Swansea UK. GCMS
was performed on a Hewlett Packard 6890 GC fitted with a J &
W scientific DB-5MS column (30 m x 0.25 mm internal diameter)
and a Micromass GCT Premiere detecting in the range m/z 50–
800 in EI+ mode with scanning once a second with a scan time of
0.9◦s. Injections were performed in split mode (split ratio 5 : 1) at
50 ◦C. Chromato◦grams were begun with an oven temperature of
50 ◦ C rising at 4 C min-1 for 25 min (up to 150 ◦C) and then at
20 C min-1 for 5 min (250 ◦C final temperature).
To a stirred suspension of sodium hydride (60% dispersion◦in
mineral oil, 824 mg, 20.6 mmol) in anhydrous THF (40 cm3) at 0 C
under an N2 atmosphere was added dropwise, triethyl 2-fluoro-2-
phosphonoacetate (4.19 cm3, 20.6 mmol) followed immediately by
a solution of 6-methyl-5-hepten-2-one (8) (3.34 cm3, 22.7 mmol)
in anhydrous THF (10 cm3). The complete reaction mixture was
stirred for 14 h under N2. Water (50 cm3) and diethyl ether (50 cm3)
were added, and the organic layer was separated. The aqueous
layer was extracted with diethyl ether (2 ¥ 20 cm3). The combined
ethereal extracts were washed with water (2 ¥ 100 cm3) and
saturated NaCl solution (100 cm3), dried (MgSO4), filtered and
then concentrated under reduced pressure. The resulting ester was
a 1 : 1 mixture of E and Z isomers and was reduced without further
purification. To a stirred solution of the crude ester in anhydrous
THF (100 cm3) at -78 ◦C, was added, dropwise (over 3 min),
diisobutylaluminium hydride (1.0 M solution in hexanes, 51.5 cm3,
51.5 mmol). The reaction mixture was allowed to stir at -78 ◦C for
1 h, then 0 ◦C for 1 h and was then quenched by careful addition of
saturated aqueous sodium potassium tartrate (150 cm3). The re-
sulting mixture was then extracted with diethyl ether (3 ¥ 100 cm3).
The combined organic extracts were dried over anhydrous MgSO4,
filtered and then concentrated under reduced pressure. Purification
of the residue by flash column chromatography on silica gel with
hexane and ethyl acetate (4 : 1) as eluent gave the E isomer
10 (1.72 g, 48%) followed by the Z-isomer 9 as a colourless oil
(1.79 g, 51%); TLC Rf 0.14 (hexane : EtOAc = 4 : 1); HRMS (ES+,
[M + NH4]+) found 190.1602, C10H21FNO requires 190.1602; nmax
(thin film)/cm-1 3341.8, 2918.9, 1704.5, 1449.9, 1378.1, 1157.6 and
1011.2; dH (500 MHz, C2HCl3) 1.63 (3 H, s, CH3), 1.69 (3 H, d,
=
J
H-F 7.5, CH3C CF), 1.71 (3 H, d, J 0.5, CH3), 1.81 (1 H, b, OH),
2.10 (4 H, m, CH2CH2), 4.23 (2 H, d, JH-F 25.0, CH2OH) and 5.13
2
=
(1 H, t, J 6.0, C CH); dC (125 MHz, C HCl3) 15.32 (d, J
6.25,
C-F
=
CH3C CF), 17.62 (CH3), 25.64 (CH3), 25.97 and 29.78 (d, JC-F
6.5, CH2CH2), 57.95 (d, JC-F 31.5, CH2OH), 116.00 (d, JC-F 15.0,
=
=
CH3C CF), 123.67 (C CH), 132.08 (quaternary C) and 151.79
2
=
(d, JC-F 240.0, CH3C CF); dF (282 MHz, C HCl3) -121.33; m/z
(CI+) 190.1 (100%, [M + NH4]+) and 172.2 (20, M+).
Thin layer chromatography was performed on pre-coated
aluminium plates of silica G/UV254 (Fluka) TLC visualizations
were performed with 4.2% ammonium molybdate and 0.2% ceric
sulfate in 5% H2SO4, 0.1 berberine hydrochloride in HCl/EtOH or
UV light. Flash chromatography was performed according to the
method of Still.31 Reverse phase HPLC was performed on a system
comprising of a Dionex P680 pump and a Dionex UVD170U
detector unit.
(E)-3,7-Dimethyl-2-fluoroocta-2,6-dien-1-ol (10)
TLC Rf 0.21 (hexane : EtOAc = 4 : 1); HRMS (ES+,
[M + NH4]+) found 190.1600, C10H21ONF requires 190.1602; nmax
(thin film)/cm-1 3345.4, 2920.1, 2860.4, 2359.0, 1704.4, 1449.8,
1378.3, 1267.8, 1149.3, 1021.2, 908.3 and 830.2; dH (500 MHz,
C2HCl3) 1.61 (3 H, s, CH3), 1.70 and 1.71 (6 H, CH3CCF and
terminal CH3 overlap), 1.88 (1 H, b, OH), 2.04–2.12 (4 H, m,
968 | Org. Biomol. Chem., 2009, 7, 962–975
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CH2CH2), 4.18 (2 H, d, JH-F 25.00, CH2OH) and 5.10 (1 H, tt,
J 7.0, J 1.5, CHCH2CH2); dC (125 MHz, C2HCl3) 13.46 (d, JC-F
9.0, CH3C CF), 17.62 (CH3), 25.61 (CH3), 26.40 (d, JC-F 2.5) and
(2Z,6Z)-Ethyl 3-diethylphosphoryl-7,11-dimethyl-6-fluoro-
dodeca-6,10-dienoate (12)
=
A stirred suspension of NaH (0.07 g, 2.79 mmol) in anhydrous
diethyl ether (50 cm3) was cooled to 0 ◦C as a solution of b-
keto ester 11 (0.66 g, 2.31 mmol) in diethyl ether (10 cm3) was
added slowly. The mixture was then stirred at 0 ◦C for 30 min
until effervescence ceased. Diethyl chlorophosphate (0.51 cm3,
3.49 mmol) was added slowly to the resulting clear, colourless
solution and stirring was continued at 0 ◦C for an additional
2 h. The reaction was quenched by addition of saturated aqueous
ammonium chloride (20 cm3). The mixture was diluted with water
(20 cm3), and the organic layer was separated. The aqueous layer
was then extracted with diethyl ether (2 ¥ 15 cm3). The combined
ethereal extracts were washed with water (2 ¥ 15 cm3) and brine
(15 cm3), dried over MgSO4 then filtered and then concentrated
under reduced pressure. Purification of the crude product by
flash column chromatography on silica gel with hexane and ethyl
acetate (2 : 1) as eluent gave 12 as a pale yellow oil (0.94 g,
97%); TLC Rf 0.14 (hexane : EtOAc = 2 : 1); HRMS (ES+,
[M + H]+) found 421.2144, C20H35FO6P requires 421.2155; nmax
(thin film)/cm-1 2979.7, 2925.4, 2353.0, 1727.5, 1663.9, 1443.4,
1368.1, 1280.1, 1207.5, 1147.1, 1035.2, 991.5 and 800.6; dH
(500 MHz, C2HCl3) 1.19 (3 H, t, J = 7.0, CO2CH2CH3), 1.30 (6 H,
29.78 (d, JC-F 5.0, CH2CH2), 57.63 (d, JC-F 31.25, CH2OH), 115.62
=
=
(d, JC-F 15.0, CH3C CF), 123.26 (C CH), 132.96 (quaternary C)
and 152.66 (d, JC-F 242.5, CH3C CF); dF (282 MHz, C2HCl3)
=
-119.5 (t, JH-F 24.5); m/z (CI+) 190.1 (100%, [M + NH4]+) and
172.2 (15, M+).
(Z)-Ethyl 7,11-dimethyl-6-fluoro-3-oxo-dodeca-6,10-dienoate (11)
A stirred solution of the alcohol 9 (0.62 g, 3.62 mmol) and
triethylamine (1.03 cm3, 7.40 mmol) in anhydrous THF (40 cm3)
under N2 was cooled to -45 ◦C and then methanesulfonyl chloride
(0.37 cm3, 4.80 mmol) was added. The resulting milky mixture
◦
was stirred at -45 C for 45 min and then a solution of lithium
bromide (1.29 g, 14.8 mmol) in THF (10 cm3) was added via
cann◦ula at -45 ◦C. The resulting suspension was allowed to warm
to 0 C and stirred for an additional 1 h. Ice cold water (20 cm3)
and hexane (20 cm3) were added, the two layers were separated,
and the aqueous layer was extracted with hexane (2 ¥ 15 cm3).
The combined organic layers were washed with saturated aqueous
NaHCO3 (15 cm3) and brine (15 cm3) then dried over Na2SO4
and filtered. Concentration of the solvent gave the intermediate
bromide as a light yellow oil which was used in the next step
without further purification.
=
dt, J 7.0, J 1.0, 2 ¥ POCH2CH3), 1.51 (3 H, d, J 2.5, CH3C CF),
=
=
1.53 (3 H, s, CH3C CH), 1.61 (3 H, s, CH3C CH), 1.96–2.00
=
(4 H, m, C CHCH2CH2), 2.44–2.54 (4 H, m, CFCH2CH2),
To a stirred suspension of NaH (0.27 g, 11.3 mmol) in anhydrous
THF (100 cm3) was added ethyl acetoacetate (1.30 cm3, 10.2 mmol)
4.07 (2 H, q, J 7.0, CO2CH2CH3), 4.20 (4 H, quintet, J 7.0,
=
2 ¥ POCH2CH3), 5.02 (1 H, m, C CHCH2) and 5.30 (1 H, s,
◦
dropwise at 0 C. After 10 min, n-BuLi (2.5 M, 4.29 cm3,
2
=
C CHCO2); dC (125 MHz, C HCl3) 14.19 (CO2CH2CH3), 15.43
(d, JC-F 6.5, CH3C CF), 16.01 (POCH2CH3), 16.07 (POCH2CH3),
10.7 mmol) was added slowly over 3 min, during which time the
colourless solution gradually turned yellow. After stirring for an
additional 10 min at 0 ◦C, a solution of the above bromide in
THF (2 cm3) was added. The clear solution turned to a cloudy
yellow suspension. After stirring for 30 min at 0 ◦C, hydrochloric
acid (3 M, 5.0 cm3) was added followed by water (20 cm3) and
diethyl ether (20 cm3) before the organic layer was separated. The
aqueous layer was extracted with diethyl ether (2 ¥ 15 cm3) and
then the combined ethereal extracts were washed with water (2 ¥
15 cm3) and brine (15 cm3). After drying over MgSO4 the solution
was filtered and concentrated under reduced pressure. Purification
of the crude product by flash column chromatography on silica
gel with hexane and ethyl acetate (4 : 1) as eluent gave 11 as a
pale yellow oil (0.95 g, 98%); TLC Rf 0.40 (hexane : EtOAc =
4 : 1); HRMS (ES+, [M + Na]+) found 307.1672, C16H25FNaO3
requires 307.1685; nmax (thin film)/cm-1 2968.6, 2921.1, 1746.8,
1719.6, 1650.0, 1445.6, 1368.1, 1317.8, 1235.8, 1156.0, 1077.9,
1036.4 and 832.6; dH (500 MHz, C2HCl3) 1.21 (3 H, t, J 7.15,
=
=
=
=
17.58 (CH3C CH), 25.62 (CH3C CH), 25.94 (d, JC-F 30.0,
CFCH2CH2), 26.22 (d, JC-F 1.5, C CHCH2CH2), 29.70 (d, JC-F
=
7.5, C CHCH2CH2), 32.47 (CFCH2CH2), 59.88 (CO2CH2CH3),
64.77 and 64.83 (2 ¥ POCH2CH3), 106.07 (d, JC-P 7.5, COCHCO2),
=
=
=
113.16 (CH3C CFCH2), 123.92 ((CH3)2C CHCH2), 131.71 (qua-
ternary C), 151.05 (d, JC-F 241.5, CH3C CF), 160.04 (d, J 7.5,
quaternary C) and 163.54 (quaternary C); dF (282 MHz, C2HCl3)
-114.32 (t, JH-F 26.0); dP (202 MHz, C2HCl3) -8.66; m/z (ES+)
421.2 (100%, [M + H]+).
(2E,6Z)-Ethyl 6-fluoro-3,7,11-trimethyldodeca-2,6,10-
trienoate (13)
A stirred suspension of CuI (0.84 g, 4.43 mmol) in anhydrous
diethyl ether (50 cm3) under argon was cooled to 0 ◦C and
MeLi (1.6 M in diethyl ether, 5.54 cm3, 8.86 mmol) was added
dropwise. A yellow precipitate formed immediately, and the
mixture was stirred for 30 min at 0 ◦C, during which time the yellow
precipitate disappeared and a near colourless solution remained.
This solution was then cooled to -78 ◦C and a solution of 12
(0.93 g, 2.21 mmol) in diethyl ether (5 cm3) was added slowly
over 5 min via a cannula. The resulting yellow solution was
=
=
CH2CH3), 1.52 and 1.53 (6 H, m, CH3C CF and CH3C CH),
1.61 (3 H, s, CH3), 1.98 (4 H, m, CH2CH2), 2.45 (2 H, dt, JH-F
22.0, JH-H 7.5, CFCH2CH2), 2.67 (2 H, t, J 7.5, CFCH2CH2),
=
3.38 (2 H, s, O CCH2CO2), 4.12 (2 H, q, J 7.0, CH2CH3)
and 5.01 (1 H, m, CHCH2CH2); dC (125 MHz, C2HCl3) 14.06
=
(CH2CH3), 15.41 (d, JC-F 6.5, CH3C CF), 17.59 (CH3), 22.57
◦
(d, JC-F 30.0, CFCH2CH2), 25.64 (CH3), 26.16 and 29.65 (d, JC-F
stirred for 3 h at -78 C, iodomethane (1.38 cm3, 22.1 mmol)
=
=
7.5, CH2CH2C CF), 39.62 (CH2CH2C CF), 49.36 (COCH2CO2),
added and the mixture was stirred for a further 15 min at
-78 ◦C. The yellow orange mixture was poured into an ice-
cooled mixture of saturated aqueous ammonium chloride and
concentrated ammonia (1 : 1, 10 cm3) and the organic layer
separated. The aqueous layer was extracted with diethyl ether
=
61.38 (CH2CH3), 112.41 (d, JC-F 17.5, CH3C-CF), 123.55 (C CH),
=
=
=
131.73 (C CH), 151.50 (d, JC-F 239.0, C CF), 167.00 (ester C O)
2
=
and 201.59 (ketone C O); dF (282 MHz, C HCl3) -114.72 (t, JH-F
25.5); m/z (ES+) 307.2 (100%, [M + Na]+).
This journal is
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(2 ¥ 20 cm3). The combined ethereal extracts were washed with
water (2 ¥ 15 cm3) and brine (15 cm3), dried over MgSO4, filtered
then concentrated under reduced pressure. Purification of the
crude product by flash column chromatography on silica gel with
hexane and ethyl acetate (9 : 1) as eluent gave 13 as a pale yellow
oil (0.56 g, 95%); TLC Rf 0.45 (hexane : EtOAc = 9 : 1); HRMS
(EI+, M+) found 282.1988, C17H27O2F requires 282.1995; nmax
(thin film)/cm-1 2965.6, 2925.3, 2855.1, 1717.5, 1649.0, 1447.3,
1381.8, 1272.6, 1223.3, 1149.5, 1049.6 and 855.9; dH (500 MHz,
C2HCl3) 1.28 (3 H, t, J 7.0, CO2CH2CH3), 1.57 (3 H, d, J 2.5,
(2E,6Z)-Trisammonium 6-fluoro-3,7,11-trimethyldodeca-
2,6,10-trien-1-yl diphosphate (1b)
A stirred solution of 14 (208 mg, 0.868 mmol) and triethylamine
(242◦mm3, 1.74 mmol) in anhydrous THF (10 cm3) was cooled to
-45 C and then methanesulfonyl chloride (87 mm3, 1.13 mmol)
was added dropwise. The resulting milky white mixture was stirred
at -45 ◦C for 45 min and then a solution of lithium bromide
(301 mg, 3.47 mmol) in THF (5 cm3) was added via a cannula at
-45 ◦C. The suspension was allowed to warm to 0 ◦C and stirred
for addition 1 h. Cold water (10 cm3) and hexane (10 cm3) were
added. The two layers were separated, and the aqueous layer was
extracted with hexane (2 ¥ 10 cm3). The pooled organic layers
were washed with saturated aqueous NaHCO3 (10 cm3) and brine
(10 cm3) and then dried over anhydrous Na2SO4. Concentration
of the solvent under reduced pressure gave the required bromide
as a light yellow oil which was used without further purification.
To a stirred solution of the bromide in anhydrous acetonitrile
(10 cm3) under N2 was added freshly recrystallized tris(tetra-
n-butylammonium) hydrogendiphosphate (1.80 g, 2.00 mmol)
prepared by the method of Davisson et al.19 The reaction mixture
was stirred for 2 h and then the solvent was removed under
reduced pressure. The resulting residue was then dissolved in
1 : 49 (v/v) isopropyl alcohol and 25 mM aqueous ammonium
hydrogencarbonate (2 cm3, ion-exchange buffer). The pale yellow
solution was slowly passed through a column containing 30 equiv.
of DOWEX 50W-X8 (100–200 mesh) cation-exchange resin that
had been pre-equilibrated with two column volumes of ion-
exchange buffer. The column was eluted with two column volumes
of ion-exchange buffer at a flow rate of one column volume per
15 min. The clear light yellow eluent was freeze dried to yield a
fluffy white solid, which was purified by reverse phase HPLC (150 ¥
21.2 mm Phenomenex Luna C-18 column, eluting under isocratic
conditions with 10% B for 20 min, a linear gradient to 60% B over
25 min, then a linear gradient to 100% B over 5 min and finally
with 100% B for 10 min; solvent A: 25 mM NH4HCO3 in water,
solvent B: CH3CN, flow rate 5.0 cm3 min-1, detecting at 220 nm)
to give 1b as a white solid (188 mg, 48% for two steps); HPLC tR =
36.45 min; HRMS (ES-, [M - H]-) found 399.1145, C15H26FO7P2
requires 399.1143; nmax (KBr disc)/cm-1 2929.9, 1707.6, 1494.4,
1456.3, 1200.2, 1124.7, 1088.8, 1041.5, 914.1, 809.9, 720.8 and
595.0; dH (500 MHz, 2H2O at pH 8.5 buffered with N2H4O2H) 1.45
=
=
CH3C CF), 1.61 (3 H, s, CH3C CHCH2CH2), 1.69 (3 H, s,
=
=
CH3C CHCH2CH2), 2.05–2.08 (4 H, m, (CH3)2C CHCH2CH2),
=
2.19 (3 H, d, J 1.0, CH3C CHCO2), 2.32 (2 H, t, J 7.5,
CFCH2CH2), 2.37–2.44 (2 H, m, CFCH2CH2), 4.15 (2 H, q,
=
J 7.0, CH2CH3), 5.11 (1 H, b, (CH3)2C CHCH2) and 5.69
(1 H, s, C CHCO2); dC (125 MHz, C2HCl3) 14.29 (OCH2CH3),
=
=
=
15.47 (d, JC-F 6.5, CH3C CF), 17.58 (CH3C CHCH2), 18.66
=
=
(CH3C CHCO2), 25.64 (CH3C CHCH2), 26.23 (d, JC-F 2.5,
=
(CH3)2C CHCH2CH2), 26.87 (d, JC-F 30.0, CFCH2CH2), 29.67
=
(d, JC-F 7.5, (CH3)2C CHCH2CH2), 37.74 (CFCH2CH2), 59.48
=
(OCH2CH3), 112.21 (d, JC-F 16.5, CH3C CFCH2), 116.24
=
=
(C CHCO2), 123.99 ((CH3)2C CHCH2), 131.68 (quaternary C),
=
152.06 (d, JC-F 240.0, CH3C CF) and 158.32 and 166.65 (quater-
nary C); dF (282 MHz, C2HCl3) -113.40 (t, JH-F 24.5); m/z (EI+)
282.2 (2%, M+) and 69.1 (100).
(2E,6Z)-6-Fluoro-3,7,11-trimethyldodeca-2,6,10-trien-1-ol (14)
To a stirred solution of 13 (387 mg, 1.37 mmol) in anhydrous THF
(10 cm3) at -78 ◦C, was added, dropwise, diisobutylaluminium
hydride (1.0 M solution in hexanes, 3.43 cm3, 3.43 mmol).
The reaction mixture was allowed to stir at -78 ◦C for 1 h,
then 0 ◦C for 1 h before being quenched by careful addition
of saturated aqueous sodium potassium tartrate (15 cm3). The
resulting mixture was then extracted with diethyl ether (3 ¥
10 cm3). The combined organic extracts were dried over anhydrous
MgSO4, filtered and then concentrated under reduced pressure.
Purification of the residue by flash column chromatography on
silica gel with hexane and ethyl acetate (4 : 1) as eluent gave 14 as
a colourless oil (0.30 g, 91%); TLC Rf 0.32 (hexane : EtOAc =
2 : 1); HRMS (EI+, M+) found 240.1897, C15H25FO requires
240.1889; nmax (thin film)/cm-1 3327.8, 2919.0, 1706.3, 1670.5,
1449.1, 1381.5, 1288.4, 1189.6, 1142.0, 1110.8, 1075.8, 1006.2,
924.5 and 829.9; dH (500 MHz, C2HCl3) 1.46 (1 H, s, OH), 1.49
=
=
=
(3 H, d, J 2.0, CH3C CFCH2), 1.49 (3 H, s, CH3C CHCH2CH2),
=
1.56 (3 H, s, CH3C CHCH2O), 1.61 (3 H, s, CH3C CHCH2CH2),
=
1.96 (4 H, m, (CH3)2C CHCH2CH2), 2.10 (2 H, t, J 7.5,
=
=
(3 H, d, J 2.5, CH3C CF), 1.53 (3 H, s, CH3C CHCH2OH), 1.61
CFCH2CH2), 2.26 (2 H, td, J 25.0, J 7.5, CFCH2CH2), 4.34
=
=
(3 H, s, CH3C CHCH2CH2), 1.62 (3 H, s, CH3C CHCH2CH2),
=
(2 H, t, J 6.5, CHCH2O), 5.06 (1 H, b, (CH3)2C CHCH2)
=
1.97 (4 H, m, (CH3)2C CHCH2CH2), 2.12 (2 H, t, J 7.5,
2
=
and 5.35 (1 H, t, J 7.5, C CHCH2O); dC (125 MHz, H2O at
CFCH2CH2), 2.22 (2 H, m, CFCH2CH2), 4.06 (2 H, d, J
pH 8.5 buffered with N2H4O2H) 14.57 (d, JC-F 6.5, CH3C CF),
=
=
7.0, CH2OH), 5.03 (1 H, b, (CH3)2C CHCH2) and 5.36 (1 H,
dt, J 7.0, J 1.0, C CHCH2OH); dC (125 MHz, C2HCl3)
=
=
15.60 (CH3C CHCH2CH2), 16.95 (CH3C CHCH2CH2), 24.90
=
=
=
(CH3C CHCH2O), 25.45 (CH3)2C CHCH2CH2), 26.63 (d, JC-F
=
=
15.47 (d, JC-F 6.5, CH3C CF), 16.18 (CH3C CHCH2CH2),
=
25.0, CFCH2CH2), 28.86 (d, JC-F 7.5, (CH3)2C CHCH2CH2),
=
=
17.61 (CH3C CHCH2OH), 25.65 (CH3C CHCH2CH2), 26.28
35.94 (CFCH2CH2), 62.47 (d, JC-P 5.0, CH2O), 112.22 (d, JC-F
=
(CH3)2C CHCH2CH2), 27.28 (d, JC-F 29.0, CFCH2CH2),
=
16.5, CH3C CFCH2), 120.56 (d, JC-P 9.0, CHCH2O), 124.14
=
29.65 (d, JC-F 7.5, (CH3)2C CHCH2CH2), 36.44 (CFCH2CH2),
=
((CH3)2C CHCH2), 133.75 and 141.06 (quaternary C) and 153.48
=
59.29 (CH2OH), 111.48 (d, JC-F 17.5, CH3C CFCH2), 124.06
(d, JC-F = 236.5, CH3C CF); dF (282 MHz, 2H2O at pH 8.5
=
=
(CHCH2OH), 124.08 ((CH3)2C CHCH2), 131.67 and 138.64
buffered with N2H4O2H) -113.69 (t, JH-F 24.5); dP (202 MHz, 2H2O
at pH 8.5 buffered with N2H4O2H) -6.95 (d, JP-P 21.0), -10.38 (d,
JP-P 21.0); m/z (ES-) 399.1 (100%, [M - H]-).
=
(quaternary C) and 152.96 (d, JC-F 241.5, CH3C CF); dF
(282 MHz, C2HCl3) -112.73 (t, JH-F 27.0); m/z (EI+) 240.2
(6%, M+) and 202.2 (100).
970 | Org. Biomol. Chem., 2009, 7, 962–975
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Ethyl 4-(benzyloxy)-3-oxobutanoate (16)33
(4 : 1) as eluent to give 18 as a yellow oil (crude yield 7.25 g,
73%). The product was used in next step without further
purification.
Benzyl alcohol (8.28 cm3, 80.0 mmol) was added dropwise to a
stirred suspension of sodium hydride (3.21 g, 80.0 mmol) in toluene
(200 cm3), slight cooling was required with an ice bath to maintain
ambient temperature. After hydrogen evolution ceased, the thick
slurry was allowed to stir for 2 h. Ethyl 4-chloroacetoacetate (15)
(5.44 cm3, 40.0 mmol) was then added dropwise over 20 min and
the reaction mixture was stirred for 16 h. Water (100 cm3) was
then carefully added and the resulting mixture was extracted with
diethyl ether (3 ¥ 50 cm3). The organic phases were combined,
washed with brine (50 cm3), dried (MgSO4) filtered and then
concentrated under reduced pressure to give the title compound as
a pale yellow oil. Purification of the crude product by flash column
chromatography on silica gel with hexane and ethyl acetate (4 : 1)
as eluent gave 16 as a pale yellow oil (7.69 g, 82%); TLC Rf 0.31
(hexane : EtOAc = 4 : 1); nmax (thin film)/cm-1 2982.3, 1747.6,
1722.4, 1657.6, 1496.4, 1453.7, 1399.9, 1367.4, 1319.0, 1229.5,
1140.9, 1100.1, 1032.9, 743.3 and 700.3; dH (500 MHz, C2HCl3)
To a stirred suspension of NaH (washed with hexane to remove
mineral oil, 0.37 g, 15.2 mmol) in anhydrous THF (50 cm3) at 0 ◦C
was added a solution of the THP ether 18 (3.50 g, 15.2 mmol) in
THF (5 cm3). The reaction mixture was stirred for 1 h whilst slowly
warming to room temperature and then 1-bromo-3-methyl-but-2-
ene (2.30 g, 15.2 mmol) was added and the reaction was allowed
to stir for 16 h. Water (50 cm3) was then carefully added and the
resulting mixture was extracted with ethyl acetate (3 ¥ 30 cm3). The
organic phases were combined, washed with brine (30 cm3), dried
(MgSO4), filtered and then concentrated under reduced pressure.
Purification of the crude product by flash column chromatography
on silica gel with hexane and ethyl acetate (5 : 1) as eluent gave
19 as a light yellow oil (2.34 g, 53%); TLC Rf 0.33 (hexane :
EtOAc = 5 : 1); HRMS (ES+, [M + NH4]+) found 316.2122,
C16H30NO5 requires 316.2118; nmax (thin film)/cm-1 2940.1, 1721.2,
1443.2, 1374.3, 1323.6, 1267.3, 1203.0, 1130.1, 1077.2, 1038.6,
968.8, 906.1, 871.5 and 816.6; dH (500 MHz, C2HCl3) 1.20 (3 H,
td, J 7.0, J 2.0, CH2CH3), 1.56 (3 H, s, CH3), 1.60 (3 H, s, CH3),
=
=
1.27 (3 H, t, J 7.0, CH2CH3), 3.56 (2 H, s, O CCH2C O), 4.17
=
(2 H, s, CH2OCH2C O), 4.19 (2 H, q, J 7.0, CH2CH3), 4.61
(2 H, s, PhCH2O) and 7.34 (5 H, m, Ar-CH); dC (125 MHz,
=
1.47–1.77 (6 H, m, (CH2)3), 2.46–2.53 (2 H, m, C CHCH2CH),
2
=
=
C HCl3) 14.07 (CH2CH3), 46.08 (O CH2C O), 61.41 (CH2CH3),
3.44 and 3.72 (2 H, m, (CH2)3CH2O), 3.54 (1 H, dt, J 27.0, J 7.5,
=
73.51 (PhCH2O), 74.82 (CH2OCH2C O), 127.94, 128.12 and
=
=
=
O CCHC O), 4.07–4.28 (4 H, m, O CCH2O and CH2CH3), 4.55
=
128.56 (Ar-CH), 136.94 (Ar-C quaternary), 167.00 (ester C O)
=
(1 H, dt, J 13.0, J 3.5, OCHO) and 5.06 (1 H, m, CH3C CH); dC
+
+
=
and 201.72 (ketone C O); m/z (CI ) 254.2 (100%, [M + NH4] ).
(125 MHz, C2HCl3) 14.07 (d, J 1.5, CH2CH3), 17.72 (CH3), 18.81
(d, J 29.0, (CH2)3CH2), 25.23 (CH2CH2CH2), 25.72 (CH3), 26.46
Ethyl 4-hydroxy-3-oxobutanoate (17)20
=
(d, J 6.5, C CHCH2CH), 30.08 (OCHCH2CH2CH2), 55.14 (d, J
=
=
18.0, O CCHC O), 61.19 (CH2CH3), 61.92 ((CH2)3CH2O), 71.63
To a stirred solution of 16 (7.70 g, 32.6 mmol) in anhydrous
ethanol (80 cm3) was added 10% palladium on activated charcoal
(3.08 g, 2.89 mmol Pd) and the solution stirred under a hydrogen
atmosphere for 16 h. The solution was filtered through a short
=
(COCH2O), 98.46 (OCHO), 119.87 (CH3C CHCH2), 134.60
=
=
=
(CH3C CHCH2), 169.19 (ester C O) and 203.40 (ketone C O);
m/z (CI+) 316.3 (15%, [M + NH4]+) and 102.1 (100).
R
pad of Celiteꢀ and the filtrate was concentrated under reduced
pressure. Purification of the crude product by flash column
chromatography on silica gel with hexane and ethyl acetate (1 : 2)
as eluent gave 17 as a pale yellow oil (4.52 g, 95%); TLC Rf 0.37
(hexane : EtOAc = 1 : 2); nmax (thin film)/cm-1 3455.5, 2984.9,
1720.7, 1623.8, 1408.9, 1370.3, 1321.2, 1269.3, 1152.7, 1026.9,
943.9, 858.7 and 810.5; dH (500 MHz, C2HCl3) 1.29 (3 H, t, J
7.0, CH2CH3), 3.02 (1 H, br, OH), 3.51 (2 H, s, COCH2CO), 4.20
(2 H, q, J 7.0, CH2CH3) and 4.39 (2 H, s, CH2OH); dC (125 MHz,
C2HCl3) 14.03 (CH2CH3), 45.36 (COCH2CO), 61.84 (CH2CH3),
68.54 (CH2OH); 166.46 (CO2CH2CH3) and 202.49 (COCH2OH);
m/z (CI+) 164.1 (100%, [M + NH4]+).
6-Methyl-1-(tetrahydro-2H-pyran-2-yloxy)hept-5-en-2-one (20)
To a stirred solution of 19 (2.5 g, 8.40 mmol) in ethanol (80 cm3)
and water (30 cm3) was added potassium hydroxide (3.53 g,
63 mmol) and the solution stirred for 24 h. The solvent was
removed under reduced pressure and the residue was dissolved
in ethyl acetate (50 cm3). This solution was washed with water
(50 cm3) and brine (50 cm3), dried (MgSO4), filtered and then
concentrated under reduced pressure. Purification of the crude
product by flash column chromatography on silica gel with
hexane and ethyl acetate (5 : 1) as eluent gave 20 as a light
yellow oil (0.95 g, 52%); TLC Rf 0.36 (hexane : EtOAc =
5 : 1); HRMS (CI+, [M + NH4]+) found 244.1906, C13H26NO3
requires 244.1907; nmax (thin film)/cm-1 2941.5, 1720.2, 1442.3,
1380.2, 1263.6, 1203.2, 1127.8, 1074.4, 1037.8, 969.1, 905.9, 871.4
and 815.6; dH (400 MHz, C2HCl3) 1.54–1.89 (6 H, m, (CH2)3),
1.62 (3 H, s, CH3), 1.68 (3 H, s, CH3), 2.28 (2 H, q, J 7.5,
3-Ethoxycarbonyl-6-methyl-1-(tetrahydro-2H-pyran-2-yloxy)-
hept-5-ene-2-one (19)
To a stirred solution of 17 (6.30 g, 43.2 mmol) in anhydrous
CH2Cl2 (80 cm3) under an atmosphere of nitrogen was added 3,4-
dihydro-2H-pyran (7.87 cm3, 86.3 mmol) and p-toluenesulfonic
acid (0.41 g, 2.16 mmol) and the solution stirred for 16 h. The
solvent was removed under reduced pressure, and the resulting
oil was diluted with ethyl acetate (50 cm3) and water (50 cm3).
The separated organic layer was washed with saturated aqueous
sodium hydrogencarbonate (50 cm3) and brine (50 cm3), dried
(MgSO4) and concentrated to afford the crude THP ether as
an amber oil, which was partially purified by flash column
chromatography on silica gel with hexane and ethyl acetate
=
=
C CHCH2CH2), 2.49 (2 H, m, C CHCH2CH2), 3.51 and 3.81
=
(2 H, m, (CH2)3CH2O), 4.17 (2 H, AB system, J 17.0, O CCH2O),
=
4.65 (1 H, t, J 3.5, OCHO) and 5.08 (1 H, m, CH3C CH);
dC (100 MHz, C2HCl3) 17.67 (CH3), 19.14 (OCHCH2CH2CH2),
=
22.07 and 39.16 (C CHCH2CH2), 25.29 (OCHCH2CH2CH2),
25.70 (CH3), 30.26 (OCHCH2CH2CH2), 62.28 ((CH2)3CH2O),
=
=
72.06 (O CCH2O), 98.69 (OCH(CH2)3), 122.61 (CH3C CH),
+
=
=
132.91 (CH3C CHCH2) and 208.60 (C O); m/z (CI ) 244.2 (10%,
[M + NH4]+) and 102.1 (100).
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1-Hydroxy-6-methylhept-5-ene-2-one (21)
25 as a minor by-product (56 mg, 6%) followed by 25 (E isomer)
as a pale yellow oil (640 mg, 68%); Rf 0.27 (hexane : EtOAc =
9 : 1); HRMS (ES+, [M + H]+) found 235.0327, C9H1679BrO2
requires 235.0328; nmax (thin film)/cm-1 2977.6, 1713.6, 1648.9,
1438.4, 1368.1, 1272.6, 1217.4, 1147.1, 1041.7, 951.3, 865.9 and
810.7; dH (500 MHz, C2HCl3) 1.30 (3 H, t, J 7.0, CH2CH3), 2.06
To a stirred solution of 20 (0.95 g, 4.20 mmol) in ethanol
(40 cm3) under an atmosphere of N2 was added pyridinium p-
toluenesulfonate (0.11 g, 0.42 mmol) and the solution was stirred
at room temperature for 16 h and then at 50 ◦C for a further 1 h.
The solvent was then removed under reduced pressure and the
residue was dissolved in ethyl acetate (40 cm3). The solution was
washed with water (40 cm3) and brine (40 cm3), dried (MgSO4),
filtered and then concentrated under reduced pressure. Purification
of the crude product by flash column chromatography on silica
gel with hexane and ethyl acetate (4 : 1) as eluent gave 21 as a
light yellow oil (0.57 g, 96%); TLC Rf 0.24 (hexane : EtOAc =
4 : 1); HRMS (ES+, [M + NH4]+) found 160.1330, C8H18NO2
requires 160.1332; nmax (thin film)/cm-1 3440.6, 2969.2, 2917.1,
1720.4, 1441.8, 1405.8, 1378.2, 1276.3, 1068.1, 996.0 and 829.2; dH
(500 MHz, C2HCl3) 1.62 (3 H, s, CH3), 1.68 (3 H, s, CH3), 2.32 (2 H,
=
(2 H, m, BrCH2CH2), 2.18 (3 H, d, J 1.0, CH3C CH), 2.32 (2 H,
t, J 8.0, BrCH2CH2CH2), 3.42 (2 H, t, J 7.5, BrCH2), 4.16 (2 H,
=
q, J 7.0, CH2CH3) and 5.72 (1 H, m, C CH); dC (125 MHz,
2
=
C HCl3) 14.30 (CH2CH3), 18.67 (CH3C CH), 30.25 (BrCH2CH2),
32.61 (BrCH2), 38.99 (BrCH2CH2CH2), 59.61 (CH2CH3), 116.65
+
=
=
=
(C CH), 157.51(C CH) and 166.57 (C O); m/z (CI ) 252.0
(100%, [M(79Br) + NH4]+) and 254.0 (100, [M(81Br) + NH4]+).
Z-isomer; Rf 0.35 (hexane : EtOAc = 9 : 1); HRMS (ES+,
[M + H]+) found 235.0328, C9H16O279Br requires 235.0328; nmax
(thin film)/cm-1 2978.7, 1713.4, 1649.9, 1439.6, 1366.7, 1349.0,
1222.7, 1149.6, 1044.4 and 868.0 cm-1; dH (500 MHz, C2HCl3)
=
=
q, J 7.0, C CHCH2CH2), 2.44 (2 H, t, J 7.0, C CHCH2CH2), 3.17
(1 H, t, J 7.0, OH), 4.23 (2 H, d, J 7.0, CH2OH) and 5.05 (1 H,
=
1.28 (3 H, t, J 7.0, CH2CH3), 1.91 (3 H, d, J 1.5, CH3C CH),
2.05 (2 H, m, BrCH2CH2), 2.74 (2 H, t, J 8.0, BrCH2CH2CH2),
m, CH3C CHCH2); dC (125 MHz, C2HCl3) 17.63 (CH3), 22.39
=
3.44 (2 H, t, J 7.0, BrCH2), 4.15 (2 H, q, J 7.0, CH2CH3) and 5.71
=
=
(C CHCH2CH2), 25.62 (CH3), 38.48 (C CHCH2CH2), 68.26
2
=
(1 H, d, J 1.5, C CH); dC (125 MHz, C HCl3) 14.30 (CH2CH3),
=
=
=
(CH2OH), 121.94 (C CH), 133.57 (C CH) and 209.55 (C O);
=
18.65 (CH3C CH), 31.39 (BrCH2CH2), 32.24 (BrCH2CH2CH2),
m/z (CI+) 160.2 (45%, [M + NH4]+) and 127.1 (100).
=
=
33.24 (BrCH2), 59.58 (CH2CH3), 117.19 (C CH), 158.22 (C CH)
79
+
=
and 166.16 (C O); m/z (CI+) 252.0 (60%, [M( Br) + NH4] ) and
5-Bromopentan-2-one (24)21,22
254.0 (60, [M(81Br) + NH4]+), 174.0 (100).
To a stirred solution of a-acetylbutyrolactone (23) (6.46 cm3,
60.0 mmol) in toluene (40 cm3) was added hydrobromic acid (48%
in H2O, 10.2 cm3, 90.0 mmol). The mixture was then heated at
80 ◦C for 14 h under N2. Water (30 cm3) and diethyl ether (30 cm3)
were added and the organic layer was separated. The aqueous layer
was extracted with diethyl ether (2 ¥ 20 cm3) and the combined
ethereal extracts were washed with water (2 ¥ 20 cm3) and brine
(20 cm3), dried (MgSO4), filtered and then concentrated under
reduced pressure. Purification by flash column chromatography
on silica gel with hexane and ethyl acetate (4 : 1) as eluent gave 24
as a pale yellow oil (8.7 g, 88%); Rf 0.25 (hexane : EtOAc = 4 : 1);
HRMS (EI+, [M(79Br)]+) found 163.9829, C5H979BrO requires
163.9831; nmax (thin film)/cm-1 2963.8, 1714.7, 1434.6, 1367.2,
1301.3, 1247.1, 1179.0, 908.6 and 735.0; dH (500 MHz, C2HCl3)
2.12 (2 H, quintet, J 7.0, CH2CH2CH2), 2.17 (3 H, s, CH3), 2.65
(E)-6-Bromo-3-methyl-hex-2-en-1-ol (26)
To a stirred solution of 25 (1.50 g, 6.40 mmol) in anhydrous THF
(60 cm3) at -78 ◦C (dry ice-acetone bath) under an atmosphere
of nitrogen was added dropwise diisobutylaluminium hydride
(1 M solution in hexanes, ◦15.4 cm3, 15.4 mmol). The resulting
mixture was stirred at -78 C for 2 h and then allowed to warm
◦
to 0 C. Saturated aqueous potassium sodium tartrate (50 cm3)
and diethyl ether (50 cm3) were added. The mixture was stirred at
room temperature for another 30 min, and the organic layer was
separated. The aqueous layer was extracted with diethyl ether (2 ¥
30 cm3) and the combined ethereal extracts were washed with brine
(30 cm3), dried (MgSO4), filtered and then concentrated under
reduced pressure. Purification by flash column chromatography
on silica gel with hexane and ethyl acetate (2 : 1) as eluent
gave 26 as a light yellow oil (1.20 g, 97%); Rf 0.29 (hexane :
EtOAc = 2 : 1); nmax (thin film)/cm-1 3327.9, 2936.0, 1668.1, 1437.5,
1382.1, 1284.6, 1244.0, 1202.3, 1092.2, 1000.9 and 865.9 cm-1; dH
(500 MHz, C2HCl3) 1.61 (3H, s, CH3), 1.92 (2 H, quintet, J 7.0,
BrCH2CH2), 2.11 (2 H, t, J 7.0, BrCH2CH2CH2), 3.33 (2 H, t,
=
(2 H, t, J 7.0, CH2C O) and 3.45 (2 H, t, J 6.5, BrCH2); dC
(125 MHz, C2HCl3) 26.37 (CH2CH2CH2), 30.07 (CH3), 33.28
+
=
=
(BrCH2), 41.43 (CH2C O) and 207.30 (C O); m/z (EI ) 165.9
(40%, [M(81Br)]+), 164.0 (40, [M(79Br)]+) and 92.8 (100).
(E)-Ethyl 6-bromo-3-methyl-hex-2-enoate (25)
J 7.0, BrCH2), 4.10 (2 H, d, J 7.0, CH2OH) and 5.39 (1 H, m,
2
=
To a stirred suspension of sodi◦um hydride (0.12 g, 4.80 mmol) in
anhydrous THF (10 cm3) at 0 C was added dropwise triethyl 2-
phosphonoacetate (0.95 cm3, 4.80 mmol) and a solution of 24
(0.66 g, 4.00 mmol) in anhydrous THF (5 cm3). The reaction
mixture was stirred for 14 h under an atmosphere of nitrogen.
Water (20 cm3) and diethyl ether (20 cm3) were added and the
organic layer was separated. The aqueous layer was extracted
with diethyl ether (2 ¥ 15 cm3) and the combined ethereal extracts
were washed with water (2 ¥ 10 cm3) and brine (10 cm3), dried
(MgSO4), filtered and then concentrated under reduced pressure.
Purification by flash column chromatography on silica gel with
hexane and ethyl acetate (9 : 1) as eluent gave the Z isomer of
C CH); dC (125 MHz, C HCl3) 16.19 (CH3), 30.63 (BrCH2CH2),
33.18 (BrCH2), 37.71 (BrCH2CH2CH2), 59.29 (CH2OH), 124.61
+
79
+
=
=
(C CH), 137.76 (C CH); m/z (EI ) 192.0 (100%, [M( Br)] ) and
194.0 (100%, [M(81Br)]+).
(E)-(6-Bromo-3-methylhex-2-en-1-yloxy)-tetrahydro-
2H-pyran (27)
To a stirred solution of 26 (2.82 g, 14.6 mmol) and 3,4-dihydro-
2H-pyran (2.68 cm3, 29.2 mmol) in CH2Cl2 (80 cm3) under N2 at
0 ◦C was added p-toluenesulfonic acid (0.14 g, 0.74 mmol), and the
mixture stirred for 16 h whilst warming to room temperature. The
972 | Org. Biomol. Chem., 2009, 7, 962–975
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solution was then diluted with diethyl ether (80 cm3) and washed
with a saturated aqueous sodium hydrogencarbonate solution
(50 cm3). The aqueous layer was extracted with diethyl ether
(2 ¥ 50 cm3) and the combined ethereal extracts were washed
with water (50 cm3) and brine (50 cm3), dried (MgSO4), filtered
and then concentrated under reduced pressure. Purification by
flash column chromatography on silica gel with hexane and ethyl
acetate (9 : 1) as eluent gave 27 as a yellow oil (3.72 g, 86%); Rf 0.23
(hexane : EtOAc = 9 : 1); HRMS (ES+, [M(79Br) + NH4]+) found
294.1065, C12H2579BrNO2 requires 294.1063; nmax (thin film)/cm-1
2940.1, 2865.2, 1669.3, 1439.8, 1383.6, 1353.0, 1245.5, 1199.8,
1116.8, 1076.2, 1023.2, 905.6, 868.8 and 813.8 cm-1; dH (500 MHz,
warm to -20 ◦C over 1 h and maintained at -20 ◦C for 2 h.
The reaction was then quenched by addition of diethyl ether
(20 cm3) and water (20 cm3). The aqueous layer was extracted
with diethyl ether (3 ¥ 20 cm3) and the combined ethereal extracts
were washed with brine (20 cm3), dried (MgSO4) filtered and
then concentrated under reduced pressure. Purification by flash
column chromatography on silica gel with hexane and ethyl acetate
(30 : 1) as eluent gave 29 as a light yellow oil (0.39 g, 30% 3
steps); TLC Rf 0.33 (hexane : EtOAc = 30 : 1); HRMS (ES+,
[M + Na]+) found 347.2360, C20H33FNaO2 requires 347.2357; nmax
(thin film)/cm-1 2937.9, 1739.2, 1667.5, 1443.3, 1380.6, 1261.1,
1200.2, 1134.2, 1117.5, 1075.9, 1024.1, 974.6, 904.5, 869.4 and
813.4; dH (500 MHz, C2HCl3) 1.43–1.78 (6 H, m, (CH2)3), 1.53
(3 H, s, CH3), 1.60 and 1.62 (6 H, 2 ¥ s, 2 ¥ CH3), 1.98–2.17 (8 H,
C2HCl3) 1.60–1.85 (6 H, m, CH(CH2)3), 1.69 (3 H, s, CH3 CH),
=
2.00 (2 H, quintet, J 7.0, BrCH2CH2), 2.19 (2 H, t, J 7.0,
BrCH2CH2CH2), 3.40 (2 H, t, J 7.0, BrCH2), 3.51 and 3.88 (2 H,
=
m, 2 ¥ C CHCH2CH2), 3.42 and 3.80 (2 H, m, (CH2)3CH2O),
=
=
m, (CH2)3CH2O), 4.01 and 4.23 (2 H, m, C CHCH2O), 4.62 (1 H,
3.93 (1 H, dd, J 12.0, J 7.0, C CHCH2O), 4.15 (1 H, dd, J 12.0,
2
=
=
J 6.5, C CHCH2O), 4.55 (1 H, t, J 3.5, OCHO), 4.77 (2 H, d,
m, OCHO) and 5.42 (1 H, m, C CH); dC (125 MHz, C HCl3) 16.34
=
(CH3), 19.61, 25.49, and 30.66 ((CH2)3CH2O), 30.71 (BrCH2CH2),
JH-F 48.0, CH2F), 5.03 (1 H, m, (CH3)2C CH), 5.30 (1 H, m,
=
=
33.25 (BrCH2), 37.80 (BrCH2CH2CH2), 62.32 ((CH2)3CH2O),
C CHCH2O) and 5.35 (1 H, td, J 7.5 and 3.0, FCH2C CH);
dC (125 MHz, C2HCl3) 16.39 (CH3), 17.69 (CH3), 19.61, 25.51
and 30.72 ((CH2)3), 25.67 (CH3), 25.94 (d, JC-F 1.25), 26.88, 34.81
=
=
63.58 (C CHCH2O), 97.95 (OCHO), 121.98 (C CH) and 138.13
+
79
+
=
(C CH); m/z (EI ) 294.2 (50%, [M( Br) + NH4] ), 296.2 (50,
[M(81Br) + NH4]+) and 102.1 (100).
and 39.58 (d, JC-F 2.5) (2 ¥ C CHCH2CH2), 61.28 ((CH2)3CH2O),
63.58 (C CHCH2O), 79.71 (d, JC-F 158.75, CH2F), 97.87 (OCHO),
=
=
=
=
121.29 (C CHCH2O), 123.85 (CH3)2C CH), 130.72 (d, JC-F 10.0,
2-[(2E,6Z)-3,11-Dimethyl-7-fluoromethyl-dodeca-2,6,10-
trien-1-yloxy]-tetrahydro-2H-pyran (29)
=
=
FCH2C CH), 131.78 and 139.27 (2 ¥ C CH) and 134.65 (d, J
2
=
14.0, FCH2C CH); dF (282 MHz, C HCl3) -214.38 (t, JH-F 48.5);
To a stirred solution of 21 (0.57 g, 4.01 mmol) in anhydrous
CH2Cl2 (30 cm3) under an atmosphere of nitrogen was added
2,6-lutidine (1.11 cm3, 9.56 mmol) and the solution was cooled to
0 ◦C. Trifluoromethanesulfonic anhydride (0.81 cm3, 4.81 mmol)
was added dropwise and the solution was stirred at 0 ◦C for 45 min
after which time the solvent was removed under reduced pressure.
The residue was dissolved in ethyl acetate (20 cm3) and washed with
10% aqueous CuSO4 solution (20 cm3), 10% aqueous potassium
hydrogencarbonate (20 cm3) and brine (20 cm3). After drying over
anhydrous MgSO4 the solution was filtered and then concentrated
under reduced pressure to give the intermediate triflate as a red
oil. This was immediately dissolved in anhydrous THF (30 cm3)
and the flask flushed with nitrogen. Tetrabutylammonium fluoride
(1 M solution in THF, 8.02 cm3, 8.02 mmol) was added drop-wise,
and the solution was stirred for 1.5 h. The solution was then
concentrated under reduced pressure and directly purified by flash
column chromatography on silica gel with hexane and ethyl acetate
(19 : 1) as eluent. Due to the volatility of the fluoro ketone 22, the
solvent was not evaporated totally and the product was used in
next step directly.
A mixture of 27 (6.05 g, 21.8 mmol) and triphenylphosphine
(8.59 g, 32.8 mmol) in anhydrous acetonitrile (100 cm3), under an
atmosphere of nitrogen, was heated under reflux for 14 h. After
cooling, the solvent was concentrated under reduced pressure to
afford an oily mixture. Excess triphenylphosphine was removed
by triturating the mixture with anhydrous diethyl ether, and the
sticky residue was dried in vacuo for 5 h to afford the intermediate
phosphonium salt 28 as a viscous oil. To a stirred solution of
triturated 28 (1.51 g, 2.80 mmol) in anhydrous THF (40 cm3)
under N2 at -78 ◦C, was added lithium hexamethyldisilazide (1 M
solution in THF, 2.80 cm3, 2.80 mmol), the mixture was stirred
for 30 min at -78 ◦C, and then the solution of 22 prepared earlier
was added dropwise. The whole reaction mixture was allowed to
m/z (CI+) 342.4 (20%, [M + NH4]+) and 102.1 (100).
(2E,6Z)-7-Fluoromethyl-3,11-dimethyl-dodeca-2,6,10-
triene-1-ol (30)
This compound was prepared and purified in a manner identical
to that described for the alcohol 21, using the THP ether 29 (0.16 g,
0.49 mmol) to give the title compound as a colourless oil (0.11 g,
94%); TLC Rf 0.21 (hexane : EtOAc = 4 : 1); HRMS (EI+, M+)
found 240.1889, C15H25FO requires 240.1889; nmax (thin film)/cm-1
3337.4, 2966.8, 2920.9, 1667.4, 1445.1, 1380.2, 1241.6, 1103.5,
978.2, 825.5 and 741.7; dH (500 MHz, C2HCl3) 1.62 (3 H, s, CH3),
1.69 (3 H, CH3), 1.70 (3 H, CH3), 2.07–2.24 (8 H, m, 2 ¥ CH2CH2),
4.16 (2 H, d, J 7.0, CH2OH), 4.86 (2 H, d, JH-F 48.0, CH2F),
=
=
5.11 (1 H, m, (CH3)2C CH) and 5.42 (2 H, m, 2 ¥ C CHCH2);
dC (125 MHz, C2HCl3) 16.25 (CH3), 17.64 (CH3), 25.67 (CH3),
25.96 (d, JC-F 2.5), 26.86, 34.82 (d, JC-F 1.5) and 39.43 (d, JC-F
2.5) (2 ¥ CH2CH2), 59.30 (CH2OH), 79.72 (d, JC-F 160.0, CH2F),
=
=
123.81 (CH3)2C CH), 124.06 (C CHCH2OH), 130.68 (d, JC-F 9.0,
=
=
FCH2C CH), 131.85 and 138.78 (2 ¥ C CH) and 134.73 (d, JC-F
2
=
15.0, FCH2C CH); dF (282 MHz, C HCl3) -214.03 (t, JH-F 48.5);
m/z (EI+) 240.2 (1%, M+) and 69.1 (100).
(2E,6Z)-Trisammonium 3,11-dimethyl-7-fluoromethyl-
dodeca-2,6,10-trien-1-yl diphosphate (1c)
This compound was prepared in a manner identical to that for
the diphosphate 1b, using the alcohol 30 (0.10 g, 0.42 mmol) to
give the 1c as a white fluffy solid (85.2 mg, 45%); HPLC tR
=
36.17 min; purity 98.20% by analytical RP HPLC; HRMS (ES-,
[M - H]-) found 399.1124, C15H26FO7P2 requires 399.1138; nmax
(KBr disc)/cm-1 2898.3, 1729.0, 1669.0, 1494.4, 1457.6, 1412.6,
1199.9, 1123.0, 1079.8, 1022.1, 909.2, 804.5, 721.0 and 597.1; dH
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(500 MHz, 2H2O at pH 8.5 buffered with N2H4O2H) 1.47 (3 H, s,
CH3), 1.55 (3 H, s, CH3), 1.57 (3 H, s, CH3), 1.96–2.13 (8 H, m, 2 ¥
CH2CH2), 4.32 (2 H, t, J 6.5, CH2O), 4.83 (2 H, d, JH-F 47.0, CH2F),
the following signals 1.18 (s), 1.36 (br s), 1.39 (very broad), 1.50 (s),
1.65 (s), 1.66 (s), 1.70–1.80, 1.84–2.34 (broad spread of multiplets),
2.62–2.69 (br m), 4.51 (br s), 4.55 (br s), 4.66–4.90 (br m), 4.97–
5.04 (br m) and 5.20–5.32 (br m); GC-MS tr 29.1 min; m/z (EI+)
222.2 (20%, M+), 207.2 (68, [M - CH3]+), 202.2 (42, [M - HF]+),
193.1 (8, [M - CH3 - CH2]+), 187.2 (61, [M - CH3 - HF]+), 179.1
(60, [M - CH3 - CH2CH2]+), 173.1(20), 165.1 (12), 159.1 (91),
145.1 (60), 131.1 (61), 126.1 (25), 119.1 (60), 105.1 (100), 91.1 (95),
79.1 (69) and 67.1 (48).
=
=
5.05 (1 H, b, (CH3)2C CH), 5.32 (1 H, t, J 6.5, C CHCH2O) and
2
=
5.44 (1 H, m, FCH2C CH); dC (125 MHz, H2O at pH 8.5 buffered
with N2H4O2H) 15.63 (CH3), 17.98 (CH3), 24.85 (CH3), 25.47 (d,
JC-F 2.5), 26.13, 34.10 and 38.85 (d, JC-F 4.0) (2 ¥ CH2CH2), 62.48
(d, JC-P 16.5, CH2O), 81.01 (d, JC-F 151.5, CH2F), 120.27 (d, JC-P
=
=
=
9.0, C CHCH2O), 124.03 ((CH3)2C CH), 132.60 (FCH2C CH),
=
=
133.77 and 142.03 (2 ¥ C CH) and 134.09 (d, JC-F 12.5, C CH2F);
dF (282 MHz, 2H2O at pH 8.5 buffered with N2H4O2H) -210.48 (t,
Cope rearrangement of 3c
J
H-F 48.0); dP (122 MHz, 2H2O at pH 8.5 buffered with N2H4O2H)
-7.54 (d, JP-P 21.0) and -10.38 (d, JP-P 21.0); m/z (ES-) 399.1
(100%, [M - H]).
The NMR sample containing the product isolated after incubation
of 14-fluoro-farnesyl diphosphate 1c ◦with aristolochene synthase
(as described above) was heated at 90 C for 2 h. After cooling 1H,
13C, and 19F NMR analysis was then performed on the product
along with COSY and HSQC analysis. Some minor impurities
were evident and so the compound was purified by preparative thin
layer chromatography (eluting with pentane, Rf = 0.6) yielding 31c
as a colourless oil, 4 mg. dH (500 MHz, C2HCl3) 1.31–1.37 (1 H,
Incubation of 1b and 1c with PR-AS
Incubation of the FPP analogues was carried out using a
modification to the procedure of Miller et al.8 Silylated glassware
was used throughout to minimise the risk of any acid catalysed
rearrangements of terpenoid products taking place.
1
1
m, of CH2), 1.44–1.54 (3 H, m, CH2 and CH2), 1.66 (3 H, s,
2
2
1
2
CH3), 1.68 (3 H, s, CH3), 1.89–1.96 (3 H, m, CH and of CH2),
6-Fluorogermacrene A (3b)
2.05 (1 H, dt, J 11.5 and 5.0, CH2CHCH2), 4.42 and 4.60 (1 H,
1
2 ¥ d, AB system, JHH 9.5, of CH2F), 4.51 and 4.69 (1 H, 2 ¥
2
To a stirred solution of 6-fluorofarnesyl diphosphate (1b) (10 mg,
22 mM) in assay buffer (25 mM Tris, 5 mM MgCl2 and 5 mM
EDTA, 15% (v/v) glycerol, 22 cm3) was added aristolochene
synthase solution (250 mm3 of a 40 mM solution) followed by
pentane (5 cm3). This mixture was gently agitated in a shaker at
room temperature in a sealed flask for 24 h. A further portion
of enzyme solution was added and shaking was continued for a
further 24 h. Pentane (10 cm3) was added and the layers were
separated. The aqueous layer was further extracted with pentane
(2 ¥ 20 cm3) then the pooled pentane layers were dried over a
mixture of anhydrous Na2SO4 and basic alumina. The solution
was then filtered through glass wool and then the pentane was
carefully removed by evaporation under a stream of argon. 3b was
isolated as a colourless oil (~2 mg, 40%) with two minor impurities
being evident from GCMS and NMR spectrometry.
GCMS, tr = 27.4 (major product m/z (EI+) = 222), 28.0 (m/z
(EI+) = 222) and 29.4 min (m/z (EI+) = 240); 1H NMR spectrum
was identical to that reported by Faraldos et al. for the opposite
enantiomer,10 except with minor impurities evident as discussed
in the text, (see Fig. 3); dF (282 MHz, C2HCl3) -112.9 (t, JHF 46);
m/z (EI+) 222.2 (20%, M+), 207.2 (28, [M - CH3]+), 202.2 (4, [M -
HF]+), 193.2 (15, [M - CH3 - CH2]+), 187.2 (16, [M - CH3 - HF]+),
179.2 (48, [M - CH3 - CH2CH2]+), 173.2 (3), 165.1 (12), 159.1 (21),
151.1 (18), 145.1 (11), 139.1 (17), 125.1 (28), 119.1 (29), 105.1 (28),
99.1 (24), 93.1 (42), 85.0 (11), 79.1 (35) and 68.1 (100).
1
2
1
2
=
=
d, AB system, JHH 9.0, of CH2F), 4.57 (1 H, br s, of C CH2),
4,82 (1 H, br s, of C CH2), 5.00 (1 H, d, JHH 8.5, of HC CH2),
1
2
1
1
2
=
=
5.03 (1 H, br s, of HC CH2) and 5.73 (1 H, dd, JHH 11 and 17.5,
2
CH CH2); dF (282 MHz, C HCl3) -231.2 (t, JHF 48.0); m/z (EI+)
2
=
222.2 (3%, M+), 207.2 (80, [M - CH3]+), 202.2 (1, [M - HF]+),
193.1 (12, [M - CH3 - CH2]+), 189.2 (41), 179.1 (42), 173.1 (4),
165.1 (15), 161.1 (22), 147.1 (38), 133.1 (40), 126.1 (35), 121.1 (50),
119.1 (50), 111.1 (18), 105.1 (79), 93.1 (100), 79.1 (80) and 67.1
(52).
Acknowledgements
This work was supported by the UK’s Engineering & Physical
Sciences Research Council grant number EP/D069580/1. We
thank Dr. R. Jenkins for help with GC-MS and NMR experiments
and the EPSRC national mass spectrometry service, Swansea, for
some of the mass spectroscopic analyses.
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