Stereochemistry of eudesmane cation formation during catalysis by aristolochene synthase from Penicillium roqueforti

Article abstract of DOI:10.1039/b804198a

The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital and density functional theory calculations, a proton transfer mechanism has recently been proposed, in which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2) proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the elimination and protonation reactions was investigated by the analysis of the reaction products from incubation of 1 and of [12,12,12,13,13,13- ²H₆]-farnesyl diphosphate (15) with aristolochene synthase from Penicillium roqueforti (PR-AS) in H₂O and D₂O. The results reveal proton loss from C12 during the reaction and incorporation of another proton from the solvent. Incubation of 1 with PR-AS in D₂O led to the production of (6R)-[6-²H] aristolochene, indicating that protonation occurs from the face of the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds, along which protons could be delivered from the solvent to the active site.

Full text of DOI:10.1039/b804198a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Stereochemistry of eudesmane cation formation during catalysis by
aristolochene synthase from Penicillium roqueforti†
David J. Miller,a Jiali Gao,b Donald G. Truhlar,b Neil J. Young,a Veronica Gonzaleza and Rudolf K. Allemann*a
Received 11th March 2008, Accepted 28th March 2008
First published as an Advance Article on the web 28th April 2008
DOI: 10.1039/b804198a
The aristolochene synthase catalysed cyclisation of farnesyl diphosphate (1) has been postulated to
proceed through (S)-germacrene A (3). However, the active site acid that reprotonates this neutral
intermediate has so far proved difficult to identify and, based on high level ab initio molecular orbital
and density functional theory calculations, a proton transfer mechanism has recently been proposed, in
which proton transfer from C12 of germacryl cation to the C6,C7-double bond of germacryl cation (2)
proceeds either directly or via a tightly bound water molecule. In this work, the stereochemistry of the
elimination and protonation reactions was investigated by the analysis of the reaction products from
incubation of 1 and of [12,12,12,13,13,13-2H6]-farnesyl diphosphate (15) with aristolochene synthase
from Penicillium roqueforti (PR-AS) in H2O and D2O. The results reveal proton loss from C12 during
the reaction and incorporation of another proton from the solvent. Incubation of 1 with PR-AS in D2O
led to the production of (6R)-[6-2H] aristolochene, indicating that protonation occurs from the face of
the 10-membered germacrene ring opposite the isopropylidene group. Hence these results firmly
exclude proton transfer from C12 to C6 of germacryl cation. We propose here Lys 206 as the general
acid/base during PR-AS catalysis. This residue is part of a conserved network of hydrogen bonds,
along which protons could be delivered from the solvent to the active site.
cascade that leads to the generation of two 6-membered rings,
Introduction
three chiral centres, and two double bonds with high regio- and
Tens of thousands of terpene natural products have been described
stereospecificity (Scheme 1). Concurrent to diphosphate expulsion
to date, all of which derive from only a small number of linear
precursors. The 15-carbon isoprenoid, farnesyl diphosphate (FPP,
PR-AS facilitates attack of C1 in FPP (1) by the C10, C11 double
bond to produce germacryl cation (2). Proton loss from C12 leads
1) is the biogenetic precursor of more than 300 different sesquiter-
pene hydrocarbon scaffolds in plants, bacteria and fungi.1–6
Cyclisation of 1 (and other isoprenyl diphosphates) is catalysed by
terpene synthases that rely on a common mainly a-helical structure
known as the class I terpene synthase fold to promote one of
the arguably most complex biochemical reactions.4,5 Unlike most
other biochemical reactions, the chemistry of terpene cyclases
is mainly dependent on the generation and stabilisation of
carbocationic intermediates. A common mechanistic feature of
all class I terpene synthases is the initial, metal ion-dependent
cleavage of the alkyl diphosphate bond generating a putative
carbocation intermediate prior to cyclisation. In the absence of
the diphosphate group, a strong acid would be required to activate
the olefinic substrate.
Aristolochene synthase from Penicillium roqueforti (PR-AS)
chaperones its linear achiral substrate FPP along a complex
reaction pathway to produce (+)-aristolochene in a cyclisation
aSchool of Chemistry, Main Building, Cardiff University, Park Place, Cardiff,
UK CF10 3AT. E-mail: allemannrk@cf.ac.uk
bDepartment of Chemistry and Minnesota Supercomputing Institute, Uni-
versity of Minnesota, 207 Pleasant St SE, Minneapolis, MN, 55455-0431,
USA
1
† Electronic supplementary information (ESI) available: H NMR spec-
trum of (+)-aristolochene isolated from incubation of PR-AS and FPP. See
DOI: 10.1039/b804198a
Scheme 1
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to the production of (S)-germacrene A (3),7,8 which was then
postulated to undergo reprotonation of the C6, C7 double bond
and a further cyclisation to form the bicyclic eudesmane cation
(4). Successive 1,2 hydride shift and methyl migration followed by
loss of HSi on C8 completes the generation of (+)-aristolochene
(7).7
A difficulty with the above mechanism through germacrene
A appears to be the reprotonation of the neutral intermediate
germacrene A, a step that requires strong acid to generate
eudesmane cation (4), which has been shown to be stabilised
through interactions with the p-system of the indole ring of Trp
334.9 Several candidates have been proposed for the acid that
reprotonates germacrene A (3), including a proton shuttle from
the solvent to Tyr 92 in the active site by way of Arg 200, Asp
203, and Lys 206,8 an unprecedented active site oxonium ion,10
or the diphosphate itself,11 but it has so far proved difficult to
find conclusive experimental support for any of these proposals.
We have recently published results from density functional theory
(DFT), semi-empirical and high level molecular orbital theory
calculations in the gas phase to study the cyclisation of FPP along
with molecular docking and modeling studies in the active site of
PR-AS, employing combined quantum mechanical and molecular
mechanical (QM/MM) methods. This has led to the proposal
of an alternative reaction pathway, in which protonation of the
C6,C7-double bond is accomplished by intramolecular proton
transfer from C12 of 2 (Scheme 2, pathway a) rather than through
the involvement of a general acid thereby bypassing the neutral
intermediate germacrene A (Scheme 1).12 In this mechanism the
direct intramolecular proton transfer had a computed barrier of
about 22 kcal mol−1, which was further lowered to 16–20 kcal
mol−1 by PR-AS. We now report results from experimental and
computational studies to test this intramolecular proton transfer
mechanism as well as an alternative pathway through a proton
shuttle from the isopropyl group of germacryl cation (2) to
the C6,C7-double bond via an enzyme bound water molecule
(Scheme 2, pathway b).
Scheme 2
Results and discussion
Direct proton transfer mechanism
Our computational analysis revealed that intramolecular proton
transfer from C12 in germacryl cation was a thermodynamically
feasible reaction for the generation of eudesmane cation during
PR-AS catalysis.12 To test whether the enzymatic reaction fol-
lowed this pathway, [12,12,12,13,13,13-2H6]-farnesyl diphosphate
(15) required preparation. This was achieved as outlined in
Scheme 3.
The procedure was a modification of that used to prepare
[12,12,12-2H3]-FPP.7 8-Hydroxygeranyl acetate 8 was brominated
Scheme 3 Reagents and conditions: (i) PBr3, CH2Cl2, 5 ◦C; (ii) PhSO2Na, DMF, 70% 2 steps; (iii) NaOH, MeOH–H2O, 91%; (iv) 3,4-DHP, p-TsOH,
CH2Cl2, 90%; (v) (EtO)2P(O)CH2CO2Et, NaH, 57%; (vi) LiAlH4, Et2O, 47%; (vii) MsCl, NEt3, LiBr, THF, −45 ◦C; (viii) n-BuLi, THF, HMPA, −78 ◦C,
36%; (xi) LiBHEt3, PdCl2dppf, THF, 58%; (x) PPTS, EtOH, 50 ◦C, 65%; (xi) MsCl, NEt3, LiBr, CH2Cl2, −45 ◦C then (Bu4N)3HP2O7, CH3CN followed
+
by NH4 cation exchange, 53%.
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with PBr3 in CH2Cl2 and then transformed into phenylsulfone 9a
by treatment with sodium benzenesulfinate in DMF. It was then
necessary to remove the acetate functionality by saponification
with methanolic sodium hydroxide and then to reprotect the
alcohol with a THP group to give 9b in 82% yield over these two
steps. In parallel to this d6-acetone 10 was transformed into hex-
adeuterated ester 11 by treatment with triethyl phosphonoacetate
and sodium hydride in anhydrous THF. Reduction of the ester
with DIBAL-H13 was followed by transformation into bromide 12
by treatment with MsCl and NEt3 at −45 ◦C in THF followed by
addition of LiBr. All these steps proceeded in moderate to good
yields. Coupling of bromide 12 and sulfone 9b was achieved by
treatment of a mixture of the two in anhydrous THF containing
10% HMPA with n-BuLi at −78 ◦C. Sulfone 13 was isolated
in 36% yield after chromatography. Final synthesis of 15 was
achieved by reductive desulfonylation of 13 in 58% yield using
LiBHEt3 solution as reductant and PdCl2dppf as catalyst followed
by acid catalysed removal of the THP group in ethanol and then
diphosphorylation of the resulting hexadeuterofarnesol 14 using
conditions previously reported.14,15
Incubation of 15 with PR-AS in the presence of 5 mM Mg2+
was followed by GC-analysis of the hexane-extractable reaction
products. This indicated the same distribution that has been
observed in incubations with unlabelled FPP. In addition to the
main product aristolochene, approximately 8% of germacrene
A was observed (Fig. 1). Direct proton transfer from C12 to
2
the C6,C7-double bond of 2 would generate H6-aristolochene
and a molecular ion peak m/z of 210 in the mass spectrum of
the product. However, the mass spectrum of the main reaction
product (and also the germacrene A by-product) was characterised
by a molecular ion peak m/z of 209 (Fig. 1) indicating that
conversion of germacryl cation to aristolochene occurred with
the replacement of one deuteron with a proton. This finding
is in agreement with an earlier report7 in which the reaction
products from an incubation of [12,12,12-2H3]-FPP with AS from
Aspergillus terreus were analysed by 2H-NMR spectroscopy and a
single olefinic peak at dD 4.71 ppm was identified.
Fig. 1 (a) Total ion chromatogram of the hexane-extractable products
arising from incubation of [12,12,12,13,13,13-2H6]-FPP 15 with PR-AS
and (b) the mass spectrum of the aristolochene product at 24.76 min.
removed from C12 to provide a possible model for the structure
of the transition state TS (Scheme 2). A relaxed potential energy
surface scan was performed using Gaussian03, revision B.0317 and
AM118 with the distance between the hydroxonium proton closest
to the C6,C7 p-bond varied and all other geometric parameters
allowed to adjust freely resulting in an approximate transition state
geometry. As had been observed for the direct proton transfer,12
there was broad agreement between the results obtained using
DFT (mPW1PW19 and MPWB1K20) and semi-empirical methods
(AM118 and PM321). Conversion of 2 to eudesmane cation (4)
via water mediated proton transfer was exergonic for all methods
(Table 1). The free energy of the transition state TS is between
15 and 18 kcal mol−1 higher than that of germacryl cation (2)
at the DFT level of theory, while semiempirical methods yielded
somewhat higher barriers. Compared to the results obtained for
direct proton transfer, the energy barrier was lowered by 7.4 and
11 kcal mol−1 for mPW1PW and MPWB1K (Table 1). The semi-
empirical methods, on the other hand, showed a slight increase in
the activation free energy (0.4 and 2.2 kcal mol−1 for AM1 and
PM3 respectively) but this may be due to the inadequacies in the
semi-empirical method.
Proton shuttle mechanism—gas phase calculations
The above observations ruled out the direct intramolecular proton
transfer from C12 to the C6,C7-double bond (Scheme 2a) previ-
ously suggested based on computation.12 However, the potential
absence of deuterium incorporation at C6 does not exclude a
transfer of the label from C12 to the C6,C7-double bond, but
may simply suggest an indirect mechanism in which the transfer
occurs by way of an active site general acid such as a bound water
molecule12 or the pyrophosphate group.11 A water molecule that
could serve as a proton relay had been identified in the model of
the transition state in the active site of PR-AS (Scheme 2, pathway
b) between the isopropyl group and the double bond. Such a
water molecule seemed ideally placed to deliver a proton to C6 of
germacryl cation from the Si-face.
To investigate this shuttle mechanism a set of semi-empirical
molecular orbital theory and DFT calculations were performed.
The starting point for the calculations was based on the previously
identified minimum energy structure for germacryl cation (2).12
Using Molden,16 a hydroxonium ion was placed in close proximity
to the lower face of the ten-membered ring and a proton was
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Table 1 Gibbs energy changes DG◦
of reaction intermediates and
transition state for the conversion of germacryl cation (2) to eudesmane
298
cation (4) via the potential proton shuttle mechanisma
DG◦298/kcal mol−1
2
TS
4
AM1
PM3
mPW1PW
MPWB1K
2.6
3.5
3.8
1.1
31.0
24.5
21.6
16.1
−16.2
−14.7
−25.2
−38.6
a For comparison to previous calculations, all energies are reported relative
to that of the calculated free energy of farnesyl cation (set to zero kcal
mol−1).12 A water molecule was also included in these calculations. The
Pople 6-31+G(d,p) basis set was used for the DFT calculations (mPW1PW
and MPWB1K).
Stereochemistry of eudesmane cation (4) generation
These computational results suggest that proton transfer from C12
to the C6,C7-double bond via a shuttle involving a water molecule
may be energetically more favourable than direct intramolecular
transfer. The incubations of PR-AS with [12,12,12,13,13,13-2H6]-
FPP described above are in agreement with such a water shuttle
mechanism since the expected kinetic isotope effect and potential
exchange with bulk solvent might result in the loss of the label.
To test whether proton incorporation at C6 was observed during
PR-AS catalysis, preparative scale incubation of FPP with PR-AS
in buffer made up in D2O (the pH was adjusted to 7.1 instead of
7.5 because of the 0.4 difference between pD and the measured
pH for solutions containing a high percentage of D2O) resulted
in the isolation of aristolochene characterised by a molecular ion
peak of 205 according to GC-MS analysis (Fig. 2) suggesting
deuteron uptake from the solvent. 1H-NMR spectroscopic analysis
of the product indicated the absence of the multiplet at dH = 2.02–
2.10 ppm (Fig. 3), which is one of the signals observed for the
two protons on C6 of aristolochene (Fig. 3a; Table 2). 2H-NMR
analysis of the same material showed a resonance at 2.1 ppm (ESI†)
indicating that a proton is delivered to C6 ultimately from the
solvent during the cyclisation reaction. Steady-state measurements
of the turnover of FPP by PR-AS in H2O and D2O showed a kinetic
solvent isotope effect of 1 indicating that the rate-limiting step does
not involve proton transfer. It is worthy of note that the 2H-NMR
spectrum of this material also showed a resonance at 5.3 ppm
(ESI†). This may correspond to the C8-proton of aristolochene
Fig. 2 (a) Total ion chromatogram of hexane extractable products
generated from incubation of PR-AS, FPP and Mg2+ in buffered 2H2O
and (b) EI+ mass spectrum of aristolochene eluting at 25.69 min.
7
possibly suggesting that the elimination of HSi from C8 in the
final step of PR-AS catalysis is a reversible process under the
reaction conditions used here. However, the appearance of this
peak is not matched by the disappearance of the corresponding
1
proton resonance in the H-NMR spectrum although there is a
small reduction in the integration of this peak relative to that of
2
the C12 resonance. The H-NMR spectrum is also noisy due to
the low level of material coupled with the inherent insensitivity of
2H-NMR spectroscopy and so the integration of these two peaks
may be unreliable. Furthermore there is not a significant quantity
of doubly deuterated aristolochene (m/z = 206) observed in the
mass spectrum (Fig. 2) so it seems most likely that deuterium
incorporation into C8 is happening at a low level at best.
Fig. 3 (a) 1H NMR spectrum (500 MHz) of and assignments for
aristolochene (7) isolated from incubation of FPP with PR-AS and Mg2+
;
the vinylic region of the spectrum (4.5–5.5 ppm) is not shown (see ESI†); (b)
1H NMR spectrum (500 MHz) of aristolochene isolated from incubation
of FPP with PR-AS and Mg2+ in 2H2O; the arrow indicates that a proton
on C6 is absent from the spectrum in this material. The carbon atoms in
aristolochene are numbered as in the parent molecule FPP (Scheme 1).
Solvent proton uptake to C6 is in agreement with both
proton transfer from C12 via a bound water molecule to the
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Table 2 Assignments for the 500 MHz 1H NMR spectrum of aristoloch-
ene (7)a
resulted in the enhancement of the resonances for protons on
C1 (dH 1.69–1.72), C4 (dH 1.24–1.32), C10 (dH 2.11–2.18), C15 (dH
0.77) and importantly to the signal at dH 2.02–2.10 ppm. Analysis
of the conformation of aristolochene, which is determined by
the equatorial-like orientation of the isopropylidene substituent
(Fig. 3), identified this resonance to be from HRe on C6, since
the axial proton is closest to C14 and therefore likely to display
a NOE. It is interesting to note that protonation of germacrene
A during catalysis by tobacco epi-aristolochene synthase (TEAS)
also occurs from the Re-face of the double bond at C6.22 Despite
minimal sequence similarity and strikingly different active site
contours,11 TEAS and PR-AS may use similar strategies to achieve
the protonation of (R)- and (S)-germacrene A for the generation
of stereochemically different products.
dH/ppm
Integration
Multiplicity
Assignment
0.77
0.90
1.10
3 H
3 H
1 H
1 H
1 H
2 H
1 H
3 H
1 H
1 H
2 H
d, J 6.5 Hz
CH3-15
CH3-14
H-1 (axial)
CH-3
H-4
CH2-5
s
t, J 13 Hz
1.18–1.21
1.24–1.31
132–1.38
1.61–1.65
1.67
1.69–1.72
1.76–1.84
1.92–1.96
m
m
m
m
s
m
m
m
H-4
CH3-13
H-1 (equatorial)
H-9
H-9 and H-6
(equatorial)
H-6 (axial)
H-10
2.02–2.10
2.11–2.18
4.64
1 H
1 H
2 H
1 H
m
m
m
d, J 5.5
CH2-12
CH-8
5.25
Conclusions
a The compound is numbered according to the numbering of the parent
molecule FPP (1). Assignments are based on COSY, HSQC, HMBC as
well as the observed coupling constants. All resonances are dH (500 MHz)
in ppm. Entries are chemical-shift range followed by multiplicity in
parentheses and coupling constants.
The results described here indicate that protonation of the C6,C7-
double bond in (S)-germacrene A occurs from the Re-face at C6
and hence rule out proton transfer from C12 to C6 either by
direct proton transfer or by a mechanism involving a mediating
water molecule. This observation is also in agreement with a large
body of experimental work that suggests that germacrene A is an
intermediate during AS catalysis.8,23,24
C6,C7-double bond or direct protonation of the double bond
from other solvent exchangeable groups such as amino acid side
chains, the diphosphate group or an as of yet unidentified water
molecule bound on the opposite side of the germacryl ring system.
Transfer via a bound water molecule however would necessarily
lead to a specific stereochemical outcome in that the proton must
be delivered from the lower (Si) face of the germacryl ring. In
this scenario, incubation of FPP with PR-AS in D2O should lead
to (6S)-[6-2H] aristolochene (Scheme 2), while protonation of C6
from the Re-face would exclude the water mediated proton transfer
(Scheme 2).
To distinguish the chemical shifts of the two diastereotopic
protons on C6, a sample of (+)-aristolochene generated from
a preparative scale incubation of 1 with PR-AS was analysed
by 1H difference NOE NMR experiments (Fig. 4). Selective
saturation of the resonance for the protons on C14 (dH 0.90)
The identity of the active site acid remains, therefore, elusive.
Tyr 92 had initially been proposed as the active site general acid
responsible for the protonation of germacrene A. However, site
directed mutagenesis experiments, in which Tyr 92 was replaced
with Phe, revealed that the mutant still produced substantial
amounts of aristolochene thereby ruling out Tyr 92 as the
obligatory active site acid.8,10 The elucidation of the X-ray crystal
structure of AS from Aspergillus terreus in which a diphosphate
ion was found bound to one of the subunits of the tetrameric
enzyme, together with comparisons with trichodiene synthase
and the monoterpene cyclase bornyl-diphosphate synthase, led
to the proposal that the diphosphate anion might be positioned to
function as a possible general acid/base during AS catalysis.11 In
the absence of evidence for this proposal, other than the proximity
of the diphosphate group to the C6,C7-double bond, we propose
here Lys 206 as the potential general acid/base during PR-AS
catalysis. Preliminary results from an analysis of the effects of
replacing Lys 206 are in agreement with this proposal. Substitution
with glutamine led to a reduction of the catalytic efficiency of
approximately 3 orders of magnitude, while only a 4-fold reduction
was observed when Lys 206 was replaced by arginine.25
Based on the X-ray structure of PR-AS, a hydrogen-bonding
network from the side chain of Lys 206 to Asp 203 and Arg 200
was identified.26 The hydrogen bonding network might provide
a proton shuttle from Arg 200 at the solvent exposed top of the
active site to the double bond at C6,C7 of germacrene A.8 Since the
e-amino group of Lys 206 is approximately equidistant to carbons
6 and 8 of germacrene A and eudesmane cation, it could not only
serve as the active site general acid but also abstract a proton
from 6 to produce 7. It is worthy of note that despite only 61%
sequence identity between the two fungal aristolochene synthases
from P. roqueforti and A. terreus, this network of hydrogen bonds
is conserved with Lys 181, Asp 178 and Arg 175 of AT-AS taking
the positions of Lys 206, Asp 203 and Arg 200 in PR-AS. A full
Fig. 4 (a) 600 MHz 1H NMR spectrum of PR-AS generated aristolochene
and (b) difference NOE spectrum of the same material with saturation of
the CH3-14 resonance. NOEs are evident to protons on C10 and C15 and
one proton each on C6, C1 and C4. The proton showing a NOE to CH3-14
on C6 is that replaced by deuterium when FPP and PR-AS are incubated
in buffered D2O (Fig. 3).
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study of the effects of replacing the residues in the network of
amino acids in PR-AS is currently under way.
(2.41 cm3, 25.6 mmol) in a dropwise fashion and stirring was
continued for 1 h. The solution was concentrated under reduced
pressure and the residue was partitioned between diethyl ether
(100 cm3) and a saturated sodium hydrogencarbonate solution
(100 cm3). The separated ethereal layer was successively washed
with water (100 cm3) andbrine (100 cm3) thendried over anhydrous
MgSO4 and filtered under reduced pressure. After concentration
under reduced pressure, the crude intermediate bromide was
dissolved in anhydrous dimethylformamide (50 cm3) and to this
stirred solution under nitrogen was added benzenesulfinic acid
sodium salt (3.82 g, 23.3 mmol). Stirring was continued for
16 h then the solution was diluted with diethyl ether (200 cm3).
The organic solution was successively washed with water (3 ×
200 cm3) and brine (200 cm3) then dried over anhydrous MgSO4.
After filtration and concentration under reduced pressure the oily
product was chromatographed on silica gel (eluting with 1 : 1
hexane–ethyl acetate) to give 9a as a colourless oil (5.46 g, 70%);
mmax/cm−1 (thin film) 3060 (w), 2971 (m), 2920 (m), 1732 (vs), 1669
(w), 1447 (s), 1385 (m), 1367 (m), 1307 (vs), 1234 (vs), 1134 (vs),
1086 (s), 1024 (m), 954 (m), 884 (w); dH (500 MHz, C2HCl3) 1.66
Experimental
General procedures
All chemicals were purchased from Sigma-Aldrich. Tetrahy-
drofuran (THF) and diethyl ether were distilled from
sodium–benzophenone ketyl under nitrogen. Acetonitrile,
dichloromethane, toluene and triethylamine were distilled from
calcium hydride under nitrogen. Ecoscint scintillation fluid was
purchased from National Diagnostics. 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. 31P and 13C NMR spectra
were measured on a Bruker Avance 500 NMR spectrometer
and are reported as chemical shift downfield from 85% H3PO4
and tetramethylsilane respectively, coupling constants in Hertz
where appropriate and assignment. Assignments are made to the
limitations of COSY, DEPT 90/135, gradient HSQC and gradient
HMBC spectra. 2H NMR spectra were recorded on a Jeol Eclipse
+300 NMR spectrometer. NOE difference spectra were recorded
on an Inova 600 MHz spectrometer using a selective excitation
pulsed field gradient 1D-NOESY pulse sequence. 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 recorded on a Micromass Q-
Tof micro mass spectrometer.
=
and 1.72 (2 × 3 H, 2 × s, 2 × CH3C CH), 1.88–1.91 and 2.03–2.15
(4 H, m, CH2CH2), 2.02 (3 H, s, CH3CO), 2.18 (2 H, s, O2SCH2),
=
4.57 (2 H, d, J 7.5, OCH2), 5.05 (1 H, t, J 6.5, C CH), 5.26 (1 H, tq,
=
J 7.5 and 1, C CH), 7.54–7.57, 7.64–7.68 and 7.85–7.87 (5 H, m,
2
=
ArCH); dC (125 MHz, C HCl3) 16.4 and 16.7 (2 × CH3C CH),
21.0 (CH3CO), 26.5 and 38.4 (CH2CH2), 61.2 and 66.2 (SCH2
and OCH2), 118.8, 128.5, 128.9, 133.5 and 135.4 (ArCH and 2 ×
=
=
C CH), 123.8, 138.5 and 141.2 (quaternary ArC and 2 × C CH),
+
+
=
171.1 (C O); m/z (ES ) 337 (100%, [M + H] ).
(E,E) 8-Benzenesulfonyl-3,7-dimethyl-1-(tetrahydro-2-
pyranyloxy)-octa-2,6-diene (9b)
A solution of 9a (5.27 g, 14.4 mmol) in a 10% solution of NaOH in
9 : 1 methanol–water (50 cm3) was stirred for 16 h. The ethanol was
removed under reduced pressure and the residue was partitioned
between ethyl acetate (50 cm3) and water (50 cm3). The separated
aqueous layer was extracted with ethyl acetate (2 × 50 cm3) and the
pooled organics were washed with brine (150 cm3) then dried over
anhydrous MgSO4. After concentration under reduced pressure
the residual oil was chromatographed on silica gel (eluting with
1 : 1 hexane–ethyl acetate) to give the intermediate alcohol as a
colourless oil (3.87 g, 91%).
Thin layer chromatography was performed on pre-coated alu-
minium plates of silica G/UV254 (Fluka). Flash chromatography
was performed according to the method of Still.27 Reverse phase
HPLC was performed on a system comprising of a Dionex P680
pump and a Dionex UVD170U detector unit.
Expression and purification of AS and AS-F112A in E. coli
To a stirred solution of the crude alcohol (3.69 g, 12.5 mmol) in
dichloromethane (50 cm3) was added 3,4-dihydropyran (2.29 cm3,
25 mmol) followed by p-toluene sulfonic acid (238 mg, 1.25 mmol).
Stirring was continued for 24 h then the solution was concentrated
under reduced pressure and the residue was dissolved in ethyl
acetate (50 cm3). This solution was washed successively with
saturated sodium hydrogencarbonate solution (50 cm3), water
(50 cm3) and brine (50 cm3). After drying over anhydrous MgSO4,
the solution was filtered and concentrated under reduced pressure.
The oily residue was chromatographed on silica gel (eluting with
3 : 1 hexane–ethyl acetate) to give 9b as a colourless oil (4.27 g,
90%); mmax/cm−1 (thin film) 2939 (s), 2866 (s), 1732 (w), 1667 (w),
1586 (w), 1446 (s), 1386 (m), 1355 (m), 1307 (s), 1260 (m), 1200
(m), 1134 (s), 1086 (s), 1023 (s), 904 (m), 869 (m), 814 (m), 742
(m), 726 (m), 690 (s); dH (500 MHz, C2HCl3) 1.54 and 1.69 (2 ×
Enzymes were produced in E. coli BL21(DE3) harbouring a
cDNA for AS under the control of◦the T7 promoter as previously
described.28 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. Proteins were then extracted from the inclusion bodies
and purified following our established protocols.8,28 Each enzyme
was pure as judged by SDS-gel electrophoresis.
(E,E) O-Acetyl 3,7-dimethyl-8-phenylsulfonyl-octa-2,6-diene-1-ol
(9a)
To a stirred solution of 829 (5.00 g, 23.3 mmol) in anhydrous
CH2Cl2 at 5 ◦C (ice–water bath) under N2 was added PBr3
3 H, 2 × s, 2 × CH3C CH), 1.44–1.52, 1.63–1.67, 1.73–1.80 and
=
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=
1.97–2.01 (10 H, m, OCH2(CH2)3 and C CHCH2CH2), 3.42–
3.46 and 3.79–3.84 (2 H, m, OCH2(CH2)3), 3.65 (2 H, s, SO2CH2),
was added as a solution in anhydrous THF (10 cm3) via a cannula
and this mixture was stirred at 5 ◦C (ice–water bath) for 1 h. The
reaction was quenched by the addition of cold brine (20 cm3) and
the mixture was extracted with diethyl ether (3 × 20 cm3). The
pooled ethereal extracts were washed with brine (50 cm3) then
dried over anhydrous MgSO4 and filtered under reduced pressure.
The ether was carefully evaporated to give the crude bromide as
a pale yellow oil that was immediately dissolved in anhydrous
THF (10 cm3) and added, via a cannula, to a solution of 9b
=
3.91 (1 H, dd, J 7 and 12, C CHCH2O), 4.15 (1 H, dd, J 6 and
12, C CHCH2O), 4.53 (1 H, t, J 3, OCHO), 4.96 (1 H, t, J 7,
C CH), 5.20 (1 H, t, J 6.5, C CH), 7.45–7.78 (5 H, m, ArCH);
=
=
=
2
=
dC (125 MHz, C HCl3) 16.3 and 16.7 (2 × CH3C CH), 19.7, 25.5,
26.6, 30.7 and 38.5 (CH2CH2 and OCH2(CH2)3), 62.4, 63.6 and
66.2 (SO2CH2 and 2 × OCH2), 98.0 (OCHO), 121.1, 128.5, 128.9,
=
133.5 and 135.7 (3 × ArCH and 2 × C CH), 123.6, 138.5 and
+
139.1 (quaternary ArC and 2 × C CH); m/z (ES ) 379 (100%,
(185 mg, 0.490 mmol) in anhydrous THF (10 cm3) and HMPA
=
3
[M + H+].
(2 cm ) under N2. Freshly activated powdered 4 A molecular
˚
sieves (∼200 mg) were added and the solution was cooled to
−78 ◦C (dry ice–acetone bath). To this stirred mixture was slowly
added, over 30 min, n-BuLi (2.5 M in hexanes, 196 mm3, 0.490
mmol) and then the mixture was stirred for 16 h whilst slowly
warming to room temperature. The reaction was quenched by the
addition of saturated ammonium chloride solution (20 cm3) and
the mixture was extracted with diethyl ether (3 × 20 cm3). The
pooled ethereal extracts were washed with water (2 × 50 cm3)
and brine (50 cm3) then dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (eluting with 4 : 1 hexane–ethyl
acetate) to give 13 as a colourless oil (77 mg, 36%); mmax/cm−1 (thin
film) 2936 (m), 2869 (m), 2130 (w), 1448 (m), 1302 (s), 1198 (m),
1142 (s), 1083 (s), 1020 (s), 722 (s); dH (500 MHz, C2HCl3) 1.53 and
1.58 (2 × 3 H, 2 × s, 2 × CH3), 1.43–1.59, 1.62–1.78 and 1.88–2.02
(10 H, m, OCH2(CH2)3 and CH2CH2), 2.50 (1 H, ddd, J 7, 11.5 and
15, CH2CHSO2), 2.74 (1 H, ddd, J 4, 7 and 15, CH2CHSO2), 3.40
(1 H, dd, J 4 and 11.5, CH2CHSO2), 3.42–3.45 and 3.79–3.83 (2 H,
Ethyl 3,3,3-[2H,2H,2H]-methyl [4,4,4-2H,2H,2H]-but-2-eneoate
(11)
To a stirred solution of triethyl phosphon◦oacetate (15.24 g,
68.0 mmol) in anhydrous THF (100 cm3) at 5 C (ice-water bath)
under N2 was added sodium hydride (80% dispersion in mineral
oil, 0.918 g, 30.6 mmol) in portions. After effervescence had ceased,
d6-acetone (100 atom% Sigma-Aldrich, 1.5 cm3, 20.4 mmol) was
added and the solution was stirred for 16 h whilst slowly warming
to room temperature. Water (100 cm3) was carefully added and
the mixture was extracted with diethyl ether (3 × 100 cm3).
The pooled ethereal extracts were washed with saturated sodium
hydrogencarbonate solution (200 cm3), water (200 cm3) and brine
(200 cm3) then dried over anhydrous MgSO4 and filtered under
reduced pressure. The title compound was obtained as a colourless
liquid by fractional distillation at atmospheric pressure collecting
the fraction boiling at 154–156 ◦C (5.17 g, 57%); mmax/cm−1 (thin
film) 2982 (m), 2130 (w), 1714 (s), 1634 (m), 1446 (w), 1366 (w),
1218 (s), 1154 (m), 1097 (m), 1055 (m), 788 (w); dH (500 MHz,
C2HCl3) 1.29 (3 H, t, J 7, CH2CH3), 4.16 (2 H, q, J 7, CH2CH3),
=
m, OCH2(CH2)3), 3.90 (1 H, dd, J 7 and 12, C CHCH2), 4.14
=
(1 H, dd, J 6 and 12, C CHCH2), 4.53 (1 H, dd, J 3 and 4, OCHO),
=
=
4.79 (1 H, t, J 7, C CH), 4.96 (1 H, t, J 7, C CH), 5.19 (1 H, t, J
5.69 (1 H, s, C CH); dC (125 MHz, C2HCl3) 14.30 (CH3), 59.4
7, C CH), 7.43–7.46, 7.53–7.56 and 7.73–7.75 (5 H, m, ArCH);
dC (125 MHz, C2HCl3) 13.7, 16.3 (2 × CH3), 19.7, 24.1, 25.5, 26.4,
=
=
+
=
=
=
(CH2), 116.2 (C CH), 156.1 (C CH), 166.7 (C O); m/z (EI )
134 (71%, M+), 116 (50), 106 (68), 89 (100), 61 (70).
30.7 and 38.5 (OCH2(CH2)3 and CH2CH2), 62.4 (OCH2(CH2)3),
=
63.7 (C CHCH2O), 74.1 (SO2CH), 98.0 (OCHO), 118.8, 121.0
=
and 135.4 (3 × C CH), 128.7, 128.9 and 133.3 (ArCH), 126.9,
(E,E) 12,12,12-(2H,2H,2H)-8-Benzenesulfonyl-3,7-dimethyl-11-
(2H,2H,2H)-methyl-1-(tetrahydro-2-pyranyloxy)-dodeca-2,6,10-
triene (13)30
=
134.6, 138.1 and 139.3 (ArC quaternary and 3 × C CH); m/z
(ES+) 451 (100%, [M + H]+).
To a stirred solution of 11 (536 mg, 4.00 mmol) in anhydrous
CH2Cl2 at −78 ◦C (dry ice–acetone bath) under N2 was added
DIBAL-H solution (1.0 M in hexanes, 12 cm3, 12 mmol) and the
mixture was stirred for 1 h at −78 ◦C. The reaction was quenched
(E,E) 12,12,12-[2H,2H,2H]-3,7-Dimethyl-11-[2H,2H,2H]-methyl-
dodeca-2,6,10-triene-1-ol (14)31,32
To a stirred solution of 13 (94 mg, 0.216 mmol) in anhydrous
THF (10 cm3) under N2 was added PdCl2dppf (35 mg, 43.2 mmol)
and the solution was stirred under N2 for 30 min. To this stirred
mixture was added, dropwise, lithium triethylborohydride (1.0 M
solution in THF, 432 mm3, 0.432 mmol), the solution immediately
turned a deep red colour. The complete mixture was stirred under
N2 for 2 h then water (10 cm3) was carefully added to quench
the reaction. The mixture was extracted with diethyl ether (3 ×
20 cm3) then the pooled ethereal extracts were washed with water
(2 × 20 cm3) and brine (20 cm3). The organic solution was then
dried over anhydrous MgSO4, filtered and evaporated to give a pale
red oil that was passed through a short column of silica (eluting
with 19 : 1 hexane–ethyl acetate) to yield the intermediate THP
ether as a colourless oil (39 mg, 58% crude yield). This was used
directly in the next step without further purification. HRMS (CI+,
[M + NH4]+) found 330.3270, C20H322H6NO2 requires 330.3279.
R
by the addition of celiteꢀ and Na2SO4·10H2O (4 g each) and then
this mixture was stirred for 16 h. The resulting suspension was
filtered through a thin pad of celite and the pad was washed with
several portions of CH2Cl2. After concentration under reduced
pressure the intermediate alcohol was isolated as a colourless oil
(174 mg, 47% crude yield) and used without further purification
in the next step (m/z (EI+) 92.1 (60%, M+), 74.1 (48, [M − H2O]+),
63.1 (100).
To a stirred solution of the crude hexadeuterated alcohol
(7.46 mmol) in anhydrous THF (10 cm3) under N2 was added
anhydrous triethylamine (1.25 cm3, 8.95 mmol) and the mixture
was cooled to −45 ◦C (dry ice–acetone bath). Methanesulfonyl
chloride (754 mm3, 9.70 mmol) was added and the solution
(turning to a white milky suspension) was stirred at −45 ◦C (dry
ice–acetone bath) for 45 min. Lithium bromide (2.59 g, 29.8 mmol)
2352 | Org. Biomol. Chem., 2008, 6, 2346–2354
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=
The crude THP ether (38 mg, 0.121 mmol) was dissolved in
freshly distilled ethanol and to this stirred solution was added
pyridinium p-toluenesulfonate (2.8 mg, 12 mmol). This stirred
mixture was heated at 50 ◦C for 24 h. After cooling, the ethanol was
removed under reduced pressure and the residue was immediately
purified by flash chromatography on silica gel (eluting with 3 : 1
hexane–ethyl acetate) to yield the title compound as a colourless oil
(18 mg, 65%); HRMS (M+) found 228.2360, C15H202H6O requires
228.2360; mmax/cm−1 3319 (br s), 2927 (s), 2856 (s), 2222 (w), 1661
(w), 1444 (m), 1379 (m), 1010 (s); dH (500 MHz, C2HCl3) 1.53
(3 H, s, CH3), 1.62 (3 H, s, CH3), 1.87–2.07 (8 H, m, 2 × CH2CH2),
1.60 (3 H, s, CH3C CHCH2O), 1.85–2.03 (8 H, m, 2 × CH2CH2),
=
4.35 (2 H, t, J 5.5, CH2O), 5.09 (2 H, m, 2 × C CH), 5.33 (1 H,
2
=
t, J 7.0, C CH); dC (125 MHz; H2O at pH 8.5 buffered with
N2H4O2H) 15.3 and 15.7 (2 × CH3), 25.7, 26.40, 26.9, 38.5 and
38.9 (2 × CH2CH2), 62.6 (CH2O), 120.0, 124.0 and 124.3 (3 ×
2
=
=
C CH), 136.7, 142.8 and 143.0 (3 × C CH); dP (202 MHz; H2O
at pH 8.5 buffered with N2H4O2H) −6.80 (1 P, d, JPP 22.0) and
−10.28 (1 P, d, JPP 22.0); m/z (ES−) 387.0 (100%, [M − H]−).‡
Incubation of FPP and analogues with aristolochene synthase
Incubations of FPP and deutero analogue 15 with aristolochene
synthase were carried out as previously described.33
=
4.08 (2 H, d, J 7, CH2OH), 5.01–5.06 (2 H, m, 2 × C CH),
5.35 (1 H, t, J 7, C CH); dC (125 MHz, C2HCl3) 15.9 and 16.2
=
Briefly, enzyme (25 mM) was incubated with FPP (10 mM,
100 mm3) in a volume of 500 mm3 containing 25 mM Tris (pH 7.5),
5 mM MgCl2, 5 mM 2-mercaptoethanol and 15% glycerol at 25 ◦C,
overlayed with 100 mm3 of hexane. The reactions were stopped by
addition of 100 mM EDTA solution (100 mm3), then extracted
with hexane (3 × 3 cm3). The hexane extracts were vortexed with
silica (50 mg) then the decanted solvent was removed under a
gentle stream of nitrogen. Samples were analysed by GC-MS
performed on a Hewlett Packard 6890 GC fitted with a J & W
scientific DB-5MS column (30 m × 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).
(2 × CH3), 26.0, 26.6, 39.2 and 39.7 (2 × CH2CH2), 59.4 (OCH2),
=
123.3, 123.8 and 124.3 (3 × C CH), 134.7, 135.4 and 139.8 (3 ×
+
+
+
=
C
CH); m/z (EI ) 228.2 (3%, M ), 210.2 (18, [M − H2O] ), 135.1
(35), 93.1 (100).
(E,E) 12,12,12-[2H,2H,2H]-3,7-Dimethyl-11-[2H,2H,2H]-methyl-
dodeca-2,6,10-trien-1-yl diphosphate trisammonium salt (15)14,15
The title compound was prepared as previously described for other
farnesyl diphosphate analogues.14,15 To a stirred solution of N-
chlorosuccinimide (11.6 mg, 86.8 mmol) in anhydrous CH2Cl2
(2 cm3) under N2 at −30 ◦C (dry ice–acetone bath) was added
dimethyl sulfide (6 mm3, 86.8 mmol). This solution (that rapidly
turned into a milky-white suspension) was stirred for 15 min at
◦
◦
−30 C then briefly warmed to 0 C in an ice–water bath. After
cooling to −40 ◦C (dry ice–acetone bath), 14 (16.5 mg, 72.4 mmol)
was added as a solution in anhydrous CH2Cl2 (2 cm3) via a
cannula. This solution was stirred for 16 h whilst slowly warming
to room temperature. Brine (5 cm3) was added and the mixture was
extracted with hexane (3 × 10 cm3). The pooled hexane extracts
were dried over anhydrous MgSO4 then filtered and concentrated
under reduced pressure. The resulting intermediate chloride was
used immediately without further purification.
For incubations carried out in D2O, the enzyme, substrate and
buffers were made up as normal then lyophilised and redissolved
in the required volume of 100 atom% D2O. This was repeated twice
and then the buffer was adjusted to pH 7.1 by addition of DCl.
Activity assays
Enzyme assays were carried out essentially as previously
described.15,33 Assays (250 mm3 final volume) were initiated by ad-
dition of purified AS solution (1 mM, 25 mm3, final concentration
100 nM). Assays contained 0.1–5 mM [1-3H]-farnesyl diphosphate
(240 000 dpm nmol−1), 20 mM Tris, 5 mM MgCl2, 5 mM 2-
mercaptoethanol and 15% glycerol and were pre-warmed to 37 ◦C
prior to addition of enzyme solution. After incubation for 4 min,
each assay was stopped by addition of 100 mM EDTA (100 mm3)
and overlayed with hexane (500 mm3). After vortexing for 10 s,
the hexane was removed and the sample extracted with hexane in
the same way (2 × 500 mm3). The pooled hexane extracts were
vortexed with silica (50 mg), the sample was centrifuged at 13 000
rpm for 5 min and then the hexane was decanted into a scintillation
vial containing 15 cm3 of Ecoscint and analysed for radioactivity.
KM and kcat values were determined by a non-linear fit of the
data to the equation V = Vmax[S]/(KM + [S]) using Sigmaplot
for Windows Version 10.0. For assays under normal conditions
and in D2O, KM (0.8 lM) and kcat (0.02 s−1) were identical within
experimental error and so no kinetic solvent isotope effect was
measurable.
To a stirred solution of this material in anhydrous acetonitrile
3
˚
(2 cm ) and freshly activated, powdered, 4 A molecular sieves
(100 mg), under N2, was added tris (tetra-n-butylammonium)
hydrogendiphosphate14,15 (128 mg, 142 mmol). The complete
reaction mixture was stirred for 16 h and then solvent was
removed under reduced pressure and the resulting opaque residue
was dissolved in 2 cm3 of 1 : 49 (v/v) isopropyl alcohol and
25 mM ammonium hydrogencarbonate solution (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 equilibrated with two column
volumes of ion-exchange buffer. The column was eluted with two
column volumes of the same buffer at a flow rate of one column
volume per 15 min. The clear light yellow eluent was lyophilized to
dryness to give a solid, which was purified by reverse phase HPLC
(150 × 21.2 mm Phenomenex Luna column, eluting with 10% B
for 20 min, then a linear gradient to 60% B over 25 min and finally
a linear gradient to 100% B over 5 min; solvent B: CH3CN, solvent
A: 25 mM NH4HCO3 in water, flow rate 5.0 cm3/min, detecting at
220 nm) to give 7 as a white solid (17 mg, 53%); HPLC tR 46.2 min;
mmax/cm−1 (KBr disc) 2848 (br m), 1497 (s), 1453 (s), 1406 (s), 1201
(s), 1120 (s), 1093 (s), 1026 (m), 905 (s), 797 (m); dH (500 MHz;
‡ Note that this compound has recently been prepared using a different
methodology.22
2
2
2
=
H2O at pH 8.5 buffered with N H4O H) 1.50 (3 H, s, CH3C CH),
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Gas phase calculations
10 B. Felicetti and D. E. Cane, J. Am. Chem. Soc., 2004, 126, 7212–7221.
11 E. Y. Shishova, L. Di Costanzo, D. E. Cane and D. W. Christianson,
Biochemistry, 2007, 46, 1941–1951.
12 R. K. Allemann, N. J. Young, S. Ma, D. G. Truhlar and J. Gao, J. Am.
Chem. Soc., 2007, 129, 13008–13013.
13 H. V. Thulasiram, R. M. Phan, S. B. Rivera and C. D. Poulter, J. Org.
Chem., 2006, 71, 1739–1741.
14 V. J. Davisson, A. B. Woodside, T. R. Neal, K. E. Stremler, M.
Muehlbacher and C. D. Poulter, J. Org. Chem., 1986, 51, 4768–4779.
15 D. J. Miller, F. Yu, N. J. Young and R. K. Allemann, Org. Biomol.
Chem., 2007, 5, 3287–3298.
The starting point for the calculations was based on the previously
identified minimum energy structure for the germacryl cation.12
Using Molden,16 a hydroxonium ion was placed in close proximity
to the lower face of the ten-membered ring and a proton was
removed from C12 to provide an approximate model for the
structure of the transition state. A relaxed potential energy surface
scan was performed using Gaussian03, revision B.0317 and AM118
with the distance between the hydroxonium proton closest to the
C6, C7 p-bond varied and all other geometric parameters allowed
to adjust freely.
From this simulation, an approximate transition state geometry
was obtained. Transition state searches, using the harmonic os-
cillator approximation, were performed using the AM1,18 PM3,21
mPW1PW19 and MPWB1K20 methods. To account approximately
for anharmonicity, all vibrational modes below 100 cm−1 were
raised to 100 cm−1 in all calculations.
16 G. Schaftenaar and J. H. Noordik, J. Comput. Aided Mol. Des., 2000,
14, 123–134.
17 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, J. J. A. Montgomery, T. Vreven, K. N. Kudin, J. C.
Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci,
M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M.
Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E.
Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, R. C. A. J. Austin, C.
Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth,
P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S.
Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y.
Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W.
Chen, M. W. Wong, C. Gonzalez and J. A. Pople, Gaussian 03, Revision
B.03/D.05, 2004.
The Gibbs free energy change (DG298) for the formation of the
TS was defined as the difference in free energy between the TS and
the sum of free energies of germacryl cation and an isolated water
molecule.
Starting geometries for these calculations are available from the
authors on request.
18 M. J. S. Dewar, E. G. Zoebisch, E. F. Healy and J. J. P. Stewart, J. Am.
Chem. Soc., 1985, 107, 3902–3909.
Acknowledgements
19 C. Adamo and V. Barone, J. Chem. Phys., 1998, 108, 664–675.
20 Y. Zhao and D. G. Truhlar, J. Phys. Chem. A, 2004, 108, 6908–6918.
21 J. J. P. Stewart, J. Comput. Chem., 1989, 10, 209–220.
22 D. J. Schenk, C. M. Starks, K. R. Manna, J. Chappell, J. P. Noel and
R. M. Coates, Arch. Biochem. Biophys., 2006, 448, 31–44.
23 D. J. Miller, F. Yu and R. K. Allemann, ChemBioChem, 2007, 15,
1819–1825.
We would like to thank Dr Rob Jenkins, Cardiff University for
help with mass spectrometry and NMR spectroscopy, Dr Matt
Crump, Bristol University for help with the NOE experiment and
the EPSRC and BBSRC for funding. This work was supported in
part by NSF grant CHE-0704974.
24 F. Yu, D. J. Miller and R. K. Allemann, Chem. Commun., 2007, 4155–
4157.
25 S. Forcat, S. E. Taylor and R. K. Allemann, unpublished.
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