A 1,6-ring closure mechanism for (+)-δ-cadinene synthase?

Article abstract of DOI:10.1021/ja211820p

Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphosphate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene cyclases, DCS carries out this transformation with >98% fidelity but, as a consequence, leaves no mechanistic traces of its mode of action. The formation of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate (3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-hydride shift would then relocate the carbocation within the transient macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in (+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene, respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS incubations with enantiomerically pure [1- ²H₁](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence of multiple mechanistic pathways.

Full text of DOI:10.1021/ja211820p

Article
pubs.acs.org/JACS
A 1,6-Ring Closure Mechanism for (+)-δ-Cadinene Synthase?
Juan A. Faraldos, David J. Miller, Veronica Gonzalez, Zulfa Yoosuf-Aly, Oscar Cascon, Amang Li,
́
́
́
and Rudolf K. Allemann*
School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom
S
* Supporting Information
ABSTRACT: Recombinant (+)-δ-cadinene synthase (DCS) from Gossypium
arboreum catalyzes the metal-dependent cyclization of (E,E)-farnesyl diphos-
phate (FDP) to the cadinane sesquiterpene δ-cadinene, the parent hydrocarbon
of cotton phytoalexins such as gossypol. In contrast to some other sesquiterpene
cyclases, DCS carries out this transformation with >98% fidelity but, as a
consequence, leaves no mechanistic traces of its mode of action. The formation
of (+)-δ-cadinene has been shown to occur via the enzyme-bound intermediate
(3R)-nerolidyl diphosphate (NDP), which in turn has been postulated to be
converted to cis-germacradienyl cation after a 1,10-cyclization. A subsequent 1,3-
hydride shift would then relocate the carbocation within the transient
macrocycle to expedite a second cyclization that yields the cadinenyl cation with the correct cis stereochemistry found in
(+)-δ-cadinene. An elegant 1,10-mechanistic pathway that avoids the formation of (3R)-NDP has also been suggested. In this
alternative scenario, the final cadinenyl cation is proposed to be formed through the intermediacy of trans, trans-germacradienyl
cation and germacrene D. In addition, an alternative 1,6-ring closure mechanism via the bisabolyl cation has previously been
envisioned. We report here a detailed investigation of the catalytic mechanism of DCS using a variety of mechanistic probes
including, among others, deuterated and fluorinated FDPs. Farnesyl diphosphate analogues with fluorine at C2 and C10 acted as
inhibitors of DCS, but intriguingly, after prolonged overnight incubations, they yielded 2F-germacrene(s) and a 10F-humulene,
respectively. The observed 1,10-, and to a lesser extent, 1,11-cyclization activity of DCS with these fluorinated substrates is
consistent with the postulated macrocyclization mechanism(s) en route to (+)-δ-cadinene. On the other hand, mechanistic
results from incubations of DCS with 6F-FPP, (2Z,6E)-FDP, neryl diphosphate, 6,7-dihydro-FDP, and NDP seem to be in better
agreement with the potential involvement of the alternative biosynthetic 1,6-ring closure pathway. In particular, the strong
inhibition of DCS by 6F-FDP, coupled to the exclusive bisabolyl- and terpinyl-derived product profiles observed for the DCS-
catalyzed turnover of (2Z,6E)-farnesyl and neryl diphosphates, suggested the intermediacy of α-bisabolyl cation. DCS
incubations with enantiomerically pure [1-2H1](1R)-FDP revealed that the putative bisabolyl-derived 1,6-pathway proceeds
through (3R)-nerolidyl diphosphate (NDP), is consistent with previous deuterium-labeling studies, and accounts for the cis
stereochemistry characteristic of cadinenyl-derived sesquiterpenes. While the results reported here do not unambiguously rule in
favor of 1,6- or 1,10-cyclization, they demonstrate the mechanistic versatility inherent to DCS and highlight the possible existence
of multiple mechanistic pathways.
intermediates.3h,8,9 Fluorine containing prenyl diphosphates
INTRODUCTION
■
have been used to study several terpene synthases including
aristolochene synthases from the fungi Aspergillus terreus and
Penicillium roqueforti (AT-AS and PR-AS),3b,c,g,6h tobacco 5-epi-
aristolochene synthase (TEAS),3d,h trichodiene synthase
(TS),3e,8e,10 taxadiene synthase,3a limonene and (+)-bornyl
diphosphate synthases,3f,6g and α-pinene synthase.11
δ-Cadinene synthase (DCS) from Gossypium arboreum is a
sesquiterpene cyclase that catalyzes the metal-dependent
conversion of farnesyl diphosphate (FDP, 1) to the bicyclic
hydrocarbon (+)-δ-cadinene (6) with a specificity of >98%
(Scheme 1). This transformation is the first committed step in
the biosynthesis of cotton phytoalexins such as gossypol.12
Similar to all class I terpene cyclases,6 DCS maintains the
characteristic aspartate-rich D307DXXD motif on helix D that
Terpene synthases catalyze complex reaction cascades with high
regio- and stereochemical precision involving cyclizations
(alkylations), rearrangements, and deprotonations of highly
reactive carbocations.1 Only recently has it become possible to
address experimentally the intricate mechanistic details of these
reactions through the use of substrate analogues,2,3 aza-
analogues of putative carbocationic intermediates,4 mutant
enzymes,5 and X-ray crystallography.6 Fluorine-containing
analogues of enzyme substrates have been shown to be
instrumental in mechanistic investigations,7 and in particular,
fluoro prenyl derivatives3 have provided crucial insights
regarding the cationic mechanisms of terpene synthases.8
While the small size of the fluorine atom does not appear to
significantly compromise active-site binding,3d,h,6g,h,8b,d its
intrinsic electronegativity is known to inactivate fluoro-
containing double bonds toward protonation and electrophilic
alkylation3c,d,g and to alter the stability of allylic cation
Received: December 19, 2011
Published: March 7, 2012
© 2012 American Chemical Society
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displacement of the diphosphate group of 1 yields the transient
trans, trans-germacradienyl cation (7), which further undergoes
a 1,3-hydride shift followed by proton loss from C15 to
generate the neutral hydrocarbon germacrene D (9) as the key
biosynthetic intermediate.21 A conformational change of
enzyme-bound 920 gives rise to its reactive transoid conformer,
which upon proton transfer to the exocyclic double bond and
1,6-ring closure produces the Z-configured cation 5, the final
carbocationic intermediate common to pathways (a) and (b).
Recently, pathway (b) via germacrene D has been shown to
operate in a promiscuous sesquiterpene synthase from
Medicago truncatula (MtTPS5).22
Scheme 1. Reaction Mechanisms Proposed in the Literature
for DCS Catalysis15,20
A third mechanistic possibility is also outlined in Scheme 1
(path c). In this pathway, the formation of [5-2H]- and
[11-2H]-δ-cadinene from racemic [1-2H]-farnesyl diphosphate
was explained by an initial 1,6-electrophilic ring closure that
leads to α-bisabolyl cation as the key biogenetic precursor of
hydrocarbon 6.15b In support of this proposal, it has been
reported previously that NDP (2), a reaction intermediate on
path a, is converted by DCS to (E)-β-farnesene and β-
bisabolene in addition to δ-cadinene.15c This observation is not
easily explained if a 1,10-cyclization mechanism (e.g., path a) is
followed. In addition, when [4,4,15,15,15-2H5]-nerolidyl
diphosphate was used, no isotope loss to the solvent was
observed,15b,c a result that makes unlikely the alternative 1,10-
cyclization mechanism22 via germacrene D (9) since it involves
protonation/deprotonation at C15.
Here, we describe results obtained through the use of
deuterated and fluorinated farnesyl diphosphates as mechanistic
probes to examine the proposed DCS catalyzed reaction
cascades summarized in Scheme 1. Enzymatic incubations with
fluorinated FDP analogues (Figure 1) bearing fluorine at C2
chelates two (A and B) of the three Mg2+ ions essential for
catalysis.13 However, amino acid sequence alignments as well as
X-ray structural analysis13 have revealed the absence of the
conserved second metal-binding “NSE/DTE” motif6 on helix
H. Instead, DCS possesses a second aspartate rich motif
(D451DVAE), similar to those found in “isoprenoid chain
elongation enzymes” such as farnesyl diphosphate synthase.14
Mechanistically, the consensus in the literature12a,13,15 is in
favor of the DCS-catalyzed reaction path a outlined in Scheme
1. This pathway goes through (3R)-nerolidyl diphosphate
((3R)-NDP, 2) as an enzyme-bound intermediate that after
rotation around the C2,C3 σ-bond, leads to the correct orbital
alignment for 1,10-macrocyclization and production of cis-
germacradienyl cation (3).1a A subsequent C1 → C11 1,3-
hydride shift of the original H-1si of 115c,d is followed by a 1,6-
electrophilic ring closure reaction that generates cadinenyl
cation (5), from which δ-cadinene (6) is formed after proton
loss from C6. The tertiary diphosphate 2 has been shown to be
a substrate of DCS,15b,c and its formation (or that of (2Z,6E)-
FDP, 15)16 within the active site of the enzyme has been
inferred from [1,2-13C2]acetate as well as [2-14C] and
[4-14C]mevalonate labeled feeding experiments.17,18 In closely
related biosynthetic studies, (3R)-NDP (2) has also been
identified as an enzyme-bound intermediate in (+)-1-epi-
cubenol biosynthesis,19 and more recently, 2 has been
suggested as an intermediate in (−)-δ-cadinene biosynthesis.15d
The biosynthesis of cadinane-type sesquiterpenes could also
occur without the intervention of enzyme bound NDP (2) that
ultimately allows the formation of the cis C2,C3-double bond
(FDP numbering) present in 6.20 In this alternative scenario
(path b, Scheme 1), a 1,10-cyclization via the direct
Figure 1. Farnesyl and geranyl diphosphate analogs used in the
present study.
(10 and 11) and C10 (12) led to the formation of 2F-
germacrenes (1,10-ring closure) and 10F-humulene (1,11-ring
closure) as single products, results that are consistent with a
macrocyclization reaction (paths a and b) as the biosynthetic
means of (+)-δ-cadinene production. Conversely, however, the
strong inhibition of DCS displayed by 6F-FDP (13), together
with the exclusive bisabolyl- and terpinyl-derived product
profiles observed from incubations with (2Z,6E)-farnesyl (15)
and neryl (20) diphosphates and the absence of germacrene
production (i.e., expected 1,10-cyclization activity) upon
incubation with 6,7-dihydro-FDP (17) are in better agreement
with a 1,6-ring closure mechanism (path c) by way of the (6R)-
α-bisabolyl cation23 (37). While these apparently conflicting
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mechanistic interpretations of the experiments described here
do not unambiguously show whether 1,6- or 1,10 cyclization is
the pathway followed during the DCS catalyzed turnover of
FDP (1) to (+)-δ-cadinene (6), they imply a high degree of
mechanistic versatility of DCS and may suggest the existence of
multiple pathways to 6.
macrene A, the C2,C3 cis isomer of 21. The 1H NMR spectrum
of 22 showed no major changes when a CDCl3 solution of 22
was cooled to −50 °C and then gradually warmed to +50 °C, a
result that is in good accordance with the observation that the
parent hydrocarbon helminthogermacrene A exists almost
exclusively as one conformer at room temperature in C6D6.24
In contrast, all-trans-germacrene sesquiterpenes exist as several
interconvertible conformational isomers in solution, a dynamic
effect that had been observed previously among others25 for
2F-germacrene A,3c 6F-germacrene A,3d and (+)-germacrene
A.26
RESULTS AND DISCUSSION
■
Incubation of DCS with (2Z,6E)-2F-FDP (10), (2E,6E)-
2F-FDP (11), and [15,15,15-2H3]-FDP (14). The catalytic
mechanism of DCS is thought to go by way of (3R)-NDP (2)
involving a coupled ionization-isomerization-1,10-cyclization
reaction (1 → 3, pathway a, Scheme 1).15 Hence, the presence
of a fluorine atom on C2 should prevent (or slow down), via
depletion of electron density,9 the heterolytic diphosphate ester
cleavage reaction that secures the supply of farnesyl cation.
Indeed, under steady-state kinetic conditions (2Z,6E)-2F-FDP
(10) and (2E,6E)-2F-FDP (11) were found to inhibit the
enzyme, albeit with reduced binding affinities (Ki = 70 μM and
Ki = 30 μM for 10 and 11, respectively) when compared to
FDP (KM = 3.2 μM),13 indicating a rather modest competitive
inhibition toward DCS (Supporting Information). Remarkably,
the C2-fluorinated ‘cis’ isomer 113h binds to the active site of
DCS approximately twice as tightly as its corresponding ‘trans-
configured’ analogue 10, which is structurally a better mimic of
all-trans FDP (1). These kinetic results together with the better
resemblance of diphosphate 15 (and the fluorinated analogue
11) to the presumed reaction intermediate 2 support the
potential intermediacy of (E,Z)-farnesyl diphosphate (15) or its
corresponding cation in the biosynthesis of 6.17,18,20 This
suggestion is in agreement with the observation that (3R)-NDP
(2) binds to DCS with 10-fold greater affinity than FDP (1).15c
Despite the inhibitory properties of diphosphates 10 and 11
against DCS, GC−MS analyses of individual incubations of 10
and 11 revealed that the enzyme was able to turn over these
analogues, giving in both cases single but different fluorinated
hydrocarbons characterized by their molecular ions of m/z 222.
The product generated from (2Z,6E)-2F-FDP (10) was readily
identified by GC−MS as 2-fluorogermacrene A (21, Figure 2),
These results indicate that the fluorinated diphosphates 10
and 11 were able to efficiently suppress the native isomerization
of 1 to 2 (path a). Indeed, the DCS-generated 2-
fluorogermacrenes 21 and 22 (Figure 2) were shown to
preserve the original C2,C3-double bond geometry of their
corresponding substrates 10 (trans) and 11 (cis) after the
enzymatic 1,10-cyclization reaction. The relative ease with
which 21 and 22 are formed is consistent with the possible
involvement of the 1,10-macrocyclization(s) pathway assumed
to operate in δ-cadinene biosynthesis. In particular, the
maintenance of the substrate’s Z/E-geometry of the C2,C3
double bond during the enzymatic reaction weighs in favor of
the 1,10-pathway (b, Scheme 1) that avoids the formation of a
cisoid NDP through the direct generation of trans, trans-
germacrenyl cation27 (7) and then germacrene D as the
biosynthetic intermediates.19,21 In support of pathway b, it
could be reasoned that after the enzymatic 1,10-ring closure,
the electron-withdrawing effect of the vinylic 2-fluoro
substituent might prevent the native C1 → C11 hydride shift
(7 → 8, Scheme 1) that relocates the positive charge at C1,
thus, explaining the observed accumulation of 2F-germacrene A
(21) or 2F-helminthogermacrene (22) after deprotonation.
However, since the formation of δ-cadinene along pathway b
would require protonation of the exocyclic double bond of
germacrene D (9),22 the reaction cascade would be expected to
show, in addition to proton exchange with the solvent,2g,i,22
a
dependence on pH, as observed for catalysis by TEAS,28 the
maize enzymes TPS6 and TPS11,2i the fungal cyclase Cop4,2p
and some Cop4 mutants.5o However, in contrast to Cop4,2p,5o
the catalytic cycle of DCS was not disrupted by higher values of
pH (7.5 → 10.5) and no accumulation of germacrene D was
observed (Supporting Information).29 Moreover, incubations of
[15,15,15-2H3]-FDP (14) with DCS in aqueous buffer (H2O)
led to the release of [15,15,15-2H3]-δ-cadinene with the
deuterium content intact (Supporting Information). These
observations are in agreement with those obtained with
[4,4,15,15,15-2H5]-NDP15b and seem to rule out pathway b20
(Scheme 1).
Figure 2. Structures of 2F-germacrene A (21) and 2F-helmintho-
germacrene A (22).
Incubation of 10F-FDP (12) with DCS. Similar
conclusions were reached from experiments with 10F-FDP
(12). The reduced electron density of the C10,C11-double
bond in 12, coupled to the destabilizing effect of the 10-fluoro-
substituent on the developing positive charge on C11 during
DCS catalysis, was anticipated to prevent the proposed 1,10-
cyclization reaction (Scheme 1). Consequently, the formation
of 10F-farnesenes or 10F-bisabolenes arising from the putative
(3R)-10F-NDP was expected. Diphosphate 12 acted as a
competitive inhibitor (Ki = 16.5 μM) of DCS, a result that
suggests a potentially high energy barrier for the isomerization
of 12 to (3R)-10F-NDP during DCS catalysis in support of the
DCS-catalyzed 1,10-pathway. DCS was also able to turn over
diphosphate 12 yielding a single (>95% by GC−MS)
a fluorinated cyclodecadiene sesquiterpene isolated during
previous mechanistic studies on aristolochene synthase (PR-
AS) catalysis (Supporting Information).3c For comparison,
analytical incubations of (2E,6E)-2F-FDP (11) were carried out
with DCS and PR-AS. Similarly, both enzymes were able to
turn over 11 to the same product 22 as judged by GC−MS
analysis (Supporting Information). Compared to 2-fluoroger-
macrene A (21), the product formed from 11 was characterized
by a shorter GC retention time and a much higher reluctance to
undergo a thermal Cope rearrangement. On the other hand, 21
and 22 displayed almost identical MS fragmentation patterns.
1
These results, together with H- and 19F-NMR spectroscopic
analysis, indicated that 22 was indeed 2-fluoro-helminthoger-
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fluorinated hydrocarbon (26) that failed to undergo thermal
Cope rearrangements even at temperatures as high as 250 °C,
thus, most likely ruling out a germacrene hydrocarbon as the
enzymatic product.30 The mass spectrum of this fluorinated-
hydrocarbon was different from that of an authentic sample of
enzymatically generated (E)-10F-β-farnesene (25) (Supporting
Information).31a In addition, despite the striking similarity
between the EI MS spectra of this fluorinated hydrocarbon and
(Z)-α-farnesene (Supporting Information), the chemical
generation of an authentic 3:1:1 mixture of 10F-farnesenes31b
(23−25, Figure 3) and subsequent GC/MS comparisons with
incubations of DCS with 13 were expected to yield, via 6F-3,
fluorinated germacrene hydrocarbons (e.g., 27−29, Scheme 2)
Scheme 2. Structures of 6F-Germacrenes (27−29) from a
a
Putative 6F-NDP (6F-2)
a
The detection of the possible enzymatic release of 6F-2 in the
aqueous phase was not attempted.
through premature deprotonation reactions. Intriguingly,
diphosphate 13 was not a substrate of DCS, and even after
prolonged enzymatic incubations, no turnover was observed.
Steady-state kinetic studies demonstrated that 6F-FDP (13)
was a potent competitive inhibitor of DCS with an inhibition
constant (Ki = 2.4 μM, Supporting Information) comparable to
the Michaelis−Menten constant measured for FDP (KM = 3.2
μM).13
Assuming that the C6,C7-double bond of 1 (or 13) is not
likely to participate in the production of (3R)-NDP (2) (path a,
Scheme 1),15,17,18 the strong inhibition displayed by 13
suggests that the native DCS-catalyzed reaction cascade leading
to 6 might proceed through the alternative 1,6-ring closure
pathway c (Scheme 1), as 1,10-macrocyclization-derived
products would be expected for this analogue from path a.3c,d
Hence, in this scenario, δ-cadinene (6) is formed by
nucleophilic attack of the central C6,C7-double bond of 2 on
C1 to generate the α-bisabolyl cation (Scheme 1), which has
been reported to be central to other sesquiterpene synthase-
catalyzed cyclizations of FDP.23 The destabilizing effect of the
vinylic 6-fluoro substituent toward the proposed electrophilic
1,6-alkylation would explain the inability of DCS to use 6F-
FDP (13) as a substrate.
The possibility of a 1,6-electrophilic ring closure in DCS
catalysis was further supported by incubations with racemic 6,7-
dihydroFDP (17).33 Indeed, in agreement with the results
previously obtained with the 1,6-cyclase TS,33a,b DCS
incubations of diphosphate 17 yielded (Z)-α-6,7-dihydrofarne-
sene34 as the main (64%) hydrocarbon. Conversely, 6,7-
dihydrogermacrene is the exclusive product detected with the
1,10-cyclase aristolochene synthase from A. terreus (AT-AS).35
Incubation of DCS with Geranyl (19), Neryl (20), and
2F-Geranyl (18) Diphosphates. It has been documented that
sesquiterpene synthases that can isomerize the C2,C3-double
bond of (E,E)-FDP (1) often produce cyclic monoterpenes
from GDP (19).2h,i,p,36 In contrast, rigorously ‘trans’ enzymes
such as TEAS2h or the germacrene A synthase NS1,2p produce
exclusively acyclic monoterpenes when they encounter 19.
Thus, for ‘cis’ sesquiterpene cyclases3h that utilize a coupled
ionization-isomerization-1,6-cyclization mechanism,23 neryl
cation and α-terpinyl cation1b,37 (Scheme 3) can act as good
mimics of the corresponding C15 cis-farnesyl and α-bisabolyl
Figure 3. Structures of 10F-farnesenes (23−25) and 10F-α-humulene
(26).
the DCS-generated product ruled out any of the three possible
fluorinated farnesenes. This result indicates that DCS does not
seem to convert 12 to its (3R)-10F-NDP isomer which likely
precludes the enzymatic formation of 1,6-cyclized products
from 12. Diphosphate 12 has been used previously to explore
the 1,6-ring closure mechanism catalyzed by trichodiene
synthase (TS). Surprisingly, 10F-FDP acted as a potent
inhibitor of TS,8a thus, suggesting the potential existence of a
alternative/degenerate mode of catalysis by TS.
While an unambiguous full structure determination of the
product generated from 10F-FDP (12) is outstanding,
1
preliminary H NMR (500 MHz, CDCl3) spectroscopic data
are in agreement with a α-10F-humulene hydrocarbon structure
(26, Figure 3) arising from an unprecedented DCS-mediated
1,11-macrocyclization. This suggestion is supported by the
observation of a downfield doublet of triplets signal (JH−F
=
40.4 Hz and JH−H = 8.1 Hz) at 5.00 ppm, which strongly
resembles the characteristic doublet of triplets absorbance at
5.59 ppm (JH−H = 15.8. Hz, and JH−H = 7.3 Hz) assigned to the
H-9 olefinic proton of α-humulene.32 The relative upfield H-9
shift (ca. 0.6 ppm) observed for 26 is consistent with the γ-
shielding effect of the vinylic fluoro substituent on C9. In
contrast to the characteristic 19F-NMR triplet signal (JH−F
=
24.6 Hz) at −114.0 ppm of diphosphate 12, the 19F-NMR (282
MHz) spectrum of the reaction product generated from 12
displayed a doublet (JH−F =41.6. Hz) centered at −119.9 ppm,
which further supports the identification of this enzymatic
hydrocarbon as 10F-α-humulene (26) (Supporting Informa-
tion).
Incubations of 6F-FDP (13) and 6,7-DihydroFDP (17)
with DCS. 6-Fluoro-FDP (13) has been used previously to
probe the catalytic cycles of PR-AS3c and TEAS.3d The kinetic
data obtained for TEAS revealed that the presence of the vinylic
fluorine had negligible effects on binding affinity, diphosphate
ionization, and the initial 1,10-cyclization reaction.3d For DCS,
irrespective of which 1,10-mechanism is followed (Scheme 1),
the presence of the 6-fluoro substituent should destabilize the
transition state(s) leading to cadinenyl cation 5. Therefore,
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proceed along path a (Scheme 1). GC−MS analysis of the
pentane extractable products from incubations of 15 with DCS
showed that the enzyme generated two hydrocarbon products
(Supporting Information). The MS fragmentation pattern and
GC retention time of the minor product (33%) matched that of
δ-cadinene (6).15 To identify the major product (67%), a
preparative-scale incubation of 15 with DCS was carried out.
The hydrocarbon (50% of total) and alcohol (50% of total)
products were initially separated by silica gel column
chromatography. Each fraction was then purified further by
preparative TLC to yield two hydrocarbon fractions (A and B)
and one alcohol fraction. 1H NMR spectroscopic measurements
of fraction A revealed the presence of δ-cadinene (6) of ca. 85%
purity.15d,39 Fraction B (corresponding to the major GC peak
with shorter retention time) was found to be composed of a
3:3:2 mixture of (Z)-γ-bisabolene (38), β-bisabolene (39), and
(Z)-α-bisabolene (40) as judged by 1H NMR spectroscopy and
comparison with the literature values previously reported for
Scheme 3. Monoterpenes Produced by DCS from Geranyl
(19) and Neryl (20) Diphosphates
cations, respectively. On the basis of these considerations, and
to assess the feasibility of a 1,6-ring closure, the ability of DCS
to convert 19 to cyclic monoterpenes was tested. DCS was
indeed able to catalyze the conversion of 19 to an approximate
2:3 mixture of cyclic (α-phellandrene (30), limonene (31), γ-
terpinene (32), and α-terpinolene (33)), and acyclic (myrcene
(34), (E)-β-ocimene (35), and (Z)-β-ocimene (36)) mono-
terpenes (Supporting Information). Interestingly, neryl diphos-
phate (20) produced only cyclic monoterpenes (Scheme 3)38
suggesting that the observed acyclic olefins (34−36, Scheme 3)
arise exclusively from the transoid geranyl cation, and that their
enzymatic release occurs before isomerization to neryl
cation.1b,37 In accordance with this mechanistic picture, 2-
fluorogeranyl diphosphate (18) was shown to efficiently
prevent the ionization-isomerization step essential for the
accumulation of cyclic products; indeed, 18 was a potent
competitive inhibitor of the DCS (Ki = 8 μM) (Supporting
Information). These results reflect on the ability of DCS to use
an isomerization-1,6-cyclization step and sustain a possible
reaction via α-bisabolyl cation (Scheme 1) from FDP.
these compounds.40,41 In addition, H NMR spectroscopy of
1
the alcohol fraction revealed the presence of an inseparable
2:1:1 mixture of nerolidol and the two epimeric α-bisabolols 41
and 42,42 which most likely arise from the nonspecific addition
of water to α-bisabolyl cation. It is worthy of note that this
product distribution (Scheme 4) is remarkably similar to that
previously observed from DCS incubations using NDP (2) as
the substrate.15c
The accumulation of the bisabolyl-derived olefins 38−40,
accounting for ca. 67% (GC−MS) of all cyclic hydrocarbons, is
easily explained with an initial DCS-catalyzed 1,6-ring closure
reaction that leads to the (6R)-α-bisabolyl cation (37), which is
then either deprotonated to the observed hydrocarbons 38−40,
or trapped by a molecule of water to generate the tertiary
alcohols 41 and 42 (Scheme 4). However, the observation of
considerable amounts of alcohols 41 and 42 from incubation of
15 in the absence of recombinant DCS established, for the
most part, their nonenzymatic origin. It is notable that not only
6, but also the DCS-generated hydrocarbon (Z)-γ-bisabolene
(38) as well as the α-bisabolols are terpene volatiles found in
cotton plants.15c
Mechanistically, the formation of δ-cadinene (6) together
with a variety of bisabolyl-derived hydrocarbons (38−40)
supports the possibility of a 1,6-ring closure mechanism.
However, the result is intriguing. Either DCS possesses the
ability to mediate parallel 1,10- and 1,6-ring closure reactions
along energetically similar pathways (Scheme 4), or all cyclic
enzymatic products including 6 arise from a predominant
conformation of farnesyl cation (A, Scheme 5) that allows the
1,6-cyclization mechanism observed with the surrogate
diphosphate 15. The possibility of a DCS-catalyzed 1,6-ring
closure pathway has been suggested previously15b and the
feasibility of the required 1,3-H and 1,5-H shifts involving 37
has been discussed.43 However, a detailed description of a 1,6-
mechanism consistent with both the 5,10-cis stereochemical
relationship of cadinenyl-derived hydrocarbons and the
deuterium distribution found in 6 using racemic and chiral
[1-2H1]-FDPs15 has not been provided before. In agreement
with our proposal (vide infra), Tantillo and Hong have
provided computational evidence from gas-phase calculations in
support of 1,3- and 1,5-H shifts that could interconnect, via
cation 37, the biosynthesis of amorphadiene (1,6-ring closure)
and amorphene (1,10-ring closure) derived sesquiterpenes.
These gas-phase calculations energetically favored a 1,6-ring
Incubation of DCS with (2Z,6E)-FDP (15). The possibility
of either a 1,10- or a 1,6-ring closure mechanism in DCS
catalysis was further evaluated with (2Z,6E)-FDP (15)
(Scheme 4). It had been recognized previously that
diphosphate 15 could be considered an effective ‘preisomerized’
form of (E,E)-FDP (1).2n,3h Hence, its ionization by DCS
should supply an ion pair of cisoid farnesyl cation and
diphosphate anion ready for the enzymatic reaction cascade to
Scheme 4. Sesquiterpenes Generated by DCS from (2Z,6E)-
a
FDP (15)
a
The (R)-configuration of 39−40 is assumed based on the
intermediacy of a putative (6R)-configured α-bisabolyl cation (37).
Alcohols 41 and 42 are nonenzymatic products.
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Scheme 5. DCS-Catalyzed Conversion of [1-2H1](1R)-FDP
((1R)-1) and 15 to the (6R)-α-Bisabolyl Cation (37) via
[1-2H1](3R)-NDP (2) or the Corresponding cis-Farnesyl
Cation/PPO− Ion Pair
Scheme 6. Conversion of Cation 37 to (+)-6 Involving 1,3-
and 1,5-Hydride Shifts of the H-1Si Proton of [1-2H1](1R)-
FDP
closure rather than the accepted 1,10-cyclization mechanism in
amorphene biosynthesis.44
Incubation of [6-2H1]-FDP (16) and [1-2H1](1R)-FDP
((1R)-1) with DCS. In the present study, the stereochemical
course of the DCS-catalyzed reaction was followed by
individual incubations with [6-2H1]-FDP (16) and
[1-2H1](1R)-FDP ((1R)-1), which resulted in the formation
of unlabeled 6 and [5-2H1]-6, respectively (Supporting
Information), as evidenced by GC/MS analysis. In contrast
to what was observed with 6 (base peak at m/z 161), the GC-
mass spectra of the product ([5-2H1]-6) generated from (1R)-1
had a base peak m/z of 162 (assigned to [M+-C3H7]), thus,
indicating that the original H-1Re proton of FDP was retained
on C1 during the formation of [5-2H1]-6.15d Hence, the loss of
an undeuterated isopropyl side chain from [5-2H1]-6 confirms
the migratory properties of the original H-1Si of diphosphate 1.
The formation of unlabeled 6 from incubations with [6-2H1]-
FDP (16) indicates the loss of the H-6 of FDP during catalysis.
While these (and other15) limited labeling experiments alone
cannot distinguish between a 1,6- and a 1,10-mechanism
(Schemes 1, 5 and 6), the experimental observations reported
here support an electrophilic 1,6-alkylation reaction as the
predominant pathway to (+)-δ-cadinene (6).43 Moreover, the
1,6-ring closure pathway, illustrated in Schemes 5 and 6 with
the deuterium labeled substrate (1R)-1 and unlabeled 15, is in
good agreement with previous feeding experiments with
[1,2-13C2]-acetate and [2-14C1]- and [4-14C1]-mevalonate in
cotton plants, from which a plausible role for diphosphate 15
was inferred,17,18 and with more recent studies that revealed the
(3R)-enantiomer of nerolidyl diphosphate (2, Scheme 5) as the
active substrate of recombinant (+)-DCS.15c
considering the cisoid farnesyl cation (A) as the reactive active
site intermediate from which cyclic 37 is formed.
After formation of the (6R)-α-bisabolyl cation (37), the
remaining mechanistic steps to (+)-6 (Scheme 6) resemble
those precedented in amorpha-4,11-diene synthase
(AMDS).2h,46 However, in contrast to AMDS catalysis, MM2
molecular modeling studies indicate that after the unique C1 →
C7 hydride shift to the Si face of 37 involving the original H-1Si
of FDP,2h,46 the second cyclization step (B → D, Scheme 6)
requires a conformational ring inversion (twist B → half twist
B) to obtain the exo 1,10-electrophilic cyclization onto the Si
face of carbocation B that ultimately guarantees the correct
5,10-cis stereochemistry diagnostic of cadinene sesquiterpenes.
As shown in Scheme 6, the required conformational change
could also occur at the bisabolyl cation (37) stage, but since the
C1 → C7 hydride shift occurs to the same C7 Si face of cation
37, the transient 7R-stereochemistry displayed by B (and E, see
Scheme 7) would be expected.
Scheme 7. Conversion of Cation E to Amorpha-4,11-diene
(42) and Amorphenes (43), respectively44
Accordingly, we propose that the enzyme-generated tertiary
diphosphate 2 (Scheme 5) reacts in the active site pocket of
(+)-DCS in the anti, endo conformation typically found in
mono- and sesquiterpene cyclases that bring about 1,6-
cyclizations.1a−f,36 Since biomimetic cyclizations of the related
linalyl and neryl derivatives are chemically effected mainly from
this conformation,9,45 it seems plausible to suggest that the
prenyl chain of 15 adopts a helical orientation similar to that of
NDP (2), the enzyme bound intermediate generated from
FDP. With this chirality, the 6,7-double bond of 2 (and the
double bond of 15) is ideally positioned to effect the presumed
anti 1,6-cyclization that ensures the formation of the α-bisabolyl
cation (37) with the proposed (R)-configuration at C6
(Scheme 5). An identical R-stereochemistry would be expected
The proposed exo cyclization (Scheme 6) generates the
second cyclohexane ring of D in a high-energy boat
conformation that nevertheless allows the critical 1,5-H shift
to cadinenyl cation 5 with concomitant release of strain. Finally,
proton loss from C6 of the trans-fused cadinenyl cation 5
(Scheme 6) accounts for the formation of (+)-[5-2H1]-δ-
cadinene, or δ-cadinene, when (1R)-1 or [6-2H1]-FDP (16) are
used as the respective substrates. It is worth noting that while a
similar 1,10 electrophilic cyclization onto the C1 Re face of
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cation B (twist B → C, Scheme 6) could equally explain, via
cation C, the 5,10-cis stereochemistry present in 6 (Scheme 6),
the anti stereochemical relationship between H7 and C11 in C
precludes the proposed 1,5-hydride shift and hence the
installment of the correct deuterium label in 6 observed with
d-labeled FDPs.15c,d
A corollary from this stereochemical analysis stems from the
fact that an endo 1,10-cyclization onto the Re-face of
carbocation B (twist conformer) would give rise to the cis-
fused amorphenyl cation E, the direct precursor of amorpha-
4,11-diene 42.2h,44,46 Similarly, the high-energy boat conforma-
tion of cation E (Scheme 7) allows the presumed 1,5-H shift
that accounts for amorphene (43) biosynthesis.44
humulene (26) via unprecedented 1,10- and 1,11-cyclizations,
in which the geometry of the C2,C3 double bond of the
starting diphosphates remained unchanged. This observation
contrasts with the native cyclization of FDP, in which DCS
must convert the initial trans C2,C3 π-bond via NDP (2) to cis
in δ-cadinene. While a 1,11-cyclization mechanism en route to
δ-cadinene seems unlikely, the observation of 1,10-cyclizations
from 10 and 11 and the possible dual 1,6- and 1,10-ring
closures from 2 and 15 are in agreement with the mechanistic
versatility of cadinane-producing sesquiterpene synthases,43
which do not appear to depend on a common biosynthetic
mechanism in spite of their shared ancestral origin.47
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures, gas chromatograms, mass
spectra and/or NMR spectra of key compounds, as well as
inhibition kinetics studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
In summary, the mechanism of the DCS-catalyzed cyclization
of FDP (1) to (+)-δ-cadinene (6) was studied by incubations
using a variety of substrate analogues. In agreement with a
mechanism via (3R)-NDP (2),15c prenyl diphosphates with
vinylic fluoro substituents at C2 (10, 11, and 18) were shown
to act as competitive inhibitors that prevent the initial cationic
ionization-isomerization step. Surprisingly, FDP analogues 10
and 11 with fluorine at C2 also acted as substrates of DCS and
yielded fluorinated germacrenes arising from 1,10-cyclizations
with the stereochemistry of the C2,C3 double bond remaining
intact. The isolation of 2F-germacrenes (21, 22), despite
inhibition and stereochemical arguments, seems to favor the
1,10-macrocyclization mechanism. Similarly, the inhibition
displayed by 10F-FDP (12), together with the formation of a
10F-α-humulene (26) via a DCS-mediated 1,11-cyclization of
12, suggests a 1,10-pathway (path a, Scheme 1) as the most
plausible biosynthetic pathway. On the other hand, 6-fluoro-
FDP (13) served as a potent competitive inhibitor of DCS, an
observation that is difficult to explain assuming a 1,10-
macrocyclization mechanism (Scheme 1). In addition,
diphosphate 13 was the only fluorinated C15 diphosphate that
was not turned over by the enzyme, suggesting an early
involvement of the central C6,C7-double bond of 1 during
DCS catalysis. Incubations of DCS with (2Z,6E)-FDP (15)
produced, in addition to δ-cadinene, a mixture of bisabolenes
and bisabolols arising exclusively from a 1,6-ring closure, and
resembling the product distribution previously observed when
NDP (2) was used as the substrate. Taken together, these
results are best explained with the involvement of a 1,6-
cyclization of (E,E)-FDP (1) in DCS chemistry to generate
(6R)-bisabolyl cation.23 Further support for the possible
involvement of this mechanism was provided by the
observations that C10 GDP (19) and neryl diphosphate (20)
generated a variety of 1,6-ring closure products and that 6,7-
dihydroFDP (17) yielded the acyclic cis-6,7-dihydro-α-
farnesene as the main product of the enzymatic reaction rather
than the expected 6,7-dihydrogermacrene.
■
S
AUTHOR INFORMATION
Corresponding Author
allemannrk@cardiff.ac.uk
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the UK’s Biotechnology and
Biological Sciences Research Council (BBSRC) through grant
BB/G003572/1, the UK’s Engineering and Physical Sciences
Research Council (EPSRC) through grant EP/D06958/1 and
Cardiff University. We thank Dr. Rob Jenkins (Cardiff
University) for assistance and valuable discussions regarding
GC−MS experiments, Dr Xiao-Ya Chen (Institute of Plant
Physiology and Ecology, and Institute for Biological Sciences,
Shanghai, China) for plasmid pXC1 harboring the cDNA of
(+)-δ-cadinene, and Dr Linda Field (Rothamsted Research,
U.K.) for a cDNA encoding for (E)-β-farnesene synthase.
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