Chemoenzymatic preparation of germacrene analogues

Article abstract of DOI:10.1039/c2cc35542f

A small library of novel germacrenes was generated using a combination of two plant enzymes, germacrene A synthase, and D synthase and modified farnesyl diphosphate (FDP) analogues. This chemoenzymatic approach allows the preparation of potentially valuable volatiles for biological studies. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc35542f

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 9702–9704
www.rsc.org/chemcomm
COMMUNICATION
Chemoenzymatic preparation of germacrene analoguesw
Oscar Cascon, Sabrina Touchet, David J. Miller, Veronica Gonzalez, Juan A. Faraldos and
´
Rudolf K. Allemann*
Received 31st July 2012, Accepted 14th August 2012
DOI: 10.1039/c2cc35542f
A small library of novel germacrenes was generated using a
combination of two plant enzymes, germacrene A synthase, and
D synthase and modified farnesyl diphosphate (FDP) analogues.
This chemoenzymatic approach allows the preparation of potentially
valuable volatiles for biological studies.
1
Terpenoids represent a valuable class of bioactive fine chemicals
and are therefore attractive targets for synthetic modification;
modulation of their natural properties may lead to new medicinal
and agrochemical compounds with improved properties. However,
the complexity of the hydrocarbon skeletons and the often
significant chemical instability of many terpenoids can present
2
a formidable challenge to the synthetic chemist. Synthetic
biology approaches have focused on the preparation of natural
3a
terpenoids in living organisms, but they operate with whole
biochemical pathways using fundamental biosynthetic building
blocks (e.g. isopentenyl diphosphate) and can therefore not
easily be applied to generate modified terpenes. One attractive
synthetic approach that complements current terpene synthetic
Fig. 1 Proposed biosynthesis of germacrenes A (3a) and D (5a).
Modified substrate analogues of GAS and GDS (framed).
of the acid labile germacrene A (3a) has so far hampered the
1
2
3b,c
development of a satisfactory chemical synthesis. Nevertheless,
fluorinated germacrene A analogues with improved stabilities
have previously been produced enzymatically from fluorinated
biology
and circumvents the difficult task of engineering full
metabolic pathways to generate alternative substrates in vivo,
could rely on the chemical preparation of FDP analogues as
substrates of recombinant terpene synthases to create modified
4a,c,5b
FDP analogues.
4
,5
Thus, based on the biological and potential economic significance
terpenoids. Modified FDPs have been used extensively to
study the mechanisms of the cationic reactions mediated by
of compounds 3a and 5a, germacrene A and D synthases from
1
3
4
–7
Solidago canadensis were selected to investigate their capability to
produce non-natural germacrenes from modified FDPs. To this
end, recombinant GAS and GDS were overproduced in E. coli and
(
sesqui)terpene synthases. However, despite the fact that several
4,5,8
unnatural FDPs are indeed turned over by these enzymes,
only
a few reports have explored the synthetic utility of terpene
9
synthases toward the production of valuable novel terpenoids.
9b,14
purified as previously described.
Several fluorine and methyl
modified FDPs were screened by GC-MS on an analytical
scale for substrate activity. Germacrene A analogues were
readily identified through their ability to undergo thermal
Cope rearrangements to the corresponding b-elemene analogues
Germacrene A and germacrene D synthases (GAS and
GDS) are two plant sesquiterpene synthases that catalyze the
2
+
Mg -dependent conversion of FDP (1a) to germacrene A
3a) and germacrene D (5a), respectively (Fig. 1). These two
macrocyclic sesquiterpenes have been shown to act as semio-
(
1
4
under GC-MS conditions; germacrene D analogues were
1
0
identified from their mass spectra since the presence of the
+
chemicals affecting the olfactory response of insects. While a
more stable (i.e. more abundant, 100%) [M À 43] fragment
synthesis of the rather unstable germacrene D (5a) has been
+
in the EI -MS is diagnostic of the parent 5a. Only modified
1
reported, the extreme thermal and photochemical instability
1
FDP analogues (Fig. 1, framed) that gave a relatively strong
ion count in the total ion chromatogram (GC-MS) as compared
with the natural substrate 1a were considered suitable for this
study (vide infra).
School of Chemistry and Cardiff Catalysis Institute, Cardiff
University, Main Building, Park Place, Cardiff CF10 3AT, UK.
E-mail: allemannrk@cardiff.ac.uk; Fax: +44 (0)29 208 74030;
Tel: +44 (0)29 208 79014
w Electronic supplementary information (ESI) available: Synthetic
methods, enzyme production and purification, kinetic data, analytical
data of reaction products. See DOI: 10.1039/c2cc35542f
Interestingly, in contrast to what has been observed with
4a,d,9a
other sesquiterpene synthases,
2-fluoro-FDP was not turned
over significantly by GAS or GDS. In addition, the H/F and in
particular the H/CH substitution at the C15 position of FDP
3
9
702 Chem. Commun., 2012, 48, 9702–9704
This journal is c The Royal Society of Chemistry 2012

View Article Online
was not tolerated by either enzyme. The full details and the
mechanistic implications of these observations are beyond the
scope of this manuscript and will be published elsewhere.
Optimal reaction conditions for the preparation of modified
germacrenes: initial preparative incubations using both
enzymes were shown to be inefficient and hence an optimisation
of the reaction conditions was carried out. After some experi-
mentation, conversions were found to be optimal at concentra-
product (retention time 28.5 min, approx. >50% total hydro-
carbons, ESIw) underwent a thermal rearrangement, thereby
suggesting a germacrene A analogue as the major enzymatic
product from substrate 1f. In addition, in a parallel study with
PR-AS, the same compound mixture was generated from 1f
1
(ESIw). Purification by preparative TLC and subsequent H-NMR
analysis of this sample unambiguously confirmed the structure
of the major GAS- and PR-AS-generated products as 14Me-
germacrene A (3f). Hydrocarbon 3f displayed the well documented
2+
tions of Mg , FDP and enzyme of 10 mM (5 mM for GAS),
1
2
0
.35 mM and 6 mM, respectively (ESIw). Higher concentrations
conformational flexibility exhibited by germacrene A (3a).
1
2
+
of Mg
and/or enzyme led to the formation of insoluble/
Indeed, H-NMR spectra comparisons (ESIw) with those
previously obtained for (3a) at different temperatures suggested
2
+
12
inactive FDP–Mg -complexes and/or enzyme aggregation,
which in turn resulted in less efficient turnovers. The concen-
tration of GDS (but not GAS) could be increased to 12 mM
simply by inclusion of 1% of the non-denaturing detergent
that 3d exists as an interconverting mixture of the same three
conformers observed for 3a, albeit with different relative
populations (ESIw). As with the parent hydrocarbon 3a, the
most abundant conformation corresponds to the ‘crossed
3
-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate)
CHAPS) in the assay buffer. The nature of the organic solvent,
the reaction vessel itself and the extractive work up were of
1
2
(
up–up’ (UU) configuration, but in contrast to 3a, the combined
‘parallel down–up’ DU and ‘up–down’ UD conformations of 3f
dominates (61%) over the individually more stable (i.e. more
abundant) UU conformation (39%). The present conformational
distribution relates to the apparent increase in steric bulk on C14
of 3f relative to 3a, which likely raises the energy of the UU
conformer with respect to the UD and DU conformations. For
a diagram and further explanation regarding the conformations
of 3f see ESI.w
9b
fundamental importance for optimal conversions. In the final
optimised conditions, d-chloroform was used as the organic
layer, the incubations were carried out in sealed tubes with
gentle agitation and enzymatic products were extracted
overnight using an automated rotator. The filtered and dried
d-chloroform solutions were then analysed by GC-MS and
NMR-spectroscopy. Under these conditions, the enzymatic
conversions of 1a to germacrene D (5a) and germacrene A (3a)
were 76% and 40%, respectively (Fig. 2).
Incubations of FDP analogues with GDS: 6F-FDP (1b) and
14Me-FDP (1f) were turned over efficiently by GDS each
giving a single product displaying the more abundant and
Incubations of FDP analogues with GAS: fluorinated germacrene
A analogues were obtained from incubations of GAS with
+
+
stable [M À 43] fragment in their EI -MS spectra, which is
characteristic of germacrene D (5a) through loss of the isopropyl
group within the MS-detector. Indeed, preparative incubations
6F-FDP (1b) and 14F-FDP (1d) (Fig. 2). These compounds were
identified by GC-MS through co-elution with authentic material
previously isolated from experiments with aristolochene synthase
1
19
followed by direct H- and F-NMR spectroscopy analysis
confirmed their identity as the expected germacrene D analogues
(ESIw).
4
c
from Penicillium roqueforti (PR-AS, see ESIw).
Upon incubation with GAS, 10F-FDP (1c) produced efficiently
(
30% rel.) a single fluorinated hydrocarbon that co-eluted in the
GC-MS column with an authentic sample of a-10F-humelene, a
known compound prepared previously using d-cadinene synthase
Two products (10% rel.) in an approximate 3 : 1 ratio were
observed in the pentane extracts from incubation of 10F-FDP
(1c) with GDS (ESIw). GC-MS analysis of a mixture of
4d
4d
(DCS). This result demonstrates that with diphosphate 1c, both
10-fluoro-farnesenes, prepared in a previous study, unambigu-
enzymes are able to catalyze an anti-Markovnikov 1,11-macro-
cyclisation via p-donation from the vinylic fluorine atom into the
distal C10,C11-double bond of 1c.
ously identified the major product as (E)-b-10F-farnesene
(ESIw). Interestingly, the minor component (25%) of this
mixture was also identified by co-elution as the fluorinated
a-10F-humulene previously observed in incubations of 1c with
14Me-FDP (1f) was also readily turned over by GAS
4d
yielding a mixture of at least seven hydrocarbons. The major
GAS or DCS.
a
2+
b
Fig. 2 Incubations of modified FDP (1b, d–f) under optimized conditions: GAS (6 mM), Mg (5 mM) and FDP (0.35 mM); GDS (12 mM),
2+
Mg (10 mM) and FDP (0.35 mM). Conversions were determined by GC-FID (ESIw) in pentane. Relative conversions (rel.) denote percentage
with respect to 1a.
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 9702–9704 9703

View Article Online
Both 15F-FDP (1e) and 14F-FDP (1d) were converted by
GDS as judged by GC-MS to a well-defined product under
analytical conditions; each product (5e and 5d) displayed the
4 (a) D. J. Miller, F. L. Yu and R. K. Allemann, ChemBioChem,
2
007, 8, 1819–1825; (b) D. J. Miller, F. L. Yu, N. J. Young and
R. K. Allemann, Org. Biomol. Chem., 2007, 5, 3287–3298;
c) D. J. Miller, F. L. Yu, D. W. Knight and R. K. Allemann,
Org. Biomol. Chem., 2009, 7, 962–975; (d) J. A. Faraldos,
D. J. Miller, V. Gonzalez, Z. Yoosuf-Aly, O. Cascon, A. Li and
(
+
major [M À 43] -fragment suggesting that they were indeed
germacrene D derivatives. However, prolonged preparative
incubations led to the formation of a second product apparently
arising from the initial GDS-generated product (ESIw).
Although the presence of this minor product hampered a full
NMR interpretation of the spectrum of the original enzymatic
´
R. K. Allemann, J. Am. Chem. Soc., 2012, 134, 5900–5908.
(a) J. P. Noel, N. Dellas, J. A. Faraldos, M. Zhao, B. A. Hess,
L. Smentek, R. M. Coates and P. E. O’Maille, ACS Chem. Biol.,
2010, 5, 377–392; (b) J. A. Faraldos, Y. X. Zhao, P. E. O’Maille,
J. P. Noel and R. M. Coates, ChemBioChem, 2007, 8, 1826–1833;
(c) Y. Jin, D. C. Williams, R. Croteau and R. M. Coates, J. Am.
Chem. Soc., 2005, 127, 7834–7842; (d) D. E. Cane, G. H. Yang,
Q. Xue and J. H. Shim, Biochemistry, 1995, 34, 2471–2479;
(e) D. E. Cane and Y. S. Tsantrizos, J. Am. Chem. Soc., 1996,
118, 10037–10040.
5
1
product (5e), the observation ( H NMR, 500 MHz) of a
relatively downfield (approx. 2 ppm with respect to 5a) wide
2
doublet at d
H
= 6.53 ppm ( JH–F = 86.0 Hz, CQCHF) instead
2
of the diagnostic broad doublet at d
3.0 Hz, CQCHH, exo methylene group) of 5a (ESIw) is
consistent with the major product being 15F-germacrene D
H
= 4.77 ppm ( JH–H =
1
3c
6 (a) D. J. Miller and R. K. Allemann, Nat. Prod. Rep., 2012, 29, 60–71;
b) E. Y. Shishova, F. L. Yu, D. J. Miller, J. A. Faraldos, Y. X. Zhao,
1
(
R. M. Coates, R. K. Allemann, D. E. Cane and D. W. Christianson,
J. Biol. Chem., 2008, 283, 15431–15439; (c) F. L. Yu, D. J. Miller and
R. K. Allemann, Chem. Commun., 2007, 40, 4155–4157; (d) H. A.
Gennadios, V. Gonzalez, L. Di Costanzo, A. Li, F. L. Yu,
D. J. Miller, R. K. Allemann and D. W. Christianson, Biochemistry,
2009, 48, 6175–6183; (e) J. A. Aaron, X. Lin, D. E. Cane and
D. W. Christianson, Biochemistry, 2010, 49, 1787–1797;
19
(5e, Fig. 2). Surprisingly, the F-NMR spectrum of this
mixture displayed three absorbances, two identical doublets
2
(
J
H–F = 86.0 Hz) at d
F
= À136.1 (minor) and À138.6 (major)
2
ppm, respectively, plus a downfield triplet ( JH–F = 46.0 Hz)
at À184.8 ppm due to the very minor peak observable by
GC-MS. Thus, the major (and only) enzymatic product is
most likely produced by GDS as a mixture of two geometric
(f) R. P. McAndrew, P. P. Peralta-Yahya, A. De Giovanni,
J. H. Pereira, M. Z. Hadi, J. D. Keesling and P. D. Adams, Structure,
2011, 19, 1876–1884; (g) J. A. Faraldos, A. K. Antonczak,
V. Gonzalez, R. Fullerton, E. M. Tippmann and R. K. Allemann,
´
1
5F-germacrene D isomers (5e) (Fig. 2) that is not resolved by
J. Am. Chem. Soc., 2011, 133, 13906–13909.
(a) D. W. Christianson, Chem. Rev., 2006, 106, 3412–3442;
GC-MS. This observation implies that the corresponding
tightly bound carbocation (4e) possesses sufficient mobility
within the active site of GDS to allow a not completely specific
proton-loss to generate the observed isomeric mixture of 5e.
In summary, the results presented here provide insight into
aspects of the reaction mechanisms employed by GAS and
GDS and describe a general chemoenzymatic approach for the
synthesis of non-natural terpenoids that are otherwise not
easily accessible by classical chemical synthesis or synthetic
biology. Indeed, these results show that GAS and GDS can
turn over a variety of modified FDPs to germacrene A and D
analogues often with synthetically acceptable conversions and
in sufficient amounts for biological testing as semiochemicals.
This work was supported by the United Kingdom’s
Biotechnology and Biological Sciences Research Council
through grants BB/G003572/1 and BB/H01683X/1 and by
Cardiff University. We thank Dr Rob Jenkins, Cardiff University,
for assistance with NMR spectroscopy.
7
(
b) M. Koksal, Y. H. Jin, R. M. Coates, R. Croteau and
D. W. Christianson, Nature, 2011, 469, 116–120; (c) M. Koksal,
H. Y. Hu, R. M. Coates, R. J. Peters and D. W. Christianson, Nat.
Chem. Biol., 2011, 7, 431–433.
8
9
(a) J. A. Faraldos, B. Kariuki and R. K. Allemann, J. Org. Chem.,
2
010, 75, 1119–1125; (b) J. A. Faraldos and R. K. Allemann, Org.
Lett., 2011, 13, 1202–1205; (c) J. A. Faraldos, B. M. Kariuki and
R. M. Coates, Org. Lett., 2011, 13, 836–839.
(a) L. S. Vedula, Y. X. Zhao, R. M. Coates, T. Koyama,
D. E. Cane and D. W. Christianson, Arch. Biochem. Biophys.,
2007, 466, 260–266; (b) Z. Yoosuf-Aly, J. A. Faraldos, D. J. Miller
and R. K. Allemann, Chem. Commun., 2012, 48, 7040–7042.
0 (a) W. S. Bowers, C. Nishino, M. W. Nielson, M. E. Montgomery
and L. R. Nault, Science, 1977, 196, 680–681; (b) R. Mozuraitis,
M. Stranden, M. I. Ramirez, A. K. Borg-Karlson and
H. Mustaparta, Chem. Sens., 2002, 27, 505–509; (c) T. J. A.
Bruce, et. al., Pest Manage. Sci., 2005, 61, 1115–1121; (d) M. A.
Birkett, et. al., Phytochemistry, 2008, 69, 1710–1715.
1
1
1 S. L. Schreiber and R. C. Hawley, Tetrahedron Lett., 1985, 26,
5971–5974.
12 J. A. Faraldos, S. Wu, J. Chappell and R. M. Coates, Tetrahedron,
007, 63, 7733–7742.
2
1
3 (a) I. Prosser, A. L. Phillips, S. Gittings, M. J. Lewis, A. M. Hooper,
J. A. Pickett and M. H. Beale, Phytochemistry, 2002, 60, 691–702;
(b) C. O. Schmidt, H. J. Bouwmeester, S. Franke and W. A. Konig,
Chirality, 1999, 11, 353–362; (c) N. Bulow and W. A. Koning,
Phytochemistry, 2000, 55, 141–168.
Notes and references
1
2
J. S. Glasby, Encyclopedia of Terpenoids, Wiley, Chichester, 1982.
A. J. Minnaard, J. B. P. A. Wijnberg and A. De Groot, Tetrahedron,
1999, 55, 2115–2146.
3
(a) N. Misawa, Curr. Opin. Biotechnol., 2011, 22, 627–633; (b) P. A.
Ajikumar, et. al., Science, 2010, 330, 70–74; (c) P. J. Westfall, et. al.,
Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 111–118.
14 M. J. Calvert, P. R. Ashton and R. K. Allemann, J. Am. Chem.
Soc., 2002, 124, 11636–11641.
15 A. M. Adio, Tetrahedron, 2009, 65, 1533–1552.
9
704 Chem. Commun., 2012, 48, 9702–9704
This journal is c The Royal Society of Chemistry 2012