Conformational analysis of (+)-germacrene A by variable-temperature NMR and NOE spectroscopy

Article abstract of DOI:10.1016/j.tet.2007.04.037

(+)-Germacrene A, an important intermediate in sesquiterpene biosynthesis, was isolated in pure form from a genetically engineered yeast and was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR, ¹H NMR, and ¹³C NMR data. Variable-temperature 500 MHz ¹H NMR spectra in CDCl₃ showed that this flexible cyclodecadiene ring exists as three NMR-distinguishable conformational isomers in a ratio of about 5:3:2 at or below ordinary probe temperature (25 °C). The conformer structures were assigned by ¹H NMR data comparisons, NOE experiments, and vicinal couplings as follows: 1a (52%, UU), 1b (29% UD), and 1c (19%, DU).

Full text of DOI:10.1016/j.tet.2007.04.037

Tetrahedron 63 (2007) 7733–7742
Conformational analysis of (D)-germacrene A by variable-
temperature NMR and NOE spectroscopy
Juan A. Faraldos,a Shuiqin Wu,b Joe Chappellb and Robert M. Coatesa,
*
aDepartment of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA
bDepartment of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546-00991, USA
Received 22 December 2006; revised 5 April 2007; accepted 12 April 2007
Available online 19 April 2007
Abstract—(+)-Germacrene A, an important intermediate in sesquiterpene biosynthesis, was isolated in pure form from a genetically engi-
neered yeast and was characterized by chromatographic properties (TLC, GC), MS, optical rotation, UV, IR, 1H NMR, and 13C NMR data.
Variable-temperature 500 MHz 1H NMR spectra in CDCl3 showed that this flexible cyclodecadiene ring exists as three NMR-distinguishable
conformational isomers in a ratio of about 5:3:2 at or below ordinary probe temperature (25 ꢀC). The conformer structures were assigned by
1H NMR data comparisons, NOE experiments, and vicinal couplings as follows: 1a (52%, UU), 1b (29% UD), and 1c (19%, DU).
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
alcohol patchoulol,12 and the bicyclic hydrocarbons aristolo-
chene13 and 5-epi-aristolochene.7c,14 A small amount of ger-
macrene A (7.5%) is in fact released by aristolochene
synthase.15a Further evidence for the intermediacy of germa-
crene A in the production of bicyclic sesquiterpenes has been
obtained through site-directed mutagenesis of aristocholene
and tobacco epi-aristolochene synthases and isolation of ma-
jor amounts of the monocyclic precursor,14d,15 and by forma-
tion of dihydrogermacrene A using the substrate analogue
6,7-dihydrofarnesyl diphosphate.16
The germacrene sesquiterpenes, (+)-germacrene A (1) and
(+)-hedycaryol (2), are structurally characterized by the flex-
ible trans,trans-cyclodeca-1(10),4(5)-diene ring system, and
are believed to be important intermediates in the biosynthe-
sis of several classes of sesquiterpenes including the germa-
cranolides, eudesmanes, eremophilanes, spirovetivanes, and
guaianes (Scheme 1).1 The biosynthesis of the bicyclic ses-
quiterpenes can be easily rationalized by enzymatic proton-
ation of either the D1(10) or D4 double bond of 1 and 2
followed by cyclization (intramolecular C5–C10 or C1–C5
bond formation), rearrangements, and final quenching of
the carbocations by deprotonation or nucleophilic capture
of a water molecule as the final step.2
Both (7R)- and (7S)-germacrene A enantiomers are known
and presumed to be precursors of the stereochemically
related sesquiterpenes through cyclization and oxidative me-
tabolism. Both enantiomers are found in many essential
oils,17 indicating the widespread occurrence of the germa-
crene synthases that produce and release the enantiomeri-
cally pure 10-membered ring sesquiterpenes. To date,
germacrene A synthases have been isolated, purified, and
characterized from chicory roots,3a,18 and Ixeris dentata,19
and the cyclases from lettuce,20 goldenrod (Solidago cana-
densis),21 Artemisia annua,22 and Crepidiastrum sonchifo-
lium23 have been cloned and expressed in Escherichia coli.
(+)-Germacrene A (1) is considered to be the biogenetic
precursor of (+)-costunolide (3) in chicory,3 the simplest
member of the large group of naturally occurring germacra-
nolides,4 and an important natural product itself owing to its
role as a common precursor of all germacranolide-derived
lactones (i.e., guaianolides and eudesmanolides).5 In addi-
tion, germacrene A has often been postulated as an inter-
mediate in the biosynthesis of phytoalexins6 including
capsidiol,7 debneyol,7a,b,c costunolide,3a,8 vetispiradienes
(lubimin9 and solavetivone10), rishitin,9 and lettucenin A.8,11
The isolation of pure (E,E)-germacrenes from natural sour-
ces is often complicated by the sensitivity of these sesquiter-
penes to acidic conditions and elevated temperatures. During
distillation and GC analysis germacrene A is known to un-
dergo facile Cope rearrangement to b-elemene.24 In some in-
stances, the absolute configuration of both (+)-1 and (ꢁ)-1
has been established by means of Cope rearrangement on
chiral GC columns able to distinguish the b-elemene
Germacrene A (1) has also been proposed as an enzyme-
bound intermediate in the biosynthesis of the sesquiterpene
* Corresponding author. Tel.: +1 217 333 4280; fax: +1 217 244 8024;
e-mail: rmcoates@uiuc.edu
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.04.037

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J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
O
3
O
10
Germacranolides
9
6
1
4
8
7
2
3
13
11
5
15
R
R
(+)-1
H
Eudesmanes
Eremophilanes
OPP
Farnesyl-PP
OH
R
2
Germacrenes
H
R
Guaianes
Spirovetivanes
Scheme 1. Sesquiterpene families derived from (E,E)-farnesyl diphosphate through (7R)-germacrene A (1) and (7R)-hedycaryol (2).
enantiomers.3a,25 Adsorption on silica gel induces transan-
nular cyclization of (+)-germacrene A to a mixture of
eudesmanes: (ꢁ)-selin-11-en-4-ol, (ꢁ)-a-selinene, (+)-
selinene, and (+)-selina-4,11-diene.26 The instability of ger-
macrene A upon storage at freezer temperatures has also
been reported.24a,c,27
The present work was initiated by the isolation of consider-
able amounts of (+)-germacrene A (1) from an engineered
yeast, the lack of reliable characterization data in the litera-
ture, and the inability to obtain an authentic sample for direct
comparisons. GC analysis showed a major, sharp peak, pre-
ceded by a significantly smaller, hump-shaped peak having
a retention time similar to that of germacrene A.3a This
GC behavior is typically attributed to the occurrence of
Cope rearrangement on the column.3a,20,24,26 Although the
MSy and 13C NMR data were very similar to the limited
(ꢁ)-Germacrene A was first isolated in pure form from the
Gorgonian Eunicea mammosa24a and subsequently from
a soft coral of the genus Lobophytum.28 (ꢁ)-Germacrene
A of terrestrial origin was extracted from the aphid Therioa-
phis maculala and identified as an alarm pheromone.24c,29
The major sesquiterpene component (10 mg/inset) of the de-
fensive secretions of soldiers termites proved to be (ꢁ)-1,30
and in some instances, this fact has been used as a chemo-
taxonomic marker.31 To our knowledge, the occurrence of
(+)-germacrene A seems to be limited to higher plants, and
few reports describe its isolation.26,32
1
literature data, the presence of broad H NMR signals pre-
vented assessment of purity and confident structure and
absolute configurational assignments.
In view of the prominent position of germacrene A in sesqui-
terpene biosynthesis and the conflicting data in the literature,
we decided to investigate the behavior of this C15 cyclodeca-
diene sesquiterpene by means of variable-temperature NMR
spectroscopy.
The cyclic sesquiterpene was first characterized by Wein-
heimer et al.24a by means of IR and MS data, 60 MHz proton
NMR (CCl4) spectra, and an optical rotation [a]2D5 ꢁ3.2 (c
14.4, CCl4).y Nishino et al.24c published the first 13C NMR
data (25 MHz, CDCl3) for terrestrial (ꢁ)-1 and reported
that the 100 MHz 1H NMR spectrum (CDCl3, 30 ꢀC) of ger-
macrene A showed broad signals pointing to the possibility
of a mixture of conformers. High field 1H and 13C NMR data
were recently published by Adio et al.26 using an impure
sample of (+)-1 isolated from S. canadensis.33
2. Results and discussion
2.1. Engineering yeast for germacrene A biosynthesis
As part of our continuing effort to dissect the catalytic fea-
tures of terpene synthases by molecular comparisons be-
tween distinct synthases,40 we carried out a bioinformatic
screen of the lettuce genomic database (http://compositdb.
ucdavis.edu/database) and uncovered a putative, but unique
terpene synthase gene. While the lettuce gene showed se-
quence homology to 5-epi-aristolochene synthase, it also
showed similarity to several other well-characterized ter-
pene synthases including germacrene synthases isolated
from chicory (Cichorium intybus),18 lettuce (Lattuca sat-
iva),20 and goldenrod (S. canadensis).21 To functionally
characterize this putative terpene synthase, we isolated
a full-length cDNA copy of the mRNA using an RT-PCR
method,41 inserted it into a modified Yep352-URA3 yeast
expression vector under the transcriptional control of a mod-
ified ADH promoter, and transformed the yeast strain
Cali7-1 according to Takahashi et al.42 Cali7-1 is a yeast line
genetically engineered for high-level production of farnesyl
diphosphate, the substrate for sesquiterpene synthases.
Cali7-1 also requires an exogenous supply of uracil for
In solution, most simple (E,E)-germacrene sesquiterpenes
(e.g., germacrene B and hedycaryol) behave as several inter-
convertible conformational isomers in equilibrium,24b,34
which usually results in broadened NMR signals or even
multiple sets of NMR signals.24a,b,35 The conformation of
these flexible 10-membered ring sesquiterpenes played
a fundamental role in the proposals concerning the biogene-
sis of other types of sesquiterpenes by Ruzicka,36 Barton and
de Mayo,37 Hendrickson,38 and Parker et al.39 and is a critical
element in the current mechanisms of action of sesquiter-
pene cyclases.2
y
It should be noted that germacrene A undergoes Cope rearrangement to
b-elemene under the conditions of the MS determination. Hence the
MS data for germacrene A are actually those of b-elemene.32

J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
7735
growth owing to a genetic mutation in the endogenous
URA3 gene that is essential for uracil biosynthesis.
(m, 2H, 47%), 4.78 (d, J¼11.5 Hz, 1H, 53%), 4.66 (s, 3H,
100%), 4.62 (s, 1H, 19%), 4.58 (s, 1H, 28%), 4.56 (s, 1H,
53%), and 4.53 (d, J¼10.0 Hz, 1H, 53%) ppm.48 Broad
CH3 singlets were observed at 1.73, 1.71, 1.55, 1.48, and
1.39 ppm. The large number of individual NMR signals
(three sets) led us to consider the possibility that in solution
this sesquiterpene (1) in fact exists as a mixture of three
interconvertible conformers in ca. 5:3:2 ratio at room tem-
perature. This behavior was previously suggested for (ꢁ)-
germacrene A24c and later demonstrated for hedycaryol.35b
2.2. Isolation and characterization of germacrene A
The hexane extract from an incubation of the engineered
yeast was evaporated, and the resulting residue was re-
dissolved in pentane and filtered through a small column
of silica gel at room temperature. Further purification by pre-
parative TLC (silica gel) at room temperature afforded what
proved to be pure (+)-germacrene A (1) as a clear oil,
[a]2D5 +42.1 (CCl4). GC analysis (method A, see Section 4)
showed a major peak at tR 38 min and a small, hump-shaped
peak with a variable tR of 29–37 min, both accounting for
97% of the integration, and another small peak at tR
28 min (3%). The hump at 29–37 min in the chromatogram
is characteristic of cyclodeca-1,5-dienes and indicates the
occurrence of heat-induced Cope rearrangement to the cor-
responding b-elemenes (peak at tR 28 min) during GC anal-
ysis. When the injection port temperature was increased to
180 ꢀC (method B), the composition was b-elemene (4,
49%) and germacrene A (1, 50%). The structure and abso-
lute configuration were confirmed by Cope rearrangement
to (ꢁ)-b-elemene (4, [a]2D5 ꢁ15.8; lit.43 ꢁ11.8; lit.44 +15.4
for the enantiomer) brought about by heating a solution of
(+)-germacrene A in toluene at reflux (Scheme 2). The MS
of the b-elemene showed the molecular ion at m/z 204
(M+, 3%, C15H24) and a fragmentation pattern very similar
to that recently described for (ꢁ)-b-elemene.32 Furthermore,
the 1H and 13C NMR data of the rearrangement product thus
obtained are identical to those previously reported by Brau-
chli and Thomas.45
1
When the 500 MHz H NMR spectrum of 1 was recorded
at 50 ꢀC (Fig. 1A), the olefinic region showed broad peaks
at 5.21–5.07 (m, 1H), 5.06–4.90 (m, 1H), 4.86–4.73 (m,
1H), 4.68 (s, 2H), and 4.65–4.50 (s, 3H) revealing an equilib-
rium mixture of two conformers in ca. 4:3 ratio. In addition,
two broad CH3 singlets at high field 1.73 and 1.52 ppm (6H
and 12H) were observed. These NMR values match very
well with those previously described by Nishino et al.24c at
the same temperature: i.e. 5.2–4.75 (2H), 4.65 (1H), 4.58
(1H), 1.72 (3H), and 1.52 (6H) ppm.
1
The 500 MHz H NMR data (25 ꢀC) of germacrene A in
C6D6 (Fig. 2, Table 1) agree with the limited set of data
recently reported by Adio et al.26 However, in addition to
the peaks described by these authors, broad signals were
also observed downfield in the 5.30–4.76 ppm region
(Fig. 2), and one extra methyl singlet at higher field (1.38)
was also noticeable.
1
Table 1. 500 MHz H NMR spectral data and assignments for (+)-germa-
crene A (1) at 25 ꢀC
Protona
CDCl3 dH
C6D6 dH
b
b
5.26–5.15 (m)
5.15–5.05 (m)
5.05–4.98 (m)
4.98–4.90 (m)
5.22–5.05 (m)
Δ
Toluene
H
5.04–4.94 (m)
4.94–4.84 (m)
4.81 (s)
(+)-1
(-)-4
4.78 (br d, 11.5)
4.67 (s)
4.65–4.58 (m)
Scheme 2. Cope rearrangement of germacrene A to b-elemene.
12
4.79 (s)
The measured optical rotation of +42.1ꢀ for germacrene A in
the present work differs notably from the values (ꢁ3.2ꢀ and
ꢁ26.8ꢀ) published earlier for the (ꢁ)-enantiomer,24a,c per-
haps indicating that the germacrene A obtained previously
was impure or was a mixture of both enantiomers. The IR
spectrum was very similar to that described by Nishino
et al.24c except for a doublet absorption (1385 and
1375 cmꢁ1) characteristic of the isopropenyl group observed
at 1275 and 1261 cmꢁ1, in the present study. Although the
absence of UVabsorption above 200 nm for the (ꢁ)-enantio-
mer was noted by Weinheimer et al.,24a a weak absorption at
lmax 214 nm (log 3¼2.50) was observed for (+)-germacrene
A suggesting the possibility of a transannular interaction
between the double bonds,46 similar to those postulated for
germacrol (lmax 210 nm), germacrone (lmax 213 nm), and
costunolide (lmax 213 nm).47
1
12’
5
2,3,6,7,9
8a
13
4.78–4.69 (m)
4.68 (s)
4.52 (br d, 10.5)
2.48–1.75 (m)c
1.73–1.59 (m)
1.67 (s)
4.57 (s)
4.54 (br d, 10.0)
2.44–1.78 (m)c
1.73 (d, 2.5)
1.53 (br s)
8b
15
1.55–1.57(m)
1.45 (br s)
1.42 (br s)
1.31 (br s)
1.49 (br s)
1.40 (br s)
14
a
Assignments based on Ref. 26.
Multiplicity and J values in hertz are given in parenthesis.
Nine allylic protons.
b
c
The chemical shifts for the more intense signals in the 13C
NMR spectrum (125 MHz, 25 ꢀC) in C6D6 (see Section 4)
are almost identical to those previously recorded and
assigned.26 However, 18 other peaks now attributed to the
minor conformers were not reported, presumably because
they might have arisen from impurities known to have been
present in the sample.33 Relatively strong resonances for
three previously unreported methyl groups at 15.4, 19.9,
and 24.6 ppm observed in the 13C NMR spectrum of
The 500 MHz 1H NMR spectrum of (+)-germacrene A was
recorded at room temperature in both CDCl3 (Fig. 1B) and
C6D6 (Fig. 2) for direct comparison of the data (see Table
1) with the published values. In CDCl3, the downfield ole-
finic region showed broad peaks for the vinyl protons at dH
5.26–5.15 (s, 1H, 19%), 5.15–5.05 (s, 1H, 28%), 5.05–4.90

7736
J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
Figure 1. 1H NMR (500 MHz) spectra of (+)-germacrene A at different temperatures: (A) 50 ꢀC, (B) 25 ꢀC, (C) 0 ꢀC, (D) ꢁ20 ꢀC, and (E) ꢁ50 ꢀC. The letters a,
b, and c designate peaks for three conformers a, b, and c.

J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
7737
Figure 2. 1H NMR spectrum (500 MHz, C6D6) of (+)-germacrene A at room temperature.
14
compound 1 in the present work would then belong to one
or both of the minor conformers.
15
H3C
CH3
CH3
10
1
4
R
R
R
The 13C NMR spectrum (125 MHz, 25 ꢀC) of (+)-
germacrene A (1) in CDCl3 (see Fig. 3 and Section 4) is
very similar to that recorded in C6D6. The 36 resonances
observed contrast with the limited 13C NMR data reported
in CDCl3 (25 MHz, 25 ꢀC) by Nishino et al.,24c although
the shifts previously reported for the trigonal carbons are
in good agreement with our data. Interestingly, the peak at
37.0 ppm assigned to C9 by these authors appeared as a mi-
nor peak in both CDCl3 and C6D6. In addition, the peak at
41.4 ppm first assigned to C7 has been recently reassigned
to C9 by 2D NMR experiments in benzene.26
5
CH3
UD (parallel)
UU (crossed)
H3C
R
H3C
CH3
CH3
DU (parallel)
DD (crossed)
Figure 4. Possible conformations of germacradiene sesquiterpenes such as
germacrene A (1) and hedycaryol (2) fixed by the pseudo-equatorial posi-
tion of the relatively large substituents (R¼isopropenyl and hydroxypropyl).
The conformers are denoted as UU, UD, DU, and DD in reference to the U
(up) and D (down) orientations of C10 and C4 methyl groups on the 10-
membered ring.34,35b
2.3. Conformational analysis
As mentioned above, the 1H NMR spectrum (CDCl3) of this
(E,E)-configured sesquiterpene exhibited broad signals at
room temperature (Fig. 1B) and suggested a conformational
equilibrium in solution (Fig. 4). If the isopropenyl group of 1
is large enough to ensure an equatorial or pseudo-equatorial
position on the cyclodecadiene ring, germacrene A (1) can,
therefore, adopt any of four distinct conformations, namely
UU, UD, DU, and DD (Fig. 4).34,35b These conformations
have either a parallel (UD and DU) or crossed (UU and
DD) relationship of the double bonds, and all four con-
formers are interconvertible by rotations of the 1,10 and
Figure 3. 13C NMR spectrum (125 MHz, CDCl3) of germacrene A at room temperature. A total of 36 separate signals are attributed to germacrene A, 15 of
which are ascribed to the major conformer based on previous 2D correlations with the 1H NMR spectrum in benzene.26

7738
J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
4,5 double bonds through the ring, and by rotations of the
C6–C7–C8 segment. In the ground state, Allinger MM2 cal-
culations49 predict that conformers UD and DD of germa-
crene A should be almost isoenergetic and about 1.4–
1.5 kcal/mol higher in strain energy than the UU isomer
(Table 2), and the DU conformer is predicted to be the least
stable with a relative strain energy of 2.65 kcal/mol. These
values agree with previous MM1 calculations for germa-
crene A, which also predicted the UU and UD conformations
to be more stable and more populated (62% UU and 36%
UD) at room temperature.50 In addition, our MM2 calcula-
tions for similar (E,E)-cyclodecadiene sesquiterpenoids (Ta-
ble 2) are in good agreement with those previously reported
for germacrene B,34b,51 hedycaryol,50,52 11,12-dehydroger-
macrene A,53 and costunolide (3).34b,53
A previous variable-temperature 1H NMR study of hedycar-
yol (2) at 60 MHz by Wharton and co-workers35b showed
that this related sesquiterpene alcohol exists in solution
mainly as one crossed and two parallel conformers (DU
and UD). The latter were favored at ꢁ30 ꢀC to the extent
of 75%. The crossed and parallel conformers were distin-
guished by the relatively high field absorption of the H5
vinyl hydrogen at 4.4 ppm (d, J¼10 Hz) in the crossed
conformer due to the shielding effect of the opposing double
bond. The assignments of the methyl groups for germacrene
sesquiterpenes were established by Sathe et al.54 and corrob-
orated by Wharton et al.35b using deuterated dihydropregei-
jerenes to identify the olefinic methyl singlets (1.32 (H14)
and 1.48 (H15)) for the predominant crossed (UU) con-
former. However, evidently the lower chemical shift disper-
sion at 60 MHz was insufficient for more precise
determination of the populations of the three hedycaryol
conformers.
1
A variable-temperature 500 MHz H NMR study of (+)-
germacrene A (1) was carried out in order to verify the co-
existence of more than one conformer on the NMR time
scale. At low temperatures, the rate of rotation around the
1,10 and 4,5 double bonds would be slowed down, and in
fact less broadening and well separated multiple signals cor-
responding to a mixture of three conformers were observed
(Fig. 1, C, D, and E). At ꢁ20 ꢀC, the vinyl protons (H1, H5,
and H12) appear as six downfield resonances indicating
the presence of at least three NMR-distinguishable con-
formational isomers (a, b, and c) of similar energy in a ratio
Based on these considerations, we assigned the most stable
conformer (52% at ꢁ20ꢀ, Fig. 1 panel D) of germacrene A
1
(1), which exhibits a relatively high field H NMR signal
at 4.51 (d, J¼10.0 Hz, H5) and methyl singlets at 1.37
(H14) and 1.52 (H15) ppm, to the UU conformation (a in
Fig. 1D and 1a in Table 3), as previously suggested for
one conformer of hedycaryol and dihydropregeijerene.35b
Accordingly, the other two downfield sets of 1H NMR
signals observed in the spectra at low temperatures (Fig. 1,
Table 3) correspond to the less populated b (29%) and c
(19%) conformers of germacrene A, which are predicted
to have the parallel orientation of double bonds as demon-
strated earlier for hedycaryol.
1
of 52:29:19 (Fig. 1D, Table 3).48 In addition, the H NMR
spectrum at ꢁ20 ꢀC showed eight high field singlets for
the vinyl methyls, which were fully separated at tempera-
tures lower than 0 ꢀC (Fig. 1D and E). Interestingly, the
multiplicity of the vinyl proton centered at 4.51 ppm (broad
d) seems to be temperature-dependent, becoming a triplet
(J¼6.5 Hz) at temperatures below ꢁ30 ꢀC (Fig. 1E, a).
Similarly, the unsymmetrical triplet centered at 5.07 ppm
starts to develop as a doublet of doublets with approximate
J values of 9.5 and 6.6 Hz at temperatures lower than
ꢁ20 ꢀC (Fig. 1E, b).
NOE measurements have been used to determine the confor-
mation of germacrene-type sesquiterpenes in solution.24b
NOE data have proven effective in ascertaining the preferred
conformation of the relatively rigid germacranolides,55 in
which not all the operations (inversion of double bonds
and flip of the C6–C7–C8 segment) leading to all conformers
of a given structure are energetically possible. In a few in-
stances NOE experiments, in combination with variable-
Table 2. Relative steric energies (kcal/mol) of the four conformers (see
Fig. 4) of germacrene A and related germacrene-type sesquiterpenes calcu-
lated by the Allinger MM2 force field method in the present work
1
temperature H NMR spectra and/or molecular mechanic
calculations have been applied to establish the preferred con-
Sesquiterpene
UU
UD
DD
DU
formation of simpler and flexible germacrenes.56,57
Germacrene Aa,b
Germacrene Bc
Costunolidec
0.00 (84) 1.37 (8.3) 1.49 (6.7) 2.65(1)
0.00
0.00
0.45
1.78
3.75
0.00
0.47
0.04
4.76
0.22
0.00
1.23
4.50
1.41
2.00
In the present work, NOE measurements were complicated
by the fact that, even at low temperatures (ꢁ20 ꢀC), the flex-
ible sesquiterpene (dynamic system) was in the slow ex-
change regime on the chemical shift time scale (separate
signals from each contributing form are observable), and
consequently transfer of saturation took place.58 Thus, satu-
ration of the well separated frequency corresponding to H1
(4.78 ppm) of the major conformer a was transferred to the
signals at dH 5.01 (conformer b) and 4.94 (conformer
Hedycaryolc
Dehydrogermacrene Ac 0.04
Populations (%) at 25 ꢀC are given in parenthesis for germacrene A.a
Calculated assuming entropy differences between conformers are negligi-
a
ble.
MM3 calculations gave similar relative energies: UU (0.00), UD(1.99),
b
DD (1.82), DU (3.56).
The relative steric energies agree with literature data. See Refs. 34b,
50–53.
c
Table 3. Selected 500 MHz 1H NMR data (CDCl3, ꢁ20 ꢀC) for the (+)-germacrene A (1) conformers a (52%), b (29%), and c (19%), (dH, m, (J in Hz))
Conformer
H-12
H-120
H-13
H-14
H-15
H-1
H-5
a
b
c
4.65 s
4.65 s
4.65 s
4.54 s
4.57 s
4.51 s
1.71 s
1.70 s
1.71 s
1.37 s
1.54 s
1.68 s
1.52 s
1.46 s
1.48 s
4.78 dd (11.2, 4.0)
5.01 t (8.1)
4.94 d (12.1)
4.51 d (10.0)
5.07 dd (9.5, 6.6)a
5.21 t (7.9)
a
Multiplicity and J values at ꢁ30 ꢀC.

J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
7739
c) ppm by the exchange process (Table 3). This unexpected
phenomenon turned out to be extremely useful by verifying
the H1 chemical shift position of the less populated con-
formers b and c. In addition, a small NOE enhancement
(1%) was observed at 4.51 (H5, conformer a) ppm, thus cor-
roborating the syn relationship of the vinylic hydrogens in
the predominant chair–chair (UU) conformation of germa-
crene A, i.e., 1a (Fig. 5).
crossed DD conformer as one of the observable minor con-
formational isomers. These experimental results (in solu-
tion) contrast with the theoretical MM2 calculations,
which predict that germacrene A (1) would exist as an equi-
librium mixture of two crossed (UU and DD) and one paral-
lel (UD) set of conformers in the gas phase (Table 2).
The experimental J values for the olefinic hydrogens (H1
and H5) of conformer b (Table 3) were compared with those
predicted for the UD conformation using the Karplus equa-
tion59 and the dihedral angles (F) of ꢁ22.9ꢀ (H1H2b,
J¼8.9 Hz), ꢁ138.2ꢀ (H1H2a, J¼7.7 Hz), 33.1ꢀ (H5H6b,
J¼7.6 Hz), and 150.2ꢀ (H5H6a, J¼9.6 Hz), which were cal-
culated by the MM2 program. The calculated J values for H1
and H5 seem to be in good agreement with the observed
coupling constants for conformer b (Table 3). This agree-
ment provided the basis for a tentative assignment of the sec-
ond most populated conformational isomer (30%, 1b) as the
parallel (UD) conformation. The conformational equilibria
and NMR assignments are summarized in Figure 5. The
energy-minimized Chem3D figures of the three distinguish-
able conformers (1a, 1b, and 1c) of germacrene A are dis-
played in Figure 6. It is interesting to note that the recently
isolated isogermacrene A (isopropenyl moiety at C6) exists
as one predominant UU conformation at temperatures lower
than room temperature (e.g., 10 ꢀC).60
Saturation transfer was again observed when the downfield
resonance at dH 5.21 (H5) corresponding to the least popu-
lated conformer (c) was irradiated. In this case the saturation
was transferred to the frequencies at dH 5.07 (H5, conformer
b) and 4.51 (H5 conformer a) ppm (Table 3). This pheno-
menon was used again to corroborate chemical shift assign-
ments for the olefinic methyl singlets of a, b, and c
conformers (Table 3) by selective saturation of the signals
at dH 1.37 (H14, conformer a) and 1.54 (H14 conformer b)
and simultaneous saturation of dH 1.48 and 1.46 (H15, con-
formers c and b, respectively). In addition, six small NOE
enhancements (2–3%) were observed as follows: H5/H14
(b), H5/H14 (c), H15/H1 (b), H15/H1 (c), H14/H5 (b),
and H14/H5 (c). These results clearly verify that the two
minor conformers (b and c) of germacrene A adopt the par-
allel configurations (UD and DU) of double bonds, with anti
orientations of their vinylic protons H1 and H5. These two
minor parallel conformers account for almost 50% of the
total mixture of conformers in solution and therefore, germa-
crene A exists as a 1:1 mixture of one crossed (52%) and
two parallel forms (48%). Furthermore, the absence of
an observable NOE between H1 and H5 rules out the
X-ray crystallographic structures of silver nitrate adducts of
(E,E)-configured germacrenes such as germacrene B,61 ger-
macrone,62 pregeijerene,47b,63 and costunolide64 have shown
that the most stable (UU) conformation is also the preferred
one in the solid state. Unfortunately attempts to prepare
X-ray quality crystals of the AgNO3 adduct of (+)-
germacrene A (1) following literature procedures47b,65 were
unsuccessful.
1.54
CH3
1.48
H3C
H 4.94
(4.65, 4.51)
(4.65, 4.57)
CH2
1.70
CH3
H
5.07
CH2
1.71
CH3
5.01
H
1.68
CH3
5.21
H C
3
H
1.46
3. Conclusion
1c (19%)
1b (29%)
Cultures of a yeast strain harboring a terpene synthase gene
from lettuce produced large quantities of a pure C15 hydro-
carbon identified as (R)-(+)-germacrene A. This widely
occurring but sensitive sesquiterpene was thoroughly char-
acterized for the first time by its TLC and GC behaviors,
by MS, optical rotation, and spectral properties, and by ther-
mal rearrangement to (ꢁ)-b-elemene. Analysis of the com-
plex variable-temperature NMR spectra indicated that
germacrene A exists as a mixture of three conformational
isomers in a ratio of about 5:3:2 at temperatures at or below
ordinary probe temperatures (25 ꢀC). The most stable
1.37
CH3
1.52
(4.65, 4.54)
H3C
CH2
1.71
CH3
4.78
H
H 4.51
1a (52%)
Figure 5. The three conformers observed in the proton NMR spectra of ger-
macrene A, 1a (52%), 1b (29%), and 1c (19%), are assigned to one major
crossed form (UU) and two minor parallel arrangements (UD and DU).
The characteristic H NMR chemical shifts and assignments of hydrogens
1, 5, 12, 13, 14, and 15 of each conformer are shown by the placement of
the bold numbers (see also Table 3).
1
Figure 6. Chem3D representations of the three observable conformers of germacrene A in solution. The structures were minimized according to the MM2 force
field method.

7740
J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
conformer (52%) was assigned as the UU (up–up) form (1a)
in which the two methyl groups and isopropenyl substituent
adopt positions on the top face of the crossed cyclodecadiene
ring. The less populated conformers observed (29 and 19%)
are attributed to the parallel UD and DU (up–down and
down–up) orientations having the C4 and C10 vinyl methyl
groups on opposite faces of the cyclodecadiene core (1b and
1c). These conclusions are similar to those reached for hedy-
caryol (2), except that the UD and DU conformers predom-
inate over the UU alternative,35b a difference attributable to
the larger steric size of the hydroxypropyl substituent. The
conformer populations of germacrene A in the sesquiterpene
cyclase active sites are likely to be very different than those
in solution, and that is certainly a major factor in the product
specificities of these enzymes. The proximity and orienta-
tions of the C]C double bonds and the positions of the
ring substituents set the stage for the course of the ensuing
cyclizations and rearrangements.
conformations (UU, UD, DU, and DD) shown in Figure 4
were judged to be the most likely to be populated in the
gas phase. The UU (20.83 kcal/mol) conformation was
found to correspond to the global minimum, and the confor-
mations UD, DD, and DU were interpreted as local minima
with steric energies of 22.20, 22.32, and 23.47 kcal/mol,
respectively.
4.1.1. Production of (D)-germacrene A (1) with the engi-
neered yeast. Yeast transformation, culturing, and terpene
production were performed according to Takahashi et al.42
In brief yeast transformed with the recombinant Yep352 vec-
tor harboring the lettuce terpene synthase gene and a wild
type copy of the URA3 gene was selected for prototrophic
growth on minimal media without uracil supplementation,
and three independent colonies were selected by PCR
screening for the lettuce gene. Each colony was grown in
10 mL of minimal media at 28 ꢀC for 2–4 days before trans-
ferring the entire culture into the nutrient rich YPDE
medium (150 mL) for terpene production.42 After 7 days,
the yeast cultures were lysed by vigorous mixing (30 s)
with an equal amount of acetone (160 mL), followed by
a second 30 s mixing with an equal volume of hexane
(160 mL). The upper hexane layer was carefully transferred
to a collection vessel, and the lower aqueous layer was
extracted with additional hexane. The combined organic
extracts were concentrated to a final volume of 20 mL using
a stream of nitrogen. Large-scale production of the sesqui-
terpene product was achieved by inoculating the initial
10-mL yeast cultures into each of ten 200-mL aliquots of
the YPDE medium and by allowing the cultures to grow
for an additional 7 days. The 2 L of combined yeast culture
was extracted as described above and concentrated under
a stream of nitrogen to a final volume of 20–30 mL.
4. Experimental
4.1. General methods
Optical rotations were measured on a JASCO DIP-370 dig-
ital polarimeter at 25 ꢀC. The UV spectrum was obtained on
a Shimadzu UV-2401 PC spectrophotometer. IR spectra
were measured on a Perkin Elmer Spectrum BX, FTIR spec-
trophotometer. GC analyses were conducted on a Rtx-5
30-m fused silica capillary column (split ration ca. 100:1).
The following programs were used: Method A¼initial
temperature 50 ꢀC for 1 min, ramp 5 ꢀC/min to 130 ꢀC at
an injection temperature of 110 ꢀC. Method B¼initial tem-
perature 60 ꢀC for 3 min, ramp 4 ꢀC/min to 150 ꢀC at an
injection temperature of 180 ꢀC.
4.1.1.1. (D)-Germacrene A (1). Cold (dry ice) hexane
solutions containing germacrene A were shipped from the
University of Kentucky to the University of Illinois, and im-
mediately evaporated with a stream of nitrogen. The residue
was re-dissolved in pentane and filtered through silica gel
with additional pentane as an eluent. Fractions containing
sesquiterpene 1 (TLC Rf 0.71 (pentane)) were combined
and evaporated with a stream of nitrogen to afford a total
of 363 mg (two batches) of pure germacrene A as a clear
oil. The purity of this material was estimated to be >95%
1H and 13C NMR spectra were recorded on a Varian 500
spectrometer (500 MHz for 1H and 125 MHz for 13C).
Chemical shifts are given in parts per million using TMS
as an internal standard. CDCl3 from Aldrich Chemical Co.
was purified by filtering through basic alumina (Brockmann
˚
I, standard grade, 150 mesh, 58 A) and dried overnight over
˚
molecular sieves (4 A) in the dark. Benzene-d6 was used
without purification. The silica gel used for column chroma-
tography (Merck 60, 230–400 mesh) was purified as follows:
a suspension of 3.0 g of silica gel in 15 mL of pentane (15%
Et3N) was stirred at room temperature for 30 min, filtered
under vacuum, washed with n-pentane, and dried in an
oven (110 ꢀC). TLC was carried out with Merck 60F-254
plates with 0.25 mm thickness. Visualizations of the TLC
spots were performed by spraying with an 0.1% solution
of berberine hydrochloride in EtOH, and/or UV light.
n-Pentane (HPLC grade) was purchased from Fisher Scien-
tific and was used without further purification.
1
by H NMR spectroscopy. For characterization purposes,
small quantities (10–15 mg) were further purified by prepar-
ative TLC on silica gel using n-pentane as developing sol-
vent. Data for germacrene A: TLC Rf 0.71 (pentane); [a]D25
+42.1 (c 1.0, CCl4), lit.24c ꢁ26.8 (c 1.0, CCl4), lit.24a ꢁ3.2
(c 14.4, CCl4); UV (CH3OH) lmax nm (log 3) 214 (2.49);
IR nmax (liquid film, CH2Cl2) 3061, 1643, 1275, 1261,
1
886, 841 cmꢁ1; H NMR (see Tables 1 and 3). 13C NMR
(125 MHz, CDCl3, 25 ꢀC) 153.7 (C11), 152.3*, 138.2
(C10), 131.7 (C5), 128.9 (C4), 128.6*, 126.9*, 126.4 (C1),
125.3*, 121.8*, 108.2*, 107.5*, 107.3 (C13), 51.3 (C7),
47.9*, 45.7*, 41.7 (C9), 41.5*, 39.5 (C2 or C3), 38.8*,
37.0*, 34.8 (C6), 34.6*, 33.6 (C8), 33.4*, 32.6*, 31.5*,
30.7*, 26.7 (C2 or C3), 24.2, 21.9*, 20.3 (C12), 19.9, 16.7
(C15), 16.2 (C14), 15.4. 13C NMR (125 MHz, C6D6,
25 ꢀC) 153.6 (C11), 152.2*, 137.8 (C10), 131.9 (C5),
128.8 (C4), 126.8 (C1), 125.6*, 122.2*, 108.8*, 108.0*,
107.8 (C13), 51.8 (C7), 48.4*, 46.1*, 41.9 (C9), 41.8*,
Molecular mechanics calculations (employing the MM2
force field)49 were performed with the program CS Chem
3D UltraÒ (version 8.0) from Cambridge Soft. All calcula-
tions were carried out on a standard PC equipped with a
Pentium[R] 4 CPU 2.40 GHz processor and 512 MB
RAM. A total of 24 start geometries were modeled using
this method with relative steric energies ranging from 0.0
to 13.0 kcal/mol. After the minimization process, the four

J. A. Faraldos et al. / Tetrahedron 63 (2007) 7733–7742
7741
Phytochemistry 1988, 27, 1365–1370; (c) Whitehead, I. M.;
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39.8 (C3 or C2), 39.1*, 37.4*, 35.2 (C6), 34.9*, 33.9 (C8),
33.7*, 33.0*, 31.7*, 31.1*, 27.0 (C2 or C3)*, 24.6, 22.0*,
20.3 (C12), 19.9, 16.7 (C15), 16.2 (C14), 15.4. The asterisks
designate less intense resonances. Assignments were made
based on those in Refs. 24c and 26 (benzene-d6), and corre-
spond to the major conformer at room temperature.
4.1.1.2. (L)-b-Elemene (4). A solution of germacrene A
(1) (10.1 mg, 0.05 mmol) in 1 mL of toluene was heated at
reflux for 2 h in a flame-stretched 10-mL test tube as the
reaction vessel. After cooling, the solution was applied to
a preparative TLC plate (see Section 4.1). The toluene was
allowed to evaporate under a nitrogen stream, and the plate
was developed using n-pentane to give (ꢁ)-b-elemene
(9.9 mg, quant.) as a clear oil: TLC Rf 0.81 (pentane);
[a]D25 ꢁ15.8 (c 0.50, CHCl3), lit.43 ꢁ11.8 (c 4.63 CHCl3),
lit.44 +15.4 enantiomer (c 0.60, CHCl3); UV (CH3OH)
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1
lmax nm (log 3) 210 (2.00); H and 13C NMR data in total
agreement with Brauchli and Thomas.45 EIMS m/z (%)
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Acknowledgements
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We are very grateful to Dr. A. M. Adio for exchanges of
information and for 1D and 2D NMR spectra of an impure
sample of germacrene A isolated from S. Canadensis,26
Dr. Vera Mainz of the School of Chemical Sciences NMR
Facility at the UI for assistance with the VT and NOE
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the UI for consultations and advice concerning the MM2
calculations. This work was supported by grants from the
National Institutes of Health (GM 13956 to RMC and GM
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