Sesquiterpenes of the liverwort Scapania undulata

Article abstract of DOI:10.1016/j.phytochem.2003.10.018

The essential oil of the liverwort Scapania undulata, collected in the Harz mountains, Northern Germany, was analysed by gas chromatography (GC), GC-mass spectrometry (MS) and several new components were isolated and investigated by various NMR techniques. As new natural compounds the sesquiterpene hydrocarbons (+)-helminthogermacrene (1) [the 4Z-isomer of germacrene A (9)], (-)-cis-β-elemene (2) as a Cope-rearrangement product of 1, (+)-β-isolongibornene (3) and (-)-perfora-1,7-diene (4) could be identified. 1 has an identical mass spectrum and identical GC retention time on a non-polar stationary phase as germacrene A (9) but is considerably more stable than the latter. The Cope-rearrangement of 1 proceeds slowly at 350°C and (-)-cis-β-elemene (2) is formed together with small amounts of other diastereoisomers.

Full text of DOI:10.1016/j.phytochem.2003.10.018

Phytochemistry 65 (2004) 199–206
www.elsevier.com/locate/phytochem
Sesquiterpenes of the liverwort Scapania undulata
Adewale Martins Adioa, Claudia Paulb, Petra Klotha, Wilfried A. Koniga,*
aInstitut fur Organische Chemie, Universitat Hamburg, Martin-Luther-King-Platz6, D-20146 Hamburg, Germany
¨
¨
bThetis-IBN GmbH, Notkestrasse 85, D-22607 Hamburg, Germany
Received 18 September 2003; received in revised form 7 October 2003
Abstract
The essential oil of the liverwort Scapania undulata, collected in the Harz mountains, Northern Germany, was analysed by gas
chromatography (GC), GC–mass spectrometry (MS) and several new components were isolated and investigated by various NMR
techniques. As new natural compounds the sesquiterpene hydrocarbons (+)-helminthogermacrene (1) [the 4Z-isomer of germa-
crene A (9)], (À)-cis-b-elemene (2) as a Cope-rearrangement product of 1, (+)-b-isolongibornene (3) and (À)-perfora-1,7-diene (4)
could be identified. 1 has an identical mass spectrum and identical GC retention time on a non-polar stationaryꢀphase as germa-
crene A (9) but is considerably more stable than the latter. The Cope-rearrangement of 1 proceeds slowly at 350 C and (À)-cis-b-
elemene (2) is formed together with small amounts of other diastereoisomers.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Scapania undulata; Liverworts; Sesquiterpene hydrocarbons; Cope rearrangement; (+)-Helminthogermacrene; (+)-b-Isolongibornene;
(À)-cis-b-Elemene; (À)-Perfora-1,7-diene
1. Introduction
or extraction with diethylether revealed the presence of
a component with the mass spectrum of b-elemene,
however eluting clearly before b-elemene. Usually b-ele-
mene is formed by Cope-rearrangement of the thermally
extremely labile germacrene A (9). Although a compo-
nent with the mass spectrum and the retention time of
germacrene A was detected, an increase of the injection
port temperature of the gas chromatograph did not
generate b-elemene, but an ‘‘isomer’’ eluting before b-
elemene. The stereochemical analysis of the b-elemene
isomer (2) and its precursor 1 and the identification of 2
new sesquiterpene hydrocarbons (+)-b-isolongibornene
(3) and (À)-perfora-1,7-diene (4) is described. Interest-
ingly, 3 seems to be identical with ‘‘scapanene’’ and 4
with ‘‘undulatene’’. Both compounds were discussed in
the work of Andersen et al. (1977), and their (incomplete)
spectroscopic data are in agreement with 3 and 4,
respectively, but their structures could not be identified
at that time.
Liverworts produce a large variety of sesquiterpenes,
particularly sesquiterpene hydrocarbons, which are
important intermediates in the biosynthesis of function-
alized sesquiterpenes and may be useful reference com-
pounds in studying the product specificity of
sesquiterpene synthases, particularly after site-specific
mutagenesis. Several chemotypes of Scapania undulata
have been characterized (Asakawa, 1995). Two of them
predominate in the German Harz mountains: a chemo-
type with the major sesquiterpene constituents posses-
sing a cadinane skeleton (‘‘ent-1-epi-cubenol-type’’) and
a chemotype with (À)-longiborneol and longipinane
derivatives as major constituents (‘‘longiborneol type’’).
The constituents of the liverwort S. undulata have been
investigated before (Andersen et al., 1977). Besides some
known sesquiterpene hydrocarbons, a few compounds
were discussed which could not be fully identified. A
recent screening of the volatile constituents of the long-
iborneol-type S. undulata prepared by hydrodistillation
2. Results and discussion
* Corresponding author. Tel.: +49-40-428382824; fax: +49-40-
428382893.
E-mail address: wkoenig@chemie.uni-hamburg.de (W.A. Konig).
The essential oil of Scapania undulata, a liverwort
which is very abundant in the Harz mountains in
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.10.018

200
A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
Scheme 1.
Northern Germany, was prepared by hydrodistillation
and analysed by GC and GC–MS. In order to exclude
artefact formation by hydrodistillation also an ether
extract was prepared and investigated by the same
methods. All known constituents were identified by
comparison of their mass spectra and gas chromato-
graphic retention indices with a spectral library estab-
lished under identical experimental conditions (Joulain
and Konig, 1998; MassFinder Software and Data Bank,
Hochmuth et al., 2002). Sesquiterpenoids with unknown
mass spectra were selected for isolation by preparative
GC and spectroscopic investigation by NMR. The pre-
sence of a large amount of longiborneol in the essential
oil indicated that the investigated sample belongs to the
‘‘longiborneol-chemotype’’.
(+)-helminthogermacrene
[(+)-1,5-dimethyl-8-(1-
methylethenyl)-cyclodeca-1E,5Z-diene] (1, 1%), (+)-a-
chamigrene (2.2%), d-cadinene, E-a-bisabolene, (À)-
perfora-1,7-diene (4, 1.1%) (À)-longipinanol (7.2%, in
the ether extract, see below), maaliol, (À)-longiborneol
(30.2%), 1-epi-cubenol, 2-himachalen-7-ol (2.1%)
(Scheme 1). Longipinanol is rather labile, as already
indicated by Andersen et al. (1977), and was almost
completely degraded during hydrodistillation, but was
detected in considerable amounts in the ether extract.
The sense of optical rotation of the new compounds
was determined by polarimetricmeasurements.
Enantioselective GC using cyclodextrin phases in con-
junction with chemical conversions was employed to
assign the absolute configuration.
The following sesquiterpenes were identified in the
order of their elution from a capillary column with
polydimethylsiloxane (CPSIL-5) as constituents of the
essential oil of S. undulata (relative concentrations of
major compounds are given in parentheses, those below
1% are only listed): a-longipinene (1.6%), a-ylangene,
longicyclene, sativene, b-longipinene (5%), (À)-cis-b-
elemene (1), longifolene (19%), b-maaliene, a-barba-
2.1. (+)-Helminthogermacrene (1)
The mass spectrum of the 10-membered monocyclic
sesquiterpene hydrocarbon exhibits a molecular ion sig-
nal at m/z 204 and an elemental composition of C15H24.
The mass spectrum of 1 and the retention index on a
non-polar stationary phase are identical to germacrene
A (9). However a separation from germacrene A is
achieved on more polar stationary phases as CPSil 19.
Unlike many germacratrienes with E,E-configuration of
the olefinic double bonds in the cyclodecadiene system
which show temperature dependent NMR spectra
(multiple sets of signals) indicative of conformational
tene,
E-b-caryophyllene,
b-ylangene,
(+)-b-iso-
longibornene (3, 2.4%), isobazzanene, b-barbatene
(1.5%), a-himachalene (4.7%), ar-curcumene, iso-
bicyclogermacrene, (+)-a-amorphene, g-himachalene
(2.3%), isolepidozene (Hardt et al., 1995) Z-a-bisabo-
lene (1.6%), (+)-a-muurolene, b-himachalene (1.1%),

A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
201
equilibria in solution (Takeda, 1974; de Kraker et al.,
2001), 1 occurs essentially in one preferred conforma-
tion even at room temperature. Although the H NMR
mene diastereomer (5) and a trace of ‘‘normal’’ (À)-b-
elemene (6) in the ratio of (8:1:ꢁ0.08), respectively (in
this elution order from the dimethylpolysiloxane sta-
tionary phase CPSIL-5). This observation is in agree-
ment to investigations of Kodama et al. (1979) who
investigated the rearrangement products of all possible
geometrical isomers of hedycaryol [germacra-1(10),4-
dien-11-ol, 7]. The (E,Zꢀ)-isomer of hedycaryol is repor-
ted to rearrange at 300 C to the corresponding cis-iso-
elemol (8, Scheme 1), but 3 different diastereoisomers,
which correspond to 2 (major isomer), 5 and 6, are
1
spectrum (C6D6) showed broadened signals in the
region between ꢀ 1.84–2.24, the three downfield shifted
singlets for the methyl groups at ꢀ 1.57 (3H, s, CH3-14),
1.66 (3H, br.s, CH3-12) and 1.70 (3H, s, CH3-15) are
well resolved. In the 13C NMR spectrum the olefinic
carbon signals at ꢀ 150.6 (s) and 109.3 (t) suggested an
exo-methylene double bond, which was confirmed by
1
two signals in the H NMR spectrum at ꢀ 4.77 (1H, br.
s) and 4.85 (1H, s). The multiplet signal at ꢀ 5.30-5.44
(2H, m) was assigned to the two methine protons H-1
ꢀ
formed at 500 C, apparently by conformational equili-
bration before rearrangement. Similar observations were
made by Takeda (1974) for Cope rearrangement reac-
tions with other germacrene type sesquiterpenes, which
formed mixtures of diastereoisomers. In other cases, e.g.
germacrene A, B and C, only one type of diastereomer
is observed upon heating.
(+)-Helminthogermacrene (1) was isolated for the
first time as a natural product. It should be mentioned
that in a recent paper Prosser et al. (2002) claimed the
formation of (+)-b-elemene from its precursor (À)-ger-
macrene A from S. undulata. It can be speculated that
they really obtained (À)-cis-b-elemene (2) and not (+)-
b-elemene as no germacrene A (9) and only a minor
peak of the (À)-enantiomer of b-elemene (6) could be
detected in any of the chemotypes of S. undulata.
1
and H-5. As expected, the H NMR of 1 recorded in
acetone-d6 at À8 ꢀC and À16 ꢀC showed less broadened
and separated multiplet signals and also predominantly
one conformation since only one set of signals was
observed. All this information from 13C NMR as well as
1
HMBC, HMQC and H–1H COSY led to structure 1
for this compound. Its relative configuration resulted
from the NOESY spectrum recorded in the region ꢀ 4.4–
5.8 in which the methyl group at ꢀ 1.70 (3H, s, CH3-15)
spatially interacts with the multiplet signal at ꢀ 5.30–
5.44. This interaction suggests that H-15 is in cis-con-
figuration with the H-5 methine proton, which is only
possible for a Z-4-5 double bond. Spatial interaction of
H-12 at ꢀ 1.66 (3H, br.s) with the methylene protons was
also observed (Fig. 1). Considering the minute struc-
tural difference between 1 and germacrene A (9) the a-
orientation of H-7 in 1 was inferred from the known
absolute configuration of 9 both having the same posi-
2.2. (À)-cis-ꢁ-Elemene (2)
Compound 2 is present only in trace amounts in the
original essential oil of S. undulata. As shown above, it
can be generated from 1 by Cope rearrangement and
isolated by preparative GC by injecting it into the pre-
parative gas chromatograph at an injector temperature
of 390 ꢀC. 2 exhibits a mass spectrum undistinguishable
from b-elemene with a molecular ion signal at m/z 204
and an elemental composition of C15H24. Its structure
1
tive direction of optical rotation. The H NMR spectra
recorded in CDCl3 and the 13C NMR recorded in ace-
tone-d6 were identical with those reported for the
levorotatory enantiomer of 1 found in the defensive
secretion of the termite Amitermes wheeleri (Scheffrahn
et al., 1986) and the same enantiomer isolated from the
mycelium of the fungus Helminthosporium sativum with
its structure verified by total synthesis (Winter et al.,
1980; McMurry and Kocovsky, 1985). The absolute
configuration of (À)-helminthogermacrene was not
unambiguously established, although the a-orientation
of H-7 was concluded from the sesquiterpene congeners
(À)-sativene and (À)-longifolene isolated from the same
organism (Winter et al., 1980). Since the reacting con-
formations of 1 can be correlated with the configuration
of the products of transannular cyclization reactions,
which usually create chiral centers regio- and stereo-
specifically in reactions starting from trigonal carbon
atoms (Bulow and Konig, 2000), 1 was subjected to
acid-catalyzed and thermal (Cope) rearrangement reac-
tions. In contrast to helminthogermacrene, which is
thermally more stable, germacrene A undergoes Cope
rearrangements at room temperature (Weinheimer et
al., 1970). ꢀThermal isomerization of 1 was achieved
above 350 C to afford cis-b-elemene (2), another b-ele-
1
was elucidated based on H NMR spectra and its rela-
tive configuration was established by 1H–1H COSY,
HMBC, HSQC and NOESY-NMR in comparison with
Fig. 1. NOE correlations observed for (+)-1.

202
A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
the spectral data of authentic b-elemene (6). The 1H
NMR was recorded in both C6D6 and CDCl3 to achieve
the best resolution over the whole chemical shift range.
to the (E,E)-isomer (9), was confirmed by enantio-
selective GC.
It is also interesting to note that compound 1 did not
isomerise when treated with silica gel for 3 h, which
proves its stability in comparison to (E,E)-germacrene
A (9).
All the information from spectral data, transannular
cyclization reactions in addition to the biosynthetic
relationship suggested by Winter et al. (1980) for the
identified sesquiterpenes from S. undulata (Scheme 1) is
in agreement with the 1(10)E,4Z-configuration for the
cyclodecadiene system of compound 1 and the cis-con-
figuration of compound 2 which was unambiguously
assigned based on the NOESY correlation of H-5 and
H-14, while the absolute configuration is still tentative
and mainly based on the assumptions of Winter et al.
(1980).
1
The H NMR (C6D6) showed signals of three methyl
singlets at ꢀ 1.05 (3H s, CH3-14), 1.66 (3H, s, CH3-15)
and 1.67 (3H, s, CH3-12). Interestingly, the CH3-15 sig-
nal in 2 is slightly up-field shifted by 0.04 ppm as com-
pared to b-elemene (6). The two methylene protons at ꢀ
5.01 (ddd, 2H, H-2, J=1.26, 11.04, 17.34 Hz) formed an
ABX system with a methine proton at ꢀ 6.29 (dd, 1H, H-
1, J=11.04, 17.34 Hz). Additional structural informa-
tion was obtained from the 13C NMR spectrum of 2
which confirmed the presence of three methyl groups at ꢀ
21.38, 22.89 and 27.81, six methylene groups at ꢀ 28.02,
33.76, 42.23, 109.09, 112.90 and 113.35, three methine
groups at ꢀ 46.48, 56.24 and 143.27 and three qua-
ternary carbons at ꢀ 39.45, 147.20 and 150.29.
For the relative configuration of 2 a cis-relationship at
C-5 and C-10 was concluded from a strong NOESY
interaction of the H-5 and H-14 protons. Furthermore,
interactions between H-9a with both H-5 and H-7 con-
firmed that the three hydrogens are on the same side of
the molecule. Since 2 is formed from 1 with retention of
its configuration at C-7, a-orientation for H-7 and con-
sequently for CH3-14 is suggested (Fig. 2).
It is also noteworthy that a series of cis-fused seli-
nenes was found in the S. undulata sample investigated
by Andersen et al. (1977), as well as in the volatiles of
Helminthosporium sativum (Dorn, 1975) and Amitermes
excellens (termite), (Naya et al., 1982) which are more
closely allied to the germacrene-sativene group of
sesquiterpenes than to the more common trans-fused
selinenes.
The structure of compound 5, the diastereomer of 2,
1
was assigned based on identical GC–MS and H NMR
1
2.3. (+)-ꢁ-Isolongibornene (3)
spectra with 2. The signals of 5 in the H NMR spec-
trum (C6D6) were slightly shifted up-field with the
methine proton at ꢀ 6.22 (1H, dd, J=11.03, 17.65 Hz)
compared to ꢀ 6.29 in 2. A slight up-field shift of the
methine proton signal to ꢀ 6.23 (dd, 1H, J=11.35, 17.66
Hz) as compared to ꢀ 6.31 (dd, 1H, J=11.03, 17.65 Hz)
was also observed when the 1H-NMR of 2 was recorded
in CDCl3, while the corresponding proton of authentic
b-elemene (6) absorbs at ꢀ 5.80 (1H, dd, J=10.7, 17.3
Hz). The C-10 methyl singlet of 5 also appeared slightly
shielded at ꢀ 1.04 as compared to 1.05 in 2.
In the mass spectrum of 3 a molecular ion signal at
m/z=204, corresponding to the elemental composition
C15H24, is observed. In the 1H NMR spectrum one
exocyclic double bond can be assigned to resonances at
ꢀ 4.91 (s, 1 H, H-15a) and 4.96 (br.s., 1 H, H-15b). In
addition, three methyl singlets at ꢀ 0.83 (CH3-14), 0.95
(CH3-12) and 1.12 (CH3-13) are present. In the 13C
NMR spectrum signals of five methylene groups appear
at ꢀ 22.6 (C-9), 30.5 (C-2), 31.0 (C-1), 40.5 (C-8), 41.4
(C-10) and 104.5 (C-15). Furthermore, the signals of
three methine groups at ꢀ 46.3 (C-6), 57.0 (C-3) and 60.7
Treatment of 1 with boron trifluoride etherate in die-
thyl ether at room temperature for 3 hours gave d-seli-
nene (10) and
a few unidentified sesquiterpene
hydrocarbons. Interestingly, the comparison of 10 pro-
duced by treatment of 1 with BF3 by enantioselective
GC with authentic(+/ À)-10 using a cyclodextrin sta-
tionary phase confirmed that a mixture of the (+)- and
(À)-enantiomer in the ratio 5 : 1 was formed.
The formation of both enantiomers of 10 and the co-
ocurrence of a small amount of diastereomers of 2 sug-
gests that more than one conformation might be present
1
during the rearrangement process, although H NMR
ꢀ
ꢀ
recordings at ambient temperature, À8 C and À16 C
did not reflect any significant doubling of signals. Thus,
the conformational equilibrium in solution pre-
dominantly favoured the (+)-(E,Z)-1 configurational
isomer (Fig. 3). The existence of
a
trace
[ꢁ0.08%=(À)>(+)] of b-elemene (6), corresponding
Fig. 2. NOE correlations for (À)-2.

A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
203
(C-5) and 3 quaternary carbon signals at ꢀ 36.2 (C-11),
48.6 (C-7) and 161.6 (C-4) can be detected. The assign-
ment of these resonances to the corresponding carbon
atoms was achieved by HMBC and HSQC measure-
ments and is supported by the 1H–1H COSY NMR
data. Particularly the series of connectivities from C-14
to C-7 and further to C-8, C-9, C-10, C-11, C-12 and C-
13 can be established from the HMBC measurements
(Fig. 4) as well as the position of the geminal methyl
groups to C-11. Also from the HMBC experiment the
position of C-5 next to C-11, the position of C-2 next to
C-3 (2J coupling), the position of the methylene groups
CH2-1 and CH2-2 next to methines CH-3 and CH-6 can
be concluded. The spatial relationship of the methylene
groups CH2-1 and CH2-2 and CH2-8, CH2-9 and CH2-
10 and the spatial relationship of CH2-1 and CH-6 is
also confirmed by the NOESY experiment.
nals at ꢀ 5.40 (br.s, 1 H) and 5.45 (br.s, 1 H) were
assigned to the olefinicmethine protons H-2 and H-7,
respectively. Additional structural information was
1
obtained from the 13C NMR and by the H–1H COSY
spectrum. The connectivity derived from the HMBC
spectrum (Fig. 5) confirmed a perforane skeleton. The
proposed relative configuration at C-4, C-5 and C-11
was established by the NOESY spectrum (Fig. 6). The
1
compound exhibits identical H NMR and MS data as
the unknown compound ‘‘undulatene’’ earlier reported
from the same species by Andersen et al. (1977). Scheme 2
shows the proposed biogeneticpathway for the perfor-
ane skeleton from cis-trans-farnesyldiphosphate. Keto-
halides and hydroxyl derivatives with perforane back-
bone have earlier been isolated from the marine alga
Laurencia perforata and their structures were confirmed
by total synthesis (Gonzalez et al., 1975, 1978). Howard
The structure of 3 is closely related (but with a re-
arranged carbon skeleton) to the known b-longipinene
(11) and longiborneol (12), both major constituents of
S. undulata. The assigned name for 3 is intended to
reflect this relationship.
2.4. (À)-Perfora-1,7-diene (4)
Pefora-1,7-diene (4), a new bicyclic sesquiterpene with
perforane skeleton exhibited a molecular ion signal at
m/z 204 and an elemental composition of C15H24. The
1H NMR spectrum (C6D6) showed signals of a doublet
and three singlets for methyl groups at ꢀ 0.82 (3 H, d, H-
13, J=6.6 Hz), 0.74 (3 H, s, H-14), 1.62 (3 H, br.s, H-
12) and 1.74 (3 H, s, H-15). The downfield shifted sig-
Fig. 4. Long-range correlations in the HMBC spectrum of (+)-b-iso-
longibornene (3).
Fig. 3. Rearrangement reactions with (+)-helminthogermacrene (1).

204
A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
and Fenical (1979) reported some additional hydroxy
and acetoxy derivatives with perforane skeleton from a
related Laurencia species.
3.1.2. Preparative GC
Modified Varian 1400 and 2800 instruments, equip-
ped with stainless steel columns (1.85 m  4.3 mm) with
10% polydimethylsiloxane SE-30 on Chromosorb W-
HP or with 2.5% octakis(2,6-di-O-methyl-3-O-pentyl)-
g-cyclodextrin in OV-1701 (50%, w/w) on Chromosorb
G-HP or with 6% heptakis(6-O-tert-butyldimethylsilyl-
2,3-di-O-methyl)-b-cyclodextrin in SE-52 (50%, w/w)
on Chromosorb W-HP; FID; helium as carrier gas at a
flow rate of 240 ml/min; injector and detector tem-
3. Experimental
3.1. General experimental procedures
3.1.1. Gas chromatography
ꢀ
Orion Micromat 412 double column instrument with
25 m fused silica capillaries with polysiloxane CPSil-5
and polysiloxane CPSil-19 (Chrompack); Carlo Erba
Fractovap 2150 or 4160 gas chromatographs with 25 m
fused silica capillaries with octakis(2,6-di-O-methyl-3-
O-pentyl)-g-cyclodextrin, heptakis(2,6-di-O-methyl-3-
O-pentyl)-b-cyclodextrin or heptakis(6-O-tert.butyldi-
methylsilyl-2,3-di-O-methyl)-b-cyclodextrin in OV 1701
(50%, w/w), split injection; split ratio approx. 1:30;
FID; carrier gas 0.5 bar H2; injector and detector tem-
peratures were 200 and 250 C, respectively (Hardt and
Konig, 1994).
3.1.3. GC–MS
Electron impact (70 eV) GC–MS was carried out with
a Hewlett Packard HP 5890 gas chromatograph coupled
to a VG Analytical 70-250S mass spectrometer.
3.1.4. NMR-spectroscopy
NMR measurements were carried out with a Bruker
WM 400 (400 MHz) or a Bruker WM 500 (500 MHz)
instrument in C6D6 and/or CDCl3 using TMS as inter-
nal standard.
ꢀ
peratures were 200 ꢀC and 250 C, respectively.
3.1.5. Polarimetry
Measurements were performed with a polarimeter 341
(Perkin-Elmer) at 589 nm at 20 ꢀC. Due to the very
small amounts of isolated compounds only the sense of
optical rotation is given to avoid inaccurracies.
Fig. 5. Long-range correlations in the HMBC spectrum of (À)-per-
fora-1,7-diene (4).
Scheme 2. Proposed biogenetic pathway to perfora-1,7-diene from
Z,E-farnesyldiphosphate.
Fig. 6. NOESY correlations for (À)-perfora-1,7-diene (4).

A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
205
3.1.6. Acidic rearrangement of 1
1H), 2.48–2.54 (m, 1H), 4.60 (br.s, 1H), 4.67 (br.s, 1H),
5.21 (br.d, 1H, J=10.7 Hz), 5.33 (br.s, 1H); 13C NMR
[data taken from HSQC/HMBC, one carbon missing
(125.7 MHz, C6D6, 20 ꢀC )]: ꢀ=16.32, 24.27, 25.30,
29.52, 31.09, 33.15, 41.01, 49.49, 109.31, 124.68, 126.59,
132.02, 134.42, 150.59; 13C NMR [data taken from
To a solution of 1 in diethyl ether (5 ml) BF3 Et2O (3
ml) was added at room temp. The solution was stirred
for 5 min and allowed to stand for 3 h. Then ice cooled
aqueous 0.5 M KOH (20 ml) was added. The organic
layer was washed several times with aqueous 0.5 M
KOH, then with saturated aqueous NaCl solution and
dried over MgSO4; the solvent was concentrated and ana-
lysed with GC and GC–MS using achiral polysiloxane
and chiral cyclodextrin derived GC phases.
ꢀ
HSQC/HMBC (125.7 MHz, acetone-d6, À8 C)]: 16.06,
19.21, 24.19, 25.53, 29.97, 30.91, 33.08, 41.27, 49.99,
109.27, 124.96, 126.57, 132.43, 134.84, 151.19; MS (EI,
70 eV), m/z (rel. int.): 204 [M+] (17), 189 (19), 175 (5),
161 (34), 147 (36), 133 (18), 121 (42), 107 (43), 93 (67),
81 (58), 68 (100), 67 (52), 53 (44), 41 (68).
3.1.7. Thermal isomerization of 1
Compund 1 undergoes thermal isomerization at an
injector port temperature of 390 C of the preparative
GC instrument. The isomerized products 2, 5 and 6
were isolated using an SE 30 column.
ꢀ
3.5. (À)-cis-ꢁ-Elemene (2)
Colourless oil; RICPSIL5=1382; sense of optical rota-
1
tion (C6D6): (–); H NMR (500 MHz, C6D6): ꢀ=1.05
3.2. Plant material and essential oils
(3H, s, CH3-14), 1.27 (dt, 1H, H-9a, J=5.04, 12.93 Hz),
1.49–1.54 (m, 2H, 8-H2), 1.58–1.64 (m, 3H, 6-H2, H-9b),
1.66 (s, 3H, CH3-15), 1.67 (s, 3H, CH3-12), 1.87-1.92 (m,
1H, H-7), 1.94 (dd, 1H, H-5, J=3.46, 11.98 Hz), 4.75 (s,
1H, H-3a), 4.82 (d, 2H, 13-H2, J=13.24 Hz), 4.87 (s,
1H, H-3b), 5.01 (ddd, 2H, 2-H2, J=1.26, 5.99, 11.03
S. undulata was collected from several sites from rocks
in small rivers or submersed. Aqueous homogenates of
the fresh plant material was submitted to hydrodistillation
(2 h) using n-hexane as collection solvent. Alternatively
diethyl ether extracts were prepared from the air-dried
plant material at room temp. (24 h). Because of the greatly
differing weight the fresh material was not weighed.
1
Hz), 6.29 (dd, 1H, H-1, J=11.04, 17.34 Hz); H NMR
(500 MHz, CDCl3): 1.03 (s, 3H), 1.42 (dt, 1H, J=5.04,
12.92 Hz), 1.43–1.58 (m, 2H), 1.59–1.67 (m, 3H), 1.68 (s,
3H), 1.72 (d, 1H, J=14.5 Hz), 1.75 (s, 3H), 1.97–2.01
(m, 1H), 2.03 (dd,1H, J=3.47, 12.3 Hz), 4.65 (s, 1H),
4.71 (d, 1H, J=5.99), 4.78 (s, 1H), 5.01 (ddd, 2H,
J=1.58, 11.04, 18.9 Hz), 6.31 (dd, 1H, J=11.03, 17.65
Hz); 13C NMR [data taken from HSQC/ HMBC (125.7
MHz, C6D6 )]: ꢀ=21.38 (q, C-12), 22.89 (q, C-15), 27.81
(q, C-14), 28.02 (t, C-8), 33.76 (t, C-6), 39.45 (s, C-
10), 42.23 (t, C-9), 46.48 (d, C-7), 56.24 (d, C-5),
109.09 (t, C-13), 112.90 (t, C-2), 113.35 (t, C-3),
143.27 (d, C-1), 147.20 (s, C-4), 150.29 (s, C-11); MS
(EI, 70 eV), m/z (rel. int.): 204 [M+] (15), 189 (40),
175 (10), 162 (11), 161 (44), 147 (31), 133 (25), 121
(41), 119 (37), 107 (66), 105 (51), 95 (31), 93 (100),
91 (42), 81 (98), 79 (59), 77 (30), 68 (79), 67 (65), 55
(44), 53 (37), 41 (65).
3.3. Isolation of single constituents of the essential oils
The isolation of 1 was carried out using preparative
GC at an injector temperature of 120–140 ꢀC. The
essential oil of S. undulata was frationated using an SE-
30 column from 90 to 150 ꢀC with a heating rate of 2 ꢀC/
min. The fraction with high concentration in 1 was fur-
ther purified using prep. GC columns with heptakis(6-O
-tert-butyldimethylsilyl-2,3-di-O-methyl)-b-cylodextrin
ꢀ
(120 C, isothermal) and octakis(2,6-di-O-methyl-3-O-
pentyl)-g-cyclodextrin (125 ꢀC, isothermal) con-
secutively. The last stage of purification was achieved
using SE-52 to remove a co-eluting impurity of a-cha-
migrene of about 1%.
3.4. (+)-Helminthogermacrene (1)
3.6. (+)-ꢁ-Isolongibornene (3)
1
Colourless oil; RICPSIL5=1503; sense of optical rꢀota-
Colourless oil; RICPSIL5=1440; H NMR (500 MHz,
1
tion (CDCl3): (+); H NMR (500 MHz, C6D6, 20 C):
C6D6): ꢀ=0.83 (s, 3H, CH3-7), 0.95 (s, 3H, CH3-12/13),
1.12 (s, 3H, CH3-12/13), 1.13-1.19 (m, 1H, H-1a), 1.19-
1.25 (m, 1H, H-10a), 1.16-1.49 (m, 4H, H-2a, H-8a, H-
9a, H-10b), 1.62-1.78 (m, 3H, H-1b, H-8b, H-9b), 1.80-
1.88 (m, 3H, H-2b, H-5, H-6), 2.03 (d, 1H, H-3, J=3.8
Hz), 4.91 (s, 1H, H-15a), 4.96 (br.s, 1H, H-15b). 13C
NMR (100.6 MHz, C6D6 ): ꢀ=22.6 (t, C-9), 23.7 (q, C-
14), 30.0 (q, C-13), 30.5 (t, C-2), 31.0 (t, C-1), 32.0 (q, C-
12), 36.2 (s, C-11), 40.5 (t, C-8), 41.4 (t, C-10), 46.3 (d,
C-6), 48.6 (s, C-7), 57.0 (d, C-3), 60.7 (d, C-5), 104.5 (t,
C-15), 161.6 (s, C-4); MS (EI, 70 eV), m/z (rel. int.): 204
ꢀ=1.35–1.45 (br.s, 2H), 1.57 (s, 3H), 1.66 (br.s, 3H),
1.70 (s, 3H), 1.85–2.01 (m, 6H), 2.05–2.15 (m, 2H), 2.16–
2.25 (m, 1H), 4.77 (br.s, 1H), 4.85 (s, 1H), 5.30–5.44 (m,
2H); 1H NMR (400 MHz, CDCl3, 20 ꢀC): 1.40–1.53 (m,
2H), 1.67 (br.s, 3H), 1.71(s, 6H), 1.80–2.05 (m, 6H),
2.18–2.45 (m, 3H), 4.66 (br.s, 1H), 4.70 (br.s, 1H), 5.19–
1
5.24 (m, 1H), 5.28–5.34 (m, 1H); H NMR (500 MHz,
ꢀ
acetone-d6, À16 C): 1.25–1.40 (m, 2H), 1.45–1.58 (m,
2H), 1.68 (br.s, 3H), 1.70 (br.s, 3H), 1.72 (br.s, 3H),
1.93–2.01 (m, 3H), 2.13–2.19 (m, 2H), 2.29–2.36 (br.s,

206
A.M. Adio et al. / Phytochemistry 65 (2004) 199–206
[M+] (33), 189 (26), 175 (11), 161 (44), 147 (22), 133
(51), 119 (85), 105 (62), 93 (88), 79 (55), 69 (51), 63 (4),
55 (59), 41 (100).
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3.7. (À)-Perfora-1,7-diene (4)
Bulow, N., Konig, W.A., 2000. The role of germacrene D as a pre-
cursor in sesquiterpene biosynthesis: investigations of acid cata-
lyzed, photochemically and thermally induced rearrangements.
Phytochemistry 55, 141–168.
Colourless oil; RICPSIL5=1543 ; sense of optical rota-
1
tion (C6D6): (À); H NMR (500 MHz, C6D6): ꢀ=0.74
(s, 3H, H-14), 0.82 (d, 3H, H-13, J=6.6 Hz), 1.18 (dq,
1H, H-10a, J=1.6, 12.3 Hz), 1.47–1.54 (m, 1H, H-4),
1.62 (br.s, 3H, H-12), 1.74 (s, 3H, H-15), 1.71–1.80 (m,
3H, H-10b, H-3a, H-6a), 1.86–1.94 (m, 2H, H-3b, H-
9a), 2.00 (br.d, 1H, H-11, J=12.3 Hz), 2.16 (br.t, 1H, H-
9b, J=13.2 Hz), 2.25 (dd, 1H, H-6b, J=9.2, 14.5 Hz), 5.40
(br.s, 1H, H-2), 5.45 (br.s, 1H, H-7). 13C NMR (125.7
MHz, C6D6): d =10.9 (q, CH3-14), 16.6 (q, CH3-13), 23.1
(q, CH3-12), 24.3 (t, C-10), 25.9 (q, CH3-15), 33.7 (t, C-3),
35.0 (t, C-9), 37.2 (s, C-5), 37.8 (t, C-6), 38.9 (d, C-4),
56.2 (d, C-11), 122.9 (d, C-2), 123.9 (d, C-7), 136.3 (s, C-
1), 140.4 (s, C-8); MS (EI, 70eV), m/z (rel. int.) : 204
[M+] (12), 189 (16), 176 (30), 161(13), 147 (7), 136 (55),
135 (17), 134 (10), 133 (18), 121 (100), 119 (21), 107 (37),
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1
Colourless oil; RICPSIL5=1387; H NMR (500 MHz,
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1
(s, 3H,), 6.23 (dd, 1H, J=11.35, 17.66 Hz); MS (EI, 70
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(8), 161 (33), 147 (28), 133 (31), 121 (40), 119 (38), 107
(67), 105 (46), 93 (100), 91 (47), 81 (76), 79 (59), 68 (55),
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Acknowledgements
The financial support of DAAD (scholarship for A.
M. Adio) and of the Fonds der Chemischen Industrie is
gratefully acknowledged. We also thank Dr. V. Sinn-
well, University of Hamburg, for his advise in obtaining
NMR spectra and to A. Meiners and M. Preusse for the
GC–MS measurements.
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T.K.B., Ciereszko, L.S., 1970. Isolation of elusive (À)-germacrene-A
from the Gorgonian Eunicea mammosa. Tetrahedron Letters 497–
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