Isogermacrene A, a proposed intermediate in sesquiterpene biosynthesis

Article abstract of DOI:10.1016/j.phytochem.2004.05.024

A sesquiterpene hydrocarbon, isogermacrene A (5), which is structurally related to the gorgonanes and zieranes, was isolated from the essential oil of Saccogyna viticulosa (Hepaticae). This monocyclic compound is proposed as the biogenetic intermediate of some rare sequiterpene skeletons. In the essential oil of the liverwort Saccogyna viticulosa, collected on the island of Madeira, the new sesquiterpene hydrocarbons isogermacrene A (5) and its Cope rearrangement product iso-β-elemene (6) were identified. 5 is proposed to act as the biogenetic precursor of several new sesquiterpenes identified in the volatiles of S. viticulosa. These include iso-α-humulene, α-gorgonene, gorgona-1,4(15),11-triene and gorgon-11-en-4-ol. In addition, the Cope product of zierene, isozierene, allo-aromadendra-4(15),10(14)-diene, aromadendra-4(15),10(14)-dien-1-ol and a prenylguaiane diterpene alcohol, named viticulol, were identified as new natural products.

Full text of DOI:10.1016/j.phytochem.2004.05.024

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2261–2275
www.elsevier.com/locate/phytochem
Isogermacrene A, a proposed intermediate in sesquiterpene
biosynthesis
a,
a
b
Thomas Hackl *, Wilfried A. Ko¨nig , Hermann Muhle
a
Institut fu¨r Organische Chemie, Universita¨t Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
b
¨
Abteilung Systematische Botanik und Okologie, Universita¨t Ulm, D-89081 Ulm, Germany
Received 30 January 2004; received in revised form 28 May 2004
Available online 31 July 2004
Abstract
In the essential oil of the liverwort Saccogyna viticulosa, collected on the island of Madeira, the new sesquiterpene hydrocarbons
isogermacrene A (5) and its Cope rearrangement product iso-b-elemene (6) were identified. 5 is proposed to act as the biogenetic
precursor of several new sesquiterpenes identified in the volatiles of S. viticulosa. These include iso-a-humulene, a-gorgonene, gor-
gona-1,4(15),11-triene and gorgon-11-en-4-ol. In addition, the Cope product of zierene, isozierene, allo-aromadendra-4(15),10(14)-
diene, aromadendra-4(15),10(14)-dien-1-ol and a prenylguaiane diterpene alcohol, named viticulol, were identified as new natural
products.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Saccogyna viticulosa; Liverwort; Sesquiterpenes; Isogermacrene A; Iso-b-elemene; Cope rearrangement; Biosynthesis
1. Introduction
1962) and the recently isolated zierene (3), saccogynol
(4) (Connolly et al., 1994) and argutenol (Fraga, 2000)
are more unique members that were obtained from only
one or two sources (Fig. 1). These compounds possess
an isopropyl group in the unusual C-6 position and
apparently contain two tail-to-tail fused isoprene units.
Consequently, direct formation from the germacrenes
or formation by the usual hydride shift or methyl migra-
tion appears highly unlikely. Accordingly, a germacrene
like biogenetic intermediate (5) has been proposed,
which shares the unusual arrangement of isoprene units
(Weinheimer et al., 1968). 5 could also be considered as
a 7,11-seco-bicyclogermacrene, hence, FPP serves as the
initial biogenetic precursor. However, this hypothetic
intermediate has never been detected since its proposal.
The essential oil of the liverwort Saccogyna viticulosa
(L.) Dum. (Geocalycaceae), described as a unique
source of zierene (3) and saccogynol (4) (Connolly
et al., 1994), was reinvestigated. GC–MS screening re-
vealed the presence of the known b-gorgonene (2) and
To date more than 300 sesquiterpene skeletons are
known, which predominantly originate from enzymatic
cyclisations and further transformations of farnesyl
diphosphate (FPP) as the common acyclic precursor
(Cane, 1999). The germacrenes constitute important
intermediates between FPP and many other sesquiter-
pene structures. Nevertheless, some sesquiterpenes seem
to be an exception of the Isoprene Rule; examples are the
gorgonanes and zieranes which are rarely found in nat-
ure (Paknikar and Sood, 1973; Boeckmann and Silver,
1975). Maalioxide (1) has been identified from several
sources, mainly liverworts (Asakawa, 1995; Narayanan
et al., 1964). b-Gorgonene (2) (Weinheimer et al.,
1968), zierone (Penfold, 1926; Barton and Gupta,
*
Corresponding author. Tel.: +49-4042-838-6088; fax: +49-4042-
838-2893.
E-mail address: thomas_hackl@public.uni-hamburg.de(Th. Hackl).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.05.024

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
maalioxide (1), in addition to several unknown com-
pounds, especially in the hydrocarbon fraction. A com-
ponent (6) with a similar mass spectrum to b-elemene
(17) but a clearly different retention index was detected.
Isolation and structure elucidation of 6 revealed a new
elemene-like sesquiterpene skeleton with the isopropenyl
group in the C-6 position comparable to the gorgonanes
and zieranes. This compound was named iso-b-elemene
(6). Normally, due to their thermal lability, the elemenes
are formed by Cope rearrangement of the corresponding
germacrene precursor. Thus, it was possible to detect the
precursor of 6 by variation of the GC injection port tem-
perature in order to observe changes in the ratios of the
essential oil constituents. This compound, isogermac-
rene A (5), could be isolated in minor amounts and
was shown to be consistent with the proposed (hypo-
thetical) structure by Weinheimer et al. (1968). In addi-
tion, a new humulene-like compound related to 5,
named iso-a-humulene (7), as well as a-gorgonene (8),
gorgona-1,4(15),11-triene (9) and gorgon-11-en-4-ol
(10), two aromadendradienes, allo-aromadendra-
4(15),10(14)-diene (11) and aromadendra-4(15),10(14)-
dien-1-ol (12), the Cope rearrangement product of
zierene, isozierene (13), and a diterpene alcohol, viticulol
(14), were isolated as new natural compounds (Fig. 1).
2. Results and discussion
The investigation of the essential oil of S. viticulosa
by GC and GC–MS revealed a complex mixture of mon-
oterpenes, sesquiterpene hydrocarbons, oxygenated ses-
quiterpenoids as well as diterpenes. Several known
compounds could be identified by comparison of their
mass spectra and retention indices with a spectral library
(Joulain and Ko¨nig, 1998; Hochmuth et al., 2002) which
14
H
H
10
1
1
4
10
14
11
R
4
6
6
H
H
O
15
15
11
1
2
3: H
5
6
4: OH
14
14
14
14
H
H
1
4
1
5
10
10
10
1
6
4
6
4
6
15
H
H
HO
H
1
15
11
15
12
15
11
11
11
13
8
9
7
10
11
OH
H
H
H
HO
H
H
R
12
14
13: R = H,H
15
16
22: R = H,OH (3S)
23: R = O
H
H
H
HO
H
17
18
19
20
21
Fig. 1. Constituents of the liverwort Saccogyna viticulosa.

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2263
was established under identical experimental conditions.
The identified components are listed in their order of
elution (CPSiL-5 capillary column, relative concentra-
tions of major components [>1%] are given in parenthe-
ses): tricyclene, a-pinene, camphene, myrtenylacetate,
bicycloelemene (15), anastreptene (16), b-elemene (17),
tritomarene (18) (Warmers et al., 1998), calarene (19),
b-gorgonene (2, 12%), zierene (3, 29%), bicyclogermac-
rene (20, 4%), maalioxide (1), eudesm-11-en-4a-ol (21,
1%), saccogynol (4, 17%), isosaccogynol (22), isosacc-
ogynone (23, 4%) (Connolly et al., 1994) (Fig. 1). Un-
known components were selected for isolation and
further chemical investigation. The essential oil was
pre-fractionated by column chromatography using deac-
tivated alumina, with respect to the lability of the re-
ported constituents (Connolly et al., 1994). Structural
elucidation was performed by one- and two-dimensional
NMR experiments as well as chemical correlations. To
determine the absolute configurations, chemical conver-
sions were employed and monitored by enantioselective
GC using modified cyclodextrin phases (Ko¨nig, 1992).
2.1. (ꢀ)-Iso-b-elemene (6)
Fig. 2. AM1 MOPAC model of iso-b-elemene (6) and important
NOEs.
An early eluting hydrocarbon (6) with the molecular
formula C15H24 (M+: m/z 204) was isolated by a combina-
tion of column chromatography and preparative GC. In
the 1H NMR spectrum signals corresponding to a tertiary
methyl group at dH 1.03 (3H, s) and two olefinic methyl
groups at dH 1.64 (3H, m) and 1.71 (3H, m) were observed.
In addition seven olefinic protons at dH 4.61 (1H, m), 4.73
(1H, m), 4.79 (1H, m), 4.93 (3H, m) and 5.82 (1H, dd)
corresponded to six olefinic carbons at dC 110.1 (t),
111.5 (t), 114.9 (t), 145.2 (s), 149.7 (s) and 150.8 (d). Thus,
it was concluded that the compound contains one vinyl
and two isopropenyl groups. Considering the four units
of unsaturations and three double bonds a monocyclic
structure was assumed. Interpretation of the 2D H,H-
COSY, HMQC and HMBC spectra led to the new 1-me-
thyl-2,3-di(1-methylvinyl)-1-vinylcyclohexane structure
of 6. The signals at dH 1.92 (1H, d, J=12.0 Hz) and 2.33
(1H, ddd, J=3.6, 12.0, 12.0 Hz) were assigned to the
two adjacent methine protons H-5 and H-6, with the
J-coupling constant of 12.0 Hz indicating a trans-config-
uration. Furthermore, a long-range 4J-W-coupling
between the methyl group H-14 and H-5 indicated a
5,10-trans-fusion. The relative configuration (5S*, 6S*,
10S*) was confirmed by a NOESY spectrum. A molecular
model and important NOEs are shown in Fig. 2.
H
H
5
6
H
H
24
17
Fig. 3. Comparison of the products obtained by Cope rearrangement
of isogermacrene A (5) and germacrene A (24).
showed significant differences. The isolation of 5 was
difficult, because it eluted just shortly after zierene (3),
the major component of the essential oil. Thus, various
purification steps were necessary. Due to the lability of
5, which underwent Cope rearrangement under compa-
rable conditions as germacrene A (24), only minor
amounts could be isolated. Similar to 24 and many other
1
E,E-configured cyclodecadiene structures the H NMR
spectrum of 5 exhibited broad signals at room tempera-
ture (acetone-d6), due to a conformational equilibrium
in solution (Kulkarni et al., 1964; Weinheimer et al.,
1970; Piet et al., 1995). However, reducing the tempera-
ture to 10 ꢁC resulted in less broadened, well separated
multiplet signals. The observation of one set of signals
2.2. (+)-Isogermacrene A (5)
Isogermacrene A (5), the precursor of 6, exhibited a
molecular ion signal at m/z 204 (C15H24). The retention
index on a non-polar stationary phase was identical to
germacrene A (24) (Fig. 3) but the mass spectrum

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
indicated the predominance of only one conformation at
1
this temperature. The H NMR spectrum showed three
signals for olefinic methyl groups at dH 1.41 (3H, br s),
1.56 (3H, d), 1.68 (3H, s) and four signals for olefinic
protons at dH 4.48 (1H, m), 4.55 (1H, br s), 4.67 (1H,
br s), 4.81 (1H, m). Indicative for the structure of 5
was a signal at 2.69 (1H, m), assigned to the methine
proton H-6. A correlation signal to the olefinic proton
H-5 at dH 4.48 in the H,H-COSY spectrum confirmed
that the isopropenyl group of compound 5 is adjacent
to the C-4/C-5 double bond of the cyclodecadiene ring.
Although the complete 2D NMR data of 5 could not
be obtained, its structure could be assigned unambigu-
ously from its thermal rearrangement product 6. E-con-
figuration of the double bonds was concluded from the
high lability of 5, because much higher temperatures
are necessary for the rearrangement of an E,Z-config-
ured germacradiene (Adio et al., 2004).
Noteworthy, b-elemene (17) and iso-b-elemene (6)
exhibit a different stereochemistry. While the two isop-
ropenyl groups of 17 are cis-configured, trans-configura-
tion is found for 6 (Fig. 3). Cope rearrangement is a
stereospecific reaction that proceeds via a chair like
transition state and the configuration of the resulting
elemenes depends substantially on the structure and
predominant conformation of the corresponding germ-
acrenes (Sutherland, 1974; Houk et al., 1995; de Kraker
et al., 1998). Since a chair–chair like conformation is
preferred for the E,E-configured cyclodecadienyl sys-
tem, Cope rearrangement proceeds easily (Takeda,
1974; Terada and Yamamura, 1979, 1982). Even though
the cyclodecadienyl system is quite flexible, the conform-
ational equilibrium of the germacrenes is restricted due
to the large substituent at C-7. Hence, an equatorial
conformation predominates and Cope rearrangement
leads mainly to a single product (Kodama et al., 1979;
Piet et al., 1995; de Kraker et al., 2001). To confirm
the stereochemical relationship of 5 and 6, a semiempir-
ical molecular modelling analysis was performed using
CS MOPAC and the potential function AM1. Fig. 4
shows the calculated structure for the most stable con-
formation of 5. In agreement with the results obtained
for 24 and other germacrenes, the conformation is syn,
with respect to the two methyl groups, and the endocy-
clic double bonds are in a crossed arrangement (Takeda,
1974; Terada and Yamamura, 1979, 1982; Piet et al.,
1995). The calculated structure of 5 is consistent with
the stereochemical requirements for a Cope rearrange-
ment to 6 (Fig. 3).
Fig. 4. AM1 MOPAC model of isogermacrene A (5).
trimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin column
showed no peak separation nor broadening which
would be expected in case of different enantiomers.
From the known absolute configurations of (ꢀ)-1 and
(+)-2, S-configuration was concluded for (+)-isogermac-
rene A (5) (Paknikar and Sood, 1973; Matsuo et al.,
1974). Hence, the absolute configuration (5S,6S,10S)
was assigned to (ꢀ)-iso-b-elemene (6).
2.3. Iso-a-humulene (7)
Another unknown hydrocarbon with the molecular
formula C15H24 (M+: m/z 204) exhibited broad signals
1
in the H NMR spectrum at room temperature which
indicated a medium sized ring system. Nevertheless,
the NMR spectroscopic data could be interpreted with-
out decreasing the temperature. A broad singlet at dH
1.31 (6H) indicated the presence of two isochoric methyl
groups. In addition, two olefinic methyl groups at dH
1.38 (3H, br d) and 1.48 (3H, d) and four olefinic protons
at dH 4.61 (1H, br t), 4.98 (1H, br s), 5.02 (1H, m) und
5.48 (1H, d) were observed. The H,H-COSY spectrum
showed two endocyclic double bonds with methyl sub-
stitution, hence, an additional endocyclic double bond
was deduced. In agreement with three identified double
bonds the suspected monocyclic structure of 7 could be
confirmed. In the PENDANT spectrum four methyl
groups (dC 15.4, 16.6 and 28.4/2·C), four methylene
groups (dC 27.6, 30.3, 41.6 and 42.2), four olefinic
methine groups (dC 125.0, 128.0, 140.3 and 141.5) and
three quaternary carbon atoms (dC 38.5, 130.0 and
132.0) were assigned. All methylene protons gave down-
field shifted signals between dH 1.90 and 2.15 due to
Treatment of 5 with an acidic ion exchange resin
(Amberlystꢂ 15) at room temperature for one hour af-
forded maalioxide (1), b-gorgonene (2), and a-gorgon-
ene (8) in the ratio 1:5:2, respectively (Fig. 5).
Additional minor products were observed but could
not be identified. Coinjection of the reaction products
with natural 1, 2 and 8 on a heptakis(6-O-tert-butyl-

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2265
+
H
H
H+ / H2O
n-hexane
GC Inj.
200 ˚C
2
8
+
HO
H
5
10
H
O
1
Fig. 5. Acid induced rearrangement of (+)-isogermacrene A (5); thermal rearrangement of (ꢀ)-gorgon-11-en-4-ol (10).
their allylic positions. In the HMBC spectrum a 3J-H,C-
correlation from the isochoric methyl groups at dH 1.31
to the methyl carbons at dC 28.4 indicated their geminal
position. Additional information from the HMBC data
led to structure 7. The E-configuration of the C-10/C-11
double bond was concluded from the coupling constant
of 15.8 Hz, observed for H-11 at dH 5.48. The NOESY
spectrum indicated E-configuration for the two methyl
substituted double bonds, due to the absence of spatial
interactions between the olefinic protons and the corre-
sponding methyl group, although this could not be fur-
ther established. 7 possesses a new skeleton with an
eleven-membered monocyclic ring reminiscent to the
humulanes (Connolly and Hill, 1991). Since the geminal
methyl groups of 7 are relocated from position C-11 to
C-1 a structurally related compound to the gorgonanes,
zieranes and isogermacrene A (5) was concluded
(Fig. 6).
with carbon signals at dC 110.5 (t), 123.6 (d), 136.7 (s)
and 152.7 (s). From the H,H-COSY, HMQC and
HMBC spectra a gorgonane skeleton was elucidated.
The position of the two double bonds were assigned to
carbons C-3/C-4 and C-11/C-12 (Fig. 1) and the com-
pound was identified as the endocyclic double bond iso-
mer of b-gorgonene (2), called a-gorgonene (8).
Although the signals for the methine protons H-5 and
H-6 coincided at dH 2.25 (2H, br s) and could not be dis-
tinguished, the NOESY spectrum exhibited spatial inter-
actions to protons on both sides of the decaline ring
system, which proved a 5,6-trans-configuration. A
trans-configuration for the decaline ring system could
be shown by NOEs between the H-14 methyl protons
and the axial proton H(Si)-2 as well as H-14 and the axial
proton H(Si)-8. Treatment of (+)-2 with an acidic ion ex-
change resin yielded (+)-8 and (ꢀ)-maalioxide (1) as ma-
jor products (Fig. 7). The absolute configuration of
natural 8 (5R,6S,10S) could be established by coinjec-
tion with the rearrangement product of (+)-2 using a
heptakis(6-O-tert-butyltrimethylsilyl-2,3-di-O-methyl)-
b-cyclodextrin column. Although 8 was isolated for the
first time from a natural source, it has previously been
described as a synthetic product by treatment of maaliol
2.4. (+)-a-Gorgonene (8)
Compound 8 was assigned the molecular formula
C15H24 (M+: m/z 204). The 1H NMR spectrum exhibited
one tertiary methyl group at dH 0.88 (3H, s) and two
olefinic methyl groups at dH 1.58 (3H, s) and 1.83 (3H,
d). One exocyclic methylen group at dH 4.75 (1H, m)
and 4.79 (1H, s) and one trisubstituted endocyclic
double bond at dH 5.37 (1H, br s) were in agreement
with a catalytic amount of iodine (Buchi et al., 1959).
¨
2.5. Gorgona-1,4(15),11-triene (9)
The early eluting hydrocarbon 9 with a molecular
ion signal at m/z 202 and the molecular formula
C15H22 was isolated in minor amounts by a combination
H+/H2O
+
n-hexane
H
H
H
O
Gorgonane
Zierane
Isogermacrane
Isohumulane
Fig. 6. Rare sesquiterpene skeletons described in this work. The
depicted arrangement of isoprene units indicates the structural
relationship. Each skeleton apparently contains two tail-tail fused
isoprene units.
2
8
1
Fig. 7. Acid induced rearrangement of (+)-b-gorgonene (2) yielding
(+)-a-gorgonene (8) and (ꢀ)-maalioxide (1).

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
of preparative and semipreparative GC. 9 exhibited sim-
ilar NMR data as 2 and 8 but contains an additional
double bond. In the 1H NMR spectrum one tertiary
methyl group at dH 0.94 (3H, s), one olefinic methyl
group at dH 1.58 (3H, s) and six olefinic protons at dH
4.80 (2H, m), 4.86 (1H, d), 4.95 (1H, br s) and 5.44
(2H, m) were observed. The olefinic protons were as-
signed to two exocyclic and one endocyclic double bond,
in agreement with carbon signals at dC 108.8 (t), 111.0
(t), 123.5 (d), 140.4 (d), 145.0 (s) and 149.8 (s). Interpre-
tation of the 2D NMR data (H,H-COSY, HMQC,
HMBC) led to the identification of structure 9. The rel-
ative configuration of 9 was derived from a NOESY
spectrum. Hydrogenation of 2 and 9 and coinjection
of the reaction products on different chiral columns
established the absolute configuration (5S,6S,10S).
1.01 (3H, s) and 1.05 (3H, s) and four olefinic protons
at dH 4.87 (2H, m), 4.94 (1H, m) and 5.05 (1H, m) were
observed. The olefinic protons were assigned to two
exocyclic methylene groups in agreement with four olef-
inic carbons at dC 105.7 (t), 110.3 (t), 150.1 (s) and
157.0 (s). The observed methine protons at dH 0.51
(2H, m), corresponding to carbon signals at dC 25.1
(d) and 29.0 (d), suggested a dimethyl substituted cyclo-
propane unit. One signal at dC 17.0 (s) was assigned to
the quaternary cyclopropane carbon atom. Interpreta-
tion of the 2D H,H-COSY-, HMQC- and HMBC spec-
tra confirmed structure 11. The relative stereochemistry
was deduced from the NOESY spectrum. A conforma-
tional study applying the AM1 potential function
implemented in the CS MOPAC program was utilised
for the assignment of NOESY correlation signals. cis-
Configuration of the azulene ring system was derived
from NOE between H-2 and H-6. The cis-configured
cyclopropane ring was deduced from an interaction of
the cyclopropane methyl protons H-12 with H-6 and
H-7 and NOE between the methyl protons H-13 and
H-5, which also indicated a trans-configuration for
the methine protons H-5 and H-6. For the assignment
of the absolute configuration, samples of (ꢀ)-allo-aro-
2.6. (ꢀ)-Gorgon-11-en-4-ol (10)
Compound 10 has the molecular formula C15H26O as
shown by HRMS (M+: m/z 222.1994). The fragment ion
at m/z 204 [M ꢀ H2O]+ indicated the presence of a hyd-
roxy group. 10 was enriched from the essential oil by
column chromatography while further purification by
preparative GC could not be achieved due to rearrange-
ment reactions in the injection port. The rearrangement
products were identified by GC and GC–MS as 1, 2 and
8. Thus, a gorgonane type sesquiterpene alcohol with a
hydroxy group at position C-4 or C-11 was proposed.
madendrene
(26)
and
(+)-allo-aromadendra-
4(15),10(14)-diene (11) were catalytically hydrogenated
(Fig. 8). Two diasteromeric products (26a and 26b)
were expected for the hydrogenation of 26 and four
diasteromeric products (11a–11d) for the hydrogenation
1
The H NMR spectrum indicated the presence of two
tertiary methyl groups at dH 0.79 (3H, s) and 1.22
(3H, s) and an olefinic methyl group at dH 1.78 (3H, s)
in addition to two olefinic protons at dH 4.63 (1H, m)
and 4.79 (1H, d, J=2.5 Hz). The 15 carbon atoms of
10 were assigned by means of a PENDANT spectrum
which indicated three methyl groups (dC 19.1, 20.3 and
24.1), seven methylene groups (dC 20.0, 21.7, 34.2,
42.5, 43.2, 45.5, 112.7), two methine groups (dC 44.6,
56.8) and three quaternary carbon atoms (dC 35.7,
73.8, 153.7). Although full assignment of all signals
could not be achieved due to overlapping of various sig-
nals, the 2D NMR data (H,H-COSY, HMQC and
HMBC) confirmed the structure of gorgon-11-en-4-ol.
The relative configuration of 10 was derived from the
NOESY spectrum while the absolute configuration
(4R,5R,6S,10S) was assigned by enantioselective GC
of the reaction products 1, 2 and 8 obtained by rear-
rangement and dehydration (Fig. 5).
H
H
H
H
+
H
H
11a
11b
H2
+
+
Pd/C
H
H
H
H
11
11c
11d
H
H
H
H
H
H
H2
+
Pd/C
2.7. (+)-allo-Aromadendra-4(15),10(14)-diene (11)
The mass spectrum of compound 11 was similar to
zierene (3) with a molecular ion peak at m/z 202
(C15H22). Instead of one olefinic methyl group and
three exocyclic methylene groups in the H NMR spec-
trum of 3, signals for two tertiary methyl groups at dH
26
26a
26b
Fig. 8. Products obtained by catalytic hydrogenation of (+)-allo-
aromadendra-4(15),10(14)-diene (11) and (ꢀ)-allo-aromadendrene
(26).
1

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2267
of 11. The expected 11a and 11b constitute the optical
antipodes of 26a and 26b. Even though a complex mix-
ture of products was obtained in both cases, two of the
products had identical retention times on non-chiral
stationary phases but different retention times on a
column with heptakis(2,6-di-O-methyl-3-O-pentyl)-b-
cyclodextrin. Thus, the absolute configuration (1R,
5S,6S,7S) was derived for 11.
2.8. (+)-Aromadendra-4(15),10(14)-dien-1-ol (12)
The molecular formula of 12 was assigned as
C15H22O by HRMS (M+: m/z 218.1662). Although only
a small amount of compound 12 was isolated it could be
1
identified by interpretation of the H NMR and H,H-
1
COSY spectra. The H NMR spectrum exhibited two
tertiary methyl groups at dH 0.98 (3H, s) and 1.06
(3H, s), four olefinic protons at dH 4.81 (1H, t), 4.84
(1H, m), 4.89 (1H, br s) and 5.06 (1H, m) as well as
two cyclopropyl protons at dH 0.36 (1H, dd) and 0.50
(1H, m), in agreement with the structure of the aroma-
dendrane alcohol 12. The position of the hydroxy group
was concluded from the absence of the H-1 proton. In
the NOESY spectrum NOEs between the H-12 methyl
protons and H-6 as well as H-7 indicated a cis-fused
cyclopropane ring. In addition, NOE between the
H-13 methyl protons and H-5 confirmed a trans-config-
uration for H-5 and H-6. Although the relative configu-
ration at C-1 could not, be derived unambiguously,
missing spatial interactions between the cyclopentane
unit and the C-6 methine proton a trans-fused azulene
ring system (Fig. 9).
Fig. 9. Proposed relative configuration of aromadendra-4(10),10(14)-
dien-1-ol (12) (AM1 MOPAC model) and the observed NOEs.
2.10. (+)-Viticulol (14)
Compound 14, a diterpene alcohol of the molecular
formula C20H34O, exhibited a molecular ion peak of
low intensity at m/z 290 (<1%), but a relative intense
[MꢀH2O]+ fragment at m/z 272 (36%). In the 1H
NMR spectrum a secondary methyl group at dH
0.96 (3H, d), a tertiary methyl group at dH 1.13
(3H, s), two olefinic methyl groups at dH 1.58 (3H,
s) and 1.68 (3H, s) and three olefinic protons at dH
4.90 (1H, d), 5.02 (1H, s) and 5.24–5.30 (1H, m) were
detected. A quaternary carbon atom at dC 73.92 sug-
gested the presence of a tertiary hydroxy group. The
molecule contains four units of unsaturation and
two double bonds (dC 107.5, 125.0, 132.2, 155.5),
hence, a bicyclic structure was assumed. Analysis of
the H,H-COSY, HMQC and HMBC spectra revealed
a prenylguaiane skeleton with the two double bonds
located in the C-11/C-19 and C-14/C-15 position of
the side chain. The carbon atom containing the hyd-
roxy group was assigned to C-10. The relative config-
uration was determined by inspection of the NOESY
spectrum as (1R*,4S*,5S*,7S*,10S*). To our knowl-
edge viticulol (14) is the first diterpene with the pre-
nylguaiane skeleton isolated from liverworts.
Prenylguaianes are mainly found in algae of the genus
Dictyota and Aplysia and less frequently in corals of
the species Xenia (Breitmaier, 1999). Examples are
2.9. (+)-Isozierene (13)
During the GC investigation of the essential oil with
different injection port temperatures for monitoring var-
iations in peak areas, the assigned peak of 13 was de-
tected to change its ratio in parallel with zierene (3).
As Cope rearrangement has been described for saccogy-
nol (4) upon the attempt of its oxidation, compound 13
was suspected to be the corresponding thermal re-
arrangement product of 3 (Connolly et al., 1994). 13
exhibited a molecular ion signal at m/z 202 (C15H22).
The 1H NMR spectrum indicated signals corresponding
to one olefinic methyl group at dH 1.45 (3H, d) and four
olefinic protons at dH 4.66 (1H, dd), 4.85 (1H, d), 5.05
(1H, s) and 5.34 (1H, dd). The PENDANT spectrum
revealed one methyl group (dC 14.9), eight methylene
groups (dC 26.8, 28.5, 29.3, 30.5, 34.1, 34.8, 37.1 and
109.8), three methine groups (dC 55.6, 127.2 and 130.0)
and three quaternary carbon atoms (dC 134.3, 143.2
and 155.8), in agreement with structure 13. The forma-
tion of isozierene (13) was established by injection of a
pure sample of 3 at varying injection port temperatures.
dictyol
E (Danise et al., 1977), pachydictyol A
(Hirschfeld et al., 1973) or acutilol B (Hardt et al.,
1996).

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
3. Conclusions
ents was confirmed by the praparation of a n-pentane
extract.
From a biogenetic point of view, 5 is proposed as the
The essential oil of S. viticulosa is composed of un-
ique sesquiterpenes, especially of the rare gorgonane
and zierane type. The new sesquiterpene isogermacrene
A (5) and its Cope rearrangement product iso-b-elemene
(6) were isolated and identified. The novel diterpene
viticulol (14) could be considered as another example
of a phylogenetic relationship between bryophytes and
algae. Functionalised diterpenes with prenylsesquiter-
pene structures, such as prenylguaianes, prenylaroma-
dendranes, prenylanastreptanes or prenylbourbonanes,
with diverse biological activities such as antibacterial,
antifungal, feeding-deterrent, antifoulant or antitumoral
properties have been described (Kurata et al., 1990;
Schmitt et al., 1998; Hardt et al., 1996). So far, the bio-
logical activity of 14 has not been investigated. The
authenticity of all isolated compounds as plant constitu-
missing link between FPP and the gorgonanes and zier-
anes. Nevertheless, direct formation of 5 in a single cyc-
lisation step seems improbable, because the compound
apparently contains two tail-to-tail fused isoprene units.
A possible biosynthetic pathway for the formation of 5
would be preceded by the formation of (ꢀ)-bicycloger-
macrene (20) with subsequent enzymatically induced
ring opening of the cyclopropane unit and the deproto-
nation of the resulting E,E-isogermacra-1(10),4-dienyl
cation (27) (Fig. 10). This proposal is supported by the
identification of (ꢀ)-20 as an essential oil constituent
of S. viticulosa. To support the hypothesis of involve-
ment of an enzyme in the ring opening reaction, the pro-
ton induced rearrangement of (+)-20, obtained from a
higher plant, was investigated by GC–MS. Exposure
O
- OPP
OPP
20
43
FPP
+
[H ]
H
H
HO
H
+
- H
WMR
Ox.
7
41
27
12
11
+
- H
+
+
Cope
[H ]
- H
H
H
6
8
40
5
+
+ H O
- H
2
H
O
H
H
Cope
HO
H
H
10
2
42
13
3
+
[H ]
Ox.
H
H
H
Cope/Ox.
R
HO
H
H
O
1
4
22, 23
9
Fig. 10. Proposed biogenetic pathways for compounds 1–13, 22 and 23 from Saccogyna viticulosa (WMR=Wagner–Meerwein rearrangement).

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2269
of (+)-20 for one day to acidic ion exchange resin in n-
hexane yielded a complex mixture of hydrocarbons
and oxygenated sesquiterpenoids. Various unidentified
compounds were formed, in addition to isoledene (28),
b-elemene (17), b-maaliene (29), c-maaliene (30), a-
maaliene (31), aromadenrene (32), selina-4(15),7-diene
(33), allo-aromadendrene (34), d-selinene (35), d-cadin-
ene (36), palustrol (37), rosifoliol (38), 5-guaiene-11-ol
(39) (the compounds are listed in order of their elution
from a CPSiL5 capillary column) (Fig. 11). None of
the gorgonane or zierane type compounds described in
this work were detected. The identification of 17 indi-
cated the formation of germacrene A (24) during the
rearrangement process. In conclusion, under non-enzy-
matic conditions C-6/C-11 cleavage of the cyclopropane
system is favoured.
A biogenetic scheme for the new sesquiterpenes (5–
13), b-gorgonene (2), zierene (3) and saccogynol (4)
and a new biogenetic pathway for the formation of
maalioxide (1) is proposed (Fig. 10). Similar to the for-
mation of the eudesmane-type sesquiterpenes from
‘‘normal’’ germacrenes, protonation of 5 followed by
cyclisation would lead to the gorgonenyl cation 40 and
subsequent deprotonation would afford either 2 or 8
(Cane, 1999; Dewick, 2002). Addition of a water mole-
cule to 40 would result in the formation of gorgon-11-
en-4-ol (10). Finally, protonation of 10 should give rise
to 1. These biogenetic correlations between 1, 2, 5, 8
and 10 are in agreement with the rearrangement reac-
tions described for 5 and 10 (Fig. 5).
Fig. 6 demonstrates the strucutural relationship be-
tween iso-a-humulen (7) and 5 which both contain
tail-to-tail fused isoprene units. Therefore, 7 can not
originate from FPP directly and a Wagner–Meerwein
rearrangement from the E,E-isogermacra-1(10),4-dienyl
cation (27) to an E,E-isohumula-1,5-dienyl cation (41)
is proposed for its biogenesis.
The formation of the sesquiterpene hydrocarbons zie-
rene (3), gorgona-1,4(15),11-triene (9) and allo-aroma-
dendra-4(15),10(11)-diene (11) suggested an additional
step, due to the reduced molecular mass of 202. Epoxi-
dation of 5 could lead to intermediate 42 and subsequent
cyclisation followed by deprotonation and dehydration
would give 3 or 9, respectively. A corresponding inter-
mediate 43 may be predicted in the formation of 11. Oxi-
dation of 3 and 11 would result in the sequiterpene
alcohols saccogynol (4) and aromadendra-4(15),10(14)-
dien-1-ol (12).
It should be mentioned that another class of sesquit-
erpenes, the nardosinanes (44), are structurally related
to the compounds presented in this work (Fig. 12) (Vi-
dari et al., 1998; Melching, 1999). Although no nardos-
inane type sesquiterpenes could be detected in S.
viticulosa, a biosynthetic analogue to the formation of
eremophilanes (Cane, 1999; Calvert et al., 2002) starting
from isogermacrene A (5) may be proposed. In some
cases the formation of gorgonanes, zieranes or nardosin-
anes through an enzymatic cyclopropane cleavage of the
maalianes, aromadendranes or aristolanes has been pre-
dicted (Barton and Gupta, 1962; Paknikar and Sood,
H
H
H
28
29
30: ∆4,15
31: ∆3
32
H
H
H
H
33
34
35
36
HO
H
H
OH
OH
37
38
39
Fig. 11. Some selected products obtained by proton induced rearrangement of (+)-bicyclogermacrene (20).

2270
Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
44
Fig. 12. Nardosinane skeleton.
1973; Rucker, 1975). This concept is similar to the pro-
¨
4.1.4. Semipreparative GC
HP 6890 gas chromatograph equipped with an auto-
sampler and a 30 m megabore thickfilm capillary col-
posed biosynthesis of 5 and may not be excluded in par-
ticular cases. During a GC–MS screening, maalioxide
(1) and iso-b-elemene (6) have been detected in the
essential oil of the liverwort Lophozia ventricosa, thus,
it could be expected that 5 is also a constituent of this
plant (Lu et al., 2004).
umn (i.d. 0.53 mm, film thickness
polysiloxane DB-1701 or a 25 m megabore thickfilm
5 lm) with
capillary column (i.d. 0.53 mm) with heptakis(6-O-tert-
butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin
in
OV1701 (50%, w/w); He was used as carrier gas; injector
and detector (FID) temperatures were 200 and 250 ꢁC,
respectively; eluting fractions were trapped at ꢀ20 ꢁC
in glass tubes using the automatic fraction collector
4. Experimental
PFC 1 from Gerstel, Mulheim, Germany, and a
¨
cryostat.
4.1. General experimental procedures
4.1.1. Gas chromatography
4.1.5. NMR spectroscopy
Carlo Erba Mega 5300 or GC 8000 double column
instruments equipped with 25 m silica capillaries with
polysiloxane CPSil-5 and polisiloxane CPSil-19 (Chro-
mopack); Carlo Erba Fractovap 2150 or 4160 gas
chromatographs with 25 m fused silica capillaries
with octakis(2,6-di-O-methyl-3-O-pentyl)-c-cyclodextrin,
heptakis(2,6-di-O-methyl-3-O-pentyl)-b-cyclodextrin or
heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-
b-cyclodextrin in OV 1701 (50%, w/w), split injection;
split ratio ca. 1:30; FID; carrier gas 0.5 bar H2; injector
and detector temperatures were 200 and 250 ꢁC,
respectively.
NMR measurements were carried out with a Bruker
WM 400 (400 MHz), a Bruker DRX 500 (500 MHz)
or a Bruker DRX 700 (700 MHz) instrument in C6D6
or acetone-d6 using TMS as internal standard.
4.1.6. Polarimetry
Measurements were performed with a polarimeter
341 Perkin–Elmer at 589 nm at 20 ꢁC in n-hexane,
C6D6 or acetone-d6. To avoid inaccuracies only the
sense of optical rotation was determined due to the
small quantity of the isolated compounds.
4.1.7. Molecular modelling
4.1.2. GC–MS and GC–HRMS
Molecular models were calculated with the program
CSChem 3D Proꢂ (Version 5.0) from Cambridge Soft.
The start geometries were calculated by the molecule
mechanical MM2 force field method (Allinger, 1977).
Geometries were optimised by the semiempirical SCF
method of the implemented program CS MOPAC
Proꢂ (Version MOPAC 97) from Fujitsu, using the
potential function AM1 (Dewar et al., 1985). All calcu-
lations were executed on a standard PC equipped with
an AMD 830 MHz processor and 256 MB RAM.
Electron impact (70 eV) GC–MS and GC–HRMS
were carried out with a Hewlett–Packard HP 5890 gas
chromatograph coupled to a VG Analytical 70-250S
mass spectrometer.
4.1.3. Preparative GC
Modified Varian 1400 instrument, equipped with
stainless steel columns (1.85 m·4.3 mm) with 10% poly-
dimethylsiloxane SE-30 on Chromosorb W-HP or with
2.5% octakis(2,6-di-O-methyl-3-O-pentyl)-c-cyclodex-
trin in OV1701 (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 Chro-
mosorb W-HP; FID; He as carrier gas at a flow rate
of 120 ml minꢀ1; injector and detector temperatures
were 200 and 250 ꢁC, respectively; eluting fractions were
trapped in teflon tubes cooled with liquid nitrogen
(Hardt and Ko¨nig, 1994).
4.2. Plant material, essential oil and extract
S. viticulosa was collected in April 2003 at Ribero
Frio/Levada de Furado, Madeira. Aqueous homogen-
ates of the fresh plant material were submitted to hydro-
distillation (2 h) to yield the essential oil, which was
collected in 1 ml of n-hexane and not further quantified.
Alternatively, air dried plant material was powdered

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2271
under liquid nitrogen and extracted with n-pentane at
7 ꢁC. Because of the greatly differing weight the plant
material was not weighed.
J=6.6, 7.9, 13.6 Hz, H(Si)-2), 1.87 (1H, ddd, J=5.4,
9.8, 12.9 Hz, H(Re)-9), 2.14 (1H, m, H(Re)-2), 2.21 (1H,
ddd, J=1.0, 9.5, 9.5 Hz, H-6), 2.28 (1H, ddd, J=5.4,
5.4, 12.9 Hz, H(Si)-9), 2.46 (1H, dddd, J=1.6, 1.6, 7.9,
7.9 Hz, H-5), 2.56 (1H, ddd, J=7.9, 7.9, 7.9 Hz, H-1),
4.28 (1H, dddd, J=1.9, 1.9, 6.6, 6.6 Hz, H-3), 4.72
(2H, m, H-12), 4.83 (1H, m, H(E)-14), 4.86 (1H, m,
4.3. Isolation of single constituents of the essential oil
The essential oil from S. viticulosa was fractionated
by column chromatography over deactivated alumina
with increasing proportions of Et2O in n-pentane. Three
hydrocarbon fractions and five fractions containing oxy-
genated sesquiterpenes and diterpenes were obtained.
Each fraction was investigated by GC and GC–MS
and submitted to further isolation procedures.
H
(Z)-14), 5.03 (1H, m, H(E)-15), 5.13 (1H, m, H(Z)-15).
OH missing; 13C NMR (100.6 MHz, C6D6): d 20.6 (q,
C-13), 30.3 (t, C-8), 34.8 (t, C-7), 36.9 (t, C-9), 39.7 (t,
C-2), 46.9 (d, C-1), 48.7 (d, C-6), 49.9 (d, C-5), 74.0 (d,
C-3), 109.2 (t, C-15), 110.7 (t, C-12), 111.9 (t, C-14),
150.6 (s, C-11), 151.9 (s, C-10), 156.7 (s, C-4); MS (EI,
70 eV) m/z (rel. int.): 218 [M+] (6), 203 (38), 185 (43),
175 (31), 157 (52), 143 (34), 131 (32), 117 (37), 105
(49), 91 (79), 79 (68), 67 (46), 55 (43), 41 (100).
4.3.1. (+)-(5S,6S,10S)-b-Gorgonene (2)
1H NMR (500 MHz, C6D6): d 0.82 (3H, s, H-14), 1.09
(1H, ddd, J=4.1, 4.1, 13.2 Hz, H(Re)-9), 1.18 (1H, ddd,
J=4.4, 4.4, 13.2 Hz, H(Si)-1), 1.26 (1H, m, H(Si)-7),
1.31–1.38 (2H, m, H(Re)-1, H(Si)-9), 1.40–1.44 (1H, m,
4.3.4. Isolation of (+)-(S)-isogermacrene A (5)
(+)-(S)-1,5-Dimethyl-7-(1-methylethenyl)-cyclodeca-
1E,5E-diene. 5 was enriched by preparative GC at an
injector temperature of 140 ꢁC using a heptakis(6-O-
tert-butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin
column (110 ꢁC isothermal, 1.8 bar He) and later puri-
fied by semipreparative GC applying a DB-1701 column
(130 ꢁC isothermal) and with temperatures for injector,
transferline and switching device of 150 ꢁC. Colourless
oil (ca. 0.2 mg); RICPSiL5 =1501; sense of optical rota-
H
(Re)-8), 1.47–1.60 (3H, m, H(Re)-2, H(Si)-2, H(Si)-8),
1.61 (3H, s, H-13), 1.66–1.71 (1H, m, H(Re)-7), 1.76
(1H, d, J=11.7 Hz, H-5), 1.90 (1H, ddd, J=5.4, 5.4,
12.9 Hz, H(Re)–3), 2.21 (1H, m, H(Si)-3), 2.32 (1H, m, H-
6), 4.77–4.80 (2H, m, H(Z)-12, H(Z)-15), 4.81-4.82 (1H,
m, H(E)-12), 4.88 (1H, br s, H(E)-15); 13C NMR (100.6
MHz, C6D6): d 17.5 (q, C-14), 19.2 (q, C-13), 21.9 (t, C-
8), 24.7 (t, C-2), 35.1 (t, C-7), 36.8 (s, C-10), 38.5 (t,
C-3), 41.8 (t, C-9), 42.9 (d, C-6), 43.1 (t, C-1), 52.6 (d,
C-5), 108.4 (t, C-15), 110.9 (t, C-12), 148.3 (s, C-4),
150.3 (s, C-11); MS (EI, 70 eV) m/z (rel. int.): 204 [M+]
(5), 189 (100), 175 (3), 161 (38), 147 (42), 133 (42), 119
(30), 105 (48), 91 (55), 79 (51), 67 (41), 55 (40), 41 (58).
1
tion (acetone-d6): (+); H NMR (700 MHz, acetone-d6,
283 K): d 1.41 (3H, br s), 1.56 (3H, d), 1.68 (3H, s),
2.68–2.70 (1H, m), 4.48 (1H, m), 4.55 (1H, br s), 4.67
(1H, br s), 4.80–4.81 (1H, m); 13C NMR (data from
HMBC spectrum, 700 MHz, acetone-d6, 283 K): d
16.3, 20.4, 40.0, 42.4, 48.1, 107.7, 126.5, 129.2, 135.3,
138.1, 151.2, 4 C missing; MS (EI, 70 eV), m/z (rel.
int.): 204 [M+] (8), 189 (36), 175 (7), 161 (38), 147 (22),
133 (30), 119 (41), 107 (73), 93 (100), 81 (72), 79 (68),
67 (47), 55 (35), 41 (49).
4.3.2. (+)-(1R*,5R*,6S*)-Zierene (3)
1H NMR (500 MHz, C6D6): d 1.29–1.38 (1H, m, H(Si)
-
8), 1.40–1.49 (1H, m, H(Si)-7), 1.56–1.63 (1H, m, H(Re)-7),
1.64 (3H, s, H-13), 1.69–1.85 (3H, m, H(Re)-2, H(Si)-2,
H
(Re)-8), 1.91 (1H, ddd, J=4.6, 10.7, 12.5 Hz, H(Re)-9),
2.08 (1H, ddd, J=1.3, 9.7, 9.7 Hz, H-6), 2.20–2.31 (2H,
m, H(Re)-3, H(Si)-9), 2.35–2.43 (1H, m, H(Si)-3), 2.51–
2.56 (1H, m, H-5), 2.72 (1H, ddd, J=7.4, 7.4, 7.4 Hz,
H-1), 4.72–4.75 (3H, m, H-12, H(Z)-14), 4.84–4.85 (1H,
m, H(E)-14), 4.90–4.92 (1H, m, H(E)-15), 4.96–4.98 (1H,
m, H(Z)-15); 13C NMR (100.61 MHz, C6D6): d 20.4 (q,
C-13), 29.4 (t, C-2), 30.8 (t, C-8), 31.8 (t, C-3), 35.2 (t,
C-7), 37.5 (t, C-9), 48.8 (d, C-6), 49.6 (d, C-1), 52.7 (d,
C-5), 107.5 (t, C-15), 110.2 (t, C-12), 110.8 (t, C-14),
150.4 (s, C-11), 153.2 (s, C-10), 154.6 (s, C-4); MS (EI,
70 eV) m/z (rel. int.): 202 [M+] (27), 187 (92), 173 (53),
159 (59), 145 (60), 131 (65), 117 (44), 105 (75), 91 (100),
79 (83), 67 (38), 53 (44), 41 (97).
4.3.5. Isolation of (ꢀ)-(5S,6S,10S)-iso-b-elemene (6)
(ꢀ)-(1S,2S,3S)-1-Ethenyl-1-methyl-2,3-di(1-methyl-
ethenyl)-cyclohexane. 6 was isolated by preparative GC
using a heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-
methyl)-b-cyclodextrin column (120 ꢁC isothermal, 1.8
bar He). Colourless oil (ca. 2 mg); RICPSiL5 =1364; sense
1
of optical rotation (C6D6): (ꢀ); H NMR (500 MHz,
C6D6): d 1.03 (3H, s, H-14), 1.17–1.27 (1H, m, H(Si)-7),
1.30–1.39 (2H, m, H(Re)-8, H(Si)-8), 1.41–1.47 (2H, m,
H
(Re)-9, H(Si)-9), 1.63–1.65 (3H, m, H-13), 1.65–1.70
(1H, m, H(Re)-7), 1.71 (3H, m, H-15), 1.92 (1H, d,
J=12.0 Hz, H-5), 2.33 (1H, ddd, J=3.6, 12.0, 12.0 Hz,
H-6), 4.60–4.61 (1H, m, H(Z)-3), 4.73 (1H, m, H(Z)-12),
4.78–4.79 (1H, m, H(E)-12), 4.90–4.96 (3H, m, H-2,
H
(E)-3), 5.82 (1H, dd, J=10.4, 17.8 Hz, H-1); 13C
NMR (100.6 MHz, C6D6): d 18.4 (q, C-14), 20.0 (q, C-
13), 22.3 (t, C-8), 24.3 (q, C-15), 33.9 (t, C-7), 40.3 (t,
C-9), 40.7 (s, C-10), 45.3 (d, C-6), 56.3 (d, C-5), 110.1
4.3.3. (+)-(1R*,3S*,5R*,6S*)-Saccogynol (4)
1H NMR (500 MHz, C6D6): d 1.31–1.44 (2H, m,
(Si)-7, H(Si)-8), 1.54–1.58 (1H, m, H(Re)-7), 1.60 (3H,
H
s, H-13), 1.63–1.69 (1H, m, H(Re)-8), 1.71 (1H, ddd,

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
(t, C-2), 111.5 (t, C-12), 114.9 (t, C-3), 145.2 (s, C-4),
149.7 (s, C-11), 150.8 (d, C-1); MS (EI, 70 eV), m/z
(rel. int.): 204 [M+] (2), 189 (17), 175 (6), 161 (19), 147
(16), 135 (52), 122 (55), 107 (85), 93 (100), 81 (84), 79
(85), 67 (59), 55 (45), 41 (68).
umn (110 ꢁC isothermal, 1.8 bar He) and later purified
by semipreparative GC applying a heptakis(6-O-tert-
butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin thick-
film column (100–150 ꢁC, 1 ꢁC/min, 150 ꢁC isothermal).
1
Colourless oil (ca. 0.6 mg); RICPSil5 =1426; H NMR
(500 MHz, C6D6): d 0.94 (3H, s, H-14), 1.22 (1H, dd,
J=4.7, 12.3 Hz, H(Si)-7), 1.31 (1H, dd, J=4.4, 13.2 Hz,
H(Re)-9), 1.41–1.51 (3H, m, H(Si)-9, H(Re)-8, H(Si)-8),
4.3.6. Isolation of iso-a-humulene (7)
1,3,3,8-Tetramethylcycloundeca-1,4,8-triene. 7 was
enriched by preparative GC using a heptakis(6-O-tert-
butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin col-
umn (110 ꢁC, isothermal, 1.8 bar He) and purified by
repeating this separation step twice. Colourless oil (ca.
1.58 (3H, s, H-13), 1.65–1.69 (1H, m, H(Re)-7), 2.11
(1H, d, J=11.7 Hz, H-5), 2.41 (1H, ddd, J=3.8, 11.7,
11.7 Hz, H-6), 2.51–2.55 (1H, m, H-3), 2.76–2.81 (1H,
m, H-3), 4.79–4.81 (2H, m, H-12), 4.86 (1H, d, J=1.9
Hz, H(Z)-15), 4.95 (1H, br s, H(E)-15), 5.40–5.47 (2H,
m, H-1, H-2); 13C NMR (data from HMQC/HMBC
spectra, 500 MHz, C6D6): d 19.1 (q, C-12), 20.0 (q, C-
14), 22.0 (t, C-8), 34.6 (t, C-7), 37.3 (t, C-3), 38.4 (s,
C-10), 39.4 (t, C-9), 42.4 (d, C-6), 50.8 (d, C-5), 108.8
(t, C-15), 111.0 (t, C-12), 123.5 (d, C-2), 140.4 (d, C-1),
145.0 (s, C-4), 149.8 (s, C-11); MS (EI, 70 eV) m/z (rel.
int.): 202 (6), 187 (100), 173 (4), 159 (18), 145 (42), 131
(59), 117 (45), 105 (71), 91 (75), 79 (55), 67 (22), 55
(24), 41 (44).
1
1 mg); RICPSiL5 =1471; H NMR (500 MHz, C6D6): d
1.31 (6H, s, H-12, H-13), 1.38 (3H, s, H-14), 1.48 (3H,
d, J=1.3 Hz, H-15), 1.93-1.97 (6H, m, H-3, H-7, H-8),
2.10 (2H, br s, H-4), 4.61 (1H, br t, H-5), 4.98 (1H, br
s, H-1), 4.99-5.05 (1H, m, H-9), 5.48 (1H, d, J=15.8
Hz, H-10); 13C NMR (100.6 MHz, C6D6): d 15.4 (q,
C-14), 16.6 (q, C-15), 27.6 (t, C-4), 28.4 (q, 2·C, C-12,
C-13), 30.3 (t, C-8), 38.5 (s, C-11), 41.6 (t, C-7), 42.2
(t, C-3), 125.0 (d, C-9), 128.0 (d, C-5), 130.0 (s, C-2),
132.0 (s, C-6), 140.3 (d, C-1), 141.5 (d, C-10); MS (EI,
70 eV), m/z (rel. int.): 204 [M+] (16), 189 (5), 161 (5),
147 (4), 136 (10), 121 (100), 107 (34), 93 (94), 79 (33),
67 (24), 53 (27), 41 (126).
4.3.9. Isolation of (ꢀ)-(4R,5R,6S,10S)-gorgon-11-en-4-
ol (10)
(ꢀ)-(1R,4aS,8S,9R)-1,4a-Dimethyl-8-(1-methylethe-
nyl)-decahydro-naphthalen-1-ol. 10 was obtained by
column chromatography in a separate fraction. Colour-
less oil (ca. 2 mg); RICPSiL5 =1607; sense of optical rota-
4.3.7. Isolation of (+)-(5R,6S,10S)-a-gorgonene (8)
(+)-(1S,4aS,8aR)-4a,8-Dimethyl-1-(1-methylvinyl)-1,
2,3,4,4a,5,6,8a-octahydro-naphthalene. 8 was purified
by preparative GC with a column with heptakis(6-O-
tert-butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin
(110 ꢁC isothermal, 1.8 bar He). Colourless oil (ca. 2
mg); RICPSil5 =1488; sense of optical rotation (C6D6):
1
tion (C6D6): (ꢀ); H NMR (500 MHz, C6D6): d 0.79
(3H, s, H-14), 0.99–1.09 (2H, m, H(Si)-1, H(Re)-9),
1.16–1.20 (2H, m, H(Re)-1, H(Si)-9), 1.22 (3H, m, H-15),
1.32–1.46 (6H, m, H(Re)-2, H(Si)-2, H(Re)-7, H(Si)-7,
1
(+); H NMR (500 MHz, C6D6): d 0.88 (3H, s, H-14),
0.98–1.09 (1H, m, H(Si)-9), 1.19–1.25 (1H, m, H(Re)-7),
H(Re)-8, H(Si)-8), 1.49 (1H, d, J=11.3 Hz, H-5), 1.59–
1.65 (1H, m, H-3), 1.74–1.76 (1H, m, H-3), 1.78 (3H,
d, H-13), 2.31–2.36 (1H, m, H-6), 4.63 (1H, m, H-12),
4.79 (1H, d, J=2.5 Hz, H-12); 13C NMR (100.6 MHz,
C6D6): d 19.1 (q, C-13), 20.3 (q, C-14), 24.1 (q, C-15),
20.0, 21.7, 34.2 (3·t, C-2, C-7, C-8), 35.7 (s, C-10),
43.2 (t, C-3), 44.6 (d, C-6), 42.5, 45.5 (2·t, C-1, C-9),
56.8 (d, C-5), 73.8 (s, C-4), 112.7 (t, C-12), 153.7 (s, C-
11); MS (eI, 70 eV), m/z (rel. int.): 222 [M+] (2), 207
(14), 204 (9), 189 (100), 179 (13), 163 (23), 149 (28),
137 (29), 121 (25), 109 (47), 95 (35), 81 (43), 67 (28),
55 (29), 43 (58). HRMS: m/z=222.1994 [M+] (calc. for
C15H26O: 222.1984).
1.27–1.31 (1H, m, H(Si)-1), 1.34–1.43 (3H, m, H(Re)-1,
(Re)-8, H(Re)-9), 1.44–1.60 (1H, m, H(Si)-8), 1.64 (3H,
H
s, H-13), 1.64–1.72 (1H, m, H(Si)-7), 1.83 (3H, d, J=1.5
Hz, H-15), 1.98–2.10 (2H, m, H(Re)-2, H(Si)-2), 2.15
(2H, br s, H-5, H-6), 4.75 (1H, m, H(Z)-12), 4.79 (1H,
s, H(E)-12), 5.37 (1H, br s, H-3); 13C NMR (100.6
MHz, C6D6): d 16.8 (q, C-14), 20.4 (q, C-13), 21.7 (t,
C-8), 23.5 (t, C-2), 24.6 (q, C-15), 34.0 (s, C-10), 36.8
(t, C-7), 39.3 (t, C-1), 41.3 (t, C-9), 44.0 (d, C-5), 48.4
(d, C-6), 110.5 (t, C-12), 123.6 (d, C-3), 136.7 (s, C-4),
152.7 (s, C-11); MS (eI, 70 eV), m/z (rel. int.): 204
[M+] (7), 189 (82), 175 (3), 161 (19), 147 (17), 133 (32),
119 (38), 107 (100), 93 (65), 79 (46), 67 (27), 55 (32),
41 (49).
4.3.10. Isolation of (+)-(1R,5S,6S,7S)-allo-aromaden-
dra-4(15),10(14)-diene (11)
(+)-(1S,4aR,7aS,8S)-1,1-Dimethyl-4,7-dimethylene-
decahydro-cyclopropa[e]azulene. The same isolation
procedure as for isogermacrene A (5) was applied to ob-
tain 11. Colourless oil (ca. 1.5 mg); RICPSiL5 =1457;
sense of optical rotation (C6D6): (+); 1H NMR (500
MHz, C6D6): d 0.48–0.54 (2H, m, H-6, H-7), 1.01 (3H,
s, H-12), 1.05 (3H, s, H-13), 1.08–1.14 (1H, m, H(Si)-8),
4.3.8. Isolation of (5S,6S,10S)-gorgona-1,4(15),11-tri-
ene (9)
(4S,4aS,8aS)-8a-Methyl-5-methylene-4-(1-methylvi-
nyl)-1,2,3,4,4a,5,6,8a-octahydro-naphthalene.
9
was
enriched by preparative GC using a heptakis(6-O-tert-
butyldimethylsilyl-2,3-di-O-methyl)-b-cyclodextrin col-

Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
2273
1.63–1.69 (1H, m, H(Re)-8), 1.71–1.77 (2H, m, H(Si)-2,
H(Re)2), 2.14–2.21 (1H, m, H(Re)-3), 2.21–2.29 (1H, m,
(43), 145 (50), 131 (57), 117 (36), 105 (61), 91 (86), 79
(100), 67 (37), 53 (42), 41 (79).
H
(Si)-9), 2.30–2.40 (3H, m, H(Si)-3, H-5, H(Re)-9), 2.46
(1H, m, H-1), 4.86-4.87 (2H, m, H(Z)-14, H(Z)-15), 4.94
(1H, m, H(E)-14), 5.04–5.05 (1H, m, H(E)-15); 13C
NMR (100.6 MHz, C6D6): d 16.0 (q, C(Re)-12), 17.1 (s,
C-11), 21.7 (t, C-8), 25.1 (d, C-7), 29.0 (q, C(Si)-13),
29.5 (d, C-6), 30.0 (t, C-2), 34.0 (t, C-3), 37.2 (t, C-9),
41.4 (d, C-5), 48.3 (d, C-1), 105.7 (t, C-15), 110.3 (t, C-
14), 150.1 (s, C-10), 158.0 (s, C-4); MS (eI, 70 eV), m/z
(rel. int.): 202 [M+] (15), 187(25), 174 (13), 159 (88),
145 (51), 131 (70), 117 (54), 105 (67), 91 (100), 79 (64),
67 (33), 55 (24), 41 (70).
4.3.13. Isolation of (+)-(1R*,4S*,5S*,7S*,10S*)-viticulol
(14)
(+)-(1S*,3aR*,4S*,7S*,8aS*)-1,4-Dimethyl-7-(5-me-
thyl-1-methylene-hex-4-enyl)-decahydroazulen-4-ol. In
order to isolate compound 14 preparative GC on a hep-
takis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-b-
cyclodextrin column (150 ꢁC, 1 ꢁC/min, 180 ꢁC, 1.7 bar
He) was applied. Colourless oil (ca. 1.5 mg); RICP-
1
SiL5 =2103; H NMR (500 MHz, C6D6): d 0.96 (3H, d,
J=6.9 Hz, H-17), 1.03 (3H, s, H-18), 1.11–1.19 (1H,
m, H-3), 1.26–1.33 (1H, m, H-2), 1.35–1.41 (1H, m, H-
9), 1.58 (3H, s, H-20), 1.46–1.66 (6H, m, H-20, H-30,
H-6, H-60, H-8, H-90), 1.68 (3H, s, H-16) 1.81–1.96
(2H, m, H-1, H-4), 1.97–2.05 (1H, m, H-8Õ), 2.06–2.11
(1H, m, H-5), 2.13–2.21 (3H, m, H-7, H-12), 2.22–2.30
(2H, m, H-13), 4.90 (1H, d, J=1.3 Hz, H(E)-19), 5.02
(1H, s, H(Z)-19), 5.24–5.30 (1H, m, H-14); 13C NMR:
(100.6 MHz, C6D6) d 17.8 (q, C-18), 25.9 (q, C-16),
26.7 (t, C-2), 27.3 (t, C-6), 27.7 (t, C-13), 28.0 (t, C-8),
31.1 (t, C-3), 32.2 (q, C-17), 35.7 (q, C-20), 35.8 (t, C-
12), 35.9 (t, C-9), 40.0 (d, C-4), 40.2 (d, C-5), 42.5 (d,
C-7), 56.2 (d, C-1), 73.9 (s, C-10), 107.5 (t, C-19),
125.0 (d, C-14), 132.2 (s, C-15), 155.5 (s, C-11); MS
(eI, 70 eV), m/z (rel. int.): 272 [M ꢀ H2O]+ (36), 257
(9), 243 (3), 229 (14), 215 (6), 201 (16), 190 (24), 173
(10), 161 (59), 147 (31), 133 (27), 119 (37), 107 (56), 93
(50), 81 (44), 69 (85), 55 (36), 41 (100). HRMS:
m/z=272.2501 [MꢀH2O] (calc. for C20H32: 272.2504).
4.3.11. Isolation of (+)-aromadendra-4(15),10(14)-dien-
1-ol (12)
(+)-1,1-Dimethyl-4,7-dimethylene-decahydro-cyclo-
propa[e]azulen-4a-ol. Enrichment of 12 was achieved by
preparative GC using a heptakis(6-O-tert-butyldimeth-
ylsilyl-2,3-di-O-methyl)-b-cyclodextrin column (150 to
180 ꢁC, 1 ꢁC minꢀ1, 1.7 bar He), following further puri-
fication by using a semipreparative DB-1701 thickfilm
column (150 ꢁC isothermal). Colourless oil (0.3 mg);
RICPSiL5 =1579; sense of optical rotation (C6D6): (+);
1H NMR (500 MHz, C6D6): d 0.36 (1H, dd, J=9.1,
10.7 Hz, H-6), 0.47–0.52 (1H, m, H-7), 0.58 (1H, s,
OH), 0.98 (3H, s, H-12), 1.06 (3H, s, H-13), 1.35–1.44
(2H, m, H-2, H-8), 1.65–1.71 (1H, m, H-8Õ), 1.97 (1H,
ddd, J=7.25, 12.93, 12.93 Hz, H-2Õ), 2.18–2.28 (2H, m,
H-3, H-9), 2.39 (1H, d, J=10.7 Hz, H-5), 2.66–2.78
(2H, m, H-3Õ, H-9Õ), 4.81 (1H, t, J=1.6 Hz, H(E)-14),
4.84 (1H, m, H(Z)-14), 4.89 (1H, br s, H(Z)-15), 5.06
(1H, m, H(E)-15); MS (eI, 70 eV), m/z (rel. int.): 218
[M+] (7), 203 (16), 200 (17), 185 (23), 175 (49), 157
(50), 147 (29), 129 (43), 119 (43), 105 (67), 91 (100), 79
(61), 67 (39), 55 (51), 41 (92). HRMS: m/z=218.1662
[M+] (calc. for C15H22O: 218.1671).
4.4. Derivatization reactions
4.4.1. Acid induced rearrangement of compounds 2, 5 and
20
A solution of compound 2, 5 or 20 in 0.5 ml n-hexane
was treated with 0.4 mg of the acidic ion exchange resin
Amberlystꢂ 15 and stirred at room temperature for a
varying reaction time (1 h for 5, 24 h for 20 and 48 h
for 2). Subsequently the solutions were filtered and
investigated by GC–MS.
4.3.12. Isolation of (+)-isozierene (13)
(+)-4-Methyl-9-methylene-bicyclo[8.2.1]trideca-1(13),
4-diene. 13 was obtained during the isolation of zierene
(3) by preparative GC applying a 6-O-tert-butyldimeth-
ylsilyl-2,3-O-dimethyl-b-cyclodextrin column (120 ꢁC
isotherm, 1.8 bar He) and an injector temperature of
200 ꢁC. Colourless oil (ca. 1.5 mg); RICPSiL5 =1553;
sense of optical rotation (C6D6): (+); 1H NMR (500
MHz, C6D6): d 1.08–1.18 (1H, m), 1.45 (3H, d, J=1.0
Hz), 1.57–1.75 (4H, m), 1.86–2.01 (5H, m), 2.09–2.19
(2H, m), 2.26 (1H, ddd, J=5.3, 11.8, 11.8 Hz), 2.32–
2.39 (1H, m), 3.48–3.51 (1H, m), 4.66 (1H, dd, J=1.0,
2.6 Hz), 4.85 (1H, d, J=2.0 Hz), 5.05 (1H, s), 5.34
(1H, dd, J=7.8, 7.8 Hz); 13C NMR (100.6 MHz,
C6D6): d14.9 (q), 26.8 (t), 28.5 (t), 29.3 (t), 30.5 (t),
34.1 (t), 34.8 (t), 37.1 (t), 55.6 (d), 109.8 (t), 127.2 (d),
130.0 (d), 134.3 (s), 143.2 (s), 155.8 (s); MS (eI, 70 eV),
m/z (rel. int.): 202 [M+] (83), 187 (64), 173 (33), 159
4.4.2. Hydrogenation of compounds 11 and 26
To 0.5 mg (+)-allo-aromadendra-4(15),10(14)-diene
(11) in 1 ml n-hexane 0.5 mg of Pd-C (15%) were added
and H2 was passed through the solution for 3 min. After
stirring for 3 h at room temperature the suspension was
filtered and the resulting solution concentrated. (ꢀ)-allo-
Aromadendrene (26) was treated accordingly and the
products of both reactions were compared by GC and
GC–MS. Two products were identified that had identi-
cal retention times on non-chiral stationary phases but
different retention times on a column with hepta-
kis(2,6-di-O-methyl-3-O-pentyl)-b-cyclodextrin.

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Th. Hackl et al. / Phytochemistry 65 (2004) 2261–2275
Hochmuth, D.H., Joulain, D., Ko¨nig, W.A., 2002. Massfinder 2.3,
Acknowledgements
Terpenoids and related constituents of essential oils. Available
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states: passions and punctilios, 1935–1995. Accounts of Chemical
Research 28, 81–90.
We thank Professor R. Mues, Universita¨t des Saar-
lands, for his advice. We also thank Dr. V. Sinnwell
and Mrs. B. Claasen, University of Hamburg, for
their support in the NMR and Mrs. A. Meiners
and Mr. M. Preusse for GC–MS and GC–HRMS
measurements.
Joulain, D., Ko¨nig, W.A., 1998. The Atlas of Spectral Data of
Sesquiterpene Hydrocarbons. E.B.-Verlag, Hamburg.
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macrocyclic terpenoids by intramolecular cyclisation V: transan-
nular reactions of hedycaryol isomers. Tetrahedron Letters 19,
1687–1690.
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