Synthesis and NMR characterisation of novel highly cyclised polyprenoid hydrocarbons from sediments

Article abstract of DOI:10.1039/b008104n

We report here on the identification of two novel hexacyclic alkanes (C₃₃ and C₃₅) occurring in bitumen. The C₃₅ compound 1 was identified by comparison with a standard obtained by synthesis involving a biomimetic proton-induced extensive cyclisation of an acyclic heptaprenoid. This cascade cyclisation allows the formation of eleven asymmetric centres present in the natural compound in only one step. The C₃₃ analogue 2 was identified by NMR studies after isolation from the saturated hydrocarbon fraction of a bituminous rock. Both compounds are "orphan" molecular fossils of biological lipids of unknown origin formed by the extensive cyclisation of higher regular polyprenoids.

Full text of DOI:10.1039/b008104n

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Synthesis and NMR characterisation of novel highly cyclised
polyprenoid hydrocarbons from sediments
Emmanuelle Grosjean,a Jacques Poinsot,a Armelle Charrié-Duhaut,a Stéphanie Tabuteau,a
Pierre Adam,a Jean Trendel,a Philippe Schaeffer,a Jacques Connan,a Daniel Dessortb and
Pierre Albrecht*a
1
a Institut de Chimie, UMR 7509 du CNRS, Université Louis Pasteur, 1 rue Blaise Pascal,
67000, Strasbourg, France. E-mail: albrecht@chimie.u-strasbg.fr; Fax: (33) 3 90 24 17 64;
Tel: (33) 3 90 24 16 96
b Centre de Recherche, TotalFinaElf, Avenue Larribau, 64000, Pau, France
Received (in Cambridge, UK) 6th October 2000, Accepted 1st February 2001
First published as an Advance Article on the web 7th March 2001
We report here on the identification of two novel hexacyclic alkanes (C33 and C35) occurring in bitumen. The C35
compound 1 was identified by comparison with a standard obtained by synthesis involving a biomimetic proton-
induced extensive cyclisation of an acyclic heptaprenoid. This cascade cyclisation allows the formation of eleven
asymmetric centres present in the natural compound in only one step. The C33 analogue 2 was identified by NMR
studies after isolation from the saturated hydrocarbon fraction of a bituminous rock. Both compounds are “orphan”
molecular fossils of biological lipids of unknown origin formed by the extensive cyclisation of higher regular
polyprenoids.
Introduction
gation of the saturated hydrocarbon fraction of bituminous
rocks from the Aquitaine basin (Lameignère quarry; France)6
revealed the presence of a series of high molecular weight
polycyclic hydrocarbons possessing up to 8 rings. These sat-
urated hydrocarbons display characteristic and information-
rich mass spectra, showing two main series of fragments,
each regularly shifted by 68 Da. As the shift of 68 Da was
likely to correspond to an isoprene unit (C5H8), these com-
pounds were thought to have regular polycyclic polyprenic
structures. Thus, considering the mass spectra of the hexa-
cyclic compounds, which constitute the main series, one can
observe a common fragmentation pattern showing fragments
at m/z 191, 259, 327 and 395 which remain unchanged within
the series, whereas a second fragmentation pattern (m/z
149+14n, 217+14n, 285+14n, n = 0 for C34) was found to
depend on the molecular mass of the considered compound.
The structure proposed for the hexacyclic compounds, as
illustrated in Fig. 1(a), can easily account for the two frag-
mentation patterns observed: the first set of ions (m/z 191,
259, 327 and 395) can result from the fragmentation of the
left part of the structures whereas the second set (m/z
149+14n, 217+14n, 285+14n) increases with the length of the
side chain and can originate from the fragmentation of the
right part of the structure. Based on these hypotheses, struc-
ture 1 was ascribed to the hexacyclic C35 (n = 1, Fig. 1(a))
homologue occurring in Lameignère bitumen. As this com-
pound was present in the complex bitumen fractions in
amounts too small to allow isolation and characterisation by
NMR, synthesis of a standard has been carried out.
Highly cyclised compounds based on regular polyprenoid skel-
etons have recently been shown to occur in various sediments as
monoaromatic hydrocarbons1,2 and sulfides.3 The biological
precursors from which these molecular fossils originate remain,
however, to be discovered. According to their specific structure,
these precursors were thought to derive from the proton-
induced and/or enzymatic cyclisation of all-trans regular iso-
prenoids. The possible presence in the sedimentary record of
aliphatic hydrocarbons structurally related to these com-
pounds,1,4 as suggested by mass spectrometry analyses, was
recently confirmed by the identification of a C37 heptacyclic
alkane 3 isolated from a bituminous rock from a Spanish
quarry.5 Mass spectral investigation of the saturated hydro-
carbon fraction from the same sample further revealed an
extended series of related polycyclic polyprenoid alkanes, which
may be either complete polyprenoids or dealkylated analogues.
These assumptions have now been confirmed by unambiguous
identification of two polycyclic alkanes belonging to this series
(1, 2). The hexacyclic C35 compound 1 was characterised by
comparison with a standard obtained by synthesis involving the
biomimetic proton-induced cyclisation of an acyclic regular
heptaprenoid. A lower C33 homologue 2 was isolated from the
saturated hydrocarbon fraction of a bituminous rock from a
Spanish quarry and identified by NMR studies.
Results
Gas chromatography–mass spectrometry (GC-MS) investi-
DOI: 10.1039/b008104n
J. Chem. Soc., Perkin Trans. 1, 2001, 711–719
This journal is © The Royal Society of Chemistry 2001
711

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bonds are trans. Location of the chlorine atom at position 12
was established by the occurrence of NOEs between the vinylic
proton at position 10 and the allylic protons (α to chlorine
atom) at position 12. Head to tail coupling of C20 and C15 iso-
prenoids was achieved by reacting the chloride 7 with the
sulfur-stabilised allylic carbanion obtained by reaction of the
phenyl thioether 9 with n-butyllithium,9 yielding the C35 iso-
prenoid 10. Final deprotection was performed with lithium in
ethylamine. The resulting C35 polyprenol 11 was characterised
by 1D- and 2D-NMR studies. Interpretation of 13C and DEPT
spectra allowed us to establish that the carbon chemical shifts
of one of the geminal vinylic methyl groups has a value of 28
ppm whereas the values for all the other vinylic methyl groups
appear in the 15–18 ppm range. This is in accordance with the
fact that all the double bonds are trans14 and was further
corroborated by the interpretation of the NOESY correlation
pattern which, in all cases but one (i.e. methyl 28), does not
show any NOE between vinylic protons and vinylic methyl
groups.
Several attempts were carried out to cyclise directly the poly-
prenol 11 with fluorosulfonic acid in 1-nitropropane at Ϫ78 ЊC
as described by Vlad7c for the cyclisation of lower polyprenols,
but were unsuccessful due to predominant polymerisation and
dehydration reactions. In order to limit the extent of side-
reactions, cyclisation was finally performed on the all-trans
heptaprenoic acid methyl ester 12. The latter was obtained by
oxidation of the polyprenol 11 to the related aldehyde (MnO2)
followed by further oxidation to the corresponding methyl ester
12 (NaCN, MnO2, MeOH)15 by a mild procedure which avoids
isomerisation of the double bond located at position 2. The
cyclisation was performed by slow addition of a diluted solu-
tion of the ester 12 in 1-nitropropane to a solution of fluoro-
sulfonic acid in 1-nitropropane at Ϫ78 ЊC. Work-up of the
reaction mixture, followed by chromatography on a silica gel
column, afforded a complex mixture of monoesters comprising,
as evidenced by GC-MS analysis, numerous partly cyclised iso-
prenoid esters but also a small amount of the hexacyclic ester
13 which could be isolated in 1% yield by reversed phase
HPLC.
The structure of the ester 13 was confirmed by NMR studies
comprising homonuclear (1H–1H, COSY and NOESY) and
heteronuclear (1H–13C, HMQC and HMBC) correlation
experiments. Interpretation of the DEPT spectrum, as well as
of heteronuclear (1H–13C, HMQC and HMBC) correlation
experiments, allowed us to determine clearly the carbon
skeleton and, in particular, that each ring junction methyl group
is separated from its neighbouring methyl groups by one
common remote (3J) connection with a methine (Fig. 3). Stereo-
chemical information was obtained from the NOESY experi-
ment (Fig. 3). Occurrence of NOEs between each ring-junction
methyl group and its neighbouring methyls clearly shows that
they are all axial and located on the same side of the molecule.
Furthermore, the occurrence of NOEs between H-2/H-6 and
H-2/Hax-8 indicates that H-2 is α and, therefore, that the
methoxycarbonyl group is β. Combination of the results from
all the experiments finally allowed us to establish firmly the
structure and to assign all the 1H and 13C chemical shifts (Table
1). Additional structural information was obtained from the
interpretation of the 13C chemical shifts (Table 1). Thus, the
trans,transoid,trans stereochemistry of all the ring junctions, as
well as the fact that all the ring junction methyl groups are
located on the same side of the molecule, was further supported
by the 13C chemical shifts of these methyls. Their values, below
20 ppm, indicate therefore the occurrence of γ-gauche effects,
as expected for axial methyl groups on an all-trans polycyclic
structure.16 Moreover, the strong deshielding of the ring
junction methines and of the methylenes bearing axial protons
trans relative to axial methyl groups (57 < δ < 62 ppm and
40 < δ < 43 ppm, respectively) shows the absence of γ-gauche
effects, which is also in accordance with an all-trans structure.16
Fig. 1 (a) Generic mass spectrometry fragmentation pattern of the
hexacyclic polyprenoid saturated hydrocarbons and (b) of compound 2.
The absolute configuration chosen is that determined for related
monoaromatic compounds identified in sediments.1,2
Synthesis of hexacyclic polyprenoid C35 hydrocarbon 1 and
identification in bitumen sample
The strategy used for the preparation of the C35 hexacyclic
hydrocarbon 1 involves the extensive cyclisation of an all-trans
C35 regular heptaprenoid along its whole structure. Such
cascade cyclisations described in the literature can be either
cationic-2,7 or radical-induced.8 The method adopted here was
developed in recent years by Vlad7c and consists of a proton-
induced cyclisation using a superacid (HFSO3) generating in
our case 11 asymmetric centres at one go. Thus, it was envisaged
that the synthesis of compound 1 could be achieved by a
cascade of six successive cyclisations from the acyclic all-trans
heptaprenoic acid methyl ester 12 (Fig. 2) followed by reduction
of the methyl ester and of the residual double bond. Synthesis
of the isoprenoid ester 12 relies on a method previously
described for the synthesis of squalene and involving the head
to tail coupling of two adequately functionalized C20 and C15
isoprenoid units (9 and 7, respectively; Fig. 2).9,10 The phenyl
thioether 9 was synthesised from commercial all-trans geranyl-
geraniol 8 following standard procedures.9,10 Synthesis of the
chloride 7 from farnesol 4 (Fig. 2) first requires functionalis-
ation at the head of the isoprenoid skeleton to allow incorpor-
ation of chlorine. For this purpose, the benzyl ether-protected
farnesol 511 was oxidised regioselectively at the terminal trans
methyl group with SeO2 in the presence of tert-butyl hydro-
peroxide to give (2E,6E,10E)-1-benzyloxy-3,7,11-trimethyl-
dodeca-2,6,10-trien-12-ol 6.12 The latter was converted into the
chloride 7 by treatment with toluene-p-sulfonyl chloride in the
presence of triethylamine and 4-dimethylaminopyridine.13
NMR studies comprising homonuclear (1H–1H, COSY and
NOESY) and heteronuclear (1H–13C, HMQC and HMBC)
correlation experiments were performed on the chloride 7 in
order to confirm the location of the chlorine atom on the trans
terminal methyl group. Thus, the absence of nuclear Over-
hauser effects (NOEs) between vinylic protons and vinylic
methyl groups unambiguously confirmed that all the double
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Fig. 2 Synthesis of the hexacyclic polyprenoid hydrocarbon 1. Reagents and conditions: i, benzyl chloride, NaOH, DMSO; ii, SeO2, ButOOH,
CH2Cl2; iii, pTsCl, Et3N, DMAP, CH2Cl2; iv, MeSO2Cl, Et3N, CH2Cl2; v, PhSH, MeLi, Et2O; vi, 9, BunLi, DABCO, 7, THF, Ϫ78 ЊC; vii, Li, EtNH2,
Ϫ78 ЊC; viii, MnO2, hexane; ix, MnO2, NaCN, AcOH, MeOH; x, HFSO3, 1-nitropropane, Ϫ78 ЊC; xi, LiAlH4, THF; xii, (a) MeSO2Cl, Et3N,
CH2Cl2; (b) LiAlH4, THF; xiii, H2, PtO2, AcOEt.
Reduction of the ester 13 via alcohol 14 and alkene 15 follow-
ing standard procedures yielded an equimolar mixture (as
determined by GC) of two hexacyclic alkanes 1 and 16. All our
attempts to separate these two isomers using various reversed
phase HPLC columns were unsuccessful. Comparison of the
mass spectra of the synthetic isomers with those of the C35
hexacyclic alkane occurring in Lameignère bitumen, as well as
co-injection experiments on two GC columns coated with dif-
ferent phases clearly established that only one of the synthetic
isomers is present in the bitumen sample. Since the bitumen
sample originates from a “mature” petroleum, and has under-
gone rather high thermal stress, the thermodynamically more
stable configurations at asymmetric centres of polycyclic hydro-
carbons are strongly favoured.17 Thus, it can be proposed that
methyl 4Ј is equatorial and, therefore that the hydrocarbon 1
corresponds to the geological compound. This hypothesis is
J. Chem. Soc., Perkin Trans. 1, 2001, 711–719
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1
Table 1 13C and 1H NMR data of ester 13 (500 MHz, C6D6 , δ relative
to TMS)
Table 2 13C and H NMR data of hydrocarbon 2 (500 MHz, C6D6 ,
δ relative to TMS)
C
δC
(ppm) δH (ppm)
C δC
atoma (ppm) δH (ppm)
C δC
atoma (ppm) δC (ppm)
C δC
atoma (ppm)
atoma
δC (ppm)
1
2
3
4
4Ј
5
6
7
8
8Ј
9
10
11
12
12Ј
13
14
15
173.0
62.9
129.5
124.0
21.6
23.2
55.2
36.9
42.4
15.9
17.9
61.9
37.9
42.1
16.8
17.5
61.5
37.9
—
3.08 s
—
5.50
1.77
1.91(α), 1.91(β) 20
1.15
—
1.49(α), 1.87(β) 22
1.19 s 23
1.28(β), 1.45(α) 24
0.71
—
1.64(β), 0.82(α) 26
0.85 s 27
1.29(β), 1.46(α) 28
0.64
—
16
16Ј
17
18
19
42.2
17.9
17.6
61.3
38.2
42.1
17.7
18.9
57.0
37.9
40.2
16.8
19.2
42.6
33.6
33.8
21.8
0.81(α), 1.73(β)
0.80 s
1.31(β), 1.52(α)
0.67
2
3
4
4Ј
5
6
7
8
8Ј
9
55.4
27.9
36.9
23.2
21.2
58.6
35
44.1
21.2
17.7
62.2
37.7
42.2
17.3
17.7
59.6
37.4
0.76(α), 1.29(β) 16
1.51(β) 16Ј
0.88(α), 1.78(β) 17
0.90 18
1.21(α), 1.54(β) 19
41.4
17.7
20.3
53.6
36.8
39.6
14.3
21.6
52.3
35.3
31.5
22.6
42.6
33.2
30.8
20.3
0.81(α), 1.76(β)
0.88 s
1.09(β), 1.61(α)
0.69(α)
—
—
0.82(α), 1.73(β)
0.83 s
1.52(α), 1.34(β)
0.78
0.73
—
20
20Ј
0.88(α), 1.81(β)
0.77 s
1.28(β), 1.58(α)
0.75(α)
20Ј
21
1.14(α), 1.53(β) 21
0.93 s 22
1.36(α), 1.26(β) 23
—
1.11(β)
0.77(α), 1.68(β)
0.85 s
1.64(β), 1.41(α)
1.18(α), 1.41(β)
—
0.92 s
0.88 s
10
0.74
—
24
25
0.67(α), 1.86(β)
1.43(β), 1.56(α)
1.19(α), 1.38(β)
—
0.93 s
0.87 s
24Ј
25
11
12
12Ј
13
14
15
0.84(α), 1.73(β) 26
0.84 s 27
1.36(β), 1.53(α) 28
0.70(α)
28Ј
28Ј
—
OCH3 50.8
3.40 s
a For C numbering, see Fig. 4.
a For C numbering, see Fig. 3.
the major hydrocarbons occurring in Lameignère samples,
it was likely that these new compounds were demethylated
structures.
The fraction showed, at the beginning of the gas chromato-
gram, a predominant complex mixture of low molecular
weight hydrocarbons characteristic of strongly biodegraded
bitumens.20 The minor heavy saturated hydrocarbons, including
the polycyclic polyprenoid hydrocarbons, could, however, be
enriched by successive precipitation in acetone. A sequence of
chromatographic separation steps involving different types of
reversed phase HPLC columns was performed on the precipi-
tated fraction and allowed us to isolate approximately 1 mg of a
hexacyclic demethylated compound 2 (96% purity estimated by
GC, [M+] 452 C33H56). Characterisation of hydrocarbon 2 was
carried out by 1D- and 2D-NMR studies including homo-
nuclear (1H–1H, COSY and NOESY) and heteronuclear
(1H–13C, HMQC and HMBC) correlation experiments, which
permitted us to assign the signals of all the protons and all the
carbon atoms (Table 2) and to establish unambiguously its
structure.
NMR characterization of hydrocarbon 2. 13C-NMR 1H
noise-decoupled and DEPT spectra of compound 2 show the
presence of 7 methyl, 14 methylene, 7 methine and 5 quater-
nary carbons. Furthermore, the 1H-NMR spectrum reveals the
presence of 6 methyl singlets and one methyl doublet, which
suggests a lack of two methyls on this compound as compared
to the spectrum of the complete C35 hexacyclic regular poly-
prenoid hydrocarbon 1. The carbon skeleton was established
Fig. 3 (a) Spatial representation of 13 showing the most important
NOEs observed. (b) Carbon sequence (bold) established from inverse
1
long-range H–13C correlation experiment. The absolute configuration
of compound 13 is not known. The absolute configuration represented
in this figure is that determined for related monoaromatic compounds
identified in sediments.1,2
further corroborated by previous studies on C20 regular tricyclic
polyprenoid hydrocarbons having the same partial structure
(ring E and F) in which equatorial methyls 1 and 4Ј strongly
predominate in mature samples.18
1
using H–13C long-range (2,3J) couplings, the most intense of
which are observed from the methyl groups. Among the
methyls, two exhibit remote connections (2,3J) to each other, as
well as to the same quaternary, methine and methylene carbons,
which implies that they must be geminal to each other. The
sector formed by these geminal methyl groups (i.e., atoms 22,
26, 27, 28 and 28Ј) appears to be cut off from the remaining
part of the molecule (Fig. 4). Indeed, C-22 does not show
remote connections with any remaining methyl groups, thus
suggesting the absence of the 24Ј-Me at C-23. All the other
methyl groups (i.e. 4Ј, 8Ј, 12Ј, 16Ј, 20Ј methyl groups) are linked
two-by-two to each other through one common remote connec-
Isolation and identification of demethylated hexacyclic C33
hydrocarbon 2
In the course of investigation of bituminous rocks from a
Spanish quarry (Maestu),19 GC-MS analysis of the alkane
fraction showed clear evidence for the occurrence of the poly-
prenoid polycyclic saturated hydrocarbons, including com-
pound 1, already encountered in bitumens from Lameignère
(France). GC-MS analyses also revealed the presence of other
related series of polycyclic hydrocarbons (6 to 8 rings), showing
a similar fragmentation pattern with two sets of main frag-
ments, each regularly shifted by 68 Da. However, as the main
set of fragments was shifted by 14 Da [Fig. 1(b)] as compared to
1
tion (3J) with a methine or with a methylene (C-2). H–1H
COSY and NOESY spectra allowed us to link the sector of the
geminal methyl groups to that of the other methyl groups
(Fig. 4) and to assign the signals of the remaining atoms.
Indeed, as compared to a regular hexacyclic polyprenoid
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were observed between the methyl groups 4Ј and 8Ј as expected
for an equatorial position for 4Ј. Furthermore, the shielding of
the C-3 carbon atom combined with the deshielding of the pro-
ton at C-3, which reveal the presence of γ-gauche effects
between H-3 and Me-8Ј, is also in agreement with an equatorial
position for Me-4Ј.
Discussion
The two new hydrocarbons 1 and 2 clearly result from the dia-
genetic transformation, by various processes known to occur in
the subsurface, of polycyclic polyprenoid precursors formed by
proton-induced cyclisation of regular all-trans unsaturated
polyprenoids (Fig. 5). The presence of higher homologues of 1
and 2 bearing extended side-chains may be explained by
incomplete cyclisation of higher polyprenoids.
GC-MS analysis of several biodegraded bitumen samples
containing the newly-identified hydrocarbons and having
undergone weathering to various extents shows that the relative
amount of demethylated polycyclic polyprenoid alkanes (such
as 2 and 3) increases with the extent of weathering as compared
to the amount of the related hydrocarbons from the regular
series (such as 1).19 A similar situation is observed in the case of
hopanes21a which are ubiquitous molecular fossils of a wide-
spread family of bacterial lipids, the biohopanoids.21b Exposure
of bitumen samples to biodegradation indeed results in an
increase of the relative amount of demethylated compounds 17
as compared to their regular counterparts 18.21 By analogy,22c
Fig. 4 (a) Spatial representation of 2 showing the most important
NOEs observed. (b) Carbon sequence (bold) established from inverse
1
long-range H–13C correlation experiment. The absolute configuration
of compound 2 is not known. The absolute configuration represented in
this figure is that determined for related monoaromatic compounds
identified in sediments.1,2
hydrocarbon, interpretation of the NOESY correlation pattern
afforded evidence that the missing methyl has been replaced by
a hydrogen at C-23 (presence of NOEs between 28Ј-Me/H-23,
20Ј-Me/H-23). Moreover, NOEs observed between 28Ј-Me/H-
23, H-23/20Ј-Me, 20Ј-Me/16Ј-Me, 16Ј-Me/12Ј-Me, 12Ј-Me/8Ј-
Me reveal that 28Ј-Me, H-23, 20Ј-Me, 16Ј-Me, 12Ј-Me and 8Ј-
Me are all on the same side of the molecule, thus confirming its
regular polyprenoid structure. No correlation between methyls
8Ј, 12Ј, 16Ј, 20Ј, proton 23 and protons 6, 10, 14, 18, 22 on ring
junctions could be observed in the NOESY spectrum. This is in
agreement with the fact that all the ring junctions are trans.
The underscoring of γ-gauche effects also supports the trans,
transoid,trans stereochemistry.16 The methyl groups 20Ј-Me,
16Ј-Me, 12Ј-Me, 8Ј-Me, the methine C-23 and the methylene
groups C-5, C-9, C-13, C-17, C-21, C-25 show strong γ-gauche
interactions, on account of the strong shielding of their carbon
atoms (δ13C < 21 ppm for the methyl groups, δ13C = 35 ppm for
the methine and δ13C < 22.5 ppm for the methylenes) together
with the deshielding of the corresponding axial protons belong-
ing to the methylene groups (1.1 < δ1H < 1.4 ppm). Conversely,
low-field 13C chemical shifts (as well as corresponding high-field
1H chemical shifts) indicate the absence of γ-gauche effects.
This is the case for the methines C-6, C-10, C-14, C-18, C-22
and methylenes C-2, C-8, C-12, C-16, C-20 (52 < δ13C < 62
ppm for methine and 40 < δ13C < 55 ppm for methylene
carbons, δ1H < 0.75 ppm for axial methine protons and
δ1H < 1.1 ppm for axial methylene protons). These observ-
ations are in agreement with an axial orientation for these
quoted methyl groups, as well as for the methines C-6, C-10, C-
14, C-18, C-22 which are trans relative to the methyl groups.
The low-field 13C chemical shift value for methyl 28 is character-
istic for an equatorial geminal methyl group. Similarly, the 13C
chemical shift value of 23.3 ppm for the methyl 4Ј—as com-
pared to those of axial methyl groups which do not exceed 21
ppm—suggests an equatorial position. The equatorial position
of methyl 4Ј is particularly difficult to prove since no clear NOE
could be observed with any of the protons located on C-atoms
2, 3, 4Ј, 8Ј. This is certainly due to the fact that the H-3 signal is
very broad due to numerous couplings with its neighbouring
protons and to distortion of ring F. Nevertheless, no NOEs
it may be inferred that the demethylated polyprenoid hydro-
carbons are biotransformation products of the hydrocarbons
from the regular series. Alternatively, biodegradation may
have resulted in a preferential removal of the hydrocarbons
from the regular series, leading therefore to a relative enrich-
ment of the more resistant demethylated structures, already
present in low concentrations in the original, unweathered
bitumen.22b Concerning the origin of the latter, it can be pro-
posed either that the missing methyl groups were already
absent from the biological precursors, or that functionalized
groups were present at these positions and lost by various
diagenetic transformation processes.5 It cannot be excluded
that functionalisation of methyl groups on the precursor
polyprenoid was induced by microbial oxidation processes
occurring at the earliest stages of diagenesis.23 It is striking in
this respect that the demethylated hopanes have also lost the
methyl group located at the A–B ring junction.22a As the par-
tial structural subunit constituted by the A–B–C ring system
is common to hopanoids and polycyclic polyprenoid alkanes,
it can be inferred that this partial structure may be recognised
and demethylated by bacteria involved in the biodegradation
of bitumen exposed to atmospheric conditions. Alternatively,
the absence of the methyl group at the A–B ring junction
may increase resistance towards biodegradation not only of
hopanoids but also of various structurally related polycyclic
polyterpenoids.
Experimental
General
NMR spectra were recorded on a Bruker ARX-500 or on a
J. Chem. Soc., Perkin Trans. 1, 2001, 711–719
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Fig. 5 Possible origin of the novel highly cyclised polyprenoid hydrocarbons by diagenetic transformation of biological polycyclic polyprenoid
lipids (modified from Schaeffer et al.1)
Bruker WP 200 SY spectrometer operating at observation fre-
quencies of 500 MHz (resp. 200 MHz) for 1H and 125 MHz for
13C nuclei and data were recorded at 300 K. Chemical shifts
(δ) are reported in ppm from tetramethylsilane, using the
solvent (C6D6: δ1H 7.16, δ13C 128.0 or CDCl3: δ1H 7.26, δ13C
77.0) as internal reference. J values are given in Hz. 2D-NMR
studies including homonuclear (1H–1H, COSY and NOESY)
and heteronuclear (1H–13C, HMQC and HMBC) correlation
experiments were performed on the Bruker ARX-500
spectrometer.
Gas chromatography–mass spectrometry analyses were
carried out either on a Finnigan MAT INCOS 50 spectro-
meter connected to a Varian 3400 gas chromatograph equipped
with an on-column injector and with a DB-5 J&W column (30
m × 0.25 mm, 0.1 µm film thickness), or on a Finnigan MAT
TSQ 700 spectrometer connected to a Varian 3400 gas
chromatograph (on-column injector, DB-5 J&W column, 60
m × 0.25 mm, 0.1 µm film thickness). Mass spectra were pro-
duced at 70 eV and helium was used as carrier gas. Co-injection
experiments were carried out using both an apolar column
(DB5 J&W, 60 m × 0.25 mm, 0.1 µm film thickness) and a more
polar column (DB17 J&W, 30 m × 0.25 mm, 0.15 µm film
thickness).
sonication allowed extraction of the major part of the soluble
low molecular weight constituents and consequently enrich-
ment of the remaining precipitated alkane fraction in polycyclic
polyprenoid hydrocarbons. For the isolation of the hexacyclic
compound 2 ([M+] 452, C33H56), a sequence of chromato-
graphic separation steps on different HPLC columns was
performed on the precipitated alkane fraction. Separation on a
Du Pont Zorbax ODS column (250 × 21.2 mm, 8 µm, 80 Å;
CH3OH–CHCl3 50 : 50 v/v; 20 ml minϪ1) allowed us to obtain
a fraction enriched in compound 2. Additional fractionation by
reversed phase HPLC (Du Pont Zorbax ODS 250 × 9.4 mm, 5
µm, 60 Å; CH3OH–CHCl3 55 : 45 v/v; 5 ml minϪ1; Vydac C18
250 × 4.6 mm, 5 µm, 300 Å; CH3OH–CHCl3 65 : 35 v/v; 1 ml
minϪ1) led to the isolation of 1 mg of compound 2 with a purity
of 96% (GC).
Compound 2: MS (EI, 70 eV): 452(M+, 59%), 437(36),
313(62), 287(3), 271(4), 259(6), 245(76), 231(9), 217(30), 203(7),
191(19), 177(42), 163(28), 149(56), 135(28), 121(37), 109(63),
95(90), 81(100), 69(56), 55(59).
Synthesis of hexacyclic polyprenoid alkane 1
(2E,6E,10E)-1-Benzyloxy-3,7,11-trimethyldodeca-2,6,10-
triene 5. To a solution of sodium hydroxide (4 g; 0.1 mol)
Probe mass spectra were recorded on the Finnigan MAT
TSQ 700 spectrometer.
in DMSO (100 ml) were added 15
g (0.07 mol) of
farnesol [(2E,6E,10E)-3,7,11-trimethyldodeca-2,6,10-trien-1-
ol] 4. After 2 h at room temperature, a solution of benzyl
chloride (10 g; 0.08 mol) in DMSO (20 ml) was slowly added.
The reaction mixture was kept under argon at room temper-
ature for 12 h after which the mixture was poured into water
and extracted with CH2Cl2 (3×). The crude extract was
adsorbed on to silica gel and chromatographed on a silica gel
column (CH2Cl2–hexane 50 : 50 v/v) yielding 19.8 g (91%) of
(2E,6E,10E)-1-benzyloxy-3,7,11-trimethyldodeca-2,6,10-triene
Separation of alkane fractions. Isolation of hexacyclic alkane 2
The crushed rock sample (Maestu quarry, Spain; Lameignère
quarry; France) was extensively extracted with CH2Cl2–
CH3OH (1 : 1, v/v). Chromatography of the organic extract
on a silica gel column yielded the saturated hydrocarbon frac-
tion using hexane as eluent. Successive washing of the alkane
fraction from the Spanish sample with acetone followed by
716
J. Chem. Soc., Perkin Trans. 1, 2001, 711–719

View Article Online
5. NMR δH (CDCl3, 200 MHz): 1.60 (6H, s, 2 × CH3), 1.65 (3H,
s, CH3), 1.68 (3H, s, CH3), 1.92–2.17 (8H, m, H allylic), 4.03
(2H, d, J 6.5 Hz, H-1), 4.51 (2H, s, -CH2-O-CH2-Phe), 5.06–
5.12 (2H, m, H vinylic), 5.41 (1H, t, J 6.0 Hz, H-2), 7.27–7.36
(5H, m, H aromatic). MS (EI, 70 eV): 312(M+, 1%), 221(15),
204(12), 191(25), 189(24), 161(21), 135(41), 121(45), 107(47),
91(100), 69(94), 55(18).
tion mixture was quenched with water and extracted with
diethyl ether (3×). Chromatography of the crude mixture on
silica gel (hexane–CH2Cl2 8 : 2 v/v) gave 1.0 g (58%) of
(2E,6E,10E,14E)-1-phenylsulfanyl-3,7,11,15-tetramethylhexa-
deca-2,6,10,14-tetraene 9. NMR δH (CDCl3, 200 MHz): 1.60
(12H, s, 4 × CH3), 1.69 (3H, s, CH3-4Ј), 1.93–2.09 (12H, m, H
allylic), 3.55 (2H, d, J 7.5 Hz, H-1), 5.05–5.15 (3H, m, H
vinylic), 5.32 (1H, t, J 7.5 Hz, H vinylic), 7.32–7.44 (5H, m, H
aromatic). MS (EI, 70 eV): 382(M+, 8%), 313(9), 273(13),
257(2), 229(8), 204(10), 177(16), 149(18), 135(21), 109(26),
95(29), 81(92), 69(100).
(2E,6E,10E)-1-Benzyloxy-3,7,11-trimethyldodeca-2,6,10-
trien-12-ol 6. Selenium dioxide (180 mg, 1.6 mmol) was added
to a mixture of a 70% aqueous solution of tert-butyl hydroper-
oxide (7.3 g; 81 mmol) and CH2Cl2 (25 ml). The mixture was
stirred for 30 min at room temperature and a solution of
(2E,6E,10E)-1-benzyloxy-3,7,11-trimethyldodeca-2,6,10-triene
5 (8.4 g; 27 mmol) in CH2Cl2 (20 ml) was added. After 72 h at
room temperature, toluene was added and the mixture concen-
trated under reduced pressure, diluted with water and extracted
with CH2Cl2. Chromatography on a silica gel column (CH2Cl2–
diethyl ether 9 : 1 v/v) yielded 2.8 g (32%) of (2E,6E,10E)-1-
benzyloxy-3,7,11-trimethyldodeca-2,6,10-trien-12-ol 6. NMR
δH (CDCl3, 200 MHz): 1.60 (3H, s, CH3), 1.65 (6H, s, 2 × CH3),
1.92–2.17 (8H, m, H allylic), 3.97 (2H, s, H-12), 4.03 (2H, d,
J 6.5 Hz, H-1), 4.50 (2H, s, -CH2-O-CH2-Ph), 5.12 (1H, m, H
vinylic), 5.30–5.40 (2H, m, H vinylic), 7.29–7.36 (5H, m, H
aromatic). MS (EI, 70 eV): 328(M+, <1%), 310(<1), 219(3),
189(6), 161(7), 138(12), 123(21), 107(23), 95(41), 91(100),
69(35), 55(10).
(all-E)-1-Benzyloxy-3,7,11,15,19,23,27-heptamethyl-13-
phenylsulfanyloctacosa-2,6,10,14,18,22,26-heptaene 10. A solu-
tion of (2E,6E,10E,14E)-1-phenylsulfanyl-3,7,11,15-tetra-
methylhexadeca-2,6,10,14-tetraene 9 (500 mg; 1.3 mmol) and
1,4-diazabicyclo[2.2.2]octane (180 mg; 1.6 mmol) in anhydrous
THF (10 ml) was treated with n-butyllithium (1.9 mmol) under
argon at Ϫ78 ЊC. After 30 min, a solution of (2E,6E,10E)-1-
benzyloxy-3,7,11-trimethyl-12-chlorododeca-2,6,10-triene
7
(695 mg; 2 mmol) in anhydrous THF (5 ml) was added to the
red solution. The reaction mixture was allowed to warm to
room temperature. After 3 h, the reaction mixture was cooled to
Ϫ78 ЊC and the reaction quenched with methanol. Extraction
with diethyl ether followed by chromatography over silica gel
(CH2Cl2–hexane 1 : 1 v/v) yielded (all-E)-1-benzyloxy-3,7,11,
15,19,23,27-heptamethyl-13-phenylsulfanyloctacosa-2,6,10,14,
18,22,26-heptaene 10 (480 mg; 53%). NMR δH (CDCl3, 200
MHz): 1.35 (3H, s, CH3), 1.59 (15H, s, 5 × CH3), 1.64 (3H, s,
CH3-4Ј), 1.68 (3H, s, CH3), 1.80–2.12 (22H, m, H allylic), 3.95–
4.07 (3H, m, H-13 and H-1), 4.50 (2H, s, -CH2-O-CH2-Phe),
4.94–5.23 (6H, m, H vinylic), 5.40 (1H, t, J 6.5 Hz, H-2), 7.21–
7.43 (10H, m, H aromatic). MS (EI, 70 eV): 692(M+, 1%),
623(0.5), 601(0.3), 583(5), 475(29), 407(8), 381(33), 339(5),
271(13), 173(45), 121(47), 105(89), 69(100).
(2E,6E,10E)-1-Benzyloxy-3,7,11-trimethyl-12-chlorododeca-
2,6,10-triene 7. 4-Dimethylaminopyridine (1.1 g; 9 mmol),
toluene-p-sulfonyl chloride (2.2 g; 11 mmol) and triethylamine
(2 ml; 1.4 g; 14 mmol) were added to a solution of (2E,6E,10E)-
1-benzyloxy-3,7,11-trimethyldodeca-2,6,10-trien-12-ol 6 (3.0 g;
9 mmol) in anhydrous CH2Cl2 (20 ml). The solution was kept
under argon at room temperature for 12 h. The mixture was
then poured into water and extracted with CH2Cl2. The crude
extract was chromatographed on a silica gel column (CH2Cl2)
to give (2E,6E,10E)-1-benzyloxy-3,7,11-trimethyl-12-chloro-
dodeca-2,6,10-triene 7 (2.2 g, 69%). NMR δH (CDCl3, 500
MHz): 1.59 (3H, s, CH3-4Ј), 1.64 (3H, s, CH3-8Ј), 1.72 (3H, s,
CH3-12Ј), 1.98–2.26 (8H, m, H allylic), 4.01 (2H, s, H-12), 4.03
(2H, d, J 7.0 Hz, H-1), 4.50 (2H, s, -CH2-O-CH2-Phe), 5.12 (1H,
t, J 5.5 Hz, H-6), 5.41 (1H, t, J 7.0 Hz, H-2), 5.50 (1H, t, J 6.5
Hz, H-10), 7.25–7.36 (5H, m, H aromatic); δC (CDCl3, 125
MHz): 14.13 (C-12Ј), 15.96 (C-4Ј), 16.52 (C-8Ј), 26.32 and 26.66
(C-5 and C-9), 39.04 and 39.73 (C-4 and C-8), 52.70 (C-12),
66.50 (C-1), 71.99 (-O-CH2-Phe), 120.64 (C-6), 124.35 (C-2),
128.30–129.20 (C-aromatic), 130.67 (C-10), 131.53 (C-11),
134.50 (C-3), 142.67 (C-7). MS (EI, 70 eV): 347(M+, 1%),
329(1), 311(4), 293(3), 241(33), 239(93), 225(1), 205(20),
203(100), 171(34), 135(42), 107(44).
(all-E)-3,7,11,15,19,23,27-Heptamethyloctacosa-2,6,10,14,18,
22,26-heptaen-1-ol 11. Small portions of lithium containing 1%
sodium (115 mg; 18 mmol) were added to dry ethylamine (10.5
ml) under argon at Ϫ78 ЊC. The resulting blue solution was
allowed to warm to 0 ЊC to ensure complete dissolution of
lithium. After 1 h, the reaction mixture was cooled to Ϫ78 ЊC
and a solution of (all-E)-1-benzyloxy-3,7,11,15,19,23,27-hepta-
methyl-13-phenylsulfanyloctacosa-2,6,10,14,18,22,26-heptaene
10 (250 mg; 0.36 mmol) in anhydrous THF was added. After 2
h, the reaction was quenched with sodium benzoate at Ϫ78 ЊC
(disappearance of the blue colour) and the mixture allowed to
warm to room temperature. Methanol was added and the mix-
ture poured into water. Extraction with CH2Cl2 followed by
chromatography over silica gel (CH2Cl2) afforded (all-E)-
3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-
heptaen-1-ol 11 (146 mg, 82%). NMR δH (CDCl3, 500 MHz) in
accordance with published data in the literature:24 1.60 (18H, s,
CH3-8Ј, CH3-12Ј, CH3-16Ј, CH3-20Ј, CH3-24Ј, CH3-28Ј), 1.68
(6H, s, CH3-4Ј, CH3-28), 1.97–1.99 (10H, m, H allylic), 2.00–
2.12 (14H, m, H allylic), 4.15 (2H, d, J 6.5 Hz, H-1), 5.11
(6H, m, H vinylic, H-6, H-10, H-14, H-18, H-22, H-26), 5.32
(1H, t, J 6.5 Hz, H vinylic, H-2); δC (CDCl3, 125 MHz):
15.76 (C-8Ј, C-12Ј, C-16Ј, C-20Ј, C-24Ј), 16.04 (C-4Ј), 17.43
(C-28Ј), 25.44 (C-28), 26.10–26.50 (C-5, C-9, C-13, C-17,
C-21, C-25), 39.32–39.48 (C-4, C-8, C-12, C-16, C-20, C-24),
59.18 (C-1), 123.10–124.20 (C-2, C-6, C-10, C-14, C-18, C-22,
C-26), 131.00 (C-27), 134.65–135.20 (C-7, C-11, C-15, C-19,
C-23), 139.64 (C-3). MS (EI, 70 eV): 494(M+, 3%), 476(4),
407(3), 339(5), 271(9), 203(16), 189(18), 161(25), 135(38),
84(95), 69(100).
(2E,6E,10E,14E)-1-Phenylsulfanyl-3,7,11,15-tetramethyl-
hexadeca-2,6,10,14-tetraene 9. (1) Triethylamine (760 mg; 7.5
mmol) and, after 30 min, methanesulfonyl chloride (855 mg; 7.5
mmol) were added at room temperature under argon to a solu-
tion of (2E,6E,10E,14E)-3,7,11,15-tetramethylhexadeca-2,6,10,
14-tetraen-1-ol 8 (1.3 g; 4.5 mmol) in anhydrous CH2Cl2 (25
ml). After 16 h, the reaction was quenched with water and
extracted with CH2Cl2 (3×), dried over MgSO4 and the solvent
removed under reduced pressure. The crude extract was rapidly
chromatographed on silica gel (CH2Cl2) and immediately used
without further purification.
(2) A solution of methyllithium in THF (12.5 mmol; 1 M
solution) was added dropwise under argon to a cold solution
(0 ЊC) of thiophenol (1.1 g; 10 mmol) in anhydrous diethyl ether
(20 ml). After 1 h, the crude mixture obtained from step (1),
dissolved in 20 ml anhydrous diethyl ether, was slowly added.
The resulting mixture was allowed to warm to room temper-
ature and was stirred under argon for a further 12 h. The reac-
(all-E)-3,7,11,15,19,23,27-Heptamethyloctacosa-2,6,10,14,18,
22,26-heptaenoic acid methyl ester 12. (1) A suspension of man-
ganese dioxide (870 mg; 10 mmol) in a solution of (all-E)-
J. Chem. Soc., Perkin Trans. 1, 2001, 711–719
717

View Article Online
3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-
heptaen-1-ol 11 (100 mg; 0.2 mmol) in hexane (20 ml) was
stirred for 1 h at 0 ЊC. Filtration of the reaction mixture over
silica gel using CH2Cl2 yielded crude (all-E)-3,7,11,15,19,23,27-
163(28), 135(42), 121(72), 107(80), 95(100), 81(95), 69(88),
55(80).
Hexacyclic C35 alkanes 1 and 16. The hexacyclic alkene 15
(1.3 mg; 2.7 µmol) dissolved in ethyl acetate was stirred with
platinum dioxide under a H2 atmosphere for 6 h. After purging
with argon and filtration of the reaction mixture through a
small silica gel column, the mixture was subjected to reversed
phase HPLC (Du Pont Zorbax ODS 250 × 4.6 mm, 5 µm;
acetone), yielding 900 µg (69%) of a mixture of the two isomers
separable by GC (1 : 1 ratio) but not by HPLC. NMR spectra
were measured on the mixture of two isomers 1 and 16. NMR
δH (C6D6, 500 MHz): 0.83 (3H, s, CH3), 0.84 (3H, s, CH3), 0.85
(6H, s, 2 × CH3), 0.86 (3H, s, CH3), 0.88 (3H, s, CH3), 0.93 (3H,
s, CH3), 0.90 (d, J 7.0 Hz, CH3 isomer a), 1.09 (d, J 6.5 Hz, CH3
isomer b), 1.10 (3H, d, J 6.5 Hz, CH3); isomer 1: MS (EI, 70
eV): 480(M+, 36%), 465(18), 395(12), 327(22), 299(38), 259(48),
231(80), 191(64), 163(100), 123(50), 109(62), 95(86), 81(82),
69(78), 55(72); isomer 16: MS (EI, 70 eV): 480(M+, 42%),
465(22), 395(72), 327(28), 299(20), 259(38), 231(42), 191(48),
163(56), 123(58), 109(64), 95(100), 81(98), 69(96), 55(64).
heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-al
which
was used without further purification.
(2) Crude (all-E)-3,7,11,15,19,23,27-heptamethyloctacosa-
2,6,10,14,18,22,26-heptaen-1-al was added to a suspension of
manganese dioxide (870 mg; 10 mmol) in methanol (20 ml).
Sodium cyanide (100 mg; 2 mmol) and a few drops of acetic
acid were added. After 17 h, the reaction mixture was centri-
fuged to remove the manganese dioxide and the organic phase
was concentrated, water was added and the mixture extracted
with CH2Cl2. The mixture was chromatographed over silica
gel (CH2Cl2–hexane 1 : 1 v/v) to give (all-E)-3,7,11,15,19,23,
27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenoic
acid
methyl ester 12 (84 mg; 80%) as a colourless oil. NMR
δH (CDCl3, 200 MHz): 1.48 (3H, s, CH3), 1.59 (15H, s, 5 CH3),
1.62 (3H, s, CH3), 1.79–2.09 (24H, m, H allylic), 2.18 (3H, br s,
CH3-4Ј), 3.65 (3H, s, -O-CH3), 4.92–5.24 (6H, m, H vinylic),
5.67 (1H, m, H vinylic, H-2). MS (EI, 70 eV): 522(M+, 7%),
491(3), 453(7), 385(4), 341(3), 285(4), 257(7), 203(16), 189(23),
161(32), 135(58), 121(50), 95(38), 81(75), 69(100).
Acknowledgements
We thank the Centre National de la Recherche Scientifique
and Elf Exploration Production for doctoral fellowships (A.
Charrié-Duhaut, E. Grosjean) and financial support; the
ECODEV-CNRS program for financial support; Dr N. D.
Ungur (Institute of Chemistry, Moldova Academy of Sciences,
Kishinev) for helpful discussions; E. Mastio-Motsch and P.
Wehrung, Université Louis Pasteur, Strasbourg, for mass spec-
tral analysis; R. Graff, Université Louis Pasteur, Strasbourg,
for NMR measurements.
Hexacyclic ester 13. A solution of (all-E)-3,7,11,15,19,23,27-
heptamethyloctacosa-2,6,10,14,18,22,26-heptaenoic
acid
methyl ester 12 (124 mg; 0.24 mmol; 0.015 M) in dry
1-nitropropane was slowly added (5 ml minϪ1) to a solution of
fluorosulfonic acid (12 mmol; 0.5 M) in dry 1-nitropropane at
Ϫ78 ЊC under argon. After 2 h, the reaction was quenched with
triethylamine (disappearance of the initial yellowish colour).
Extraction with CH2Cl2 followed by chromatography over silica
gel (CH2Cl2) yielded 60 mg of a crude mixture (“monoester”
fraction). Further fractionation by reversed phase HPLC (Du
Pont Zorbax ODS 250 × 9.4 mm, 5 µm; acetone–water 95 : 5
v/v) allowed isolation of 1.2 mg (1%) of the hexacyclic ester 13,
whose structure was unambiguously established by 1D- and
2D-NMR studies including homonuclear (1H–1H, COSY and
NOESY) and heteronuclear (1H–13C, HMQC and HMBC)
correlation experiments (see text and Table 1). MS (EI, 70 eV):
522(M+, 10%), 507(3), 448(4), 396(20), 327(86), 281(7), 259(49),
191(70), 177(27), 163(37), 121(61), 109(65), 95(100), 81(96),
69(98), 55(56).
References
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Hexacyclic alcohol 14. Lithium aluminium hydride in excess
was added to a solution of the ester 13 (2.5 mg; 4.8 µmol) in
anhydrous tetrahydrofuran (5 ml). The mixture was stirred and
refluxed under argon for 2 h. Extraction with CH2Cl2 (3×) fol-
lowed by thin-layer chromatography (SiO2, CH2Cl2) yielded
2 mg (85%) of the alcohol 14. NMR δH (C6D6, 500 MHz):
0.78 (3H, s, CH3), 0.80 (3H, s, CH3), 0.84 (6H, s, 2 × CH3), 0.89
(3H, s, CH3), 0.91 (3H, s, CH3), 0.93 (3H, s, CH3), 1.76 (3H, s,
CH3-4Ј), 3.73 (1H, dd, J 11.0 and 4.0 Hz, H-1), 3.81 (1H, dd,
J 11.0 and 3.0 Hz, H-1), 5.51 (1H, m, H-4). MS (EI, 70 eV):
494(M+, 12%), 479(5), 396(38), 381(12), 341(8), 327(18),
259(66), 191(38), 175(18), 163(30), 149(28), 121(56), 95(94),
81(100), 69(96).
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Hexacyclic alkene 15. Triethylamine and methanesulfonyl
chloride were added (under argon) in excess to a solution of the
hexacyclic alcohol 14 (2 mg; 4 µmol) in dry CH2Cl2. After 4 h,
the reaction mixture was poured on ice and extracted with
CH2Cl2 (3×). The mixture obtained was rapidly filtered through
silica gel (CH2Cl2). The resulting crude methanesulfonate
derivative was reduced under argon with an excess of lithium
aluminium hydride in dry THF (reflux). After 2 h, the reaction
mixture was poured on to ice and extracted with CH2Cl2. The
residue was chromatographed on silica gel (TLC, hexane) to
give 1.3 mg (67%) of the hexacyclic alkene 15. MS (EI, 70 eV):
478(M+, 5%), 464(4), 396(38), 327(15), 259(78), 191(20),
14 (a) R. M. Coates, D. A. Ley and P. L. Cavender, J. Org. Chem., 1978,
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