Mono- and dicyclic unsaturated triterpenoid hydrocarbons in ...

Mono- and dicyclic unsaturated triterpenoid hydrocarbons
in sediments from Lake Masoko (Tanzania) widely extend
the botryococcene family
Romain de Mesmay a,b , Pierre Metzger b, *, Vincent Grossi c , Sylvie Derenne b
a
Laboratoire de Microbiologie, Géochimie et Ecologie Marines, UMR CNRS 6117, Centre d’Océanologie de Marseille (OSU),
Faculté des Sciences de Luminy, case 901, 13288 Marseille cedex 09, France
b
Laboratoire de Chimie Bioorganique et Organique Physique, UMR CNRS 7618, BIOEMCO, Ecole Nationale Supérieure de Chimie de Paris,
75231 Paris cedex 05, France
c
PaléoEnvironnements et PaléobioSphère, UMR CNRS 5125, Université Lyon 1, Campus de la DOUA, Bâtiment Géode, 69622 Villeurbanne cedex, France
article info
Article history:
Received 23 July 2007
Received in revised form 19 December 2007
Accepted 4 January 2008
Available online 18 March 2008
1. Introduction
abstract
Botryococcus braunii is a green colonial microalga, previously
known as a member of Chlorophyceae but recently
reclassified in the Trebouxiophyceae (Senousy et al.,
2004). It is widely distributed in freshwater lakes, reservoirs
or ponds, and in some brackish waters and ephemeral
lakes. Isolated strains as well as wild populations are characterized
by an unusual production of oil containing
numerous hydrocarbons [see Metzger and Largeau (1999)
for a review]. According to the type of hydrocarbons produced,
three different races of B. braunii have been recog-
* Corresponding author. Tel.: +33 1 44 27 67 17; fax: +33 1 43 25 79 75.
E-mail address: pierre-metzger@enscp.fr (P. Metzger).
0146-6380/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2008.01.024
Organic Geochemistry 39 (2008) 879–893
Contents lists available at ScienceDirect
Organic Geochemistry
journal homepage: www.elsevier.com/locate/orggeochem
A mixture of C33–C37 botryococcenes and partially reduced derivatives was isolated from
ca. 32,000 year old sediment from Lake Masoko, a freshwater crater lake in the Rungwe
Range area (Tanzania). Botryococcenes and derivatives accounted for 246 lg/g dry sediment
and for >92% of the hydrocarbon fraction; 1D and 2D nuclear magnetic resonance
spectroscopy (NMR) and mass spectrometry allowed the structure of the dominant botryococcene
(43% of hydrocarbon fraction) to be established, after purification using high performance
liquid chromatography (HPLC). The compound is a novel tetraunsaturated
dicyclic C34 botryococcene and is named C34 masokocene. Overall, the structures of six
other novel botryococcenes and four partially reduced derivatives were tentatively
assigned. The structures of the new biomarkers, three dicyclic C34–C36 botryococcenes
(or masokocenes) and seven monocyclic C34–C37 analogues are discussed along with their
biosynthetic relationship. The high abundance of such polyunsaturated compounds preserved
in 32,000 year old sediment from the lake indicates an aquatic ecosystem dominated
at the time by the green alga Botryococcus braunii, as well very good preservation
of the organic matter.
Ó 2008 Elsevier Ltd. All rights reserved.
nized. Race A produces odd C 23–C 33 n-alkadienes and
trienes (Metzger et al., 1985a,1986), race B C30–C37 triterpenoid
hydrocarbons with the general formula C nH 2n-10,
called botryococcenes (Metzger et al., 1985a, 1985b), and
race L synthesizes mainly the tetraterpene trans, translycopadiene
(C40H78; Metzger and Casadevall, 1987; Zhang
et al., 2007 and references therein). The respective hydrocarbon
signatures of races A and L in sediments, crude oils
and oil shales have only been clearly identified in some rare
cases (e.g., Gatellier et al., 1993; Derenne et al., 1997; Grice
et al., 1998; Adam et al., 2006; Zhang et al., 2007), while the
contribution of race B is much more documented. This is
certainly related to the one to one correspondence between
botryococcenes and B. braunii race B. The precise structural
identification of the typical biomarkers of B. braunii race B is

880 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
not always easy to perform due to the common co-occurrence
of complex mixtures of analogues difficult to purify
using HPLC. To date, among the fifty botryococcenes tentatively
identified using gas chromatography-mass spectrometry
(GC-MS) analysis of lipids of isolated strains of B.
braunii or oils extracted from natural samples (e.g. Wake
and Hillen, 1981; Metzger et al., 1985a,b, 1988; Okada
et al., 1995; Metzger and Largeau, 1999 and references
therein), only twenty structures have been fully characterized
(Okada et al., 1997; Metzger and Largeau, 1999 and references
therein). Likewise, a few botryococcenes and
botryococcanes of fossil origin have been identified (Huang
and Murray, 1995; Huang et al., 1995, 1996; Grice et al.,
1998; Smittenberg et al., 2005). The reduced C34 botryococcane
was first discovered in a Sumatran crude oil (Moldowan
and Seifert, 1980) and then in Australian coastal
bitumens (McKirdy et al., 1986). Some C31 and C33 botryococcanes
were found in Maoming Oil Shale, China (Brassell
et al., 1986) and sulfurized cyclobotryococcanes in hypersaline
sediments from the Dead Sea Basin, Israel (Grice et al.,
1998); both cyclic and acyclic botryococcenes, together
with partially reduced counterparts, were discovered in
sediments from crater lakes in Kenya (Huang et al., 1995,
1996, 1999; Huang and Murray, 1995) and China (Fuhrmann
et al., 2003) and several C34 botryococcenes were
present in large amounts in recent sediments from a crater
lake in Galápagos (Zhang et al., 2007).
The C 30 botryococcene (m; letters in bold refer to the
structures in the Appendix), the precursor of all botryococcenes
and derivatives, is synthesized in the chloroplast
via the non-mevalonate pathway (Sato et al., 2003; Okada
et al., 2004). It arises from condensation of two farnesyl
units via presqualene pyrophosphate, which is also a precursor
for squalene. From this intermediate, rearrangement
of the cyclopropyl cation (path a, Fig. 1) or ring
opening of the cyclopropyl ring (path b, Fig. 1) and subsequent
reaction with NADPH, lead to the formation of squalene
or C 30 botryococcene, respectively (Huang and
Poulter, 1989; Okada et al., 2004). Higher homologues of
botryococcene and squalene are derived from their respective
C30 precursors by successive methylation with S-adenosylmethionine,
as indicated by bold arrows in Fig. 1 for
C30 botryococcene (Metzger et al., 1987; Achitouv et al.,
2004). Afterwards, botryococcenes are excreted to the outer
walls, forming a dense oily matrix (Metzger et al., 1987),
whose structural element is a chemically resistant biopolymer,
the algaenan derived from a polyacetal network bearing
polymethylsqualene derivatives (Metzger et al., 2007).
Cyclization of the terminal moieties of botryococcenes may
also occur (Metzger et al., 1985b; David et al., 1988; Huang
et al., 1988; Huang and Poulter, 1988; Huang et al., 1995,
1996). Accordingly, the same central pattern exhibiting 1)
the quaternary C-10 bearing a methyl and the D 26 exomethylene
unsaturation, 2) the trans D 11 double bond
and 3) the methyl group at C-13, is present in all botryococcenes.
Moreover, while C31–C34 methylated squalenes
are implicated via their diol derivatives in the synthesis
of the structural element of the outer walls, the biological
role of botryococcenes is not obvious. However, as their
accumulation (accounting generally for 30–40% of the dry
wt; Metzger et Largeau, 1999) ‘‘results in the colonies predominating
in the upper regions of the water column,
where little incident radiation is lost to the water mass”
(Wake and Hillen, 1980), they may allow occupation of
some ecological niches suitable for the algal growth.
In the course of our study of the lipid biomarkers from a
30 metre sediment core from Lake Masoko, southern Tanzania,
preliminary analysis of a few samples revealed the
presence of a wide variety of botryococcenes, most of unknown
structure. We report here the structural identification
of three dicyclic C 34–C 36 and seven monocyclic C 34–C 37
botryococcenes and partially reduced counterparts isolated
from a ca. 32,000 year old sediment layer. The possible
influence of some physicochemical and environmental
factors on the distribution and abundance of these algal
biomarkers in Lake Masoko is currently being studied on
a core section corresponding to the last 32,000 years (de
Mesmay et al., 2007b).
OP P
b
a
path a path b
+ +
3 7
26
10
11
16 20
Squalene C 30 Botryococcene
Fig. 1. Simplified scheme of squalene and C 30 botryococcene biosynthesis from presqualene diphosphate (adapted from Huang and Poulter, 1989) and sites
of methylation (bold arrows).

2. Experimental
2.1. Site description
Lake Masoko is an oligotrophic Maar lake (9°20 0 S–
33°45 0 E, 800 m above sea level, 36.5 m deep) in the Rungwe
Range. Owing to its small catchment area and absence
of river input, it is strongly sensitive to climate change. It
provides one of the most continuous Late Quaternary
lacustrine sedimentary records from Africa over the last
45,000 yr (Williamson et al., 1999; Gibert et al., 2002; Garcin
et al., 2006a,b, 2007). Multidisciplinary studies have
detailed the hydrology (Bergonzini et al., 2001; Delalande
et al., 2005), sedimentary assemblage of charcoal (Thevenon
et al., 2003), pollen (Vincens et al., 2003) and diatoms
(Barker et al., 2003). Earlier studies of pigments and ligninderived
phenol assemblages (Merdaci, 1998) outlined the
remarkable preservation of organic matter in the sediments.
More information on site description is given elsewhere
(de Mesmay et al., 2007a and references therein).
2.2. Sample description
Three cores (30 m long in total, M96-A, -B and -C) were
collected from the central area of Lake Masoko using a sedidrill-Mazier
cable corer in order to combine the most continuous
cored section and to construct a detailed composite
lithostratigraphic record from the last 45,000 yr BP (Garcin
et al., 2006a). Cores were stored at 4 °C until analysis. Samples
representative of different climatic and environmental
periods were investigated for bulk and molecular content.
We present here results from the sample containing the
highest amount of botryococcenes (1856–1871 cm interval;
31.7–32.1 14 C cal. kyr BP). It corresponds to an organic-rich
silty mud deposited during the last glacial period
between ca. 34,000 and 28,000 cal. yr BP. It consists primarily
of silty organic mud, enriched in detrital titanomagnetite
originating from the surrounding catchment soil and
littoral area (Williamson et al., 1999). The occurrence of
numerous mm to cm scale turbidites containing few organic
macrorests (plant tissue, charcoal fragments) and abundant
herbaceous pollen point to relatively arid, low lakelevel
conditions (Garcin et al., 2006a). The total organic carbon
(TOC; 6% dry wt) and fossil pigment contents of this
interval suggest a relatively oligotrophic environment and/
or oxic depositional environment (Merdaci, 1998). A C/N
ratio of 21.9 suggests a mixture of terrestrial and aquatic
sources (Tyson, 1995; Huang et al., 1999). Assuming that
all the iron occurs as pyrite, the sulfur and iron contents
(total S 0.2% dry wt; Fe ca. 0.06%) may suggest the presence
of some organosulfur compounds in the sample. However,
it must be noted that molecular sulfur (S8) is present in TIC
traces of sediment extracts. Very similar values for sulfur
and iron contents were obtained for two more recent samples
(13,000 and 500 yr).
2.3. Lipid extraction and separation
Fresh sediment (14 g) was ultrasonically extracted
(10 min.) with CH3OH (100 mL 2), CH3OH/CH2Cl2 (1:1, v/
v, 100 mL 2) and CH 2Cl 2 (100 mL 4). Extracts were com-
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 881
bined and evaporated under reduced pressure. The remaining
water was removed via azeotropic evaporation with
CH 3OH. The dry extract (110 mg) was chromatographed
over silica gel (Merck silica gel 60). Hydrocarbons, aromatic
compounds, alcohols and polar compounds were eluted
with n-hexane, n-hexane/CH2Cl2 (4:1, v/v), CH2Cl2/diethyl
ether (9:1, v/v) and CH 3OH/CH 2Cl 2 (1:1, v/v), respectively.
2.4. Hydrogenation
Hydrogenation of an aliquot of the hydrocarbon fraction
was carried out (18 h) in heptane under H2 (20 atm) in the
presence of a catalyst (Rh/C 5%). The reaction mixture was
centrifuged; the supernatant was collected and concentrated
under a stream of N2 and analyzed using GC-MS.
2.5. Ozonolysis
An aliquot of the hydrocarbon fraction in 1 ml CS2 was
reacted with ozone at 78 °C until the characteristic blue
colour of O3 persisted. Excess O3 was eliminated by bubbling
N 2 through the cold solution. The ozonides were reduced
by addition of 5 mg triphenylphosphine and the
reaction mixture was allowed to warm to room temperature.
Solvent was evaporated under reduced pressure and
the compounds were analysed using GC-MS.
2.6. GC and GC-MS
GC was carried out with an Agilent 6890 gas chromatograph
equipped with a flame ionisation detector (FID) and a
RTX-5 Sil MS column (30 m 0.25 mm; 0.5 lm film thickness).
The oven temperature was programmed from 60 to
130 °C at20°C/min and to 300 °C (35 min) at 4 °C/min. He
was the carrier gas (constant flow, 24.2 ml/min). Hydrocarbons
were quantified via external calibration using squalane
as standard. Hydrocarbons and their derivatives were
identified using GC-MS with an Agilent 6890 N chromatograph
equipped with a splitless injector and coupled to an
Agilent 5973 mass spectrometer operating at an ionization
energy of 70 eV and a range of m/z 40–700. The chromatograph
was equipped with the same capillary column as described
for GC and the same temperature programme was
used. The constant carrier gas flow (He) was 1 mL/min.
2.7. Purification of C34 botryococcene and spectroscopic
analysis
The hydrocarbon fraction was further purified using
isocratic high performance liquid chromatography (HPLC)
with a Waters 600E instrument fitted with a differential
Waters 2414 refractometer thermostated at 30 °C. The
mixture was fractionated into three sub-fractions using a
5 lm XTerra TM MS C18 column (4.6 250 mm) and repeated
injection (20 ll, 5% in CHCl 3) and elution with CH 3CN at a
flow rate of 3 ml/min. The first sub-fraction afforded C34
dicyclobotryococcene c (t R 24 min.). High resolution electron
ionization mass spectrometry (HR-EIMS, 70 eV) analysis
was performed with a Jeol MS 700 via direct inlet.
NMR spectra were recorded with a Bruker Avance 400
spectrometer operating at 400.1 and 100.6 MHz for 1 H
and 13 C, respectively. Spectra were recorded in CDCl3.

882 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
Chemical shifts were referenced relative to the residual
proton signal (7.24 ppm) or the central line of the 13 C multiplet
(77.0 ppm) of CDCl 3. Assignment of individual resonances
was achieved using a combination of 1D and 2D
( 1 H- 1 H and 1 H- 13 C) experiments. Multiplicity of each 13 C
nucleus was determined using DEPT (enhanced polarisation
transfer) spectra.
3. Results and discussion
3.1. Characterization of botryococcenes and reduced
analogues
3.1.1. Composition of hydrocarbon fraction
Examination of the hydrocarbon fraction isolated from
the 1856–1871 cm depth sediment sample using GC-MS
revealed a dominance (93% of total hydrocarbons; Table
1; Fig. 2A) of botryococcenes (CnH2n-10) ranging from C33
to C37 and of partially reduced counterparts (CnH2n-8, CnH2n-6 and CnH2n-2), together with a minor series of C17–C35
n-alkanes. The botryococcenes and related compounds
accounted for more than 246 lg/g dry sediment and
4 mg/g TOC (total organic carbon). The major compound
was an unknown C34 component (c) accounting for ca.
43% of the total hydrocarbons, each of the other components
accounting for 87%).
3.1.2. Characterization of C34 masokocene c
The high resolution EI spectrum of the dominant botryococcene
c displays M +. at m/z 466.4530, consistent with
aC34H58 compound (calcd. 466.4522, D +0.8 mmu). The
Table 1
Composition, quantification and ozonolysis products of hydrocarbons from Lake Masoko sediment
Hydrocarbon lg/g d.s. a
Formula e
MW Structure
low resolution EI mass spectrum is characterised by a major
ion at m/z 177, likely corresponding to a fragmentation
at the central quaternary carbon (Fig. 2B). Catalytic hydrogenation
led to the formation of four botryococcanes
C 34H 66 (H3, Fig. 3A) occurring in a 3/23/28/4 ratio, and
which exhibited identical mass and fragment ions
(Fig. 3C). This indicated the presence of two rings in c
and suggested that stereochemical isomerisation occurred
during hydrogenation. The mass spectrum of H3 shows
fragments at m/z 444/445 corresponding to the loss of
the C-10 ethyl, at m/z 236/237 corresponding to loss of
the long alkyl chain at C-10 and at m/z 292/293 corresponding
to loss of the short alkyl chain at C-10.
Detailed inspection of the 1 H, 13 C, DEPT, COSY (correlation
spectroscopy), HMQC (heteronuclear multiple quantum
correlation) and HMBC (heteronuclear multiple bond
correlation) NMR spectra of c (Table 2) and comparison
with NMR data from other botryococcenes indicated the
presence of a terminal vinyl [dH 5.75 (H-26), 4.92 (H-
27b), 4.90 (H-27a); d C 147.3 (C-26), 110.9 (C-27)] bound
to the quaternary carbon C-10 (dC 41.8). The HMBC experiment
further established that the latter quaternary carbon
is correlated with an olefinic proton (H-11, dH 5.26) of trans
disubstituted unsaturation [J 11,12 = 16.0 Hz; H-12: d H 5.12].
Moreover, long range correlations showed that a methine
carbon (CH-13, d H 2.03, d C 37.1) bearing a methyl (Me-
28, dH 0.94, dC 21.2) is connected to this olefin. This C-10
to C-13 substructure I (with methyls at C-10 and C-13
and a vinyl at C-10, Fig. 4), is characteristic for all botryococcene
structures (Metzger et al., 1985b); it arises from
the 1’-3 condensation of two farnesyl units (Huang and
Poulter, 1989). The downfield region of the 1 H NMR spec-
lg/g TOC b
Relative% c
Ozonolysis d
C33H56 452 0.5 8.7 0.2
C34H58 466 0.3 4.4 0.1
C34H58 466 trace
C34H66 474 a 0.8 13 0.3 O3 + O1
C34H62 470 b 1.1 17 0.4 O11 + O1
C34H58 466 c 114 1878 43.2 O11 + O9
C34H58 466 d 12 191 4.4 O11 + O9
C34H58 466 0.8 13 0.3
C34H58 466 0.5 8.7 0.2
C35H60 480 e 26 422 9.7 O11 + O10
C36H62 494 f1, f2 5.8 96 2.2 O11 + O6
C36H64 496 g1, g2 36 569 13.5 O11 + O4
C36H62 494 0.5 8.7 0.2
C37H64 508 h 10.6 174 4.0 O11 + O7
C37H64 508 7.9 130 3.0 O11 + O7
C37H66 510 i 18 291 6.7 O11 + O5
C37H64 +C37H66 508/510 3.2 52 1.2
C37H64 508 1.1 17 0.4
C36H62 494 j 6.6 109 2.5 O11 + ?
C37H66 510 0.3 4.4 0.1
Other botryococcenes and n-alkanes 20 339 7.4
R hydrocarbons 266 4346 100.0
a Dry sediment.
b Total organic carbon.
c Composition determined using GC.
d Compounds from ozonolysis.
e Compounds listed in elution order.

elative intensity (%)
100
50
0
relative intensity
n-C 27
40 42 44
69
33 34
a b
trum also shows a broad singlet for two protons (d H 5.03)
of two trisubstituted double bonds [dC 138.3 (quaternary
carbons C-6 and C-17) and 132.7 (tertiary carbons C-24
and C-29)] belonging to two identical substructures II
(Fig. 4). The HMBC correlations (Table 2 and Fig. 4) established
that II contain cyclohexenyl rings bearing three
methyls, of which two are geminal [for example, in the left
hand moiety of the molecule: Me-1 (dH 0.78, dC 23.4) and
Me-23 (d H 0.95, d C 29.5)] at C-2 (d C 34.5) and a third at
C-3 [Me-31 (dH 0.86, dC 16.2)]. Such a trimethylated cyclohexenyl
moiety was previously found in cyclobotryococcene
k, isolated from a strain of B. braunii from the Ivory
Coast and grown under laboratory conditions (David
et al., 1988). The relative stereochemistry of the methyls
in c was deduced from the correlations observed in the
NOESY (nuclear Overhauser enhancement spectroscopy)
NMR spectra (Fig. 4), which suggested that Me-31 is pseudo-axial.
The connection of each substructure II with the
rest of the molecule was drawn from the HMBC experiment.
So, in the ‘‘left” moiety of the molecule, cross peaks
of H-7 and protons of Me-32 at C-7, to C-6 indicated the
connection C-6/C-7. Furthermore, the two bond HMBC correlations
H-7/C-8, H-8/C-9, and H-9/C-10 and the long
range correlations H-7/C-9 and H-25/C-9 allowed us to
connect the ‘‘left” structural moiety of c to the quaternary
95
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 883
123
c
149
n-C 28
d
177
178
34 34
203
n-C 29
231
e
259
g1
f2
f1
36
177 285
123
-2H
-2H
231
h
i
j
c
259 -2H
-2H 285
n-C 31
retention time (min.)
50 100 150 200 250 300 350 400 450
g2
285
37 37*
37
37
M+. -CH 3
314 341 355 396 423
123
M +.
451 466
Fig. 2. (A) Total ion chromatogram of hydrocarbon fraction from 1856–1871 cm depth sediment interval from Lake Masoko [C xx are n-alkanes with xx carbon
atoms; numbers indicate the carbon number of botryococcenes and reduced counterparts whose structures have not been established in this work, cf. Table
1, * = coelution between one C37 botryococcene (C37H64) and one partially reduced counterpart (C37H66)], (B) EI mass spectrum of C34 masokocene c.
m/z
carbon C-10. In the right hand part of the molecule, 1 H
NMR data and HMBC correlations showed that the cylohexenyl
ring is bound at C-17 to a tertiary methine carbon
C-16 which bears the methyl group Me-33. Finally, crosspeaks
of H-15 to C-14 and C-13 allowed connection of
the second cyclohexenyl moiety II. Major fragment ions
at m/z 177 (base peak) and 123 in the mass spectrum of
c support this structural pattern (Fig. 2B).
Ozonolysis has proved to be a powerful tool for the structural
determination of botryococcenes and derivatives
(Huang et al., 1996). Application to an aliquot of the total
hydrocarbons resulted in two predominating compounds:
O9 (C 16H 28O 3)andO11 (C 17H 28O 4) resulting from oxidation
of masokocene c (Table 1; Figs. 5 and 6A). Mass fragmentation
patterns of O9 and O11 (Fig. 6E and G) were helpful for the
characterization of other dicyclic triterpenoid hydrocarbons
(see below). The generic name masokocene is proposed for
all these dicyclobotryococcenes. HPLC purification of c afforded
a coeluting minor botryococcene d (ca. 10% of the mixture)
which exhibited a mass spectrum identical to that of
c. It is very likely that d is a stereoisomer of c.
3.1.3. C 35 and C 36 masokocenes e and j
GC-MS analysis of the intact and the hydrogenated
hydrocarbon fractions clearly indicated that compounds e

884 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
relative intensity
%
100
50
0
%
100
50
0
%
100
50
0
relative intensity
43
33
71
C 28
125
85 111
H1
C 29
H2
42 44 46 48 retention time (min.)
125
141 167
236/237
211
and j are higher homologues of the C34 masokocene c (Figs.
2 and 7). The EI mass spectrum of e is consistent with a
C35H60 botryococcene (M +. at m/z 480; Fig. 7A). It exhibits
prominent fragments at m/z 123 and 177, suggesting the
236
294/295
294
446/447
H2
337 386 431 446
M +. -C2H5 50 100 150 200 250 300 350 400 450 m/z
H3
H3
%
100
50
0
H3
H3
C 31
H4
H4
H5
H6
H6
71
85
111
43
236
141
167
197
253
322
295 349 391 435 459 474
69
83
125
139
43
167
236
306
195 277
251 335363389 429 459
125
322/323
474/475
-C2H5 (on C-21)
306/307 277
458/459
139
125
236/237
125
459
H4
236/237
H5
50 100 150 200 250 300 350
M
400 450 m/z 50 100 150 200 250 300 350 400 450 m/z
+. %
100
50
-C2H5 0
M +. -C2H5 43
71
85
111
236
141
167
197
336
267295 363 419 464 488
125
125
236/237
336/337
488/489
H6
69
111
83 139
125
125
153
167
236/237
320/321
472/473
H7
153
473
50 100 150 200 250 300 350 400 450 m/z
43
50 100 150
195 236
251
200 250
M
293
473
320 363 388415
300 350 400 450 m/z
+. M -C2H5 +. %
100
50
-C2H5 0
43
69
83
111
125
139 167
125
209
236
236/237
H7
-CH3 (on C-21)
292/293 277
444/445
H3
292
277
321349 379
M
445
429
+. -C2H5 50 100 150 200 250 300 350 400 450 m/z
Fig. 3. (A) Total ion chromatogram of hydrogenated hydrocarbon fraction from 1856–1871 cm depth sediment interval from Lake Masoko (C xx are
n-alkanes with xx carbon atoms). (B–G) EI mass spectra of botryococcanes H2-H7.
presence of a trimethyl substituted cyclohexenyl ring in
the left hand part of the molecule, as in c. The presence
of two rings in e is supported by the mass spectra of the
several diastereomers of C35 botryococcanes H5 formed
125

Table 2
1 H [400 MHz; dH (J, Hz)] and 13 C [100 MHz; d C] NMR data of C 34
masokocene c in CDCl 3 at 300 K
Position
1
H(dH)
13
C(dC) HMBC (H ? C)
1, 30 0.78 (6H, s) 23.4 2, 3, 23, 24 a
2, 21 34.5
3, 20 1.41 (2H, m) 38.5 1, 2, 4, 5, 23, 31 a
4, 19 1.26-1.42 (4H, m) 28.1 6 a
5, 18 1.86 (H-5a, H-18a, m) 24.9 3, 4, 6, 24 a
1.78 (H-5b, H-18b, m) 3, 4, 6, 24 a
6, 17 138.3
7 1.90 (1H, m) 41.4 6, 8, 9, 32
8 1.12-1.23 (2H, m) 29.6 7, 9, 10
9 1.24 (2H, m) 39.0 8, 10, 11, 25, 26
10 41.8
11 5.26 (1H, d, 16.0) 135.8 9, 10, 12, 13, 25, 26
12 5.12 (1H, dd, 16.0, 7.7) 133.9 10, 11, 13, 26, 28
13 2.03 (1H, m) 37.1 11, 12, 14, 28
14 1.10-1.25 (2H, m) 35.1 12, 13, 15, 16, 28, 33
15 1.15-1.30 (2H, m) 32.7 13, 14, 16, 33
16 1.94 (1H, m) 40.9 14, 15, 17, 18, 29, 33
22, 23 0.95 (6H, s) 29.5 1, 2, 3, 24 a
24, 29 5.03 (2H, s) 132.7 1, 2, 3, 5, 7, 23 a
25 1.01 (3H, s) 23.8 9, 10, 11, 12, 26, 27
26 5.75 (1H, dd, 17.3, 10.7) 147.3 9, 10, 11, 25
27 4.90 (H-27a, dd, 17.3, 1.5) 110.9 10, 26
4.92 (H-27b, dd, 10.7, 1.5) 10, 26
28 0.94 (3H, d, 6.4) 21.2 11, 12, 13, 14, 15
31, 34 0.86 (6H, d, 6.4) 16.2 2, 3, 4 a
32 0.94 (3H, d, 6.4) 19.8 b
5, 6, 7, 8
33 0.94 (3H, d, 6.4) 19.9 b
15, 16, 17, 18
a
Only correlations concerning proton on the left hand part of molecule
given.
b
Signals interchangeable.
25
10
27
28
13
31
substructure I substructure II
2
6
24
HMBC
1
3
5
23
24
7
32
by stereochemical isomerization during hydrogenation
(Fig. 3A and E). A fragment ion at m/z 458/459 (due to
the loss of the ethyl at C-10) supports the presence of
two rings in H5. Moreover, fragment ions at m/z 236/237
and 306/307, originating from C-10/C-11 and C-9/C-10
cleavage, respectively, indicate the presence of one ring
in each side of H5. The ion at m/z 306/307 in the H5 spectrum
indicates that the additional carbon is situated on the
right hand side of the molecule compared to H3. The position
of this additional carbon in e was deduced from its
ozonolysis products. Comparison of the mass spectrum of
O10 (C17H30O3) with that of O9 (C16H28O3; Fig. 6E and F)
indeed indicates the presence of an ethyl in O10, (M +. -
C2H5; fragment at m/z 253). McLafferty rearrangement
leading to ion at m/z 86 indicates that the ethyl group is
likely carried by carbon C-21. In the mass spectrum of e
(Fig. 7A), the loss of an ethyl is also suggested by an ion
at m/z 451. Thus, on the basis of all these mass spectral
data we propose structure e for the C35 masokocene.
The EI spectrum of the C 36 botryococcene j (C 36H 62,M +. at
m/z 494, Fig. 7B) exhibits an intense ion at m/z 465 due to
the loss of an ethyl. The hydrogenated fraction contains several
C36 botryococcanes (C36H70, H7) with identical mass
spectra (e.g. Fig. 3A and G). Diagnostic ions at m/z 236/237
and 125 suggest the presence of a trimethylated cyclohexyl
in the left moiety of the molecule as in H3 and H5. By comparison
with H5, the ion at m/z 320/321 indicates that the
additional carbon is located in the right part of the molecule.
An intense peak at m/z 153 (57%) is consistent with a cyclohexane
ring substituted with one more carbon in H7 than in
H5. The mass spectrum of j exhibits a M + -Et fragment (m/z
23 Me
1
Me
Me
31
NOE
Fig. 4. Selected HMBC and NOE correlations in NMR analysis of C 34 masokocene c.
26
10
11
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 885
12
c
16
29
17
21
O 3
Fig. 5. Ozonolysis products of C 34 masokocene c.
H
O
O
12
H
16
O
+
O
H
O9
17 18
24
O11
H
O
21 29
26
O
2
10
24 11
6
O
Me
H
H

886 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
relative intensity
%
100
50
0
%
100
50
0
%
100
50
0
43
57
69
86
A
relative intensity
O1
O4
O
H
113
109 123
O4 O5
O2 O3
40 60 80 100 120 140 160 180 200 220 240 m/z
43
55 71
83
95
100
113
O7
127
141
465) twice more intense than the similar ion in the spectrum
of e (35% instead of 16%), suggesting that more than
one ethyl group occurs in j. Unfortunately, GC-MS analysis
of the ozonolysis products did not allow identification of
the compound resulting from the cleavage of the right half
part of the molecule, likely due to coelution with triphenyl
+H
O6 O7 O8
18 20 22 24 26 time (min.)
B C
113
226
141
141
151 168 179 197
O
H
155
161
240
O
57
226
236
M +.
254
D E
184
225
207
197 240 M
250
+. -H2O 40 60 80 100 120 140 160 180 200 220 240 260 m/z
55
O10
H
O
+H
113
+H
113
O
184
155
225
O
+H 71
100
F G
43
69
40 60 80 100 120 140 160 180 200 220 240 260 m/z
85
95 141
123
113
151
169
267
198 226
253 M
282
+.
+H +H
+H
86
254 198
141 141 254
156
M +. -CH3 M +. -C2H5 O
+H
169
127
156
+H
86
+H
21
86
O
H
43
%
100
50
0
O9
43
55
55
71
83
O10
100
O11
O5
M +.
109
123
179
240
137 155168 189 207 225 250 268
113
40 60 80 100 120 140 160 180 200 220 240 260 280
43
83
127
95
109
H
O9
H
O
+H
156
155
141
167
M
253
184 212 240
+. M -CH3 250
+. -H2O M +.
268
40 60 80 100 120 140 160 180 200 220 240 260 m/z
43
55
71
72
84
95
113
109 127
O11
O
phosphine, the reagent used for the reduction of the polyozonides.
However, taken with these mass spectral data,
the strong structural relationship between the C34-C35 masokocenes
and the C 36 analogue allows us to assign compound
j as a C36 dicyclic botryococcene with two geminal
ethyls on carbon C-21.
O
113
240
113
O
155
155
225
156
+H
O
71
+H
100
+H
72
O
H
+H +H
240 184
141 127
+H
240
72
H
184
+H +H
+H
H
O
141 197
72
137
155
169
184
222
M
281
250
40 60 80 100 120 140 160 180 200 220 240 260 m/z
+. -CH3 296
M+.
Fig. 6. (A) Total ion chromatogram of ozonised hydrocarbon fraction from Lake Masoko sediment at 1856–1871 cm depth. (B-G) EI mass spectra of
compounds O4, O5, O7, O9, O10 and O11.
%
100
50
0
%
100
50
0
O
127
155
H
197
-H
141
O
84
43

elative intensity
%
100
50
0
%
100
50
%
100
50
0
0
43
69
109
95 123
149
177
50 100 150 200 250 300 350 400 450 m/z
41
41
55
A
C
137
95
109
123
149
177
123
203
231
245 285 465
451
M
299 480
328355 423
+.
M +. -CH3 M +. -C2H5 123
3.1.4. C34 partially reduced botryococcenes
The occurrence of a C 34 octahydrobotryococcene a (Table
1; Fig. 2A) is revealed by its mass spectrum (M +. m/z
474). The corresponding C 34 botryococcane (H1) was found
in the hydrogenated sample. The exact molecular structure
of H1 and its acyclic nature is given by comparison of its
mass spectrum and retention time with an authentic standard
prepared by catalytic hydrogenation of an acyclic C 34
botryococcene (Metzger et al., 1985b). Combination of the
ozonolysis fragments O1 and O3, whose mass spectra are
published (Huang et al., 1996), allows us to establish the
position of the double bonds in a at C-11/C-12 and C-26/
C-27. This compound is 1,6,17,21-octahydrobotryococcene
previously isolated from Sacred Lake, Kenya (Huang and
177
203
231
245 285 479
M
315 369 411 452
494
+.
M +. -CH3 50 100 150 200 250 300 350 400 450 m/z
E -2H
F
69
95
123
149
177
123
177
177 299
-2H -2H
-2H
231
203
231
245 285
-2H
231
-2H -2H
285
231
-2H
285
-2H
50 100 150 200 250 300 350 400 450 m/z
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 887
259
-2H
285
h
e
451
f1
137
465
M +.
M +. -CH3 493
329 369 397 427 465
508
%
100
50
0
%
100
50
0
%
100
50
0
B
43
D
41
55
55
81
123
95 123
163
151
149
177
123
203
177
123
203
231
245 285 313 343 381 435
231
177 313
-2H -2H
259 285 317344 385 426453
M
465
+. -C2H5 M
494
+.
50 100 150 200 250 300 350 400 450 m/z
177
231
g1
481 M
496
+.
M +. -CH3 50 100 150 200 250 300 350 400 450 m/z
69
109
123
149
177
203
123
231
Murray, 1995) and from upper sediments of lake Masoko
(de Mesmay et al., 2007a).
Although we failed to detect any monocyclic C34 botryococcene
in the extract, two diastereomers of a C 34 monocyclic
botryococcane were identified in the hydrogenated
sample (compounds H2; Fig. 3A and C). The structure was
assigned on the basis of coinjection and comparison with
previous mass spectral data (David et al., 1988; Grice
et al., 1998). These hydrogenated compounds might be derived
from a partially reduced botryococcene (b) in the original
fraction (Fig. 2A) and whose mass spectrum is very
similar to those of two partially reduced botryococcene isomers,
C34H62 of undetermined structures, detected in a Chinese
lacustrine sediment core (Fuhrmann et al., 2003). In
231
-2H
177
-2H
231
259
j
-2H
465
285
-2H
-2H -2H
285
-2H
151
43
259
287 331
M
510
385 427 467
50 100 150 200 250 300 350 400 450 m/z
+.
M
495
+. -CH3 Fig. 7. (A and B) EI mass spectra of C 35 and C 36 masokocenes e and j. (C and D) C 36 monocyclic botryococcene f1 and its partially reduced counterpart g1.
(E and F) C 37 monocyclic botryococcene h and its partially reduced counterpart i.
i
467

888 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
the present case of b, the molecular ion at m/z 470 is consistent
with a tetrahydrobotryococcene (C34H62). Fragment
ions at m/z 123, 177 and 231 suggest the presence of a
trimethylated cyclohexenyl ring in the left half part of the
molecule. In addition, a diagnostic ion at m/z 259 resulting
from the cleavage of the C-12/C-13 bond indicated that
two additional unsaturations were present at C-11 and
C-26. The ozonides O11 and more especially O1, although
not specific for the degradation of b (Table 1), also support
structure b as an unprecedented tetrahydrogenated
derivative of k (David et al., 1988), with the C-17/C-29
and C-21/C-22 double bonds being hydrogenated.
3.1.5. C36 and C37 monocyclic botryococcenes and partially
reduced counterparts
GC-MS analysis of the hydrogenated hydrocarbon fraction
revealed, in addition to the aforementioned botryococcanes,
the presence of two C 36 (H4) and two C 37 (H6)
monocyclic botryococcanes (Fig. 3A). Each of these two couples
exhibited strictly identical mass spectra, with diagnostic
ions indicative of the presence of a ring in the left part of
the molecule (ions at m/z 236/237, Fig. 3D and F). The mass
spectra of H4 and H6 show similarities to that of H2. Two
(respectively three) additional carbons are situated on the
right hand moiety of the molecules (ions at m/z 322/323
for H4, Fig. 3D and at m/z 336/337 for H6, Fig. 3F).
C36 botryococcanes H4 probably result from the catalytic
hydrogenation of two partially coeluting C 36 botryococcenes
(M +. at m/z 494, f1 and f2) with identical mass spectra
(Figs. 2A and 7C) and two dihydrogenated counterparts (g1
and g2), also partially coeluting (Fig. 2A) with identical
spectra (M +. at m/z 496; Fig. 7C). Fragments at m/z 123,
177, 231 and 285 in f1, f2, g1 and g2 confirmed the occurrence
of a trimethyl cyclohexenyl ring in the left hand moeity
and of two unsaturations at C-11 and C-26. The
structures of the right hand parts of the compounds were
deduced from the identification in the ozonolysis products
of the di- and tri-oxygenated compounds O4 and O6, respectively
(Table 1). The McLafferty fragment at m/z 86 in their
spectra (e.g. Fig. 6B for O4) suggests the presence in the
structure of an ethyl ketone exhibiting a methyl group in
the a position. Consequently, an ethyl group would be present
at ‘‘C-21” (numbered according to botryococcene structures)
in f1, f2, g1 and g2. Moreover, the molecular formulae
of O4 and O6 (C16H30O2 and C16H26O3, respectively) established
that a C2 moiety was lost from the right hand part
during ozonolysis, suggesting the presence of a CH3CH=C
pattern in the C36 botryococcenes f1 and f2, and the dihydro
derivatives g1 and g2. Finally, the McLafferty fragment at m/
z 170 in the spectrum of O6 indicates the presence of a ketone
at ‘‘C-17” (data not shown), and the two discrete ions
at m/z 113 and 141 in the spectrum of O4 show the presence
of a methyl at ‘‘C-17” (Fig. 6B).
These results allow us to tentatively assign f1 and f2 as
monocylic C 36 botryococcenes and g1 and g2 as their dihydro
derivatives. The occurrence of two pairs of isomers with
identical mass spectra is probably due to the existence of
stereoisomers with respect to the C-21/C-22 double bond
stereochemistry. Based on semi-empirical topological
methods for the prediction of the chromatographic retention
of alkene isomers (Heinzen et al., 1999; Junkes et al.,
2002), it can be assumed that the stereochemistry of the
C-21 double bond is E in f1 and g1 and Z in f2 and g2.
Similarly, botryococcanes H6 were probably produced by
the catalytic hydrogenation of C37 botryococcenes (C37H64,
M +. at m/z 508) and dihydro counterparts (C 37H 66, M +. at
m/z 510) (Table 1 and Fig. 3). Among the ozonolysis products,
only the di- and tri-oxygenated O5 and O7, respectively
(Table 1) could be related to C37 hydrocarbon
precursors. MS comparisons showed that O5 (C 17H 32O 2)
and O7 (C17H28O3) were higher homologues of O4 and O6,
respectively. Moreover, a base peak at m/z 43 in the spectrum
of O7 (Fig. 6D) suggested that the carbon skeleton
probably has a terminal isopropyl group. The occurrence
of an isopropyl group in O5, justasinh and i, was also supported
by the presence in the spectra of significant ions at
m/z [(M-43) + ; Figs. 6C and 7D, E]. The presence of a
CH3CH=C pattern at C-21 in h and i is supported by the presence
of a ketone a to the isopropyl group both in O5 and O7
and by biogenetic considerations (see below). The combination
of O7 with O9,allowsustoproposeh as a higher homologue
of f1 and f2 with one more carbon. The spectra of O5
and O7 exhibit some identical fragments (m/z 100, 113, 155,
225, 240), but an ion at m/z 184 in that of O7, due to McLafferty
rearrangement, and two discrete ions at m/z 113 and
155 in that of O5, indicate that O5 and O7 differ by way
of the presence of a methyl group and a ketone, respectively.
These data suggest that i and h only differ in the presence
of an exomethylene and a methyl group at C-17,
respectively. In contrast to the C36 homologues, h (respectively
i) does not appear as a mixture of two compounds
in the GC chromatogram. Nevertheless, h (respectively i)
may occur as a mixture of stereoisomers that are not separated
under the GC conditions used.
3.2. Occurrence of masokocenes in ancient ecosystems
Dicyclobotryococcenes have been recently reported to
occur in sediments of a Norwegian fjord (Smittenberg
et al., 2005) and in freshwater wetlands of the Florida Everglades
(Gao et al., 2007), but their structures were not
unambiguously determined. On the other hand, a hypothetical
dicyclobotryococcene exhibiting a planar structure
similar to that of c had already been assumed to be the precursor
of a major dicyclobotryococcane (tentatively assigned
as H3) detected after Raney nickel desulfurisation
of a polar lipid fraction from Miocene/Pliocene immature
hypersaline sediments (Sdom Formation near the Dead
Sea; Grice et al., 1998). The authors supposed that c was
incorporated into a macromolecular matrix via sulfurisation.
The lack of c among the extractable biomarkers of
the Sdom formation was explained by its ease of sulfurisation
due to the presence of four double bonds. The strong
dominance of intact masokocene c in a ca. 32,000 year
old sediment indicates excellent conditions of preservation
in Lake Masoko sediments, even though the compound
might be sensitive to sulfurisation as well as to oxidative
or reductive attack. Although B. braunii race B is known
to produce large amounts of hydrocarbons, the lack of a
hydrocarbon biomarker from other sources in Lake Masoko
ca. 32000 years ago is a clue for an ecosystem dominated at
the time by blooms of B. braunii. This situation strongly

contrasts with the present day, which shows a minor contribution
of botryococcenes to the sedimentary hydrocarbons
and a lack of masokocene c (de Mesmay et al., 2007b).
3.3. Hypothetical biogenetic relationship between cyclic
botryococcenes
As in all botryococcenes, non-isoprenoid carbons (C-31
to C-37) likely arise from successive methylation of the
C30 botryococcene precursor m, by methyl transfer from
S-adenosylmethione. In the C 35, C 36 and C 37 compounds,
permethylation occurs on the same terminal isoprene unit
(i.e. addition of two to four carbons), like that reported for
two acyclic counterparts (Galbraith et al., 1983; Metzger
l
Me +
+
H
pat h I
pat h II
+
et al., 1985b). Two plausible biosynthetic pathways for
the cyclisation leading to masokocene c may be proposed
(Fig. 8). Cyclisation could be initiated either by methylation
(path I) or by protonation of a previously methylated
precursor (path II), as suggested for the biosynthesis of
monocyclic botryococcenes in some strains of B. braunii
(Metzger et al., 1985b; David et al., 1988; Huang and Poulter,
1988). Abiotic cyclisation of unsaturated hydrocarbons
(highly branched isoprenoids, HBIs) has been observed in
laboratory simulations of diagenetic reactions (Belt et al.,
2000). In Lake Masoko sediments, abiotic cyclisation of
botryococcenes cannot therefore be excluded entirely.
Botryococcenes in Lake Masoko ca. 32,000 years ago can
be divided into three types: i) monocyclic botryococcenes
+
-H +
Fig. 8. Plausible biosynthetic pathways for cyclisation leading to masokocene c.
reduction
methylation
methylation
methylation
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 889
cyclisation
cyclisation
cyclisation
cyclisation
a
methylation
methylation
methylation
f1, f2
h
cyclisation
reduction
b
cyclisation
cyclisation
reduction
g1, g2
reduction
i
Fig. 9. Hypothetical biogenetic relationship between acyclic, monocyclic and dicyclic botryococcenes and their reduced derivatives isolated from Lake
Masoko sediment.
k
c,d
e
j

890 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
(f1, f2 and h), ii) masokocenes (i.e. dicyclic botryococcenes:
c, d, e and j) and iii) partially reduced botryococcenes
(a, b, g1, g2 and i, Fig. 9). It is noteworthy that all the
monocyclic botryococcenes and their derivatives have a
cyclohexenyl moiety in the left hand side of the molecule.
No monocyclic compound with one ring on the right side
of the molecule has been characterized in Lake Masoko
sediments, whereas the occurrence of dicyclic compounds
indicates that both sides of the molecule can be cyclized.
Except for a, all the structures in Lake Masoko are cyclic,
whereas botryococcenes found in sediments or pure strain
cultures are mostly acyclic (Metzger and Largeau, 1999). B.
braunii microalgae in Lake Masoko 32,000 years ago produced
nearly exclusively cyclic botryococcenes, which is
quite intriguing. The virtual lack in this sample of acyclic
botryococcenes, likely to be the precursors of cyclic botryococcenes,
suggests an efficient mechanism of cyclisation.
The absence of compound k and of the monocyclic C 35 botryococcene,
probable precursors for masokocenes c and e,
respectively, means that the second cyclisation is also efficient.
The occurrence of f1 and f2 suggests that their cyclisation
to j is probably less efficient due to the steric effect
of the methyl and ethyl groups on the C-21/C-22 double
bond. The stronger steric effect in h may prevent further
cyclisation to C37 masokocene.
In all partially reduced botryococcenes in the sample (i.e.
a, b, g1, g2 and i), the most easily reducible double bond (C-
26/C-27) is left unchanged compared with the corresponding
botryococcene. Moreover, only one peak for each compound
a and b was detected from GC analysis (Fig. 2A), suggesting
the formation of only one diastereoisomer for a and b via biotic
reduction of parent botryococcenes. However, in the light
of the recent work of Hebting et al. (2006) on the preservation
pathway of sedimentary organic carbon, an abiotic process
cannot be entirely excluded. For the other partially reduced
botryococcenes g1 and g2, we attribute the occurrence of
two stereoisomers to the Z and E stereochemistry of the C-
21/C-22 double bond rather than to hypothetical diastereisomers
that would be formed by an abiotic process (Hebting
et al., 2006). We then assess that all partially reduced compounds
in the ca. 32,000 year old sediments from Lake Masoko
arise from a biotic process. Huang and Murray (1995)
and Huang et al. (1996) reported similar observations on
some reduced botryococcenes found in sediment from
Sacred Lake (Kenya), and suggested that they could originate
either from a variant population of B. braunii race B or from a
microbial reduction. Moreover, the possibility of a microbial
reduction during early diagenesis was recently proposed to
explain the occurrence of partially reduced cyclic and acyclic
botryococcenes in soils of the Everglades wetlands (Gao et al.,
2007). In the present case, it is noteworthy that the only acyclic
structure in this sample is the partially reduced C34 botryococcene
a. This could suggest an in vivo competition
between reduction and cyclisation during biosynthesis. Cyclisation
cannot occur after reduction of double bonds C-1/C-2,
C-6/C-24, C-17/C-29 or C-21/C-30. Partial reduction of l to a
prevents cyclisation occurring in the left hand moiety. Furthermore,
increasing steric hindrance due to the successive
effects of the methylation of the same isoprenoid unit results
in a strong decrease in the proportion of dicyclic masokocenes
(from 100% of C 35,downto14%ofC 36 and no C 37).
4. Conclusions
Biomarkers specific for the alga B. braunii race B are the
main constituents of the hydrocarbon fraction extracted
from a ca. 32,000 year old sediment interval from Lake Masoko,
Tanzania. Thanks to GC-MS and NMR and chemical
degradation, ten new cyclic botryococcenes and partially
reduced derivatives were identified. Three C34 to C36 dicyclobotryococcenes,
named masokocenes, were characterized,
along with seven C34 to C37 monocyclic compounds.
The structures indicate that the monocyclic botryococcenes
(CnH2n-10) are likely intermediates in the biosynthesis of the
dicyclic analogues, while the partially reduced botryococcenes
(C nH 2n-2, C nH 2n-6 and C nH 2n-8) are likely end products.
The study widely extends the number of molecular structures
within the botryococcene family.
From a biogeographical point of view, the study also reinforces
the idea that botryococcene-producing B. braunii
would be a rather common colonizer of crater (Huang
et al., 1999; Zhang et al., 2007) and maar (Fuhrmann et al.,
2003) lakes, just like reservoirs (Wake and Hillen, 1981),
dams (Metzger et al., 1985a; David et al., 1988; Metzger
et al., 1988; Jaffé et al., 1995) and also water tanks (Wolf
et al., 1985; Okada et al., 1995), under almost all latitudes
and from the sea level up to alpine zones. Known as a freshwater
alga, race B of B. braunii has been also reported to be
present in some marine environments (e.g. Grice et al.,
1998; Smittenberg et al., 2005). Physical and chemical conditions
favouring its growth in some lakes, leading sometimes
to enduring blooms (e.g. Wake and Hillen, 1981; Metzger
et al., 1985a; Townsend, 2001), are still poorly understood.
Although the preference of B. braunii race B for acidic waters
is often noted, the pH does not seem to be a critical parameter
since this microalga has been found in environments with
pH up to 8.6. Besides, it would appear from the literature (e.g.
Swale, 1968; Wake and Hillen, 1980, 1981; Metzger et al.,
1985a; Huang et al., 1999; Reynolds, 2000; Townsend,
2001; Zhang et al., 2007), that it generally grows in rather
small oligotrophic lakes (ca 1–2 km 2 or less) with a small
catchment area. However, the alga can be also present in
some great lakes like Michigan (Wolf and Cox, 1981). Studies
are currently in progress to determine the physicochemical
and environmental factors that could be at the origin of the
variation of the distribution and abundance of botryococcenes
observed in the sediments of Lake Masoko.
Acknowledgments
M. Delalande, D. Williamson and L. Bergonzini are
thanked for helpful comments and discussions. We also
thank Yongsong Huang and Hans-Peter Nytoft reviews and
constructive comments. The work was supported by the
Centre National de la Recherche Scientifique (CNRS) through
the CLEHA research programme from ECLIPSE-INSU and by
the Institute of Resource Assessment (IRA) at University of
Dar es Salaam. We are grateful to C. Fosse (ENSCP, Paris)
for exact mass determination and to M.-N. Rager (ENSCP,
Paris) for NMR spectral measurements. This paper is contribution
2 of the Rungwe Environmental Science Observatory
Network (RESON) and contribution 07.50 of UMR 5125 PEPS.

Appendix
Associate Editor—S. Schouten
R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 891

892 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893
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