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.


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 


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



% 

100 

50 

0 

% 

100 

50 

0 

% 

100 

50 

0 


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 


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



% 

100 

50 

0 

% 

100 

50 

0 

% 

100 

50 

0 

43 

57 

69 

86 

A 


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 


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


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 


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


(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


References 

Achitouv, E., Metzger, P., Rager, M.-N., Largeau, C., 2004. C 31-C 34 

methylated squalenes from a Bolivian strain of Botryococcus braunii. 

Phytochemistry 65, 3159–3164. 

Adam, P., Schaeffer, P., Albrecht, P., 2006. C40 monoaromatic lycopane 

derivatives as indicators of the contribution of the alga Botryococcus 

braunii race L to the organic matter of Messel oil shale (Eocene, 

Germany). Organic Geochemistry 37, 584–596. 

Barker, P., Williamson, D., Gasse, F., Gibert, E., 2003. Climatic and volcanic 

forcing revealed in a 50,000-year diatom record from Lake Massoko, 

Tanzania. Quaternary Research 60, 368–376. 

Belt, S.T., Allard, W.G., Rintatalo, J., Johns, L.A., van Duin, A.C.T., Rowland, 

S.J., 2000. Clay and acid catalysed isomerisation and cyclisation 

reactions of highly branched isoprenoid (HBI) alkenes: Implications 

for sedimentary reactions and distributions. Geochimica et 

Cosmochimica Acta 64, 3337–3345. 

Bergonzini, L., Gibert, E., Winckel, A., Merdaci, O., 2001. Water and 

isotopic ( 18 O and 2 H) budget of Lake Massoko, Tanzania. 

Quantification of exchange between Lake and groundwater. 

Comptes Rendus de l’Academie des Sciences, Series IIA, Earth and 

Planetary Science 333, 617–623. 

Brassell, S.C., Eglinton, G., Fu, J.M., 1986. Biological marker compounds as 

indicators of the depositional history of the Maoming oil shale. 

Organic Geochemistry 10, 927–941. 

David, M., Metzger, P., Casadevall, E., 1988. Two cyclobotryococcenes 

from the B race of the green alga Botryococcus braunii. Phytochemistry 

27, 2863–2867. 

Delalande, M., Bergonzini, L., Beal, F., Garcin, Y., Majule, A., Williamson, D., 

2005. Contribution to the detection of Lake Masoko (Tanzania) 

groundwater outflow: isotopic evidence ( 18 O,D). Hydrological 

Sciences Journal 50, 867–880. 

de Mesmay, R., Grossi, V., Williamson, D., Kajula, S., Derenne, S., 2007a. 

Novel mono-, di- and tri-unsaturated very long chain (C 37 -C 43) n - 

alkenes in alkenone-free lacustrine sediments (Lake Masoko, 

Tanzania). Organic Geochemistry 38, 323–333. 

de Mesmay, R., Grossi, V., Metzger, P., Williamson, D., Kajula, S., Derenne, 

S., 2007b. Abrupt biogeochemical and environmental changes during 

the last 32,000 years in a tropical lake (Lake Masoko, Tanzania) 

inferred from lipid biomarkers. 23rd International Meeting on 

Organic Geochemistry, Torquay, UK. Book of Abstracts, pp. 1047- 

1048. 

Derenne, S., Largeau, C., Hetényi, M., Brukner-Wein, A., Connan, J., 

Lugardon, B., 1997. Chemical structure of the organic matter in a 

Pliocene maar-type shale: Implicated Botryococcus race strains and 

formation pathways. Geochimica et Cosmochimica Acta 61, 1879– 

1889. 

Fuhrmann, A., Mingram, J., Lücke, A., Lu, H., Horsfield, B., Liu, J., 

Negendank, J.F.W., Schleser, G.H., Wilkes, H., 2003. Variations in 

organic matter composition in sediments from Lake Huguang Maar 

(Huguangyan), south China during the last 68 ka: implications for 

environmental and climatic change. Organic Geochemistry 34, 1497– 

1515. 

Galbraith, M.N., Hillen, L.W., Wake, L.V., 1983. Darwinene: a branched 

hydrocarbon from a green form of Botryococcus braunii. 


Gao, M., Simoneit, B.R.T., Gantar, M., Jaffé, R., 2007. Occurrence and 

distribution of novel botryococcene hydrocarbons in freshwater 

wetlands of the Florida Everglades. Chemosphere 70, 224–236. 

Garcin, Y., Williamson, D., Taieb, M., Vincens, A., Mathe, P.-E., Majule, A., 

2006a. Centennial to millennial changes in maar-lake deposition 

during the last 45,000 years in tropical Southern Africa (Lake Masoko, 

Tanzania). Palaeogeography, Palaeoclimatology, Palaeoecology 239, 

334–354. 

Garcin, Y., Vincens, A., Williamson, D., Guiot, J., Buchet, G., 2006b. Wet 

phases in tropical southern Africa during the last glacial period. 

Geophysical Research Letters 33, L07703. 

Garcin, Y., Vincens, A., Williamson, D., Buchet, G., Guiot, J., 2007. Abrupt 

resumption of the African Monsoon at the Younger Dryas-Holocene 

climatic transition. Quaternary Science Reviews 26, 690–704. 

Gatellier, J.-P., de Leeuw, J.W., Sinninghe Damsté, J.S., Derenne, S., Largeau, 

C., Metzger, P., 1993. A comparative study of macromolecular 

substances of a Coorongite and cell walls of the extant alga 

Botryococcus braunii. Geochimica et Cosmochimica Acta 57, 2053– 

2068. 

Gibert, E., Bergonzini, L., Massault, M., Williamson, D., 2002. AMS- 14 C 

chronology of 40.0 cal ka BP continuous deposits from a crater lake 

(Lake Massoko, Tanzania): modern water balance and environmental 

implications. Palaeogeography, Palaeoclimatology, Palaeoecology 

187, 307–322. 

Grice, K., Schouten, S., Nissenbaum, A., Charrach, J., Sinninghe Damsté, J.S., 

1998. A remarkable paradox: Sulfurised freshwater algal 

(Botryococcus braunii) lipids in an ancient hypersaline euxinic 

ecosystem. Organic Geochemistry 28, 195–216. 

Hebting, Y., Schaeffer, P., Behrens, A., Adam, P., Schmitt, G., 

Schneckenburger, P., Bernasconi, S.M., Albrecht, P., 2006. Biomarker 

evidence for a major preservation pathway of sedimentary organic 

carbon. Science 312, 1627–1631. 

Heinzen, V.E.F., Soares, M.F., Yunes, R.A., 1999. Semi-empirical topological 

method for the prediction of the chromatographic retention of cisand 

trans-alkene isomers and alkanes. Journal of Chromatography A 

849, 495–496. 

Huang, Y., Murray, M., 1995. Identification of 1,6,17,21octahydrobotryococcene 

in a sediment. Journal of Chemical Society, 

Chemical Communications, 335–336. 

Huang, Y., Murray, M., Eglinton, G., Metzger, P., 1995. Sacredicene, a novel 

monocyclic C 33 hydrocarbon from sediment of Sacred Lake, a tropical 

freshwater lake, Mount Kenya. Tetrahedron Letters 36, 5973–5976. 

Huang, Y., Murray, M., Metzger, P., Eglinton, G., 1996. Novel unsaturated 

triterpenoid hydrocarbons from sediments of Sacred Lake, Mt Kenya, 

Kenya. Tetrahedron 52, 6973–6982. 

Huang, Y., Street-Perrott, F.A., Perrott, R.A., Metzger, P., Eglinton, G., 1999. 

Glacial-interglacial environmental changes inferred from molecular 

and compound-specific d 13 C analyses of sediments from Sacred Lake, 

Mt. Kenya. Geochimica et Cosmochimica Acta 63, 1383–1404. 

Huang, Z., Poulter, C.D., Wolf, F.R., Somers, T.C., White, J.D., 1988. 

Braunicene. A novel cyclic C 32 isoprenoid from Botryococcus braunii. 

Journal of the American Chemical Society 110, 3959–3964. 

Huang, Z., Poulter, C.D., 1988. Braunicene. Absolute Stereochemistry of 

the Cyclohexane Ring. Journal of Organic Chemistry 53, 4089–4094. 

Huang, Z., Poulter, C.D., 1989. Stereochemical studies of botryococcene 

biosynthesis: analogies between 1’-1 and 1’-3 condensations in the 

isoprenoid pathway. Journal of the American Chemical Society 111, 

2713–2715. 

Jaffé, R., Wolf, G.A., Cabrera, A.C., Carvajal-Chitty, H., 1995. The 

biogeochemistry of lipids in rivers of the Orinoco Basin. Geochimica 

et Cosmochimica Acta 59, 4507–4522. 

Junkes, B.S., Amboni, R.D.M.C., Heinzen, V.E.F., Yunes, R.A., 2002. Use of a 

semi-empirical topological method to predict the chromatographic 

retention of branched alkenes. Chromatographia 55, 75–81. 

McKirdy, D.M., Cox, R.E., Volkman, J.K., Howell, V.J., 1986. Botryococcane 

in a new class of Australian non-marine crude oils. Nature 320, 57–59. 

Merdaci, O., 1998. Changements climatiques au cours des derniers 30,000 

ans en Afrique Sud-Equatoriale (Tanzanie) par l’étude des pigments et 

phénols lacustres. PhD Thesis, University Aix-Marseille III. 

Metzger, P., Casadevall, E., 1987. Lycopadiene, a tetraterpenoid 

hydrocarbon from new strains of the green alga Botryococcus 

braunii. Tetrahedron Letters 28, 3931–3934. 

Metzger, P., Largeau, C., 1999. Chemicals of Botryococcus braunii. In: 

Cohen, Z. (Ed.), Chemicals from Microalgae. Taylor and Francis, 

London, pp. 205–260. 

Metzger, P., Berkaloff, C., Casadevall, E., Couté, A., 1985a. Alkadiene- and 

botryococcene-producing races of wild strains of Botryococcus braunii. 


Metzger, P., Casadevall, E., Pouet, M.J., Pouet, Y., 1985b. Structures of some 

botryococcenes: branched hydrocarbons from the B-race of the green 

alga Botryococcus braunii. Phytochemistry 24, 2995–3002. 

Metzger, P., Templier, J., Largeau, C., Casadevall, E., 1986. An n -alkatriene 

and some n -alkadienes from the A race of the green alga Botryococcus 

braunii. Phytochemistry 25, 1869–1872. 

Metzger, P., David, M., Casadevall, E., 1987. Biosynthesis of triterpenoid 

hydrocarbons in the B-race of the green alga Botryococcus braunii. 

Sites of production and nature of the methylating agent. 


Metzger, P., Casadevall, E., Couté, A., 1988. Botryococcene distribution in 

strains of the green alga Botryococcus braunii. Phytochemistry 27, 

1383–1388. 

Metzger, P., Rager, M.-N., Largeau, C., 2007. Polyacetals based on 

polymethylsqualene diols, precursors of algaenan in Botryococcus 

braunii race B. Organic Geochemistry 38, 566–581. 

Moldowan, J.M., Seifert, W.K., 1980. First discovery of botryococcane in 

petroleum. Journal of the Chemical Society, Chemical 

Communications, 912–914. 

Okada, S., Murakami, M., Yamaguchi, K., 1995. Hydrocarbon composition 

of newly isolated strains of the green microalga Botryococcus braunii. 

Journal of Applied Phycology 7, 555–559.

Okada, S., Murakami, M., Yamaguchi, K., 1997. Characterization of 

hydrocarbons from the Yayoi strain of the green alga Botryococcus 

braunii. Phytochemical Analysis 8, 198–203. 

Okada, S., Devarenne, T.P., Murakami, M., Abe, H., Chapell, J., 2004. 

Characterization of botryococcene synthase enzyme activity, a 

squalene synthase-like activity from the green microalga 

Botryococcus braunii, race B. Archives of Biochemistry and 

Biophysics 422, 110–118. 

Reynolds, C.S., 2000. Phytoplankton designer – or how to predict 

compositional responses to trophic-state change. Hydrobiologia 

424, 123–132. 

Sato, S., Ito, Y., Okada, S., Murakami, M., Abe, H., 2003. Biosynthesis of the 

triterpenoids, botryococcenes and tetramethylsqualene in the B race 

of Botryococcus braunii via the non-mevalonate pathway. Tetrahedron 

Letters 44, 7035–7037. 

Senousy, H.H., Beakes, G.W., Hack, E., 2004. Phylogenetic placement 

of Botryococcus braunii (Trebouxiophyceae) and Botryococcus 

sudeticus isolate Utex 2629 (Chlorophyceae). Journal of 

Phycology 40, 412–423. 

Smittenberg, R.H., Baas, M., Schouten, S., Sinninghe Damsté, J.S., 2005. The 

demise of the alga Botryococcus braunii from a Norwegian fjord was 

due to early eutrophication. The Holocene 15, 133–140. 

Swale, E.M.F., 1968. The phytoplankton of Oak Mere, Cheshire, 1963- 

1966. British Phycological Bulletin 3, 441–449. 

Thevenon, F., Williamson, D., Vincens, A., Taieb, M., Merdaci, O., Decobert, 

M., Buchet, G., 2003. A Late Holocene charcoal record from Lake 

Masoko, SW Tanzania: climatic and anthropologic implications. The 

Holocene 15, 785–792. 

Townsend, S.A., 2001. Perennial domination of phytopankton by 

Botryococcus and Peridinium in a discontinuously polymictic 


reservoir (tropical Australia). Archiv für Hydrobiologie 151, 529– 

548. 

Tyson, R.V., 1995. Sedimentary Organic Matter: Organic Facies and 

Palynofacies. Chapman and Hall, London. 

Vincens, A., Williamson, D., Thevenon, F., Taieb, M., Buchet, G., Decobert, 

M., Thouveny, N., 2003. Pollen-based vegetation changes in southern 

Tanzania during the last 4200 years: climate change and/or human 

impact. Palaeogeography, Palaeoclimatology, Palaeoecology 198, 

321–334. 

Wake, L.V., Hillen, L.W., 1980. Study of a bloom of the oil-rich alga 

Botryococcus braunii in the Darwin River Reservoir. Biotechnology and 

Bioengineering 22, 1637–1656. 

Wake, L.V., Hillen, L.W., 1981. Nature and hydrocarbon content of blooms 

of the alga Botryococcus braunii occurring in Australian freshwater 

lakes. Australian Journal of Marine and Freshwater Research 32, 353– 

367. 

Williamson, D., Jackson, M.J., Banerjee, S.K., Marvin, J., Merdaci, O., 

Thouveny, N., Decobert, M., Gibert-Massault, E., Massault, M., 

Mazaudier, D., Taieb, M., 1999. Magnetic signatures of hydrological 

change in a tropical maar-lake (Lake Massoko, Tanzania): Preliminary 

results. Physics and Chemistry of the Earth Part A 24, 799–803. 

Wolf, F.R., Cox, E.R., 1981. Ultrastructure of active and resting colonies of 

Botryococcus braunii (Chlorophyceae). Journal of Phycology 17, 395– 

405. 

Wolf, F.R., Nonomura, A.M., Bassham, J.A., 1985. Growth and branched 

hydrocarbon production in a strain of Botryococus braunii 

(Chlorophyta). Journal of Phycology 21, 388–396. 

Zhang, Z., Metzger, P., Sachs, J.P., 2007. Biomarker evidence for the cooccurrence 

of three races (A, B, and L) of Botryococcus braunii in El 

Junco Lake, Galápagos. Organic Geochemistry 38, 566–581.

Mono- and dicyclic unsaturated triterpenoid hydrocarbons in ...

Create successful ePaper yourself

Delete template?

Save as template?