FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 123–135

Monoterpene biosynthesis in Agathosma crenulata

(Buchu)
Sabine Fuchs, Sabine Sewenig and Armin Mosandl*
Institut für Lebensmittelchemie, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie-Strasse 9, 60439 Frankfurt (Main),
Germany
Received 30 August 2000
Revised 25 October 2000
Accepted 1 November 2000

ABSTRACT: Young plants of Agathosma crenulata (L.) Pillans were stem fed with aqueous solutions of 2 H2 and
18
O/2 H2 -labelled monoterpene ketone precursors. The essential oil was extracted by solid phase microextraction
and subsequently analysed with enantioselective multidimensional gas chromatography–mass spectrometry.
Both labelled pulegone precursors were converted into corresponding labelled menthone, isomenthone and
menthofuran with different enantioselectivity. Feeding experiments with 18 O/2 H-labelled pulegone proved an
enzymatic conversion of pulegone to menthofuran and the loss of the pulegone oxygen. Stereoselective analysis
of all four isopulegone stereomers using octakis(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)--cyclodextrin or
heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-ˇ-cyclodextrin allowed the detection of enantiopure (1S)-
cis- and (1S)-trans-isopulegone (>99%) in A. crenulata and buchu samples. Labelled isopulegone is discussed
as a suitable pulegone precursor. Feeding experiments regarding the biosynthesis of 3-oxo-p-menthan-8-thiol and
3-oxo-p-menthan-8-thiol acetate are also reported. Copyright  2001 John Wiley & Sons, Ltd.
KEY WORDS: Agathosma crenulata (L.) Pillans; buchu; biosynthesis; menthone; isomenthone; menthofuran;
pulegone; isopulegone; 3-oxo-p-menthan-8-thiol; 3-oxo-p-menthan-8-thiol acetate; essential oil analysis

Introduction was investigated by Köpke et al., using enantioselec-

Buchu leaf oil is obtained by steam distillation of
Agathosma betulina (Berg.) Pillans or Agathsoma crenu-
lata (L.) Pillans (Rutaceae; synonym: Barosma) shrubs
native to South Africa. Due to its powerful and
characteristic minty-fruity odour, it is frequently used
as an flavour ingredient. The main components are
limonene, menthone, isomenthone, pulegone and bifunc-
tional diosphenols, also called buchu camphors. How-
ever, the constituents responsible for the characteristic
blackcurrant odour are the minor compounds 3-oxo-
p-menthane-8-thiol and its thiol acetate.1,2 The two
buchu species differ in their essential oil composi-
tion: A. betulina is characterized by a high content
of (iso)menthone (31%), ( )-diosphenol (41%) and the
cis- and trans-3-oxo-p-menthane-8-thiol (3%); A. crenu-
lata oil contains a high content of pulegone (54%) and
higher quantities of trans-3-oxo-p-menthane-8-thiol.3
The stereochemistry of limonene, (iso)menthone, pule-
gone, 3-oxo-p-menthane-8-thiol(acetate) in A. betulina
ŁCorrespondence to: A. Mosandl, Institut für Lebensmittelchemie,
Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie-Strasse
9, 60439 Frankfurt (Main), Germany. E-mail: Mosandl@em.uni-
frankfurt.de
Contract/grant sponsor: Deutsche Forschungsgemeischaft (DFG),
Germany.
tive multidimensional gas chromatography. Limonene
was found to consist of 93% (4R)-limonene. Menthone,
isomenthone, pulegone, 3-oxo-p-menthane-8-thiol and
3-oxo-p-menthane-8-thiol acetate were found to be
>99% (1S)-configured.†4 Also, menthofuran was found
to be (1S)-configured in buchu leaf oil.5 Due to the
chemical structure and the same stereochemistry at
C-1, biosynthetic correlations between (iso)menthone,
(iso)pulegone, 3-oxo-p-menthane-8-thiol (acetate) and
menthofuran were conceivable. On the other hand,
the biosynthetic correlation between (1R)-configured
(iso)menthone, (iso)pulegone and menthofuran in pep-
permint (Mentha piperita L.) is well established (refer-
ences 6, 7 and literature cited therein). Recently, stereo-
chemical and mechanistic aspects in the biogenetic con-
version of pulegone into (iso)menthone and menthofuran
were studied by in vivo feeding experiments.8,9
This paper deals with the first in vivo feeding exper-
iments with Agathosma crenulata (oval buchu). The
essential oil was collected with solid phase microex-
traction (SPME) and subsequently analysed by enan-
tioselective multidimensional gas chromatography–mass
spectrometry (enantio-MDGC–MS). On the basis of
†For clarity, the terpene nomenclature is used in the context of the
Introduction, Results and Discussion, and for 1 H-NMR assignment.

Copyright  2001 John Wiley & Sons, Ltd.

124 S. FUCHS, S. SEWENIG AND A. MOSANDL
these results, the biosynthetic relations of menthone, iso-
menthone, menthofuran, pulegone, isopulegone, 3-oxo-
p-menthan-8-thiol, and 3-oxo-p-menthan-8-thiol-acetate
were drawn, in consideration of stereochemical and
mechanistic aspects.
Materials and Methods
Plant Material
Agathosma crenulata (L.) Pillans plants were grown up
from seeds (Silverhill Seeds, CapeTown, South Africa)
in the Botanical Garden of Frankfurt University. Authen-
tic buchu essential oil was prepared by steam distillation
from Folia Bucco (conc. EB 6; Caesar & Loretz, Hilden,
Germany). A research sample of buchu oil (50% in
ethanol) was obtained from Haarman & Reimer (Holz-
minden, Germany).
Enantioselective Gas Chromatography
(Enantio-GC)
The enantiomeric distribution of labelled pulegone was
analysed with an HP Series II gas chromatograph, equi-
pped with a duran glass capillary (30 m ð 0.23 mm i.d.)
coated with a 0.23 µm film of 15% heptakis(2,3-di-
O-methyl-6-O-tert-butyldimethylsilyl)-ˇ-cyclodextrin in
SE 52. Conditions: carrier gas, hydrogen, 130 kPa; split,
30 ml/min; injector temperature, 250 ° C; detector, FID,
260 ° C; oven temperature, 80 ° C, raised at 1 ° C/min to
200 ° C (30 min isothermal).
Gas Chromatography/Mass Spectrometry
(GC–MS)
A Fisons GC 8065 coupled to a Fisons MD 800 mass
spectrometer, equipped with a MDN-5S fused silica
column (60 m ð 0.25 mm i.d., film thickness 0.25 µm;
Supelco, Munich, Germany), was used for analysing
synthesized compounds and SPME extracts. Conditions:
carrier gas, helium, 170 kPa; split, 20 ml/min; injec-
tor temperature, 230 ° C; oven temperature, 40 ° C (5 min
isothermal), raised at 2.5 ° C/min to 260 ° C (30 min
isothermal); interface temperature, 250 ° C; ion source
temperature, 200 ° C; EI, 70 eV.
Enantioselective Multidimensional Gas
Chromatography–Mass Spectrometry
(Enantio-MDGC–MS)
System 1
A Siemens SiChromat 2 was used, with two indepen-
dent column oven programmes and a live T-switching
Copyright  2001 John Wiley & Sons, Ltd.
device, coupled to the transfer line of a Finning MAT
ITD 800, using an open split interface. GC condi-
tions were as follows. Precolumn: duran glass capillary
(30 m ð 0.23 mm i.d.), coated with a 0.23 µm film of
SE 52; carrier gas, hydrogen, 196 kPa; split, 30 ml/min;
injector temperature, 220 ° C; detector, FID, 250 ° C. Main
column: duran glass capillary (30 m ð 0.23 mm i.d.),
coated with a 0.23 µm film of 50% octakis(2,3-di-O-
butyryl-6-O-tert-butyldimethylsilyl)--cyclodextrin;10
carrier gas, hydrogen, 162 kPa; detector, ITD 800; trans-
fer line temperature, 250 ° C, open split interface, 250 ° C;
helium sweeping flow, 1 ml/min; ion trap manifold,
195 ° C; EI, 70 eV; oven temperature (menthone, iso-
menthone, isopulegone, menthofuran): precolumn, 50 ° C
(5 min isothermal), raised at 5 ° C/min to 250 ° C (15 min;
54 min isothermal); cut time, 17.5–20.0 min; main col-
umn, 60 ° C (0 min isothermal), raised at 2 ° C/min to
100 ° C (0 min isothermal), raised at 1 ° C/min to 145 ° C;
40 ° C (25 min isothermal), raised at 0.5 ° C/min to 60 ° C
(0 min isothermal), raised at 2.5 ° C/min to 145 ° C.
System 2
A Siemens SiChromat 2-8 was used, equipped with
two independent column ovens and a live T-switching
device, coupled to the transfer line of a Perkin-Elmer
ITD. GC conditions were as follows. Precolumn: fused
silica capillary (30 m ð 0.25 mm i.d.), coated with a
0.25 µm film of SE 52; carrier gas, hydrogen, 180 kPa;
split, 30 ml/min; injector temperature, 260 ° C; detec-
tor, FID, 280 ° C. Main column: fused silica capillary
(30 m ð 0.25 mm i.d.), coated with a 0.25 µm film of
heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-ˇ-
cyclodextrin (BGB Analytik, Adliswil, Switzerland);
carrier gas, hydrogen 95 kPa; detector ITD 800; transfer
line temperature, 250 ° C; open split interface, 250 ° C;
helium sweeping flow, 1 ml/min; ion trap manifold,
220 ° C; EI, 70 eV. Oven temperature: precolumn: 60 ° C
(5 min isothermal), raised at 5 ° C/min to 250 ° C; cut
time, 16.0–18.0 min (isopulegone), 20.0–22.0 min
(compound A); main column: 60 ° C (25 min isother-
mal), raised at 1 ° C/min to 100 ° C. The molecular ion
(MC ) and the fragmentation ions were given as m/z with
relative peak intensities to the base peak (%).
System 3
A Siemens Sichromat 2-8 was used, with two indepen-
dent column ovens and a live T-switching device. The
main column was coupled to the transfer line of a Finni-
gang MAT GCQ mass spectrometer with an open split
inerface (SGE). GC-conditions were as follows. Pre-
column: fused silica capillary (30 m ð 0.25 mm i.d.),
coated with a 0.25 µm film of SE 52; carrier gas,
helium, 220 kPa; split, 30 ml/min; injector temper-
ature, 250 ° C; detector, FID, 250 ° C. Main column:
Flavour Fragr. J. 2001; 16: 123–135
 MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA
fused silica capillary (30 m ð 0.25 mm i.d.), coated with
a 0.25 µm film of heptakis(2,3-di-O-methyl-6-O-tert-
butyldimethylsilyl)-ˇ-cyclodextrin (BGB Analytik); car-
rier gas, helium, 138 kPa; detector, GC-Q; transfer line
temperature, 250 ° C, helium sweeping flow, 1 ml/min;
ion source, 170 ° C; EI, 70 eV. Oven temperature: pre-
column: 80 ° C (5 min isothermal), raised at 5 ° C/min
to 250 ° C; cut time, 14.5–16.2 min (menthone, isomen-
thone); main column: 60 ° C (15 min isothermal), raised
at 1 ° C/min to 150 ° C.
1 erence 13. Oxidation of 1.2 mmol d5 -citronellol with
H-NMR
The 1 H-NMR spectra were recorded on a Bruker ARX
300, 300 MHz. CDCl3 was used as solvent. Abbre-
viations: s D singlet, d D doublet; m D multiplet, J D
spin–spin coupling constant (Hz).
Synthesis of Isopulegone
Synthesis of (2S/R,5S)-2(1-methylethenyl)-5-methyl-
cyclohexanone (isopulegone) (7, 8) and (2S/R,5R)-
2(1-methylethenyl)-5-methyl-cyclohexanone (9, 10).
3.1 mmol (1S)-3,7-dimethyl-6-octenol [(1S)-Citro-
nellol], and (1R)-3,7-dimethyl-6-octenol, respectively,
was stirred with 9.4 mmol pyridinium chlorochromate
(PCC) in 10 ml dry methylene chloride for 36 h, accord-
ing to Corey.11 Pentane/diethyl ether 1 : 1 (v/v) was
added and the suspension filtered (Celite). It was extrac-
ted with 10% HCl, 10% NaHCO3 and H2 O and dried
(Na2 SO4 ). The crude product was purified by flash chro-
matography. Chromatographic conditions: column diam-
eter, 20 mm; silica gel, 30–60 µm (Baker); eluent, pen-
tane/diethyl ether, 6 : 1 (v/v). After removal of the eluent,
0.7 mmol 9, 10 and 0.9 mmol 7, 8, respectively, were
obtained.
Synthesis of (2S,5R)-2-(1-methylethenyl)-5-methyl-
cyclohexanone (9). (1R,2S,5R)-2-(1-methylethenyl)-
5-methyl-cyclohexan-1-ol [()-isopulegol, Fluka] was
oxidized as described above (reaction time, 20 h). MS
(System 2): 8: 152 (MC , 40), 137 (32), 123 (45), 109
(100), 93 (50), 81 (99), 67 (92), 53 (25); 9: 152 (MC , 39),
137 (30), 123 (40), 109 (97), 93 (48), 81 (100), 67 (93),
53 (25). 1 H-NMR (9) [12]: 1.033 (d, 3H, H-7, J D 6.2),
1.746 (s, 3H, H-9), 1.76–2.09 (m, 6H, H-1, H-2a, H-5,
H-6), 2.373–2.429 (m, 1H, H-2b), 2.954 (dd, 1H, H-4,
J D 13.0; J D 5.5), 4.717 (s, 1H, H-10-Z), 4.933 (s, 1H,
H-10-E).
Synthesis of Deuterium-labelled Precursors
Synthesis of (5R)-2-[1-[2 H3 ]methyl[2,2,2-2 H3 ]ethyli-
dene]-5-methyl[6,6-2 H2 ]cyclohexanone (d8 -(R)-pule-
gone, 27) and (5S)-2-[1-[2 H3 ]methyl[2,2,2-2 H3 ]ethyli-
dene]-5-methyl[6,6-2 H2 ]cyclohexanone (d8 -(S)-pule-
Copyright  2001 John Wiley & Sons, Ltd.
125
gone, 28). The synthesis of 27 and 28 is described in
reference 13.
Synthesis of (5R/S)-2-[1-[2 H3 ]methyl[2,2,2-2 H3 ]-
ethylidene]-5-methyl[18 O]cyclohexanone (d6 -[18 O]-pu-
legone, 29, 30). 29, 30 was synthesized as described
in reference 8. Incomplete incorporation of 18 O led to a
mixture of 83% 29, 30 and 17% d6 -pulegone (29a, 30a).
Synthesis of (2S/R,5R/S)-2(1-methylethenyl)-5-[2 H3 ]-
methyl-[4,4-2 H2 ]-cyclohexanone (d5 -isopulegone)
(31–34). (R/S)-3,7-[9,9,9-2 H3 ]Dimethyl-6-[4,4-2 H2 ]-
octenol (d5 -citronellol) was synthesized according to ref-
PCC led to 0.06 mmol d5 -isopulegone after purification
by flash chromatography (for conditions, see above). MS
(System 2): (2S, 5S)-d5 -isopulegone, (33): 157 (MC , 28),
156 (30), 141 (13), 139 (11), 125 (23), 111 (70), 94
(30), 84 (61), 67 (100). 1 H-NMR (32, 33): 1.754 (s, 3H,
H-9), 2.428 (dd, 1H, H-2b, J D 4.0, J D 24.5), 2.960
(1H, H-4, J D 5.5, J D 13.0), 4.725 (s, 1H, H-10-Z),
4.942 (s, 1H, H-10-E); unexchanged protons were found
at 0.99–1.01 (H-7).‡
Synthesis of Deuterium-labelled Reference
Compounds
Synthesis of (2S,5R)-2-[1-[2 H3 ]-methyl-[2,2,2-2 H3 ]
ethyl]-5-methyl-[6,6-2 H2 ]cyclohexanone (d8 -(2S,5R)-
menthone, 17), (2R,5S)-2-[1-[2 H3 ]-methyl-[2,2,2-2 H3 ]-
ethyl]-5-methyl-[6,6-2 H2 ]cyclohexanone (d8 -(2R,5S)-
menthone, 18), (2R,5R)-2-[1-[2 H3 ]-methyl-[2,2,2-2 H3 ]-
ethyl]-5-methyl-[6,6-2 H2 ]cyclohexanone (d8 -(2R,5R)-
isomenthone, 19), (2S,5S)-2-[1-[2H3 ]-methyl-[2,2,2-
2
H3 ]-ethyl]-5-methyl-[6,6-2 H2 ]cyclohexanone (d8 -
(2S,5S)-isomenthone, 20). The syntheses were published
in reference 13.
Synthesis of (2S/R,5S/R)-2-isopropylidene-5-[2 H3 ]-
methyl-[4,4-2 H2 ]cylohexanone (d5 -pulegone, 25, 26)
and (2S/R,5S/R)-2-isopropyl-5-[2 H3 ]methyl-[4,4-2 H2 ]-
cyclohexanone (13–16). These syntheses were described
in reference 13.
Synthesis of (2S/R,5S/R)-2-[1-[2 H3 ]-methyl-[2,2,2-
2
H3 ]ethyl]-5-methyl-cyclohexanone (d6 -menthone/d6 -
isomenthone, 21a–24a); for details, see reference 8.
Feeding Experiments
Shoots with eight leaf pairs were cut off the young
plants and placed into the feeding solution, containing
the labelled precursor and Tween 20, 0.1 mg/ml each,
in distilled water. Blank experiments were carried out
with distilled water containing 0.1 mg/ml Tween 20.
‡Due to isomerization, d5 -pulegone was detected in the 1 H-NMR
spectra. Therefore, the signals at 1.78–2.07 could not be assigned.
Other signals were assigned by means of known d5 -pulegone 1 H-NMR
data.13
Flavour Fragr. J. 2001; 16: 123–135
126 S. FUCHS, S. SEWENIG AND A. MOSANDL
Feeding experiments for the examination of menthofuran
biosynthesis were carried out with exclusion of light.
SPME
After 24 h the plants were removed and placed in a
2 ml vial. The essential oil evaporating (1 h equilibration
time at least) was extracted by a SPME fibre (2 min for
(iso)menthone, 15 min for menthofuran and GC–MS)
coated with 100 µm film of polydimethylsiloxane, which
was installed in an SPME fibre holder for manual use
(both Supelco). For some feeding experiments (stressed
in the text) the feeding time was elongated to 4 and
7 days, respectively. The fibre was thermically desorped
in the GC injector with the split valve being opened after
2 min.
O
9 standard
8
O
O
10 7
7
8
Figure 1. Main column chromatogram of isopulegone standard mixture and A. crenulata SPME extract (system 1)
Copyright  2001 John Wiley & Sons, Ltd.
Results and Discussion
Stereodifferentiation of Isopulegone
Isopulegone standards were synthesized according to
Corey et al.,11 starting from enantiopure (R)- and
(S)-citronellol, respectively. Oxidation of (1R,3R,4S)-
()-isopulegol yielded (1R,4S)-trans-isopulegone (9).
After purification of diastereomeric cis- and trans-
isopulegone obtained from citronellol, three compounds
with identical mass spectra could be detected. The trans-
isomer was identified using enantiopure (1R,4S)-trans-
isopulegone (9). One of the remaining compounds (com-
pound A) was isolated and an 1 H-NMR spectrum was
recorded, proving this compound not to be identical with
isopulegone, due to the lack of CH2 protons (H-10).
Isopulegone
SPME extract
A. crenulata
Flavour Fragr. J. 2001; 16: 123–135
 MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA
Furthermore, this compound was not identified as a ge-
nuine compound in buchu. The third compound could
not be isolated from trans-isopulegone and for that rea-
son cis-isopulegone was identified by mass spectrum and
occurrence in buchu.3,14
Isopulegone enantiomers could be separated using
enantio-MDGC–MS systems 1 and 2. In Figure 1 a
main column chromatogram (system 1) of isopulegone
standard mixture and SPME extract of A. crenulata is
shown.
Exclusively the (1S)-cis-isopulegone (7) and (1S)-
trans-isopulegone (8) were detectable. Research samples
of buchu leaf oil (Haarmann & Reimer), SPME extract
and self-prepared steam-distilled buchu oil of folia bucco
(Caesar & Loretz), and a commercial available buchu
oil (A. crenulata) were also analysed. In all samples,
only (1S)-configured cis- and trans-isopulegone (7, 8)
were detectable. These results were not unexpected,
as pulegone, (iso)menthone, 3-oxo-p-menthane-8-thiol
(acetate) and menthofuran were detected as highly enan-
tiopure (>99%) (1S)-configured compounds in buchu
leaf oil.4,5
Analysis of A. crenulata by SPME
A GC–MS chromatogram of a SPME extract is given
in Figure 2. All interesting and characteristic monoter-
penoids were detectable. Furthermore, a good correlation
Figure 2. GC–MS chromatogram of an A. crenulata SPME extract
Copyright  2001 John Wiley & Sons, Ltd.
127
with reported A. crenulata oil consituents2,3,14 could be
seen, indicating an identical terpene pattern of the plants
grown abroad. Enantioselective analysis of the A. crenu-
lata used gave good correlation with A. betulina leaf
oil, exclusively detecting (1S)-configured (iso)menthone,
pulegone, and menthofuran (>99%).4,5 Limonene was
found to consist of 95% (4R)-limonene in A. crenulata
and in A. betulina, 93% (4R)-limonene was reported.4
Menthone and Isomenthone Biosynthesis
Because of its chemical structure and the known reduc-
tion of (1R)-pulegone to (1R)-(iso)menthone in pepper-
mint, pulegone was expected to be a suitable precur-
sor. (1R)-d8 -pulegone (27) and (1S)-d8 -pulegone (28)
were synthesized using H/D-exchange reactions. Mix-
ing feeding solutions of 27 and 28 in different amounts,
feeding solutions with different enantiomeric distribu-
tions were obtained. Because of a base peak shift from
m/z D 112 in genuine p-menthan-3-one to m/z D 115 in
d8 -labelled p-menthan-3-one (McLafferty fragmentation
[15]) (Figure 3), genuine and labelled p-menthan-3-ones
can be detected simultaneously. A typical main column
chromatogram obtained after administration and biocon-
version of d8 -pulegone (27, 28) is given in Figure 4.
A. crenulata was able to convert exogenous labelled
d8 -pulegone (27, 28) into labelled p-menthan-3-ones
Flavour Fragr. J. 2001; 16: 123–135
128 S. FUCHS, S. SEWENIG AND A. MOSANDL

Figure 3. Mass spectra of genuine menthone (1, 2) and d8 -menthone (19, 20) (GC–MS)

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 123–135
 4 Administered
precursors
O
m/z = 112
O genuine p -menthan-3-one
1 (1S)-configured -

20
D

Copyright  2001 John Wiley & Sons, Ltd.

D D
D
D O m/z = 115 D
O 17 labelled p -menthan-3-one
D3 C CD3 (1S)-configured O
D3 C CD3

D3C CD3
28
18
D
D D D
m/z = 115
D
O labelled p -menthan-3-one D
O (1R )-configured
D3C CD3
19
O
D3C CD3

D3C CD3
27

18 20
D D
m/z = 115 D D
labelled p -menthan-3-one
(1S/R)-configured O O
17 19
D3C CD3 D3C CD3

1 : 1
MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA

Flavour Fragr. J. 2001; 16: 123–135

129

Figure 4. Main column chromatograms obtained after feeding d8 -pulegone (27, 28) to A. crenulata (system 1): detection of p-menthan-3-one
130 S. FUCHS, S. SEWENIG AND A. MOSANDL

(17–20). After feeding enantiopure (1S)-d8 -pulegone
(28) and (1R)-d8 -pulegone (27), the corresponding (1S)-
d8 -p-menthan-3-one (17, 20), and (1R)-d8 -p-menthan-
3-one (18, 19), respectively, were detectable, indicat-
ing that the stereochemistry at C-1 remains unaffected.
The diastereomeric ratios of (1S,4S)-isomenthone (4)
to (1S,4R)-menthone (1), (1S,4S)-d8 -isomenthone (20)
to (1S,4R)-d8 -menthone (17), and of (1R,4S)-d8 -men-
thone (18) to (1R,4R)-d8 -isomenthone (19), as well as
the enantiomeric ratios of (1S)-d8 -pulegone (28) to

Table 1. Labelling pattern and abbreviation numbers. In d5 -labelled compounds (13–16, 25, 26, 31–34) the assignment
of configuration at C-1 is changed when compared with the corresponding unlabelled compounds. The groups at
the chiral centre are placed in order of decreasing atomic mass, but their three-dimensional arrangement at C-1 is
not affected

p-menthan-3-one W X Y Z 1S, 4R 1R,4S 1R,4R 1S, 4S

CW3

W Y d5 H H H O 1 2 3 4
Y d8 D H H O 15 16 13 14
W
d6 -[18 O] H D D O 17 18 19 20
Z H D H 18 O 21 22 23 24
d6
H D H O 21a 22a 23a 24a
X 3C CX3

pulegone W X Y Z 1R 1S
CW3

W Y
d5 H H H O 5 6
W Y d8 D H H O 26 25
d6 -[18 O] H D D O 27 28
Z H D H 18 O 29 30
d6
H D H O 29a 30a
X3C CX3

isopulegone W 1S, 4S 1S, 4R 1R,4S 1R,4R

CW3

W
W d5 H 7 8 9 10
D 33 34 31 32
O

menthofuran X Z 1R 1S

d4 H O 11 12
d4 -[18 O] D O 35 36
Z D 18 O 37 38

X 3C
X

Table 2. Data obtained after feeding d8 -pulegone to A. crenulata: detection of

p-menthan-3-one
Genuine Labelled p-menthan-3-one
p-menthan-3-one
precursor 4:1 20 : 17 18 : 19 (17,20) : (18,19)
28 85/15 87/13 n.n. –
80/20 78/22 n.n. –
28 : 27 D 75 : 25 86/14 90/10 81/19 70/30
28 : 27 D 70 : 30 83/17 81/19 84/16 62/38
84/16 82/18 84/16 63/37
n.b. 86/14 81/19 53/47
28 : 27 D 50 : 50 83/17 84/16 80/20 50/50
82/18 80/20 82/18 50/50
28 : 27 D 25 : 75 84/16 85/15 73/27 26/74
27 85/15 n.n. 83/17 –
82/18 n.n. 71/29 –

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 123–135
 MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA
(1R)-d8 -pulegone (27) and (1S)-d8 -p-menthan-3-one
(17, 20) to (1R)-d8 -p-menthan-3-one (18, 19) of the dif-
ferent feeding experiments are given in Table 2.
The diastereomeric ratio of labelled p-menthan-3-
ones 20 : 17 and 18 : 19 were comparable to the ratio
4 : 1 of genuine p-menthan-3-ones. The enantiomeric
ratio of (1S)-d8 -p-menthan-3-one (17, 20) to (1R)-d8 -p-
menthan-3-one (18, 19) remained mostly the same as in
d8 -pulegone (28 : 27). Thus, the reduction of d8 -pulegone
into corresponding d8 -p-menthan-3-one is an unspecific
process with no observable enantioselectivity.
After feeding a mixture of racemic d6 -pulegone (29a,
30a) and racemic d6 -[18 O]-pulegone (29, 30), a rather
unexpected result was obtained. As can be seen from
Figure 5, the distribution of d6 -p-menthan-3-ones and
d6 -[18 O]-p-menthan-3-ones was different. In these ex-
periments the molpeak was used for detection, as the
base peak signal at m/z D 113 of d6 -p-menthan-3-ones
was overlapped by a peak of genuine p-menthan-3-one
(1, 4).
All feeding experiments with 29a, 30a and 29, 30
are summarized in Figure 6. Although there are great
differences in the detection of d6 -p-menthan-3-ones
and d6 -[18 O]-p-menthan-3-ones, the sum of labelled
p-menthan-3-ones gave the same results as were ex-
pected after feeding experiments with d8 -pulegone.
The diastereomeric ratio as well as the enantiomeric
ratio depends on the oxygen labelling. This phenomenon
is plant-specific, because comparable feeding exper-
iments with Mentha piperita L. showed no depen-
dency on the oxygen labelling.16 These results might
Figure 5. Main column chromatogram obtained after feeding d6 -[18 O]-pulegone and d6 -pulegone to A. crenulata
(system 1): detection of p-menthan-3-one
Copyright  2001 John Wiley & Sons, Ltd.
131
hint at different p-menthan-3-one building pathways in
A. crenulata.
In order to obtain closer insight into p-menthan-3-one
bioysynthesis in A. crenulata, the precursor of pulegone
is interesting. Due to its occurrence in A. crenulata and
its known importance in Mentha piperita,5,6 isopule-
gone was investigated. As explained above, only a mix-
ture of racemic d5 -cis-isopulegone (31, 34) and racemic
d5 -trans-isopulegone (32, 33) (32, 33 × 31, 34) could
be administered. Furthermore, owing to easy isomeriza-
tion of isopulegone into pulegone, pulegone emerged
in the feeding solution. After feeding 31–34 as pre-
cursors, d5 -labelled p-menthan-3-ones (13–16) were
detectable in the same ratios and amounts as after feeding
d8 -pulegone. In accordance with these results, isopule-
gone could be a possible pulegone precursor, but further
investigations must be carried out to confirm this hypoth-
esis. The isopulegone diastereomers should be sepa-
rated and (1R)-pulegone should be used to calculate the
amount of p-menthan-3-one being generated by isopule-
gone isomerization. This internal standard method was
successfully used for the study of menthofuran biosyn-
thesis in Mentha piperita L.8
In feeding experiments for studies on 3-oxo-p-men-
thane-8-thiol (acetate) with labelled pulegone and iso-
pulegone (see below), the feeding time was elongated.
The detection of labelled p-menthan-3-ones showed a
dependency on time, with the (1S)-configurated stereoiso-
mers predominating. The detection of d5 -isopulegone
after 7 days of feeding d5 -isopulegone, showed 75%
(1R)-configured cis- and trans-d5 -isopulegone, while
Flavour Fragr. J. 2001; 16: 123–135
132 S. FUCHS, S. SEWENIG AND A. MOSANDL
Figure 6. Different distribution of p-menthan-3-ones
after feeding d6 -[18 O]-pulegone and d6 -pulegone to
A. crenulata (n D 7; n1 D 1–5)
A
B
Figure 7. Mass spectra of genuine menthofuran (A) and labelled menthofuran (B) obtained after feeding
d6 -[18 O]-pulegone and d6 -pulegone to A. crenulata
Copyright  2001 John Wiley & Sons, Ltd.
racemic cis- and trans-d5 -isopulegone was detected after
1 day of feeding.
Menthofuran Biosynthesis
Pulegone seemed also to be a suitable precursor for
menthofuran. The oxidation of pulegone to menthofu-
ran in Mentha piperita L. was proved by in vivo feeding
experiments.8 As in the detection of p-menthan-3-ones,
a shift of the Retro–Diels–Alder base peak allowed the
simultaneous detection of genuine and labelled mentho-
furan. In feeding experiments with d6 -[18 O]-pulegone
(29, 30) the origin of oxygen in menthofuran can be
determined. This mixed labelling technique was required
O
12
O
D3C D
36
Flavour Fragr. J. 2001; 16: 123–135
 MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA 133

Figure 8. Main column chromatogram obtained after feeding d6 -[18 O]-pulegone and d6 -pulegone to A. crenulata
(system 1)

to distinguish between genuine menthofuran and men- Table 3. Data obtained after feeding labelled pulegone
thofuran that has been synthesized from the precursor. to A. crenulata: detection of menthofuran
Therefore, a detectable deuterium labelling indicated de Pulegone Labelled pulegone Labelled menthofuran
novo-synthesized menthofuran, even if the 18 O-label was precursor (1S)/(1R) (1S)/(1R)
lost. A main column chromatogram and the correspond- >99/<1 >99/<1
ing mass spectra of a feeding experiment with racemic 75/25 58/42
d6 -[18 O]-pulegone (29, 30) are shown in Figures 6, 7 27, 28 70/30 66/34
61/39
and 8. 50/50 24/76
As can be seen from the mass spectrum of labelled 34/67
menthofuran, the 18 O-labelling was lost, showing the 29, 30 50/50 31/69
19/81
base peak at m/z D 112 and a molpeak at m/z D 154.
27, 28 25/75 13/87
In the case of 18 O-incorporation, m/z D 114 and m/z D <1/>99 <1/>99
156, respectively, were to be expected. Furthermore,
the mass spectrum was identical with d4 -menthofuran,
obtained after feeding d6 -pulegone to Mentha.8 This First, the stereochemistry at C-1 remained unchanged
fact proves the loss of 18 O-labelling during bioconver- during the bioconversion; labelled (1S)-pulegone and
sion of pulegone. Obviously, an enzymatic oxidation (1R)-pulegone exclusively led to labelled (1S)-mentho-
of labelled pulegone is involved in the bioconversion furan and (1R)-menthofuran, respectively. Second, an
of labelled pulegone into menthofuran, in which the enantiodiscrimination could be observed favouring label-
oxygen introduced by pulegone oxidation remains in led (1R)-pulegone.
the menthofuran molecule. These findings are in agree- As was known from analogous experiments in Men-
ment with investigations in mammals by Nelson et al.17 tha piperita L.,8 small amounts of menthofuran were
and also comparable to menthofuran biosynthesis in detectable in the pure feeding solution without any incu-
peppermint.8 bation of plant material, even if incubated in the dark.
Looking at the chromatogram in Figure 7, the amount However, this detected menthofuran showed no differ-
of labelled (1R)-menthofuran (35) is higher than that of ences in enantiomeric and 16 O/18 O ratios of the pule-
labelled (1S)-menthofuran (36), the analogue of genuine gone precursor. On the other hand, when plant material
(1S)-menthofuran (12). For further investigations of this was incubated under identical conditions, enantiodis-
enantiodiscrimination effect, feeding experiments with crimination as well as a change in the 16 O/18 O ratio
enantiopure as well as different enantiomeric ratios of d8 - was observed, indicating an enzymatic process in the
pulegone (27, 28) were carried out. Table 3 summarizes plant. According to studies in Mentha, a calibration
the results. method using (1R)-menthofuran as an internal standard

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 123–135
134 S. FUCHS, S. SEWENIG AND A. MOSANDL

is proposed. Due to the disposal of plant material, no

further details could be revealed.

O O

3-Oxo-p-menthane-8-thiol (acetate) biosynthesis

3-Oxo-p-menthane-8-thiol18,19 and its acetate20 were

found to be responsible for the characteristic cassis
flavour note in buchu leaf oil. Enantioselective analysis
using enantio-MDGC–MS proved that only the (1S)-
configured thio-compounds were detectable (>99%) in
buchu leaf oil.4 Due to the high reactivity of its ˛, ˇ- O O
unsaturated structure, labelled pulegone was tested as a
possible precursor for these sulphur-containing monoter-
penoids. Unfortunately, even if feeding labelled pule-
gone (27, 28) for 7 days, no differences between controls
and fed leafs could be observed. As a second precur-
sor, labelled d5 -isopulegone (31–34) was administered
to A. crenulata for 7 days. In this case, labelled 3-oxo-
p-menthan-8-thiol and 3-oxo-p-menthan-8-thiol acetate
O O O O
were detectable with GC–MS in the same diastere-
omeric ratio as their genuine analogues. The labelled
compounds were identified by their mass spectra, which Scheme 1. Proposed biosynthetic conversion of labelled
correspond with pulegone after the elimination of their pulegone into labelled p-menthan-3-ones and mentho-
thiol and thiol acetate groups. These findings might sug- furan in A. crenulata. For clarity, the different labelling
gest labelled isopulegone to be a suitable precursor of patterns are omitted
labelled 3-oxo-p-menthan-8-thio-compunds, rather than
labelled pulegone, but it must be considered that iso-
pulegone might be isomerized to pulegone in the feed-
ing solution. In addition, isopulegone is also discussed
as a pulegone precursor in biosynthesis. Nevertheless, O
these results give a first hint of 3-oxo-p-menthan-8-thio
compounds biosynthesis, although further investigations
must be done.
All the results presented are summarized in biosyn-
thetic pathways of Agathosma crenulata (Schemes 1, 2), (?)
taking into consideration both stereochemical and mech- O O O
anistic aspects as a basis for further investigations on
buchu essential oil biosynthesis.

Conclusions
Agathosma crenulata (oval buchu) was able to con-
vert labelled pulegone enantiomers to corresponding
labelled menthone and isomenthone in an unspecific
manner, as conclusively demonstrated by in vivo feed-
ing experiments. Also, labelled menthofuran was syn-
thesized from labelled pulegone. During this conver-
sion, an enantiodiscrimination was observed favour-
ing labelled (1R)-pulegone. Mechanistic studies with
18 2
O/ H-labelled pulegone can be explained as enzymatic
hydroxylation of labelled pulegone. Labelled isopule-
gone, which was found to be (1S)-configured (>99%),
is discussed as a precursor of labelled pulegone. In this
context, further investigations with pure diastereomers
O O
SAc
SH
Scheme 2. Proposed biosynthetic pathway of labelled
monoterpenoids in A. crenulata; observed (!);
assumed (- - !); not observed (- //- !); non-enzymatic
conversion observed (?). For clarity, the different
labelling patterns are omitted
of labelled isopulegone are interesting. Also, further
insight into 3-oxo-p-menthan-8-thio-compound biosyn-
thesis might be drawn. The method of in vivo feed-
ing experiments should also be used to reveal the

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 123–135
 MONOTERPENE BIOSYNTHESIS IN AGATHOSMA CRENULATA
common biosynthetic precursor of diosphenol and men-
thone/isomenthone, which has been suggested by Collins
and Graven in the context of chemotaxonomic studies on
buchu.14
Acknowledgements—Financial support by the Deutsche Forschungs-
gemeischaft (DFG) is gratefully acknowledged. The authors wish to
thank W. Girnus, Botanical Garden of Frankfurt University, for culti-
vating the buchu plants; Dr G. Krammer (Haarmann & Reimer), who
kindly provided a research sample of buchu oil; and Dr P. Kreis, for
kindly providing folia bucco (conc., EB 6).
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