Biogenetic studies in Mentha x piperita. 1. Deuterium-labeled monoterpene ketones: Synthesis and stereoselective analysis

Article abstract of DOI:10.1021/jf990132f

Pulegone, menthone, and isomenthone isotopomers are synthesized as regioselectively deuterated d₅- and d₈-stereoisomers. Deuterium-labeled menthone and isomenthone enantiomers are analyzed using enantioselective multidimensional gas chromatography/mass spectrometry. The deuterated stereoisomers of menthone and isomenthone are separated from the unlabeled menthone and isomenthone on a glass capillary column, coated with 50% octakis(2,3-di-O-butyryl-6-O-tertbutyldimethylsilyl)-γ-cyclodextrin in OV 1701vi as the chiral stationary phase. These deuterium-labeled monoterpene ketones are proved to be highly valuable substrates in biosynthetic studies of terpenoid compounds.

Full text of DOI:10.1021/jf990132f

J. Agric. Food Chem. 1999, 47, 3053−3057
3053
Biogen etic Stu d ies in Men th a × piper ita . 1. Deu ter iu m -La beled
Mon oter p en e Keton es: Syn th esis a n d Ster eoselective An a lysis
Sabine Fuchs, Thomas Beck, Stefan Burkardt, Martin Sandvoss, and Armin Mosandl*
Institut fu¨r Lebensmittelchemie, J ohann Wolfgang Goethe-Universita¨t Frankfurt, Marie-Curie-Strasse 9,
D-60439 Frankfurt (Main), Germany
Pulegone, menthone, and isomenthone isotopomers are synthesized as regioselectively deuterated
d5- and d8-stereoisomers. Deuterium-labeled menthone and isomenthone enantiomers are analyzed
using enantioselective multidimensional gas chromatography/mass spectrometry. The deuterated
stereoisomers of menthone and isomenthone are separated from the unlabeled menthone and
isomenthone on a glass capillary column, coated with 50% octakis(2,3-di-O-butyryl-6-O-tert-
butyldimethylsilyl)-γ-cyclodextrin in OV 1701vi as the chiral stationary phase. These deuterium-
labeled monoterpene ketones are proved to be highly valuable substrates in biosynthetic studies of
terpenoid compounds.
Keyw or d s: Deuterium-labeled monoterpene ketones; stereoselective synthesis; enantioselective
multidimensional gas chromatography/ mass spectrometry (enantio-MDGC/ MS)
INTRODUCTION
Recently, feeding experiments with deuterium-labeled
monoterpenes were found to be most efficient tools for
in vivo biogenetic studies on plants (Wu¨st et al., 1996,
1998). Extending such investigations to Mentha species,
deuterium-labeled oxygenated p-menthanes are ex-
pected to serve as suitable precursors.
In this paper different syntheses of deuterium-labeled
pulegones, menthones, and isomenthones are reported.
The deuterium-labeled menthone and isomenthone ste-
reoisomers (Figure 1) are separated from their unla-
beled analogues using enantioselective multidimen-
sional gas chromatography/mass spectrometry (enantio-
MDGC/MS).
MATERIALS AND METHODS
En a n tioselective Ga s Ch r om a togr a p h y (En a n tio-GC).
An HP 5890 Series II gas chromatograph, equipped with a
duran glass capillary (10 m × 0.25 mm i.d.) coated with 30%
F igu r e 1. Enantiomers of menthone (1, 2) and isomenthone
(3, 4).
heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-â-cyclo-
dextrin in SE 52 (film thickness ) 0.5 µm), was used for the
enantioseparation of d5-pulegone. Conditions were as follows:
carrier gas, hydrogen at 50 kPa; split, 30 mL/min; injector
temperature, 280 °C; detector, FID, 240 °C; oven temperature,
80 °C, raised at 2 °C/min to 200 °C (30 min isothermal).
Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y (GC/MS).
The GC/MS analysis of the synthesized monoterpenes was
carried out with a Fisons Instruments GC 8065, coupled to a
Fisons Instruments MD 800 mass spectrometer, equipped with
a BPX-5 fused silica column (50 m × 0.25 mm i.d., film
thickness ) 0.25 µm; SGE-Analytic, Weiterstadt, Germany;
column A) or with a fused silica capillary coated with SE 52
(30 m × 0.25 mm i.d., film thickness ) 0.5 µm; column B).
Conditions were as follows: carrier gas, helium, column A 107
kPa, column B 70 kPa; split, 30 mL/min; injector temperature,
230 °C; oven temperature, 40 °C (5 min isothermal) raised at
2.5 °C/min to 250 °C (30 min isothermal); ion source temper-
ature, 200 °C; interface temperature, 250 °C; mass range, 40-
250 amu; EI, 70 eV. The molecular ion (M+) and the fragmen-
tation ions were given as m/z with relative peak intensities to
the base peak (percent).
En an tio-MDGC/MS. The enantio-MDGC/MS analyses were
performed with a Siemens SiChromat 2, equipped with two
independent column oven programs and a live-T-switching
device. The main column was coupled to the transfer line of a
Finnigan MAT ITD 800, using an open split interface. GC
conditions were as follows: precolumn, duran glass capillary
(30 m × 0.23 mm i.d.) coated with a 0.23 µm film of SE 52;
carrier gas, hydrogen at 120 kPa; split, 8 mL/min; injector
temperature, 220 °C; detector, FID, 250 °C; oven temperature,
50 °C (5 min isothermal) raised at 5 °C/min to 250 °C (15 min
isothermal); cut times, 24.5-28.5 min; main column, duran
glass capillary (30 m × 0.23 mm i.d.) coated with a 0.23 µm
film of 50% octakis(2,3-di-O-butyryl-6-O-tert-butyldimethylsi-
lyl)-γ-cyclodextrin in OV 1701vi (Schmarr, 1992); carrier gas,
hydrogen at 100 kPa; oven temperature, 60 °C (5 min
isothermal) raised at 2 °C/min to 100 °C (0 min isothermal)
and then at 1 °C/min to 135 °C; detector, ITD 800; transfer
line, 250 °C; open split interface, 250 °C; helium sweeping flow,
1 mL/min; ion trap manifold, 230 °C; EI 70 eV.
10.1021/jf990132f CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/14/1999

3054 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Fuchs et al.
Sch em e 1. Syn th esis of th e d 8-La beled P r ecu r sor
P u legon e a n d d 8-La beled p-Men th a n -3-on e Refer en ce
Com p ou n d s, Sta r tin g fr om P u legon e En a n tiom er s
Syn th esis of d 5-La beled P u legon e. Synthesis of 6-Methyl-
5-[1,1,1,3,3-2H5]hepten-2-one, 14. Compound 13 (10.8 mmol,
1.36 g) was dissolved in a solution of 7 mmol (160 mg) of
sodium metal in 80 mL of CH3OD. This solution was stirred
for 72 h at room temperature. The CH3OD was removed by
distillation over a Vigreux column, and 8 mL of D2O was added
to the residue. This solution was extracted three times with
diethyl ether, and the combined diethyl ether extract was
washed twice with water and dried over sodium sulfate to give
8 mmol (1.05 g) of crude 14 after removal of the solvent. MS
131 (5, M+), 113 (95), 95 (60), 69 (60), 46 (100).
Synthesis of (Z)-3,7-[9,9,9-2H3]Dimethyl-2,6-[4,4-2H2]ethyl
Octadienoate (d5-Ethyl Nerate, 15a ), and (E)-3,7-[9,9,9-2H3]-
Dimethyl-2,6-[4,4-2H2]ethyl Octadienoate (d5-Ethyl Geranate,
15b). An NaH suspension (ω ) 60%, 420 mg, 10.5 mmol of
NaH) was washed three times with pentane under nitrogen
atmosphere. After addition of 9 mmol (1.6 g) of trieth-
ylphosphonoacetate (TEPA), the mixture was heated under
reflux for 1 h. Eight millimoles (1.05 g) of 14 was dissolved
in 6 mL of dry diethyl ether and added to the solution,
which was then refluxed for 6 h. Thereafter, 10 mL of H2O
was carefully added, and the aqueous phase was washed
twice with diethyl ether. The combined diethyl ether solu-
tions were washed with brine and dried over sodium sulfate.
Yield, 6.3 mmol (1.27 g) of crude 15a /15b ; diastereomeric
ratio, 15a (29%), 15b (71%), proved by GC/MS; MS (15a ) 201
(1, M+), 200 (1), 156 (7), 133 (22), 127 (29), 105 (36), 87 (36),
69 (100).
Synthesis of (R/ S)-3,7-[9,9,9-2H3]Dimethyl-6-[4,4-2H2]ethyl
Octenoate (d5-Ethyl Citronellate, 16). The reduction procedure
is deduced as reported by Semmelhack and Stauffer (1975).
Compound 15a /15b (1.8 mmol, 360 mg), dissolved in 8 mL of
dry THF, was added to a suspension of 20.1 mmol (2.88 g) of
copper bromide and 16.7 mmol (4.8 mg) of sodium dihydri-
dobis(2-methoxyethoxy)aluminate (RedAl) in 30 mL of dry
THF at -20 °C under nitrogen atmosphere. The solution was
stirred at -20 °C for 1 h and for 1 h at room temperature;
subsequently, 8 mL of H2O was added carefully. After the
addition of diethyl ether (30 mL), the slurry was filtered.
The diethyl ether phase was extracted with saturated am-
monium chloride solution, water, and brine and dried over
sodium sulfate. Yield, 1.3 mmol (0.25 g) of crude 16; MS 203
(1, M+), 202 (1), 157 (20), 113 (19), 111 (21), 98 (29), 84 (34),
69 (100).
1H NMR. A Bruker ARX 300, 300 MHz, was employed for
recording the 1H NMR spectra. CDCl3 was used as solvent;
abbrevations: s ) singlet, d ) doublet, m ) multiplet, J )
spin-spin coupling constant (Hz), a ) axial, e ) equatorial.
The terpene nomenclature, given in Scheme 1 and Figure 1,
was used for assignment.
Synthesis of (R/ S)-3,7-[9,9,9-2H3]Dimethyl-6-[4,4-2H2]octenol
(d5-Citronellol, 17). The reduction was carried out according
to the method of Ma´lek (1988). RedAl (4.3 mmol, 1,3 mg) was
reacted with a solution of 16 in 5 mL of dry diethyl ether under
nitrogen atmosphere at 0 °C. The solution was stirred 2 h at
room temperature before 1.5 mL of H2O was added. The
solution was filtered through a hydrophobic filter, yielding 0.8
mmol (0.13 g) of crude 17. MS 142 (7), 99 (13), 84 (49), 69 (100),
56 (22).
Syn th esis of d 8-La beled P u legon e. Synthesis of 5-(R)-2-
[1-[2H 3]Meth yl[2,2,2-2H 3]eth ylid en e]-5-m eth yl[6,6-2H 2]-
cyclohexanone [d8-(R)-Pulegone, 7] and 5-(S)-2-[1-[2H3]Methyl-
[2,2,2-2H3]ethylidene]-5-methyl[6,6-2H2]cyclohexanone [d8-(S)-
Pulegone, 8]. The method of Gibson (1983) was applied to
pulegone. Three millimoles (470 mg) of 5 (Fluka, Deisenhofen,
Germany) was added to a solution containing 2 mL of CH3-
OD, 0.9 mL of D2O, and 1.3 mmol (30 mg) of sodium metal.
The mixture was heated to reflux for 1.5 h, cooled, diluted with
diethyl ether, and extracted twice with water, once with brine.
After drying over sodium sulfate, the diethyl ether was
removed in a vacuum. The product was purified by flash
chromatography. Chromatographic conditions were as fol-
lows: column diameter, 20 mm; silica gel, 30-60 µm (Baker
7024-01); eluent, pentane/diethyl ether 10:1 (v/v). After re-
moval of the eluent, 1.2 mmol (190 mg) of 7 was obtained; 1.8
mmol (280 mg) of 6 (Fluka), 1.2 mL of CH3OD, 2.5 mL of D2O,
and 0.8 mmol (18 mg) of sodium metal lead to 0.4 mmol (64
mg) of 8 after purification. MS (7) 160 (82, M+), 159 (23), 142
(34), 117 (53), 114 (64), 88 (59), 82 (100), 70 (50); 1H NMR (7)
δ 1.00 (d, 3H, 7-H, J ) 6.5 Hz), 1.33 (m, 1H, 6a-H), 2.25 (m,
1H, 5a-H), 2.71 (dd, 1H, 5e-H), 1.84-2.00 (m, 1H, 6e-H). Traces
of the isopropylidene protons were found at δ 1.79 (9-H) and
1.84-2.00 (10-H). Traces of the 2-H protons were found at δ
1.84-2.00 (Tori et al., 1975; Bambrige et al., 1995).
Synthesis of 5-(R/ S)-2-Isopropylidene-5-[2H3]methyl-[4,4-
2H2]cyclohexanone (d5-Pulegone, 19). The method of Corey et
al. (1976) was used in a modified cleanup procedure. Com-
pound 17 (0.8 mmol, 0,13 g) was added to a suspension of 2.5
mmol (0.53 g) of pyridinium chlorochromate (PCC) in 3 mL of
dry methylene chloride. After 36 h of stirring, pentane/diethyl
ether 1:1 (v:v) was added and the suspension filtered (Celite).
The solution was washed with 10% HCl, 10% NaHCO3, and
H2O and dried over sodium sulfate. The crude product, d5-
isopulegone (18) (0.5 mmol, 82 mg), was dissolved in 3 mL of
ethanolic sodium hydroxide (0.14 mol/L). The solution was
refluxed for 1 h, subsequently diluted with water, and ex-
tracted with diethyl ether. The organic solution was washed
with 10% HCl, washed twice with H2O, and dried over sodium
sulfate. Purification was done by preparative thin-layer chro-
matography (TLC); chromatographic conditions were as fol-
lows: silica gel 60 F 254 (Merck); mobil phase pentane/diethyl
ether 10:1 (v/v); detection, UV 254 nm; Rf 0,4; 0.02 mmol (3
mg) of pure 19 was obtained. MS (18) 157 (16, M+), 156 (13),
126 (51), 111 (99), 94 (63), 68 (100), 67 (98), 53 (46). MS (19)
157 (15, M+), 156 (34), 141 (12), 111 (44), 85 (100), 67 (47). 1H
NMR δ 1.79 (s, 3H, 9-H), 1.99 (s, 3H, 10-H), 1.9-2.1 (m, 2H,

Deuterium-Labeled Monoterpene Ketones
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3055
F igu r e 3. Enantio-MDGC/MS analysis of a standard mixture
of unlabeled and labeled d8-menthone and d8-isomenthone
(main column chromatogram).
mmol) was added and stirred for 5 min. Water (3.5 mL) and
dry toluene (1.7 mL) were added, and the resulting layers were
mixed by vigorous stirring. The aqueous solution was extracted
with two portions of 2 mL of dry toluene. The combined toluene
solutions were extracted four times with 0.4 mL of water. All
of these extractions were done under nitrogen atmosphere. The
solvent was removed under reduced pressure. The resulting
crude (R)-BINAP was dissolved in 5 mL of dry methanol, and
this solution was used without further purification.
Synthesis of (R)-3,7-[9,9,9-2H3]Dimethyl-6-[4,4-2H2]octenol
[(R)-d5-Citronellol, 17a )] and (S)-3,7-[9,9,9-2H3]Dimethyl-6-[4,4-
2H2]octenol [(S)-d5-Citronellol, 17b] According to the Modified
Procedure of Takaya et al. (1987). Compound 24a (0.7 mmol,
110 mg) was dissolved in a nitrogen atmosphere in 50 mL of
dry methanol. (R)-BINAP in methanol (2 mL) was added. The
solution, in a stainless steel autoclave, was stirred at room
temperature for 94 h at 30 bar of hydrogen. The methanol was
removed under reduced pressure, and pentane was added.
After filtration through Celite 545, the solvent was removed,
giving 0.7 mmol (112 mg) of crude 17a . Compound 24b (1.8
mmol, 285 mg) was dissolved in 2 mL of dry methanol. The
complete solution of (R)-BINAP in 5 mL of dry methanol was
added. After stirring at 30 bar of hydrogen at room temper-
ature for 66 h and working up as described above, 0.7 mmol
(111 mg) of crude 17b was obtained.
F igu r e 2. Mass spectra of genuine isomenthone (3), d8-
isomenthone (11), and d5-isomenthone (23).
1-H, 2a-H), 2.22 (d, 1H, 5a-H, J ) 15.5 Hz), 2.51 (dd, 1H, 2e-
H, J 1,2e ) 3.5 Hz, J 2e,2a ) 14.0 Hz), 2.71 (d, 1H, 5e-H, J ) 15.5
Hz). Traces of 7-H were found at δ 1.0, of 6a-H at δ 1.3, and
of 6e-H at δ 1.8.
Separation of d5-Ethyl Nerate 15a and d5-Ethyl Geranate
15b. Cyclic preparative HPLC was employed for the separa-
tion. Chromatographic conditions were as follows: Sep Tech
TM pump, 70 mL/min; Knauer variable wavelength monitor
210 nm (system: Cyclomat, Merck, Darmstadt, Germany);
column, LiChrospher Si 60 (self-packed; Merck), 250 mm ×
50 mm i.d., 12 µm particle size; eluent, pentane/diethyl ether
40:1 (v/v). After separation of 0.82 g of 15a /15b, 1.1 mmol (219
mg) of pure 15a and 2.4 mmol (479 mg) of pure 15b were
obtained.
Synthesis of 5-(R)-2-Isopropylidene-5-[2H3]methyl-[4,4-2H2]-
cyclohexanone d5(R)-Pulegone, 19a ) and 5-(S)-2-Isopropylidene-
5-[2H3]methyl-[4,4-2H2]cyclohexanone d5(S)-Pulegone, 19b). Start-
ing from 0.7 mmol (112 mg) of 17a , 0.15 mmol (1 mg) of pure
19 was obtained after purification by TLC. The enantiomeric
distribution 19a :19b was 27:73 (enantio-GC). 17b (0.7 mmol,
111 mg) yielded 0.07 mmol (10.7 mg) of pure 19b. The
enantiomeric distribution 19b:19a was found by enantio-GC
1
to be 92:8. H NMR (19b) δ 1.79 (s, 3H, 9-H), 1.99 (s, 3H, 10-
H), 1.9-2.1 (m, 2H, 1-H, 2a-H), 2.25 (d, 1H, 5a-H, J ) 15.5
Hz), 2.50 (dd, 1H, 2e-H, J 1,2e ) 3.5 Hz, J 2e,2a ) 14.0 Hz), 2.71
(d, 1H, 5e-H, J ) 15.5 Hz). Traces of 7-H at δ 1.0, of 6a-H at
δ 1.3, and of 6e-H at δ 1.8 were found.
Synthesis of (Z)-3,7-[9,9,9-2H3)Dimethyl-2,6-[4,4-2H2]octadienol
(d5-Nerol, 24a ) and (E)-3,7-[9,9,9-2H3)Dimethyl-2,6-[4,4-2H2]-
octadienol (d5-Geraniol, 24b) [According to the Method of Ma´lek
(1988)]. RedAl (3.7 mmol, 0.75 g) was added to a stirred, ice-
cooled solution of 1.1 mmol (217 mg) of 15a in 4 mL of dry
diethyl ether under nitrogen atmosphere. After removal of the
ice bath, the solution was stirred for 1 h at room temperature.
Water (5 mL) was added carefully, and the suspension was
diluted with diethyl ether and filtered. Yield, 0.7 mmol (110
mg) of crude 24a ; 2.4 mmol (479 mg) of 15b yielded 1.8 mmol
(285 mg) of crude 24b. MS (24a ) 141 (4), 126 (5), 97 (26), 85
(12), 69 (100), 53 (12).
Synthesis of (R)-2,2′-Bis(diphenylphosphino)-1,1′-binaphth-
yl)ruthenium Diacetate [(R)-BINAP] (Kitamura et al., 1992).
Ruthenium(II) chloride-1,5-cyclooctadiene complex (0.17 mmol/
47.3 mg) and (R)-2,2′-diphenylphosphino-1,1′-binaphthalene
(0.18 mmol/112.1 mg) were dissolved under nitrogen atmo-
sphere in 7 mL of dry DMF. After heating at 160 °C for 20
min, the solution was cooled to room temperature; 3.5 mL of
a degassed methanol solution of sodium acetate (364 mg, 29.9
Syn th esis of th e Deu ter iu m -La beled Men th on e a n d
Isom en th on e Isom er s (9-12). Synthesis of (2S,5R)-2-[1-[2H3]-
Methyl-[2,2,2-2H3]ethyl]-5-methyl-[6,6-2H2]cyclohexanone [d8-
(2S,5R)-Menthone, 10]; (2R,5R)-2-[1-[2H3]Methyl-[2,2,2-2H3]-
ethyl]-5-methyl-[6,6-2H2]cyclohexanone [d8-(2R,5R)-Isomenthone,
11]; (2R,5S)-2-[1-[2H3]Methyl-[2,2,2-2H3]ethyl]-5-methyl-[6,6-
2H2]cyclohexanone [d8-(2R,5S)-Menthone, 9], and (2S,5S)-2-[1-
[2H3]Methyl-[2,2,2-2H3]ethyl]-5-methyl-[6,6-2H2]cyclohexanone
[d8-(2S,5S)-Isomenthone, 12]. The method described by Evans
and Fu (1990) was modified as follows. Compound 7 (1.8 mmol,
280 mg) was dissolved in 2.5 mL of dry THF under nitrogen.
Catecholborane (1.9 mmol, 225 mg) was added at 0 °C. The
solution was stirred for 1 h at 25 °C, quenched by the addition
of 6 mL of acetone and 1 mL of saturated ammonium chloride
solution. The diastereomeric products were purified and
separated (two times) by column chromatography [silica gel
60; 0.063-0.200 mm particle size; eluent, pentane/diethyl
ether 6:1 (v:v)]. Compounds 10 (0.12 mmol, 20 mg) and 11 (0.02

3056 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Fuchs et al.
Sch em e 2. Syn th esis of th e d 5-La beled P r ecu r sor P u legon e a n d d 5-La beled p-Men th a n -3-on e Refer en ce
Com p ou n d s, Sta r tin g fr om d 5-6-Meth yl-5-h ep ten -2-on e
mmol, 4 mg) were obtained. Compounds 9 and 12 were
obtained when the procedure was begun from 8. MS (10) 162
(5, M+), 161 (23), 144 (26), 143 (36), 115 (100), 114 (70), 100
(29), 85 (29), 70 (59), 70 (60); 1H NMR (10) δ 1.02 (d, 3H, 7-H,
J ) 6.5 Hz), 1.3-1.5 (m, 2H), 1.8-2.2 (m, 7 H). Traces of the
isopropyl proton signals were seen at δ 0.83 and 0.89. Traces
were also seen at δ 2.34.
The enantioselective generation of (R)-d5-pulegone
(19a ) and (S)-d5-pulegone (19b) was based on the
enantioselective hydrogenation of d5-nerol (24a ) and d5-
geraniol (24b), respectively (Scheme 3). Note that in the
Wittig-Horner-Emmons reaction there was a deute-
rium migration (Wilhalm and Thomas, 1967) from the
C-1 and C-3-positions of the 6-methyl-5-[1,1,1,3,3-
2H5]hepten-2-one (14) to the C-2-position of the gener-
ated esters 15a and 15b. Due to keto-enol tautomer-
ism, the deuterium in the R-position to the carbonyl
group was lost during the following reactions, as indi-
Synthesis of (2R/ S,5R/ S)-2-Isopropyl-5-[2H3]methyl-(4,4-
2H2)cyclohexanone (20-23). Compounds 20-23 were synthe-
sized from 19 as described above. MS (21, 22) 159 (4, M+),
158 (12), 143 (17), 117 (33), 116 (100), 115 (78), 99 (53), 83
(60), 70 (93).
1
cated by the H NMR spectrum of 19, where both the
RESULTS AND DISCUSSION
signals for the C-6-protons of pulegone are detected.
Because of the migration in the Wittig-Horner-Em-
mons reaction some traces of unexchanged protons at
C-4 and C-7 are found.
The required labeled reference compounds 9-12 and
20-23 were prepared by hydroboration with catechol-
borane. The base peak shift from m/z 112 to m/z 115
and a molecular ion at m/z 162 prove the formation of
9-12. The base peak shift from m/z 112 to m/z 116/115
and a molecular ion at m/z 158/159 prove the formation
of 20-23, considering the loss of deuterium during the
Wittig-Horner-Emmons reaction.
Stable isotope labeling is known to be a suitable
technique in monitoring the metabolic pathway of
biologically active compounds, using mass selective
detection. During our investigations on the biosynthesis
of monoterpenoids in plants, deuterium-labeled precur-
sors were needed. For this purpose deuterium was
introduced into the precusor pulegone in such a way
that the base peak [m/z 112, McLafferty rearrangement
(Wilhalm and Thomas, 1965)] of the unlabeled men-
thone or isomenthone was shifted to higher m/z ratios
[m/z 115 (d8), m/z 116 (d5)] in the labeled menthone or
isomenthone, as seen from Figure 2.
In addition, isotopic effects were observed in enantio-
MDGC/MS analysis. Thus, labeled p-menthan-3-ones as
well as unlabeled p-menthan-3-ones were differentiated
not only by their mass spectra but also by their elution
behavior (Figure 3).
The required precusor d8-pulegone was readily avail-
able using pulegone enantiomers (5, 6) (Scheme 1). A
comparison of the 1H NMR of labeled and unlabeled
pulegone indicated up to 95% labeling. The precursor
(R/S)-d5-pulegone (19) was synthesized, starting from
6-methyl-5-hepten-2-one (13) (Scheme 2).
CONCLUSIONS
Deuterium-labeled pulegone, menthone, and isomen-
thone stereoisomers were prepared as suitable precur-
sors for biogenetic studies, for example, on Mentha
species. To realize these investigations, deuterium iso-
topomers of pulegone, menthone, and isomenthone were
synthesized as regioselectively labeled d5- and d8-
stereoisomers.
The separation and stereodifferentiation of labeled
and unlabeled menthone and isomenthone were achieved

Deuterium-Labeled Monoterpene Ketones
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3057
LITERATURE CITED
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2-on e
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stereoselectivity on the biosynthesis of menthone and
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in the following paper (Fuchs et al., 1999).
Received for review February 9, 1999. Revised manuscript
received April 30, 1999. Accepted May 9, 1999. Financial
support from the Deutsche Forschungsgemeinschaft (DFG) is
gratefully acknowledged.
J F990132F