Biogenetic studies in Syringa vulgaris L.: Synthesis and bioconversion of deuterium-labeled precursors into lilac aldehydes and lilac alcohols

Article abstract of DOI:10.1021/jf020845p

Syringa vulgaris L. inflorescences were fed with aqueous solutions of regioselectively deuterated compounds assumed to be precursors of lilac aldehyde and lilac alcohol, respectively. Volatiles were extracted by stir bar sorptive extraction (SBSE) and analyzed using enantioselective multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS); deuterium-labeled lilac aldehydes and lilac alcohols were separated from unlabeled stereoisomers on a fused silica capillary column, coated with heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (DIME-β-CD) (30%) in SE 52 (70%), as the chiral stationary phase. Feeding experiments with [5,5-²H₂]mevalonic acid lactone 22 and [5,5-²H₂]deoxy-D-xylose 23 indicate that the novel mevalonate independent 1-deoxy-D-xylose 5-phosphate/2C-methyl-D-erythritol 4-phosphate pathway is the dominant metabolic route for biosynthesis in lilac flowers. Additionally, bioconversion of deuterium-labeled d₅-(R/S)-linalool 3, d₆-(R)-linalool 21, d₅-(R/S)-8-hydroxylinalool 6, d₅-(R/S)-8-oxolinalool 7, d₅-lilac aldehydes 8-11 and d₅-lilac alcohols 12-15 into lilac during in vivo feeding experiments was investigated and the metabolic pathway is discussed. Incubation of petals with an aqueous solution of deuterated d₅-(R/S)-linalool 3 indicates an autonomic terpene biosynthesis of lilac flavor compounds in the flower petals of lilac.

Full text of DOI:10.1021/jf020845p

J. Agric. Food Chem. 2003, 51, 463−469 463
Biogenetic Studies in Syringa vulgaris L.: Synthesis and
Bioconversion of Deuterium-Labeled Precursors into Lilac
Aldehydes and Lilac Alcohols
MIRJAM KRECK, SUSEN PU¨ SCHEL, MATTHIAS WU¨ ST, AND ARMIN MOSANDL*
Institut fu¨r Lebensmittelchemie, Johann Wolfgang Goethe-Universita¨t,
Marie-Curie Strasse 9, D-60439 Frankfurt Main, Germany
Syringa vulgaris L. inflorescences were fed with aqueous solutions of regioselectively deuterated
compounds assumed to be precursors of lilac aldehyde and lilac alcohol, respectively. Volatiles were
extracted by stir bar sorptive extraction (SBSE) and analyzed using enantioselective multidimensional
gas chromatography/mass spectrometry (enantio-MDGC/MS); deuterium-labeled lilac aldehydes and
lilac alcohols were separated from unlabeled stereoisomers on a fused silica capillary column, coated
with heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-â-cyclodextrin (DIME-â-CD) (30%) in SE 52
(70%), as the chiral stationary phase. Feeding experiments with [5,5-2H2]mevalonic acid lactone 22
and [5,5-2H2]deoxy-D-xylose 23 indicate that the novel mevalonate independent 1-deoxy-D-xylose
5-phosphate/2C-methyl-D-erythritol 4-phosphate pathway is the dominant metabolic route for bio-
synthesis in lilac flowers. Additionally, bioconversion of deuterium-labeled d5-(R/S)-linalool 3, d6-(R)-
linalool 21, d5-(R/S)-8-hydroxylinalool 6, d5-(R/S)-8-oxolinalool 7, d5-lilac aldehydes 8-11 and
d5-lilac alcohols 12-15 into lilac during in vivo feeding experiments was investigated and the metabolic
pathway is discussed. Incubation of petals with an aqueous solution of deuterated d5-(R/S)-linalool
3 indicates an autonomic terpene biosynthesis of lilac flavor compounds in the flower petals of lilac.
KEYWORDS: Syringa vulgaris L. biosynthesis; stir bar sorptive extraction (SBSE); enantioselective
multidimensional gas chromatography/mass spectrometry (enantio-MDGC/MS); deuterium-labeled mono-
terpenes
INTRODUCTION
Volatile monoterpenes play an important role as attractants
for pollinating insects such as bee and wasp species (6). Various
studies in Rosa (7), Jasminium, and Gardenia species (8, 9)
show that monoterpenes are glycosylated and accumulated in
flower buds. Glycoconjugation allows better storage within plant
vacuoles and protects plants from any toxicity of the free
aglycon (6). In a hypothetical mechanism of volatile monoter-
pene formation Watanabe et al. (8, 10) postulated that mono-
terpenes are biosynthesized and glycosylated in the leaves and
then transported via the phloem to the flowers, where they are
accumulated in the flower buds. The glycosylated compounds
are then enzymatically hydrolyzed and transformed into vol-
atile compounds during flower opening. Recently different
authors (12-16) suggested the direct emission of monoterpenes
from floral tissue after the de novo biosynthesis. Pichersky et
al. (17, 19, 20) confirmed the localization and emission of
methyl benzolate and methyleugenol genes in Clarika, snap
dragon flower. A direct incorporation of deuterium-labeled
precursor to phenylethanol in rose flower head has also been
reported.
From the earliest to present times, fragrance compositions
of lilac are important constituents in perfumery. Until now there
is no commercially available lilac flower oil, although many
attempts have been made to produce satisfactory lilac concen-
trates (1, 2). Because no natural lilac flower oil is being
produced, synthetic lilac compounds are used in perfumery. The
common lilac (Syringa Vulgaris L., Oleaceae) originates from
southeastern Europe and Asia and has been cultivated in many
parts of Europe (1). Lilac aldehydes 8-11 and lilac alcohols
12-15 have been described as characteristic monoterpenes in
S. Vulgaris L. flowers with a desirable influence on the lilac
odor quality (3). In previous studies it has been reported that
four diastereoisomers of 2-[(5′-methyl-5′vinyl)tetrahydrofuran-
2′-yl]propanal and -propanol were assigned as the genuine
structures of lilac aldehydes and lilac alcohols (Figure 1)
(3-5), but little recent research has been carried out on the
biosynthetic origin of these monoterpenes in Syringa species.
With the extension of such investigations to S. Vulgaris L.,
deuterium-labeled linalool 3, 21, 8-hydroxylinalool 6, and
8-oxolinalool 7 are expected to be suitable precursors.
In addition to the feeding experiments that were carried out
to clarify the metabolism of linalool and linalool derivatives
leading to the formation of lilac alcohol, we also investigated
the biosynthetic origin of isopentenyl diphosphate (IPP) and
* Corresponding author (telephone 0049-69-798 29202/03; fax 0049-
69-798 29207; e-mail Mosandl@em.uni-frankfurt.de).
10.1021/jf020845p CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/10/2002

464 J. Agric. Food Chem., Vol. 51, No. 2, 2003
Kreck et al.
200 °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.
Enantio-SBSE-MDGC/MS. The previously reported system was
employed for simultaneous enantioselective analysis of the volatile
monoterpenes (24).
1H NMR. A Bruker ARX 300, 300 MHz, was employed for
recording the 1H NMR spectra. CDCl3 was used as solvent. Abbreva-
tions: s, singlet; d, doublet; m, multiplet; J ) spin-spin coupling
constant (hertz).
Synthesis of d5-Labeled (R/S)-Linalool. Synthesis of 6-Methyl-5-
[1,1,1,3,3-2H5]hepten-2-one (2). The method of Keinan et al. (25) was
applied. 6-Methyl-5-hepten-2-one 1 (3.37 mmol, 425 mg) was dissolved
in a solution of 2.17 mmol (50 mg) of sodium in 25 mL of CH3OD.
This solution was stirred for 72 h at room temperature. The CH3OD
was removed by distillation over a Vigreux column, and 1 mL of D2O
was added to the residue. This solution was extracted three times with
diethyl ether, and the combined diethyl ether extracts were washed twice
with water and dried over sodium sulfate to give 1.22 mmol (181.4
mg) of crude 2 after removal of the solvent: MS, m/z 131 (4, M+),
1
113 (89), 95 (16), 69 (100), 56 (57); H NMR δ 1.60 (s, 3H, 7-H),
1.66 (s, 3H, 8-H), 2.21 (m, 2H, 4-H), 5.05 (m, 1H, 5-H).
Synthesis of 3,7-[10,10,10-2H3]Dimethyl-1,6-octadien[4,4-2H2]-3-ol
[d5-(R/S)-Linalool (3)]. The Grignard reaction was carried out according
to the method of Keinan et al. (25). A solution of 1.5 mmol (197 mg)
of vinylmagnesium bromide in dry THF was cooled in an ice bath,
and compound 2 (1.38 mmol, 181.4 mg) dissolved in 10 mL of
anhydrous diethyl ether was added with vigorous stirring. When the
addition was complete, the mixture was stirred for 1 h and then allowed
to stand for 12 h. A saturated solution of ammonium chloride was then
added to decompose the magnesium complex formed. The organic layer
was dried over anhydrous sodium sulfate, solvents were removed, and
the residue was purified by flash chromatography. Chromatographic
conditions were as follows: silica gel 60 (Merck); mobile phase, diethyl
ether/petroleum ether 1:3 (v/v). 28.9 mg of pure 3 was obtained: MS,
Figure 1. Deuterium-labeled precursors for biogenetic studies in S.
vulgaris L.
dimethylallyl diphosphate (DMAPP), the two basic precursors
of monoterpenoids (21-23). Besides the classical mevalonate
route, a mevalonate-independent pathway for the formation of
IPP and DMAPP was recently discovered in bacteria, algae,
and plants. Therefore, the de novo biosynthesis of monoterpenes
in S. Vulgaris L. was investigated by in vivo feeding experiments
using deuterium-labeled deoxy-D-xylose 23 and mevalonic acid
lactone 22 as precursors.
1
m/z 159 (2, M+), 141 (44), 126 (85), 98 (100), 85 (90), 69 (95); H
NMR δ 1.55 (s, 3H, 7-H), 1.68 (s, 3H, 8-H), 2.02-2.05 (m, 2H, 5-H),
5.03-5.24 (m, 3H, 1-H, 2-H), 5.88-5.95 (m, 1H, 6-H).
Synthesis of d5-Labeled (R/S)-8-Hydroxylinalool. Synthesis of
(R/S)-3-Acetoxy-3,7[10,10,10-2H3]dimethyl-[4,4-2H2]-1,6-octadiene (4).
The acetylation was carried out according to the method of Vidari et
al. (26). To a solution of 1.47 mmol (234.3 mg) of 3 in 25.3 mmol (2
mL) of pyridine were added 2.14 mmol (218 mg) of Ac2O and 0.053
mmol (6.5 mg) of DMAP. The mixture was stirred at 90 °C for 44 h
and then quenched by adding CH3OH and aqueous NaHSO4. The
aqueous layer was extracted with CH2Cl2, and the combined organic
layers were dried over sodium sulfate. The residue was purified by
flash chromatography on silica gel 60 (Merck) eluted with a mobile
phase of pentane/diethyl ether 7:3 (v/v). After removal of the eluent,
272.8 mg (97%) of 4 was obtained: MS, m/z 201 (1, M+), 159 (4),
141 (42), 126 (85), 98 (100), 85 (91), 69 (98).
Synthesis of (R/S)-2,6-[10,10,10-2H3]Dimethyl-[5,5-2H2]-6-acetoxy-
2,7-octadienal (5). The method of Wakayama et al. (3) was used.
Compound 4 (1.36 mmol, 272.8 mg) and selenium dioxide (1.4 mmol,
0.155 g) were dissolved in 5 mL of dioxane/ethanol 9:1 (v/v), and the
solution was heated at 80 °C for 5 h. After filtration, the solvent was
removed under reduced pressure. The residue was treated with diethyl
ether/petroleum ether 1:1 (v/v), and after removal of the solvent, the
residue was purified by flash chromatography on silica gel 60 (Merck),
with a mobile phase of petroleum ether/diethyl ether 3:2 (v/v), affording
196 mg of compound 5: MS, m/z 215 (1, M+), 155 (11), 137 (7), 126
(14), 82 (100), 74 (95).
MATERIALS AND METHODS
Gas Chromatography/Mass Spectrometry (GC/MS). The GC/MS
analysis of the synthesized monoterpenes was carried out with a Fisons
Instruments GC 8065, coupled to a Fisons Instruments MD800 mass
spectrometer, equipped with a 30 m × 0.25 mm i.d., 0.23 µm fused
silica capillary coated with SE 52. Conditions were as follows: carrier
gas, helium, 65 kPa; split, 20 mL/min; injector temperature, 230 °C;
oven temperature, 40 °C (5 min isothermal) raised at 5 °C/min to 250
°C (30 min isothermal); ion source temperature, 200 °C; interface
temperature, 250 °C; mass range, 40-250 amu; EI, 70 eV.
The molecular ion (M+) and the fragmentation ions are given as
m/z with relative peak intensities to the base peak (percent).
Enantio-MDGC/MS. The enantio-MDGC/MS analyses were per-
formed with a Siemens SiChromat 2-8, 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 ITD800, using an
open split interface. GC conditions were as follows: precolumn, 30 m
× 0.23 mm i.d., 0.23 µm, fused silica capillary coated with SE 52;
carrier gas, helium at 155 kPa; split, 20 mL/min; injector temperature,
220 °C; detector, FID, 250 °C; oven temperature, 60 °C (10 min
isothermal) raised at 3 °C/min to 250 °C (20 min isothermal); cut times,
31.5-33.0 min for lilac aldehyde and 34.5-37.5 min for lilac alcohol;
main column, 30 m × 0.23 mm i.d., 0.23 µm, fused silica capillary
coated with heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-â-
cyclodextrin (DIME-â-CD) in SE 52; carrier gas, helium at 102 kPa;
oven temperature, 60 °C (30 min isothermal) raised at 1 °C/min to
Synthesis of (R/S)-2,6-[10,10,10-2H3]Dimethyl-[5,5-2H2]-2,7-octa-
dienol [d5-(R/S)-8-Hydroxylinalool (6)]. Compound 5 (0.91 mmol, 196
mg) was dissolved in 20 mL of dry diethyl ether, and 38.5 mg (1 mmol)
of lithium aluminum hydride was added in small portions. After 60
min of stirring at room temperature, water was added and the reaction
mixture was acidified with sulfuric acid and extracted with a mixed
solvent of diethyl ether/petroleum ether 1:1 (v/v). The organic layer
was dried over sodium sulfate. After removal of the solvent, the residue
was subjected to flash chromatography on silica gel 60 (Merck), eluted

Biogenetic Studies in Syringa vulgaris L.
J. Agric. Food Chem., Vol. 51, No. 2, 2003 465
with petroleum ether/diethyl ether 1:4 (v/v). After removal of the eluent,
was purified by Kugelrohr distillation at 125-130 °C (0.3 mbar) to
+), 113 (23), 84 (38),
39.4 mg of 6 was obtained: MS, m/z 175 (1, M+), 157 (2), 139 (11),
give 0.91 g (41.3%) of 19: MS, m/z 170 (1, M
1
71 (100).
126 (7), 98 (14), 74 (100), 84 (65); H NMR δ 1.18-1.23 (m, 2-H,
Synthesis of (R)-3-Acetoxy-3,7-dimethyl-[8,8,8,9,9,9-2H6]-1,6-octa-
diene (20). The synthesis was performed analogously to the method of
Puyn et al. (29) with a modified cleanup procedure. Isopropyl
[1,1,1,3,3,3-2H6][2 -2H1]triphenylphosphoniumbromide (2.06 g, 524
mmol), synthesized according to the method of Puyn et al., was
dissolved in 30 mL of THF and cooled at 0 °C as 3.8 mL of 1.6 M
n-butyllithium (5.24 mmol) in hexane was added. After stirring for 1
h at room temperature, the solution was cooled at 0 °C, and 0.9 g (5.24
mmol) of 19 was added and stirring was continued for 3 h. After
addition of 2 mL of methanol, the mixture was concentrated under
reduced pressure, and the residue was extracted with diethyl ether/
petroleum ether 1:1 (v/v). The solvent was removed under reduced
pressure, and the residue was purified by Kugelrohr distillation at 120
°C (0.1 mbar) to give 0.409 g (38.7%) of 20: MS, m/z 202 (1, M+),
160 (2), 142 (11), 124 (28), 93 (100), 71 (38); 1H NMR δ 1.58 (s, 3H,
10-H), 1.95 (s, 3H, 12-H), 5.05-5.17 (m, 3H, 1-H, 2-H), 5.91-6.01
(m, 1H, 6-H).
8-H), 1.66 (s, 3H, 9-H), 2.04-2.07 (m, 2H, 5-H), 5.05-5.44 (m, 3H,
1-H, 2-H), 5.87-5.96 (m, 1H, 6-H).
Synthesis of d5-Labeled (R/S)-8-Oxolinalool. Synthesis of (R/S)-
2,6-[10,10,10-2H3]Dimethyl-[5,5-2H2]-2,7-octadienal [d5-(R/S)-8-Oxo-
linalool (7)]. The method of Corey et al. (27) was used. Compound 6
(0.08 mmol, 15 mg) was added to a suspension of 0.25 mmol (53 mg)
of pyridinium chlorochromate in 5 mL of dry methylene chloride. After
36 h of stirring, pentane/diethyl ether 1:1 (v/v) was added and the
suspension was filtered over Celite. The solution was washed with 10%
HCl, 10% NaHCO3, and H2O and dried over sodium sulfate. After
removal of the solvent, the residue was purified with flash chroma-
tography on silica gel 60 (Merck), eluted with petroleum ether/diethyl
ether 1:4 (v/v). After removal of the eluent, 0.02 mmol (4 mg) of 7
was obtained: MS, m/z 173 (1, M+), 155 (5), 137 (4), 126 (5), 74
(100), 85 (22).
Synthesis of d5-Labeled (R/S)-Lilac Aldehyde. Synthesis of (R/S)-
2-[(5′-Methyl-5′-Vinyl)- [4′,4′-2H2][8′,8′,8′-2H3]tetrahydrofuran-2′-yl]-
propanal [d5-Lilac Aldehyde (8a/b-11a/b)]. In dry methanol (30 mL)
catalytic amounts (∼10 mg) of sodium were dissolved, and compound
5 (1.29 mmol, 223 mg) was added dropwise. After 48 h of stirring
under an argon atmosphere at room temperature, the reaction was
stopped by adding acidic Dowex (H+-form) until the solution was
neutralized. The residue was filtered, and the organic layer was dried
over sodium sulfate. The solvent was removed with a Vigreux column,
and the residue was purified with preparative thin-layer chromatography
(TLC) on silica gel 60 (Merck), mobile phase petroleum ether/diethyl
ether 1:4 (v/v), affording 4 mg of compound 8a/b-11a/b: MS, m/z
173 (1, M+), 155 (5), 116 (27), 97 (43), 55 (100), 74 (27).
Synthesis of (R)-3,7-Dimethyl-1,6-octadien[8,8,8,9,9,9-2H6]-3-ol
[d6-(R)-Linalool (21)]. Compound 21 was synthesized analogously to
the synthesis of compound 6 described above. The crude product was
purified by flash chromatography on silica gel 60 (Merck), mobile phase
petroleum ether/diethyl ether 3:1 (v/v), to give 0.198 g (56.7%) of 21:
MS, m/z 160 (1, M+), 142 (7), 124 (15), 93 (86), 89 (16), 75 (52), 71
(100); 1H NMR δ 1.55 (s, 3H, 10-H), 1.97-2.04 (m, 1H, 2-H), 5.03-
5.25 (m, 2H, 1-H), 5.86-5.96 (m, 1H, 6-H).
[5,5-2H2]Mevalonic Acid Lactone (22). [5,5-2H2]Mevalonic acid
lactone synthesis was performed analogously to the method of Simpson
et al. (30).
[5,5-2H2]-1-Deoxy-D-xylose (23). [5,5-2H2]-1-Deoxy-D-xylose was
prepared according to the method of Jux and Boland (31).
Synthesis of d5-Labeled (R/S)-Lilac Alcohol. Synthesis of (R/S)-
2-[(5′-methyl-5′-Vinyl)-[4′,4′-2H2][8′,8′,8′-2H3]tetrahydrofuran-2′-yl]pro-
panol [d5-Lilac Alcohol (12a/b-15a/b)]. Compound 8a/b-11a/b (0.058
mmol, 10.0 mg) was dissolved in 10 mL of dry ether, and 3 mg (0.078
mmol) of lithium aluminum hydride was added in small portions. After
2 h of stirring at room temperature, water was added and the reaction
mixture was acidified with sulfuric acid and extracted with a mixed
solvent of diethyl ether/petroleum ether 1:1 (v/v). The organic layer
was dried over sodium sulfate. After removal of the solvent, the residue
was subjected to TLC on silica gel 60 (Merck), mobile phase petroleum
ether/diethyl ether 1:1 (v/v). After removal of the eluent, 0.02 mmol
(3.5 mg) of 12a/b-15a/b was obtained: MS, m/z 175 (1, M+), 157
(9), 116 (29), 96 (55), 74 (23), 55 (100).
Plant Material. During the flowering period, cuttings from S.
Vulgaris L. were made from wild and domesticated lilac bushes with
differently colored flowers. The inflorescences were 10-15 cm high,
containing 5 cm long pedicles (∼10 g of plant material).
Feeding Experiments. The deuterium-labeled precursors d6-(R)-
linalool 21, d5-(R/S)-linalool 3, d5-(R/S)-8-hydroxylinalool 6, d5-(R/S)-
8-oxolinalool 7, d5-lilac aldehydes 8-11, and d5-lilac alcohols 12-15,
respectively, were dissolved in distilled water containing 0.1 mg/mL
Tween 20 to give a solution of 0.1 mg/mL of the monoterpene in each
case. In the case of d2-mevalonic acid lactone 22 and d2-1-deoxy-D-
xylose 23, respectively, 2 mg/mL of the precursor was dissolved in
distilled water. The inflorescences were cut off and put into the feeding
solutions. In each case, blank experiments were carried out with distilled
water containing 0.1 mg/mL Tween 20. During a period of 48 h, 3
µmol of the precursor was taken up by the plant. In addition, d5-
(R/S)-linalool 3 was fed through a cut stem into green leaves (∼10 g
of plant material). During 48 h, 7 µmol of d5-(R/S)-linalool 3 was taken
up by the leaves. Furthermore, lilac flower petals were isolated and
incubated directly with a solution containing 0.1 mg/mL d5-(R/S)-
linalool 3 and 0.1 mg/mL Tween 20.
Synthesis of d6-Labeled (R)-Linalool. Synthesis of (R)-3-Acetoxy-
3,7-dimethyl-1,6-octadiene (17). Compound 17 was synthesized analo-
gously to the synthesis of compound 4 described above, using (R)-
linalool 16 as the starting material, yielding 11.36 g of 17: MS, m/z
177 (1, M+), 154 (4), 136 (28), 121 (89), 107 (41), 93 (100), 80 (91),
69 (85).
Synthesis of (R)-3-Acetoxy-6,7-epoxy-3,7-dimethyl-1-octene (18). The
synthesis was performed analogously to the method of Manning et al.
(28). Compound 17 (57.87 mmol, 11.36 g) was stirred at 0 °C in 150
mL of methylene chloride. Then 23.72 g of 42% m-chloroperoxybenzoic
acid in methylene chloride was added dropwise. After stirring for 1
min, the mixture was filtered and washed with aqueous NaHCO3, water,
and brine and then dried. The solvent was removed under reduced
pressure, and the residue was purified by Kugelrohr distillation at 125
°C (0.3 mbar) to give 10.39 g (84.6%) of 18: MS, m/z 212 (1, M+),
Isolation of Monoterpenes. Monoterpenes were extracted using the
novel stir bar sorptive extraction (SBSE) method. Theory and practice
have been described recently (32). Stir bars were obtained from Gerstel
(Mu¨hlheim, Germany) under the trade name Twister. Inflorescences,
leaves, and flowers were homogenized with 1 mL of an aqueous 20%
NaCl solution per gram of plant material and some sea sand. The
homogenate was extracted for 60 min by SBSE, followed by extraction
of the monoterpenes with 2 mL of pentane/diethyl ether 1:1 (v/v) from
the poly(dimethylsiloxane) phase and concentration of the solution to
∼20 µL. Alternatively, the Twister was desorbed in the TDS system
and subsequently analyzed by simultaneous enantioselective analysis
of the volatile compounds (enantio-SBSE-MDGC/MS). In addition to
this procedure, the volatile compounds of the homogenates were isolated
using steam distillation (Deutsches Arzneibuch, DAB 9).
1
155 (2), 137 (6), 94 (48), 79 (98), 71 (100); H NMR δ 1.26 (s, 3H,
8-H), 1.29 (s, 3H, 9-H), 1.54 (s, 3H, 10-H), 2.00 (s, 3H, 12-H), 5.11-
5.18 (m, 2H, 1-H), 5.87-6.01 (m, 1H, 6-H).
Synthesis of (R)-4-Acetoxy-4-methyl-5-hexenal (19). The method of
Manning et al. (28) was used. Compound 18 (14.2 mmol, 3 g) was
dissolved in 15 mL of dry diethyl ether, and powdered periodic acid
(14.2 mmol, 3.23 mg) was added portionwise under vigorous stirring.
The mixture was stirred for 30 min, and the organic layer was separated
and washed with water, aqueous NaHCO3, and brine. After drying,
the organic layer was removed under reduced pressure, and the residue
RESULTS AND DISCUSSION
Feeding experiments with aqueous solutions of deuterium-
labeled monoterpenes have proven to be efficient methods for

466 J. Agric. Food Chem., Vol. 51, No. 2, 2003
Kreck et al.
Figure 2. Synthesis of the precursors d5-linalool, d5-8-hydroxylinalool, d5-
8-oxolinalool, d5-lilac aldehyde, and d5-lilac alcohol.
in vivo studies of biogenesis. For this purpose deuterium was
introduced into the precursors (R/S)-linalool 3, (R/S)-8-hy-
droxylinalool 6, and (R/S)-8-oxolinalool 7 (Figures 1 and 2),
so that the base peak of the corresponding unlabeled lilac alco-
hol and lilac aldehyde (m/z 111) was shifted to higher m/z
ratios [m/z 116 (d5)] (see Figure 3A,B). (R)-d6-Linalool 21 is
labeled at the isopropylidene group, so that the fragment m/z
155 of lilac alcohol was shifted to m/z 159 (d4) and the frag-
ment m/z 153 of lilac aldehyde to m/z 157 (d4), as can be seen
in Figure 3C. During cyclization, lilac aldehyde and lilac
alcohol lose two deuterium atoms, yielding the d4-labeled
compounds.
Figure 3. Mass spectra of (A) genuine lilac compounds, (B) d5-labeled
lilac compounds, and (C) d4-labeled lilac compounds.
synthesis of lilac aldehyde and lilac alcohol with (R)-linalool
as starting material, which leads to the diastereoisomers 8b, 9b,
10a, and 11a and 12a, 13a, 14b, and 15a with 5′-(R) config-
uration. Further investigations, including X-ray structure analysis
of derivatives, are in progress.
Due to the low concentration of some volatile monoterpenes
in S. Vulgaris L., SBSE was chosen as a highly sensitive
isolation procedure of the target compounds lilac aldehydes and
lilac alcohols (32). The volatile monoterpenes were extracted
from the phase, and the concentrated solution was analyzed
using GC/MS and enantio-MDGC/MS, which allows a selective
and sensitive analysis of the chiral compounds. This method
leads to the same results as the employed direct desorption of
the Twister in the enantio-SBSE-MDGC/MS system and the
analysis of the steam distilled solution of the volatile compounds.
Only the sensitivities of the methods differ: Twister extraction
or direct Twister desorption in the TDS system are more
sensitive than direct steam distillation. In vivo feeding experi-
ments with suitable precursors were used to differentiate
between the classical cytosolic mevalonic acid pathway and the
novel plastidic mevalonate-independent 1-deoxy-D-xylose 5-phos-
phate/2C-methyl-D-erythritol 4-phosphate pathway. To deter-
mine the biosynthetic origin of the basic building units, IPP
and DMAPP [5,5-2H2]mevalonic acid lactone 22 and [5,5-2H2]-
1-deoxy-D-xylose 23 were chosen for incorporation studies,
which are frequently used for such purposes (23, 34-38). In
our in vivo feeding experiments aqueous solutions of the labeled
precursors [5,5-2H2]-1-deoxy-D-xylose 23 and [5,5-2H2]mevalonic
acid lactone 22 were administered to lilac inflorescences using
In addition, inverse isotopic effects were observed during the
enantio-MDGC/MS analysis (33). The deuterium-labeled lilac
alcohol isotopomers showed a shift of their peak maximum of
∼4 s compared with the unlabeled analytes.
The first enantioselective analysis of the eight stereoiso-
mers of lilac aldehydes 8a/b-11a/b and of lilac alcohols
12a/b-15a/b, respectively, and the main column chromato-
gram obtained from Twister extraction of lilac flowers with the
four genuine 5′-(S)-configured lilac aldehyde diastereoisomers
8a, 9a, 11b, 10b and 12b, 13b, 14a, and 15b as 5′-(S)-
configured lilac alcohol diastereoisomers is shown in Figure
4. Six of the eight stereoisomers of lilac alcohol were well
separated on this phase, only the stereoisomers 12a/b remaining
unresolved.
We were not able to assign the absolute configurations of
the eight stereoisomers, but it is clear that the stereoisomers
8a, 9a, 11b, and 10b and 12b, 13b, 14a, and 15b were 5′-(S)-
configured and their corresponding enantiomers 8b, 9b, 10a,
and 11a and 12a, 13a, 14b, and 15a were 5′-(R)-configured.
These assignments were concluded from an enantioselective

Biogenetic Studies in Syringa vulgaris L.
J. Agric. Food Chem., Vol. 51, No. 2, 2003 467
Figure 6. SBSE-enantio-MDGC/MS analysis of S. vulgaris L. fed with
(R/S)-[4,4,10,10,10-2H ]-linalool 3 (main column chromatogram): (A) m/z
5
157; (B) m/z 155.
tion of one or two units of labeled IPP/DMAPP that are
generated from administered [5,5-2H2]-1-deoxy-D-xylose 23,
leading to d2- and d4-lilac alcohols, respectively. These results
are in agreement with comparable studies of IPP incorporation
from [5,5-2H2]-labeled 1-deoxyxylose 23 into linalool (23).
When [5,5-2H2]mevalonic acid lactone 22 is administered to
lilac inflorescences, incorporation into labeled lilac aldehydes
and lilac alcohols was not detectable. This indicates that
monoterpenes in S. Vulgaris L. are not generated by the MVA
pathway. The novel plasidic DOXP/MEP pathway is the
dominant route for monoterpene biosynthesis in lilac inflores-
cences. Besides the feeding experiments that were carried out
to clarify the biosynthetic origin of IPP/DMAPP, we also
investigated the metabolism of labeled linalool and linalool
derivatives leading to the formation of lilac alcohol. These in
vivo feeding experiments were carried out with aqueous
solutions of the labeled precursors (R/S)-[4,4,10,10,10-2H5]-
linalool 3, (R)-[8,8,8,9,9,9-2H6]linalool 21, (R/S)-[5,5,10,10,10-
2H5]-8-hydroxylinalool 6, and (R/S)-[5,5,10,10,10-2H5]-8-
oxolinalool 7, administered to lilac influrescences using the cut
stem method. Figure 6 shows the enantioselective GC pattern
of lilac aldehydes and lilac alcohols obtained from the flower
extract when (R/S)-d5-linalool 3 was administered. A significant
incorporation of (R/S)-d5-linalool 3 into lilac aldehydes and lilac
alcohols is detectable for m/z 155 and 157, respectively.
Surprisingly, (R/S)-d5-linalool 3 was incorporated in all four
diastereoisomeric enantiomeric pairs of lilac aldehyde and lilac
alcohol. It is noteworthy that during incorporation of racemic
deuterium-labeled linalool an enantiodiscrimination was ob-
served. In particular, the enantiomeric pairs 13a/13b and 10a/
10b show a discrimination in favor of the nongenuine 5′-(R)-
configured enantiomers of 10a and 13a. Thus, it seems that the
metabolism of linalool leading to the formation of lilac aldehyde
and lilac alcohol exhibits a very low enantioselectivity. As a
consequence, the enantiomeric purity of the genuine monoter-
penes must result from a previous highly stereospecific enzy-
matic step. When enantiopure deuterium-labeled (R)-d6-linalool
21 (R > 98%) was fed alternatively, only the four nongenuine
5′-(R)-configured lilac aldehyde and lilac alcohol stereoisomers
were generated, corresponding to the absolute configuration at
C-3 (R) of the precursor (Figure 7). Hence, a high enantiomeric
purity of genuine linalool in S. Vulgaris species with C-3 (S)
configuration can be concluded, which leads to the genuine four
5′-(S)-configured diastereoisomers of both monoterpenes. Enan-
tioselective analysis of genuine linalool in lilac confirms this
supposition, as linalool shows a high enantiomeric purity in
favor of the (S) enantiomer (>99%) (Figure 8). This is also in
Figure 4. SBSE-enantio-MDGC/MS analysis of lilac aldehyde and lilac
alcohol stereoisomers: (A) main column chromatogram of the reference
solution, resulting in eight stereoisomers of lilac aldehyde and lilac alcohol,
respectively; (B) main column chromatogram of the genuine lilac aldehyde
8a, 9a, 10b, and 11b and lilac alcohol 12b, 13b, 14a, and 15b
stereoisomers in S. vulgaris L.
Figure 5. SBSE-enantio-MDGC/MS analysis of S. vulgaris L. fed with
[5,5-2H ]-deoxy-D-xylose (DOX) 22 (main column chromatogram): (A) m/z
2
TIC; (B) m/z 157; (C) m/z 159.
the cut stem method. Figure 5 shows the main column
chromatogram of lilac aldehydes and lilac alcohols obtained
from the flower extract when [5,5-2H2]-1-deoxy-D-xylose 23 is
administered. The four diastereoisomers (5′S)-[3′,3′,7′,7′-2H4]-
lilac aldehyde and lilac alcohol are clearly detectable and well
separated from the four genuine (5′S)-lilac aldehyde and lilac
alcohol steroisomers, due to the inverse isotope effect in gas
chromatography of deuterium-labeled compounds (38). Also,
diastereoisomers of (5′S)-[3′,3′-2H2]lilac alcohols and (5′S)-
[7′,7′-2H2]lilac alcohols are detectable in the mass spectra. The
fragmentation patterns of the deuterium-labeled lilac alcohol
and aldehyde isotopomers are in agreement with the incorpora-

468 J. Agric. Food Chem., Vol. 51, No. 2, 2003
Kreck et al.
Figure 7. SBSE-enantio-MDGC/MS analysis of S. vulgaris L. fed with
(R)-[8,8,8,9,9,9-2H ]-linalool 21 (main column chromatogram): (A) m/z 157;
6
(B) m/z 155.
Figure 9. Biogenetic pathway of the monoterpenes lilac aldehyde and
lilac alcohol in S. vulgaris L.
MEP pathway, which leads to enantiopure (S)-linalool. (S)-
Linalool must be enzymatically hydroxylated leading to 8-hy-
droxylinalool, which is subsequently oxidized to 8-oxolinalool.
Cyclization of 8-oxolinalool leads to the four 5′-(S)-configured
diasteroisomers of lilac aldehyde, which must be enzymatically
reduced to the corresponding 5′-(S)-configured lilac alcohol
stereoisomers. The biogenetic pathway is summarized in Figure
9. In addition to these investigations of the biogenetic pathways
of lilac aldehydes and lilac alcohols, our interest was focused
upon the location of biosynthesis in S. Vulgaris L. To investigate
the localization of the biosynthesis of monoterpenes in lilac,
feeding experiments with detached flower petals, which were
directly incubated in solutions of d5-(R/S)-linalool 3 and
experiments with green leaves using the cut stem method were
carried out. It is noteworthy that the incubation experiment
with flower petal leads to the same results as the feeding
experiments with inflorescences as discussed previously, namely,
enantiodiscrimination in favor of some nongenuine 5′-(R)-
configured enantiomers. When d5-(R/S)-linalool 3 was admin-
istered to green leaves using the cut stem method, no deuterated
lilac aldehydes and lilac alcohols were detectable, although the
genuine monoterpenes could be observed in green leaves.
However, glycosylated monoterpenes cannot be detected with
our method, but it is obvious that flower inflorescences and
flower petals of S. Vulgaris L. are capable of de novo bio-
synthesis of volatile monoterpenes.
Figure 8. Enantio-MDGC/MS analysis of linalool: (A) main column
chromatogram of a reference solution of (R/S)-linalool; (B) main column
chromatogram of genuine linalool in S. vulgaris L.
agreement with the incorporation of deuterium-labeled 1-de-
oxy-D-xylose 23 into the corresponding genuine 5′-(S)-
configured lilac aldehydes and lilac alcohols with high enan-
tiomeric purity.
When racemic (R/S)-[5,5,10,10,10-2H5]-8-hydroxylinalool 6
was administered to lilac inflorescences, deuterated lilac alde-
hydes and lilac alcohols were clearly detectable. Enantioselective
analysis of the deuterated monoterpenes shows that the four
genuine and the four nongenuine lilac aldehyde stereoisomers
were generated. Feeding experiments with (R/S)-[5,5,10,10,10-
2H5]-8-oxolinalool 7 led to the same results. The deuterated
monoterpene was incorporated into the four diastereoisomeric
pairs of lilac aldehyde and lilac alcohol. Thus, the metabolism
of this monoterpenol proceeds with remarkably low substrate
specifity. When the eight stereoisomers of deuterium-labeled
lilac alcohols 12-15 were administered to lilac inflorescences
using the cut stem method, no incorporation into lilac alde-
hydes could be detected. Feeding experiments with the eight
stereoisomers of deuterated d5-lilac aldehydes 8-11 showed a
significant incorporation into the eight deuterated lilac alcohol
stereoisomers. Hence, it follows that lilac aldehyde is the
biogenetic precursor of lilac alcohol. Considering these results,
the biogenetic pathway of the four lilac alcohol stereoisomers
starts with the biogenesis of IPP and DMAPP by the DOXP/
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