Biotransformation of (-)-dihydromyrcenyl acetate using the plant parasitic fungus Glomerella cingulata as a biocatalyst

Article abstract of DOI:10.1021/jf000843+

The microbial transformation pf (-)-dihydromyrcenyl acetate was investigated using the plant parasitic fungus Glomerella cingulata. As a result, (-)-dihydromyrcenyl acetate was converted to dihydromyrcenol, 3,7-dihydroxy-3,7-dimethyl-1-octene-7-carboxylate, 3,7-dihydroxy-3,7-dimethyl-1-octene, 3,7-dimethyloctane-1,2,7-triol-7-carboxylate, and 3,7-dimethyloctane-1,2,7-triol. In addition, microbial transformation of dihydromyrcenol by G. cingulata was carried out. The metabolic pathway of (-)-dihydromyrcenyl acetate is discussed.

Full text of DOI:10.1021/jf000843+

4826
J. Agric. Food Chem. 2000, 48, 4826−4829
Biotr a n sfor m a tion of (-)-Dih yd r om yr cen yl Aceta te Usin g th e P la n t
P a r a sitic F u n gu s Glom er ella cin gu la ta a s a Bioca ta lyst
Mitsuo Miyazawa,* Shin-ichi Akazawa, Hiromu Sakai, and Hirokazu Nankai
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University,
3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, J apan
The microbial transformation of (-)-dihydromyrcenyl acetate was investigated using the plant
parasitic fungus Glomerella cingulata. As a result, (-)-dihydromyrcenyl acetate was converted to
dihydromyrcenol, 3,7-dihydroxy-3,7-dimethyl-1-octene-7-carboxylate, 3,7-dihydroxy-3,7-dimethyl-
1-octene, 3,7-dimethyloctane-1,2,7-triol-7-carboxylate, and 3,7-dimethyloctane-1,2,7-triol. In addition,
microbial transformation of dihydromyrcenol by G. cingulata was carried out. The metabolic pathway
of (-)-dihydromyrcenyl acetate is discussed.
Keyw or d s: Biotransformation; microbial transformation; Glomerella cingulata; (-)-dihydromyr-
cenyl acetate; dihydromyrcenol; 3,7-dihydroxy-3,7-dimethyl-1-octene-7-carboxylate; 3,7-dihydroxy-
3,7-dimethyl-1-octene; 3,7-dimethyloctane-1,2,7-triol-7-carboxylate; 3,7-dimethyloctane-1,2,7-triol
INTRODUCTION
with 1% vanillin in 96% sulfuric acid followed by brief heating
(∼120 °C, 1 min). Gas chromatography (GC) was performed
on an HP 5890 series II Plus gas chromatograph equipped with
a flame ionization detector (FID). The column was a fused
silica capillary column [DB-5, 30 m × 0.25 mm i.d., film
thickness ) 1.0 µm (J &W Scientific, Folsom, CA)]. Chromato-
graphic conditions were as follows: column temperature, 80-
260 °C at 4 °C min-1; injector temperature, 270 °C; detector
temperature, 280 °C; carrier gas, He at 1.8 mL min-1. Yields
of individual constituents were determined by peak areas as
measured by an HP 3396 series II integrator. FAB MS was
obtained on a J EOL J MS-HX 100 mass spectrometer, and the
matrix was 3-nitrobenzyl alcohol (NBA). EI-MS measurements
were obtained using gas chromatography-mass spectrometry
(GC-MS). GC-MS was performed on an HP 5972A mass
selective detector interfaced with an HP 5890 series II Plus
gas chromatograph fitted with a column (HP-5MS, 30 m × 0.25
mm i.d., film thickness ) 0.25 µm), and the chromatographic
conditions were the same as described above. Infrared spectra
(IR) were determined with a Perkin-Elmer 1760-x IR Fourier
transform spectrometer. Nuclear magnetic resonance (NMR)
spectra were recorded on a J EOL FX-500 NMR spectrometer
(1H NMR, 500.00 MHz; 13C NMR, 125.65 MHz). Tetrameth-
ylsilane (TMS) was used as the internal standard (δ 0.00) for
1H NMR spectra measured in CDCl3. Residual CHCl3 was used
as internal reference (δ 77.00) for 13C NMR spectra measured
in CDCl3. Multiplicities were determined by DEPT pulse
sequence. Specific rotation was measured by J ASCO DIP-1000.
We have reported biotransformations of acyclic ter-
penoids using the plant parasitic fungus Glomerella
cingulata (Nankai et al., 1997, 1998a,b) and common
cutworm larvae Spodoptera litura Fabricius (Miyazawa
and Murata, 2000). In the biotransformation of acyclic
terpenoids by G. cingulata, unsaturated acyclic terpe-
noids were regioselectively oxidized at the double bond
distant from the hydroxyl or carbonyl group as a main
reaction (Nankai et al., 1998a,b), whereas saturated
acyclic terpenoids were mainly hydroxylated at the
methine of the isopropyl moiety, which is most distant
from the hydroxyl group (Nankai et al., 1997). To clarify
the mode of biotransformations of acyclic terpenoids by
G. cingulata, the biotransformation of (-)-dihydromyr-
cenyl acetate (7-hydroxy-3,7-dimethyl-1-octene-7-car-
boxylate) (1) was investigated. Compound 1 is not a
natural product but an artificial product synthesized
from R-pinene (Sprecker et al., 1986) and used in the
fragrance industry. Further derivatization of 1 is of
interest in producing novel biologically active com-
pounds, in particular, perfumery ingredients. There is,
however, no report of biotransformation of 1; therefore,
as part of our continuing program, we chose 1 as a
substrate of biotransformation by G. cingulata. To
elucidate the metabolic pathway, the microbial trans-
formation of dihydromyrcenol (2) was also investigated.
The structures of metabolites and the metabolic path-
way were established. This is the first report of the
biotransformations of 1 and 2.
P r ecu ltu r e of F u n gi. Spores of G. cingulata (the strain
had been isolated from diseased grape and was received from
Dr. Hyakumachi, M., Gifu University, J apan), which have been
preserved on potato dextrose agar (PDA) at 4 °C, were
inoculated into 200 mL of sterilized culture medium (1.5%
saccharose, 1.5% glucose, 0.5% polypepton, 0.05% MgSO4‚
7H2O, 0.05% KCl, 0.1% K2HPO4, and 0.001% FeSO4‚7H2O in
distilled water) in a 500 mL shaking flask, and the flask was
shaken (reciprocating shaker, 100 rpm) at 27 °C for 3 days
(Nankai et al., 1997).
MATERIALS AND METHODS
Gen er a l P r oced u r e. (-)-Dihydromyrcenyl acetate (1) was
purchased from Taiyo Perfumery Co., Ltd., and dihydromyr-
cenol was purchased from Nagaoka Perfumery Co., Ltd. Thin-
layer chromatography (TLC) was performed on precoated
plates [silica gel 60 F254, 0.25 mm, Merck (Darmstadt, Ger-
many)], and the compounds were visualized by spraying plates
Tim e Cou r se E xp er im en t . Precultured G. cingulata (1
mL) was transferred into a 200 mL Erlenmeyer flask contain-
ing 100 mL of medium (same medium as used in preculture)
and stirred under the same conditions as for preculture. After
3 days, the organism matured, compound 1 or 2 (50 mg) was
added to the medium, and the organism was cultivated for 10
more days. The culture medium (5 mL) was removed daily for
* Author to whom correspondence should be addressed
(fax +81-6-6727-4301; e-mail miyazawa@apch.kindai.ac.jp).
10.1021/jf000843+ CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/31/2000

Microbial Transformation of (−)-Dihydromyrcenyl Acetate
J. Agric. Food Chem., Vol. 48, No. 10, 2000 4827
Ta ble 1. 1H NMR Sp ectr a l Da ta of Com p ou n d s 2-6a
H
2
3
4
5
6
1
1′
2
3
4
5
6
7
4.96 ddd (17.0, 2.0, 1.0)
4.91 ddd (10.5, 2.0, 1.0)
5.69 ddd (17.0, 10.5, 7.5)
2.13 m
5.21 dd (17.5, 1.5)
5.06 dd (11.0, 1.5)
5.91 dd (17.5, 11.0)
5.21 dd (17.5, 1.5)
5.06 dd (11.0, 1.5)
5.92 dd (17.5, 11.0)
3.54 m
3.50 m
3.69 m
3.70 m
1.67-1.79 m
1.12-1.33 m
1.12-1.33 m
1.51-1.62 m
1.52-1.64 m
1.40-1.51 m
1.40-1.51 m
1.23-1.31 m
1.24-1.38 m
1.44-1.57 m
1.29-1.39 m
1.70-1.75 m
1.32-1.58 m
1.32-1.58 m
1.32-1.58 m
1.24-1.38 m
1.39-1.50 m
8
9
10
OAc
1.21 s
1.21 s
0.99 d (6.5)
1.42 s
1.42 s
1.28 s
1.97 s
1.22 s
1.22 s
1.29 s
1.422/1.421 s
1.422/1.421 s
0.90/0.93 d (7)
1.97 s
1.22 s
1.22 s
0.89/0.92 d (7)
a
Chemical shifts in ppm; coupling constants in Hz.
Ta ble 2. 13C NMR Sp ectr a l Da ta of Com p ou n d s 2-6a
C
2
3
4
5
6
1
2
3
4
5
6
7
8
112.5 (CH2)
144.7 (CH)
37.8 (CH)
37.1 (CH2)
22.0 (CH2)
44.0 (CH2)
71.1 (C)
29.2 (CH3)
29.2 (CH3)
20.2 (CH3)
111.6 (CH2)
145.0 (CH)
73.2 (C)
42.3 (CH2)
18.2 (CH2)
41.0 (CH2)
82.4 (C)
26.0 (CH3)
26.0 (CH3)
27.8 (CH3)
170.7 (C)
111.7 (CH2)
145.1 (CH)
73.3 (C)
42.6 (CH2)
18.7 (CH2)
44.1 (CH2)
71.1 (C)
29.3 (CH3)
29.3 (CH3)
27.8 (CH3)
64.7/65.1 (CH2)
76.1/75.5 (CH)
36.0/35.5 (CH)
33.1/32.6 (CH2)
21.2/21.4 (CH2)
40.8/40.9 (CH2)
82.34/82.31 (C)
26.1/26.0 (CH3)
26.1/26.0 (CH3)
15.2/14.5 (CH3)
170.5 (C)
64.6/65.0 (CH2)
76.0/75.3 (CH)
36.0/35.6 (CH)
32.9/33.4 (CH2)
21.4/21.7 (CH2)
43.80/43.83 (CH2)
71.12/71.08 (C)
29.1/29.2 (CH3)
29.1/29.2 (CH3)
15.4/14.6 (CH3)
9
10
COCH3
COCH3
22.4 (CH3)
22.5/22.6 (CH3)
a
Chemical shifts in ppm; multiplicities were determined by the DEPT pulse sequence.
F igu r e 2. Time course for the biotransformation of 2 by G.
cingulata: (4) dihydromyrcenol (2); (b) 3-hydroxydihydromyr-
cenol (4); (9) 3,7-dimethyloctane-1,2,7-triol (6).
F igu r e 1. Time course for the biotransformation of 1 by G.
cingulata: (O) (-)-dihydromyrcenyl acetate (1); (4) dihy-
dromyrcenol (2); (0) 3-hydroxydihydromyrcenyl acetate (3); (b)
3-hydroxydihydromyrcenol (4); (9) 3,7-dimethyloctane-1,2,7-
triol-7-carboxylate (5); (2) 3,7-dimethyloctane-1,2,7-triol (6).
mixed, the solvent was evaporated, and a crude extract (897
mg) was obtained. The extract was chromatographed on a Si-
60 column with hexane/EtOAc (stepwise 19:1 to 1:19) to yield
metabolites 2 (92 mg), 3 (19 mg), 4 (92 mg), and 5 (157 mg).
Furthermore, the high polar fraction was chromatographed
on a Si-60 column with chloroform/MeOH (stepwise 19:1 to
1:1), and 6 (68 mg) was isolated.
4 days and then every 2 days; sampling was continued for more
6 days. The culture medium was extracted with EtOAc (1 mL)
and the solvent then evaporated. This extract was analyzed
by TLC, GC, and GC-MS. The ratios between the substrate
and metabolic products were determined on the basis of GC
peak areas (Figures 1 and 2).
Biotr a n sfor m a tion of ())-Dih yd r om yr cen yl Aceta te
(1) for 4 Da ys. Precultured G. cingulata (5 mL) was trans-
ferred into a 3 L stirred fermentor containing 2 L of medium.
Cultivation was carried out at 27 °C with stirring (∼100 rpm)
for 3 days under aeration. After 3 days, the organism matured,
compound 1 (1.0 g) was added to the medium, and the
organism was cultivated for an additional 4 days.
Isola tion of Meta bolites 2)6. After the cultivation de-
scribed above, the culture medium and mycelia were separated
by filtration. The medium was saturated with NaCl and
extracted with CH2Cl2 (∼800 mL), and the mycelia were also
extracted with CH2Cl2 (∼200 mL). Both CH2Cl2 extracts were
Compound 2: oil; [R]25 -2.71° (CHCl3; c 0.5); EI-MS, m/z
D
(rel int) 141 [M - CH3]+ (0.3), 138 [M - H2O]+ (0.2), 123 [M -
CH3 - H2O]+ (9), 109 (2), 95 (6), 83 (10), 81 (7), 67 (12), 59
(100), 55 (25), 43 (36), 41 (26); IR vmax cm-1 3385, 2966, 2933,
1
1465, 1375, 1162, 909; H, 13C NMR (Tables 1 and 2).
Compound 3: oil; [R]25 +3.76° (CHCl3; c 0.4); EI-MS, m/z
D
(rel int) 154 [M - CH3COOH]+ (0.2), 139 [M - CH3COOH -
CH3]+ (6), 136 [M - CH3COOH - H2O] + (2), 131 (8), 121 (11),
109 (5), 93 (12), 81 (23), 71 (100), 68 (14), 56 (39), 43 (85); IR
1
vmax cm-1 3429, 2927, 1734, 1457, 1369, 1262, 1158; H, 13C
NMR (Tables 1 and 2).
Compound 4: oil; [R]25 +2.27° (CHCl3; c 0.3); EI-MS, m/z
D
(rel int) 154 [M - H2O]+ (0.1), 139 [M - H2O - CH3]+ (8), 136

4828 J. Agric. Food Chem., Vol. 48, No. 10, 2000
Miyazawa et al.
Sch em e 1. P ossible Meta bolic P a th w a ys of Com p ou n d 1 by G. cin gu la ta
[M - 2H2O]+ (0.8), 121 (12), 109 (5), 93 (6), 81 (30), 71 (100),
1 and 2) were assigned by comparison with the spectral
data for 1 and the previous paper (Bohlmann et al.,
1975); in addition, these spectral data corresponded to
an authentic sample of dihydromyrcenol. On the basis
of the above results, metabolite 2 was identified as
dihydromyrcenol. It had been reported that metabolite
2 was formed by chemical hydration of dihydromyrcene
(Matsubara et al., 1973); however, there was no report
of the production of 2 by biotransformation.
68 (43), 59 (36), 43 (72); IR vmax cm-1 3380, 2971, 2927, 1459,
1
1374, 1186, 1167, 1035, 918; H, 13C NMR (Tables 1 and 2).
Compound 5: oil; [R]25 +5.38° (CHCl3; c 0.5); HRFAB MS
D
(pos), m/z 233.1733 [MH]+, calcd for C12H25O4 233.1753; EI-
MS, m/z (rel int) 172 [M - CH3COOH]+ (1), 157 [M - CH3-
COOH - CH3]+ (1), 141 (15), 123 (42), 112 (5), 101 (10), 95
(11), 81 (33), 69 (33), 55 (32), 43 (100); IR vmax cm-1 3405, 2938,
2877, 1734, 1369, 1261, 1207, 1155, 1082, 1050, 1020; 1H, 13C
NMR (Tables 1 and 2).
Compound 6: oil; [R]25 +6.60° (CHCl3; c 0.5); HRFAB MS
The mass spectrum indicated that metabolite 3 had
a molecular formula of C12H22O3. From other spectral
data, 3 had an acetyl group (δH 1.97; δC 170.7, 22.4; vmax
1734, 1262 cm-1) and a tertiary hydroxyl group (δC 73.2;
vmax 3429, 1158 cm-1). The 1H and 13C NMR spectra
(Tables 1 and 2) were assigned by comparison with the
spectral data for 1 and 2. On the basis of these spectral
data, metabolite 3 was identified as 3,7-dihydroxy-3,7-
dimethyl-1-octene-7-carboxylate. Rapp and Mandery
(1988) reported that metabolite 3 was formed on the
biotransformation of linalool by Botrytis cinerea.
Metabolite 4 had a molecular formula of C10H20O2
that was estimated by its mass spectrum. Other spectral
data indicated the presence of two tertiary hydroxyl
groups (δC 71.1, vmax 3380, 1167 cm-1; and δC 73.3, vmax
D
(pos), m/z 191.1671 [MH]+, calcd for C10H23O3 191.1647; EI-
MS, m/z (rel int) 157 [M - CH3 - H2O]+ (2), 141 [M - 2CH3
- H2O]+ (10), 123 (36), 95 (7), 81 (33), 71 (32), 59 (100), 43
(89), 41 (29); IR vmax cm-1 3420, 2937, 1658, 1466, 1380, 1212,
1
1157, 1081, 1050; H, 13C NMR (Tables 1 and 2).
RESULTS AND DISCUSSION
To investigate the time course of relative abundance
between substrate and metabolites on the microbial
transformation of 1 by G. cingulata, a small amount of
1 was incubated with the organism for 10 days. Five
major metabolites and some minor metabolites were
detected by TLC, GC, and GC-MS analysis. These
metabolites were not detected by TLC and GC analysis
of the culture of G. cingulata to which no substrate (1)
had been fed or when the mixture of 1 and the culture
medium had been incubated under static conditions for
10 days. It was demonstrated that G. cingulata trans-
formed 1 into various metabolites. The time course of
relative concentration changes of substrate (1) and
metabolites (2-6) was monitored by TLC and quanti-
tatively measured by using a GC method (Figure 1).
Almost all beginning substrate 1 was consumed in 6
days. Metabolites 2-4 disappeared by 8-10 days.
Metabolite 5 increased after 3 days and then decreased
rapidly after 8 days, whereas metabolite 6 increased
rapidly after 8 days. To isolate metabolites 2-6, a large-
scale incubation of 1 using G. cingulata was carried out
for 4 days. After the biotransformation, the culture was
extracted and metabolites 2-6 were isolated from the
crude extract. The structures of 2-6 were determined
by spectral analysis.
1
3380, 1186 cm-1). The H and 13C NMR spectra (Tables
1 and 2) were assigned by comparison with the spectral
data for 1-3 and the previous paper (Williams et al.,
1980). These spectral data suggested that metabolite 4
was 3,7-dihydroxy-3,7-dimethyl-1-octene. There was a
report that metabolite 4 was synthesized from isoprene
(Takabe et al., 1975).
Metabolite 5 had a molecular formula of C12H24O4
based on its HRFAB MS. Other spectral data indicated
the presence of an acetyl group (δH 1.97; δC 170.5, 22.5/
22.6; vmax 1734, 1261 cm-1), a primary hydroxyl group
(δH 3.54; δC 64.7/65.1; vmax 3405, 1020 cm-1), and a
secondary hydroxyl group (δH 3.69; δC 76.1/75.5; vmax
1
3405, 1082 cm-1). The H and 13C NMR spectra (Tables
1 and 2) were assigned by comparison with the spectral
data for compounds 1-4. It was concluded that metabo-
lite 5 was a novel compound, 3,7-dimethyloctane-1,2,7-
triol-7-carboxylate.
The molecular formula of metabolite 2 was shown to
be C10H20O on the basis of its mass spectrum. Other
spectral data indicated the loss of the acetyl group and
the presence of a tertiary hydroxyl group (δC 71.1; vmax
Metabolite 6 had a molecular formula of C10H22O3
based on its HRFABMS. The results of the spectral data
indicated the presence of a primary hydroxyl group (δH
3.50; δC 64.6/65.0; vmax 3420, 1057 cm-1), a secondary
hydroxyl group (δH 3.70; δC 76.0/75.3; vmax 3420, 1081
1
3385, 1162 cm-1). The H and 13C NMR spectra (Tables

Microbial Transformation of (−)-Dihydromyrcenyl Acetate
J. Agric. Food Chem., Vol. 48, No. 10, 2000 4829
cm-1), a tertiary hydroxyl group (δC 71.12/71.08; vmax
of a hydroxyl group into the C-3 position of 1 proceeded
enzymatically by G. cingulata. These results were
similar to our previous results of biotransformation of
acyclic terpenoids by G. cingulata [see, for instance,
Nankai et al. (1997, 1998a,b)]. We are now considering
the investigation of the biotransformations of other
acyclic terpenoids using this fungus and the larvae of
common cutworm (S. litura) (Miyazawa and Murata,
2000).
1
3420, 1157 cm-1), and no acetyl group. The H and 13C
NMR spectra (Tables 1 and 2) were assigned by com-
parison with the spectral data for compounds 1-5. On
the basis of these spectral data, metabolite 6 was
determined to be a novel compound, 3,7-dimethyloctane-
1,2,7-triol.
The metabolic pathways for the biotransformation of
1 by G. cingulata (Scheme 1) were derived from the time
course experiment (Figure 1) and the structures of the
metabolites. The major metabolite 6 was considered to
be formed by way of two pathways (A, 1 f 5 f 6; B, 1
f 2 f 6). In route A, the substrate 1 was oxidized at
the double bond to glycol (1 f 5). This glycol (5) was
possibly formed via epoxidation and subsequent hy-
drolysis of the epoxide, although the intermediate
epoxide could not be detected. Then compound 5 was
deacetylated (5 f 6); from Figure 1, this route is
suggested to be the main metabolic pathway. Metabolite
6 was also formed through route B, that is, the deacety-
lation of 1 (1 f 2) and subsequent oxidation at the
double bond to glycol (2 f 6). To clarify the probability
that 6 was produced not only from 5 but also from 2, a
small amount of 2 (authentic sample) was incubated
with G. cingulata for 10 days. As a result, two major
metabolites were detected by TLC, GC, and GC-MS
analysis. The time course of relative concentration
changes of 2 and two metabolic products was monitored
by TLC and quantitatively measured by using a GC
method (Figure 2). Their retention time on GC and mass
spectra given by GC-MS corresponded to compounds 4
and 6, respectively. These results demonstrated that
metabolite 2 was converted into metabolites 4 and 6 by
G. cingulata. Because the rate of this conversion was
slow (Figure 2), route B (1 f 2 f 6) was considered to
be a minor pathway.
LITERATURE CITED
Bohlmann, F.; Zeisberg, R.; Klein, E. 13C-NMR-Spectren von
monoterpen. Org. Magn. Reson. 1975, 7, 426-432.
Matsubara, Y.; Fujiwara, T.; Tanaka, K. Hydration of various
terpene hydrocarbones and alcohols with cation-exchange
resin. Yuki Gosei Kagaku Kyokai Shi 1973, 31, 924-927.
Miyazawa, M.; Murata, T. Biotransformation of â-myrcene by
the larvae of common cutworm (Spodoptera litura). J . Agric.
Food Chem. 2000, 48, 123-125.
Nankai, H.; Miyazawa, M.; Kameoka, H. Hydroxylation of two
saturated acyclic monoterpenoids, tetrahydrogeraniol and
tetrahydrolavandulol, by the plant pathogenic fungus Glom-
erella cingulata. J . Nat. Prod. 1997, 60, 287-289.
Nankai, H.; Miyazawa, M.; Kameoka, H. Biotransformation
of acyclic terpenoid (2Z,6Z)-farnesol by the plant pathogenic
fungus Glomerella cingulata. Phytochemistry 1998a , 47,
1025-1028.
Nankai, H.; Miyazawa, M.; Akazawa, S.; Kameoka, H. Biotrans-
formation of (()-Lavandulol by the Plant Pathogenic Fun-
gus, Glomerella cingulata. J . Agric. Food Chem. 1998b, 46,
3858-3862.
Rapp, A.; Mandery, H. Influence of Botrytis cinerea on the
monoterpene fraction of wine aroma. Bioflavour ’87 1988,
445-452.
Sprecker, M. A.; Wilson, S. R.; Steinbach, L.; Oorourke, T. Eur.
Patent Appl. EP 170205 (International Patent Classification
C07/C33/025), 1986.
Metabolite 4 was formed via two possible pathways
(C, 1 f 2 f 4; D, 1 f 3 f 4). In route C, the substrate
1 was deacetylated (1 f 2) with subsequent introduction
of the hydroxyl group at the C-3 position of 2 to give 4.
This route was proved from the time course experiment
(Figure 2). In route D, however, first the hydroxyl group
was introduced at the C-3 position of 1 (1 f 3) and then
deacetylated (3 f 4), although it was not verified that
4 was produced from 3.
Takabe, K.; Katagiri, T.; Tanaka, J . Synthesis of (()-linalool
and (()-hydroxylinalool from isoprene. Tetrahedron Lett.
1975, 34, 3005-3006.
Williams, P. J .; Straus, C. R.; Wilson, B. New linalool deriva-
tives in Muscat of Alexandria grapes and wines. Phytochem-
istry 1980, 19, 1137-1139.
Received for review J uly 10, 2000. Revised manuscript received
J uly 10, 2000. Accepted J uly 11, 2000.
The above results demonstrated that the oxidation of
the remote double bond, deacetylation, and introduction
J F000843+