Biotransformation of citrus aromatics nootkatone and valencene by microorganisms

Article abstract of DOI:10.1248/cpb.53.1423

Biotransformations of the sesquiterpene ketone nootkatone (1) from the crude drug Alpiniae Fructus and grapefruit oil, and the sesquiterpene hydrocarbon valencene (2) from Valencia orange oil were carried out with microorganisms such as Aspergillus niger, Botryosphaeria dothidea, and Fusarium culmorum to afford structurally interesting metabolites. Their stereostructures were established by a combination of high-resolution NMR spectral and X-ray crystallographic analysis and chemical reaction. Metabolic pathways of compounds 1 and 2 by A. niger are proposed.

Full text of DOI:10.1248/cpb.53.1423

November 2005
Chem. Pharm. Bull. 53(11) 1423—1429 (2005)
1423
Biotransformation of Citrus Aromatics Nootkatone and Valencene by
Microorganisms
a
a
b
,a
*
Mai FURUSAWA, Toshihiro HASHIMOTO, Yoshiaki NOMA, and Yoshinori ASAKAWA
a Faculty of Pharmaceutical Sciences, Tokushima Bunri University; and b Faculty of Human Life Sciences, Tokushima
Bunri University; Yamashiro-cho, Tokushima 770–8514, Japan. Received June 30, 2005; accepted July 30, 2005
Biotransformations of the sesquiterpene ketone nootkatone (1) from the crude drug Alpiniae Fructus and
grapefruit oil, and the sesquiterpene hydrocarbon valencene (2) from Valencia orange oil were carried out with
microorganisms such as Aspergillus niger, Botryosphaeria dothidea, and Fusarium culmorum to afford struc-
turally interesting metabolites. Their stereostructures were established by a combination of high-resolution NMR
spectral and X-ray crystallographic analysis and chemical reaction. Metabolic pathways of compounds 1 and 2
by A. niger are proposed.
Key words nootkatone; valencene; biotransformation; Aspergillus niger; Fusarium culmorum; Botryosphaeria dothidea
We are continuing to study the biotransformation of sec-
ondary metabolites such as terpenoids and aromatic com-
pounds from crude drugs and liverworts by microorgan-
isms1—7) and mammals8,9) to obtain some functional sub-
stances such as pheromones and aromatics. We reported the
biotransformation of sesquiterpenoids such as dehydrocos-
tuslactone, costunolide, a-, b-, and g-cyclocostunolides, a-
santonin, and atractylon from crude drugs, and an amber
constituent, (ꢀ)-ambrox by microorganisms such as As-
pergillus niger.10) It has been clarified that a grapefruit essen-
tial oil containing nootkatone (1) decreases the somatic fat
ratio11) and its demand by cosmetic and fiber manufacturers
has increased. Recently, we have found that the commercially
available and cheap aromatic valencene (2) from Valencia or-
ange oil is very efficiently converted into the expensive
grapefruit aromatic, nootkatone (1) by biotransformations
using Chlorella sp.12) and Mucor sp.13) In continuation of the
biotransformation of the chemical constituents isolated from
crude drugs into biologically active compounds, the biotrans-
formations of nootkatone (1) and valencene (2) were exam-
ined using Aspergillus niger, Fusarium culmorum, and
Botryosphaeria dothidea. This paper deals with the structural
elucidation of metabolites of 1 and 2 converted by the three
fungi.
Biotransformation of Nootkatone (1) by Aspergillus
niger A. niger was inoculated and cultivated in a rotary
(100 rpm) in Czapek-pepton medium (pH 7.0) at 30 °C for
7 d. (ꢁ)-Nootkatone (1) (80 mg/200 ml) was added to the
medium and further cultivated for 7 d. The crude metabolites
obtained from the culture broth by EtOAc extraction were
chromatographed on silica gel (n-hexane–EtOAc gradient) to
give two metabolites, 12-hydroxy-11,12-dihydronootkatone
(3) (10.6%, isolated yield) and C-11 stereomixtures of 11,12-
dihydroxy-11,12-dihydronootkatone (nootkatone-11,12-diol)
(4, 5) (51.5% isolated yield), respectively.
1.09 (3H, s)], two secondary methyl [dH 0.93 (3H, d, Jꢂ
7.1 Hz), 0.97 (3H, d, Jꢂ6.9 Hz)], a primary alcohol [dH 3.55
(1H, dd, Jꢂ6.3, 10.4 Hz), 3.63 (1H, dd, Jꢂ5.8, 10.4 Hz); dC
65.9 (t)], and a ketone [dC 199.7 (s)]. Compound 3 showed
correlations between (i) H-12/C-7, C-11, and C-13; and (ii)
H-13/C-7, C-11 and C-12 in the HMBC spectrum. On the
basis of the above spectral data and careful analysis of its 2D
NMR spectrum, the structure of metabolite 3 was established
to be 12-hydroxy-11,12-dihydronootkatone. The relative con-
figuration at C-11 remained to be clarified.
The HR-EI-MS of nootkatone-11,12-diol (a mixture of 4
and 5) showed a peak for [Mꢁ] at m/z 252.1730, indicating
the molecular formula of C15H24O3. The FT-IR spectrum of
the mixture indicated the presence of a hydroxyl (3358 cmꢀ1)
1
and a conjugated ketone (1652 cmꢀ1) group. The H- and
13C-NMR spectra showed that 4 and 5 were a mixture of
stereoisomers at C-11, the isolation of which was very diffi-
cult by any separation method. The structure of 11,12-diol
was confirmed by the formation of a methyl ketone (6) on
oxidation with NaIO4. Treatment of the mixture (4, 5) with
1,1ꢃ-thiocarbonyldiimidazole gave the thiocarbonates 7 and
8, which were easily separated by HPLC in the ratio of 3 : 2.
Teresa et al.14) and Haines and Jenkins15) reported that the
configuration at C-11 of 11,12-diol (9) must be “R,” because
the thiocarbonate (10) used to prepare 9 showed a negative
Cotton effect [308 nm (De ꢀ0.21)]. However, both thiocar-
bonates 7 and 8 showed negative Cotton effects [7: 319 nm
(De ꢀ0.33); 8: 310 nm (De ꢀ0.70)] in CD spectra, and thus
the absolute configuration at C-11 of 4 and 5 could not be
characterized by their CD spectra. Finally, the absolute struc-
ture of 4 was established based on X-ray crystallographic
analysis of the thiocarbonate (7) as the S configuration at C-
11, as shown in Fig. 1. Thus compounds 4 and 5 were deter-
mined to be (11S)- and (11R)-nootkatone-11,12-diol, respec-
tively. 11,12-Epoxide (11) obtained by epoxydation of
nootkatone (1) with mCPBA was biotransformed by A. niger
for 1 d to 11,12-dihydroxy-11,12-dihydronootkatone (4, 5) in
good yield (81.4%). 1-Aminobenzotriazole, an inhibitor of
cytochrome P450, inhibited the oxidation process of 1 into
compounds 3—5. From the above results, possible metabolic
pathways of nootkatone (1) are depicted in Fig. 2.
Compound (3) {[a]D +133.3° (CHCl3)} was obtained as a
single isomer at C-11, for which the molecular formula
C15H24O2 was established in high-resolution electron-impact
mass spectroscopy (HR-EI-MS) ([M]ꢁ m/z 236.1778). The
FT-IR and UV spectra of 3 indicated the presence of a hy-
droxyl (3398 cmꢀ1) and a conjugated ketone (1657 cmꢀ1;
1
lmax 238 nm) group. The H- (Table 1) and 13C-NMR (Table
Biotransformation of Nootkatone (1) by F. culmorum
and B. dothidea F. culmorum was inoculated and cultivated
2) spectra of 3 showed the presence of a tertiary methyl [dH
∗ To whom correspondence should be addressed. e-mail: asakawa@ph.bunri-u.ac.jp
© 2005 Pharmaceutical Society of Japan

1424
Vol. 53, No. 11
of a hydroxyl (3359 cmꢀ1) and a conjugated ketone
1
(1652 cmꢀ1) group. The H- (Table 1) and 13C-NMR (Table
2) spectra of 12 showed the presence of a secondary alcohol
[dH 4.47 (1H, ddd, Jꢂ1.9, 5.2, 12.1 Hz); dC 69.0 (d)] and a
ketone [dC 199.8 (s)]. The carbon signals at the C-8, C-9, and
C-10 positions of 12 appeared at a lower field (ꢁ9.2, ꢁ36.0,
and ꢁ1.0 ppm) in comparison with those of nootkatone (1),
as shown in Table 2. Compound 12 showed correlations be-
tween (i) H-9/C-1, C-5, and C-10 in the HMBC spectrum
and NOEs between H-9/H-1, H-7, H-8a and H-14 in
the NOESY, respectively. Thus the structure of 12 is 9b-
hydroxynootkatone.
The biotransformation of nootkatone (1) was examined
using the plant pathogenic fungus B. dothidea separated from
the fungus which grows on the peach. (ꢁ)-Nootkatone (1)
was cultivated with B. dothidea (peach PP8402) for 14 d
to afford 11,12-dihydroxy-11,12-dihydronootkatone (4, 5)
(54.2%) and 7a-hydroxynootkatone (13) (20.9%). The ratio
of compounds 4 and 5 was determined to be 3 : 2 by HPLC
analysis of their thiocarbonates (7, 8).
Compound 13 {[a]D ꢁ118.8° (CHCl3)} was obtained as a
colorless oil, for which the molecular formula C15H22O2 was
established by HR-EI-MS ([M]ꢁ m/z 234.1610). The FT-IR
1
and H- and 13C-NMR (Table 2) spectra of 13 resembled
those of nootkatone except for the presence of a tertiary alco-
hol [dC 74.5 (s)]. The carbon signals at the C-6, C-7, and C-8
positions of 13 appeared at a lower field (ꢁ3.3, ꢁ34.2, and
ꢁ4.3 ppm) in comparison with those of nootkatone (1), as
shown in Table 2. The relative structure of compound 13 at
C-7 was determined by NOEs between (i) H-12/H-8b; and
(ii) H-13/H-6b in the NOESY spectrum.
The conversions of nootkatone (1) to 11S- and 11R-
nootkatone-11,12-diol (4, 5) by various microorganisms and
rabbits are summarized in Table 3. The biotransformations of
1 by A. niger and B. dothidea resembled that after oral ad-
ministration of 1 to rabbits16) since the ratio of major metabo-
lites (4, 5) in rabbits was similar to that in the former two
fungi. Recently, we have reported that the biotransformation
of hinesol5) and a-santonin10) by A. niger is similar to that
with their oral administration to mammals. It is noteworthy
that the biotransformation of 1 by F. culmorum stereospecifi-
cally afforded 11R-nootkatone-11,12-diol (5).
Biotransformation of Valencene (2) by A. niger Va-
lencene (2) from Valencia orange oil was cultivated with A.
niger for 5 d to afford seven metabolites 3 (1.0%), 4 and 5
(13.5%), 14 (1.1%), 15 (1.5%), 16 (2.0%), and 17 (0.7%), re-
spectively. The ratio of compounds 4 (11S) and 5 (11R) was
determined to be 1 : 3 by HPLC analysis of their thiocarbon-
ates (7, 8).
stationary in Czapek-pepton medium (pH 7.0) at 30 °C for
7 d. (ꢁ)-Nootkatone (1) was added to the medium and fur-
ther cultivated for 7 d to afford (11R)-11,12-dihydroxy-
11,12-dihydronootkatone (5) (47.2%) and 9b-hydroxy-
nootkatone (12) (14.9%).
Compound 5 was stereospecifically obtained at C-11 by
biotransformation of 1. The purity of compound 5 was deter-
mined to be ca. 95% by HPLC analysis of the thiocarbonate
(8).
Compound 12 {[a]D ꢁ131.3° (CHCl3)} was obtained as
a colorless oil, for which the whose molecular formula
C15H22O2 was established using HR-EI-MS ([M]ꢁ m/z
234.1612). The FT-IR spectrum of 12 indicated the presence
Compound 14 {[a]D ꢀ71.1° (CHCl3)} was obtained as a
single isomer at C-11, for which the molecular formula
C15H24O2 was established by HR-EI-MS ([M]ꢁ m/z
236.1749). The FT-IR and UV spectra of 14 indicated the
presence of a hydroxyl (3350 cmꢀ1) and a conjugated diene
1
(lmax 235 nm) group. Its H- (Table 4) and 13C-NMR (Table
5) spectra showed the presence of a conjugated diene [dH
5.60 (m), 5.94 (1H, br d, Jꢂ9.6 Hz), 5.38 (m); dC 121.9 (d),
126.0 (d), 128.4 (d), 142.4 (s)] and 11,12-diol [dH 3.45, 3.60
(each 1H, d, Jꢂ11.0 Hz); dC 68.6 (t), 74.6 (s)]. The stere-
ostructure of 14 was determined by the careful analysis of
the 2D NMR spectra (HMBC, NOESY) and the above-men-

November 2005
1425
a)
Table 1. 600 MHz 1H-NMR Spectral Data of Compounds 1, 3, 5, 12 and 13 in CDCl3
Position
1
3
5
12
13
1
3a
5.77 br s
2.29 dd
5.75 br s
2.29 dd
5.76 br s
2.30 dd
6.25 br s
2.31 dd
5.81 br s
2.32 dd
(13.7, 17.0)
2.23 ddd
(0.8, 4.1, 17.0)
2.20 m
(14.0, 17.3)
2.22 ddd
(0.8, 4.4, 17.3)
1.99 m
(14.0, 17.3)
2.24 ddd
(0.8, 4.4, 17.3)
2.02 m
(14.0, 16.8)
2.25 ddd
(0.8, 4.4, 16.8)
2.06 m
(14.0, 17.0)
2.22 ddd
(0.8, 4.4, 17.0)
1.33 m
3b
4
6a
1.98 ddd
(3.0, 3.0, 12.9)
1.14 dd
(12.9, 12.9)
2.33 m
1.92 m
1.35 m
2.52 m
2.38 m
1.91 ddd
(2.7, 2.7, 13.2)
0.96 dd
(13.2, 13.2)
1.83 m
1.87 m
1.22 m
2.48 m
2.35 m
2.13 ddd
(2.7, 2.7, 12.9)
1.06 dd
(12.9, 12.9)
1.97 m
1.87 m
1.18 m
2.50 m
2.37 m
1.97 ddd
(2.5, 2.5, 13.2)
1.14 dd
(12.9, 12.9)
2.41 m
2.23 m
1.42 m
4.47 ddd
(1.9, 5.2, 12.1)
4.76 br s
1.95 dd
(3.0, 14.3)
1.47 d
6b
(14.3)
7
8a
8b
9a
9b
12
1.85 m
1.79 m
2.24 m
2.24 m
4.86 br s
4.72 br s
3.55 dd
3.46 d
(6.3, 10.4)
3.63 dd
(5.8, 10.4)
0.93 d
(11.0)
3.62 d
(11.0)
1.09 s
4.75 br s
1.74 br s
4.78 br s
1.75 s
5.06 br s
1.81 br s
13
14
15
(7.1)
0.97 d
(6.9)
1.09 s
0.97 d
(6.9)
1.12 s
0.99 d
(6.9)
1.10 s
0.98 d
(6.9)
1.13 s
0.95 d
(6.9)
1.31 s
a) Chemical shifts from TMS (multiplicity, J in Hz) in CDCl3.
Table 2. 150 MHz 13C-NMR Spectral Data of Compounds 1, 3, 5, 12 and
a)
13 in CDCl3
C
1
3
5
12
13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
124.6
199.6
42.0
40.4
39.3
43.9
40.3
31.6
33.0
170.4
149.0
109.2
20.8
14.9
16.8
124.5
199.7
42.1
40.5
39.2
41.3
34.3
30.7
33.1
171.1
40.2
65.9
13.6
15.0
16.9
124.5
199.8
42.0
40.6
39.2
38.6
39.2
27.9
32.9
170.8
74.2
68.4
19.9
15.0
16.8
120.7
199.8
41.8
40.8
39.7
43.8
38.3
40.8
69.0
171.4
147.8
109.2
20.7
15.1
17.6
124.5
199.6
41.8
41.5
39.1
47.2
74.5
35.9
28.9
171.0
151.7
109.8
19.1
14.9
18.7
Fig. 1. ORTEP Drawing of Compound 7
a) Chemical shifts from TMS in CDCl3.
Table 3. Conversion of Nootkatone to 11S and 11R-11,12-Diol by Mi-
croorganisms and Rabbit
Metabolites (ratioa) of 4 and 5)
11S-11,12-diol (4) 11R-11,12-diol (5)
Aspergillus niger
Fusarium culmorum
Botryosphaeria dothidea
Rabbit
3
1
3
3
2
20
2
2
a) Product: Ratio was determined by HPLC of thiocarbonates of 11,12-diol.
Fig. 2. Possible Metabolic Pathway of (ꢁ)-Nootkatone (1) by Aspergillus
niger

1426
Vol. 53, No. 11
Table 4. 600 MHz 1H-NMR Spectral Data of Compounds 14—17 in
a)
CDCl3
H
1
14
15
16
17
5.94 br d
(9.6)
6.87 d
(9.9)
6.00 br d
(9.9)
2.10 d
(16.2)
2.26 d
(16.2)
2
5.60 m
1.95 m
5.87 d
(9.9)
5.59 br d
(9.9)
3.91 br d
(8.8)
Fig. 3. ORTEP Drawing of 18
3a
5.90 br s
3b
2.06 m
1.53 m
tioned spectral data, except for the configuration at C-11.
Compound 15 {[a]D ꢀ50.1° (CHCl3)} was obtained as a
single isomer at C-11, for which the molecular formula
C15H22O3 was established using HR-EI-MS ([M]ꢁ m/z
250.1562). The FT-IR and UV spectra of 15 indicated the
presence of a hydroxyl (3417 cmꢀ1) and a,b,g,d-unsaturated
ketone (1666 cmꢀ1; lmax 289 nm) group which was con-
4
2.42 q
(6.9)
1.32 dq
(8.8, 6.9)
5
6a
2.35 m
1.21 m
1.86 br d
(12.4)
1.05 dd
1.99 m
1.33 dd
1.92 m
1.05 dd
6b
1.23 m
(12.4, 12.4) (12.1, 12.1) (12.4, 12.4)
7
2.03 m
2.14 m
1.83 m
5.38 m
1.99 m
2.32 m
1.99 m
5.99 dd
(1.9, 5.2)
1.96 m
2.15 m
1.81 m
5.50 dd
(2.7, 5.2)
1.68 m
1.57 m
1.33 m
1.42 m
1
firmed by the H- (Table 4) and 13C-NMR (Table 5) spectral
8a
8b
9a
data, indicating the presence of an a,b,g,d-unsaturated ke-
tone [dH 5.87 (1H, d, Jꢂ9.9 Hz), 5.99 (1H, dd, Jꢂ1.9,
5.2 Hz), 6.87 (1H, d, Jꢂ9.9 Hz); dC 125.1 (d), 132.5 (d),
141.3 (s), 145.2 (d), 201.5 (s)] and 11,12-diol [dH 3.48, 3.62
(each 1H, d, Jꢂ11.0 Hz); dC 68.5 (t), 74.6 (s)]. The stereo-
chemistry at C-11 of 15 was established to be R configura-
tion by the careful analysis of its 2D NMR spectra and the X-
ray crystallographic analysis of 3,5-dinitrobenzoate (18) pre-
pared from 15, as shown in Fig. 3.
9b
12
1.59 m
3.43 d
(11.0)
3.55 d
(11.0)
1.11 s
1.95 br s
3.45 d
(11.0)
3.60 d
(11.0)
1.12 s
0.90 d
(7.4)
3.48 d
(11.0)
3.62 d
(11.0)
1.14 s
1.11 d
(6.9)
3.42 d
(11.0)
3.56 d
(11.0)
1.09 s
1.07 d
(6.9)
13
14
Compound 16 {[a]D ꢁ17.4° (MeOH)} was obtained as a
single isomer at C-11, for which the molecular formula
C15H24O3 was established using HR-EI-MS ([M]ꢁ m/z
252.1708). The FT-IR and UV spectra of 16 indicated the
presence of a hydroxyl (3368 cmꢀ1) and a conjugated diene
15
0.89 s
1.01 s
0.92 s
0.87 s
a) Chemical shifts from TMS (multiplicity, J in Hz) in CDCl3.
Table 5. 150 MHz 13C-NMR Spectral Data of Compounds 14—17 in
a)
CDCl3
1
(lmax 237 nm). The H- (Table 4) and 13C-NMR (Table 5)
spectra of 16 showed the presence of a conjugated diene [dH
5.50 (1H, dd, Jꢂ2.7, 5.2 Hz) 5.59 (1H, br d, Jꢂ9.9 Hz), 6.00
(1H, br d, Jꢂ9.9 Hz); dC 124.2 (d), 128.6 (d), 129.4 (d),
141.3 (s)], a secondary alcohol [dH 3.91 (1H, br d, Jꢂ
8.8 Hz); dC 71.6 (d)], and 11,12-diol [dH 3.42, 3.56 (each 1H,
d, Jꢂ11.0 Hz); dC 68.6 (t), 74.6 (s)]. The relative structure of
compound 16 at C-3 was determined based on NOEs be-
tween H-3/H-14 and H-15 in the NOESY spectrum.
C
14
15
16
17
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
128.4
126.0
32.2
39.0
36.0
34.9
36.5
27.2
121.9
142.4
74.6
68.6
19.4
14.8
17.1
145.2
125.1
201.5
52.6
39.4
35.1
35.6
27.7
132.5
141.3
74.1
68.5
19.6
7.2
129.4
128.6
71.6
48.0
37.0
34.9
35.1
27.0
124.2
141.3
74.3
68.3
19.1
10.6
17.9
54.0
200.3
126.3
164.9
47.7
22.8
44.7
21.9
39.7
37.5
74.3
68.1
19.2
21.8
16.5
Compound 17 {[a]D ꢁ52.0° (CHCl3)} was obtained as a
single isomer at C-11, for which the molecular formula
C15H24O3 was established using HR-EI-MS ([M]ꢁ m/z
252.1736). The FT-IR and UV spectra of 17 indicated the
presence of a hydroxyl (3410 cmꢀ1) and an a,b-unsaturated
1
ketone (1653 cmꢀ1; lmax 242 nm) group. The H- (Table 4)
19.2
and 13C-NMR (Table 5) spectra of 17 showed the presence of
an a,b-unsaturated ketone [dH 5.90 (1H, br s); dC 126.3 (d),
164.9 (s)] and 11,12-diol [dH 3.43, 3.55 (each 1H, d, Jꢂ
11.0 Hz); dC 68.1 (t), 74.3 (s)]. The relative structure of 17
was established by HMBC and NOESY spectral analysis, as
shown in Fig. 4. Compound 17 may be obtained by the re-
arrangement of the C-5 methyl group into C-10.
a) Chemical shifts from TMS (multiplicity, J in Hz) in CDCl3.
Compounds 14—17 could be biosynthesized by elimina-
tion of a hydroxyl group of 2-hydroxyvalencene (19, 20).
The reduction of nootkatone (1) with NaBH4 and CeCl3 gave
2a-hydroxyvalencene (19) in 87% yield, and the Mitsunobu
reaction of 19 with p-nitrobenzoic acid, triphenylphosphine,
and diethyl azodicarboxylate gave nootkatol (2b-hydroxyva-
Fig. 4. Important HMBC and NOESY Spectra of Compound 17

November 2005
1427
was CDCl3. MS spectra were measured on a JEOL JMS HX-100 or a JEOL
AX-500 spectrometer. The specific rotation was recorded on a JASCO DIP-
140 polarimeter. X-Ray crystallographic analysis was carried out using a
Mac Science MXC18 diffractiotometer. Slica gel 60 for column chromatog-
raphy was purchased from Merck. TLC was carried out on silica gel 60 F254
,
precoated (layer thickness 0.25 mm, Merck) with n-hexane–EtOAc (1 : 1).
High-performance liquid chromatography (HPLC) was carried out on a Shi-
madzu LC-6A Liquid Chromatograph.
Materials (ꢁ)-Valencene ([a]D ꢁ84.6°, cꢂ1.0) (2) and (ꢁ)-nootkatone
([a]D ꢁ193.5°, cꢂ1.0) (1) which were isolated from the essential oil of Va-
lencia orange and grapefruit, respectively, were provided from Takasago In-
ternational Co., Ltd. (Japan). Purity was checked based on the 600 MHz 1H-
NMR spectrum, GC-MS, and optical rotation. The absolute configurations
of (ꢁ)-nootkatone (1) and (ꢁ)-valencene (2) were already determined to be
1 and 2, respectively.17,18)
Microorganisms and Media A. niger was isolated from soil in Osaka
prefecture and was identified based on its physiologic and morphologic fea-
tures. A Czapek-pepton medium [1.5% sucrose, 1.5% glucose, 0.5%
polypepton, 0.1% K2HPO4, 0.05% KCl and 0.001%FeSO4·7H2O in distilled
water (pH 7.0)] was used for the biotransformation of nootkatone by A.
niger, B. dothidea and B. theobromae.
Biotransformation of Nootkatone (1) by A. niger An Erlenmeyer flask
(500 ml) containing 200 ml of Czapek-pepton medium was inoculated with a
suspension of A. niger and incubated at 30 °C for 2 d in a rotary shaker oper-
ating at 100 rpm. After full growth of the microorganisms, a solution of (ꢁ)-
nootkatone (1) (80 mgꢄ5; 400 mg) was added to the culture medium of A.
niger. The incubation was then continued for a further 7 d at 30 °C. After the
completion of the incubation time, the culture was filtered in vacuo and the
broth was extracted twice with EtOAc (each 200 ml) under stirring. The
EtOAc layers were dried over MgSO4 and the solvent was evaporated in
vacuo to give the crude extract (494 mg) which was chromatographed on sil-
ica gel (50 g) with a gradient solvent system of n-hexane–EtOAc by increas-
ing the amount of EtOAc stepwise in 5% increments to afford 12-hydroxy-
11,12-dihydronootkatone (3) (4.6 mg; 10.6%) from n-hexane–EtOAc (1 : 1)
C-11 mixtures of 11,12-dihydroxy-11,12-dihydronootkatone (4, 5) (238.2
mg; 51.5%) from n-hexane–EtOAc (1 : 4) fraction.
Fig. 5. Possible Metabolic Pathway of (ꢁ)-Valencene (2) into 14—16 by
Aspergillus niger
lencene) (20) obtained in 42% yield. Nootkatol with calcium
antagonistic activity was isolated from Alpinia oxyphylla
MIQ.17) Compound 19 was biotransformed for 5 d by A. niger
to give three metabolites, 4 and 5 (6.4%), 14 (34.6%), and 15
12-Hydroxy-11,12-dihydronootkatone (3): Colorless oil, [a]D22 ꢁ133.3°
(cꢂ0.75, CHCl3); EI-MS: m/z 236 (Mꢁ), 218, 203, 175, (100), 161, 147,
121; HR-EI-MS: m/z 236.1778 (Mꢁ), C15H24O2 requires 236.1777; FT-IR
(5.5%), respectively. Compound 20 was biotransformed for (KBr) cmꢀ1: 3399 (OH), 2926, 1657 (CꢂO), 1298, 1040; UV (MeOH) lmax
1
nm (log e): 238 (4.19); H- and 13C-NMR (600, 125 MHz) spectral data are
5 d by A. niger to give three metabolites, 4 and 5 (21.8%), 15
(5.5%), and 16 (10.4%), respectively. Both ratios of 4 (11S)
and 5 (11R) obtained from 19 and 20 were 1 : 3. From the
above results, plausible metabolic pathways of valencene (2)
and 2-hydroxyvalencene (19, 20) by A. niger are shown in
Fig. 5.
In summary, the grapefruit fragrance, nootkatone (1) used
in the modern cosmetic and fiber business was biotrans-
formed by A. niger and B. dothidea to give 11S- and 11R-
nootkatone-11,12-diol (4, 5) in the ratio of 3 : 2. The bio-
transformation of 1 by F. culmorum stereospecifically af-
forded 11R-nootkatone-11,12-diol (5). Biotransformation of
(ꢁ)-valencene (2), which can be cheaply obtained from Va-
lencia oranges, afforded the structurally interesting metabo-
lites 14—17 together with 3—5. Because biotransformation
of 2-hydroxyvalencene (19, 20) by A. niger gave metabolites
14—16, the biosynthetic pathway (Fig. 5) was confirmed.
The metabolites 3—5, 12, and 13 of (ꢁ)-nootktone (1) and
shown in Tables 1 and 2, respectively.
11S- and 11R-nootkatone-11,12-diol (4, 5) colorless oil, [a]D22 ꢁ130.5°
(cꢂ0.81, CHCl3); EI-MS: m/z 252 (Mꢁ), 234, 221 (100%), 206, 178, 161,
121; HR-EI-MS: m/z 252.1730 (Mꢁ), C15H24O3 requires 252.1725; FT-IR
(KBr) cmꢀ1: 3358 (OH), 2938, 2866, 1652 (CꢂO), 1063; UV (MeOH) lmax
nm (log e): 238.5 (4.16); 1H-NMR (CDCl3): compound 4 (11S); d 0.97 (3H,
d, Jꢂ6.9 Hz, H-14), 1.11 (3H, s, H-13 and H-15), 3.46 (1H, d, Jꢂ11.0 Hz),
3.60 (1H, d, Jꢂ11.0 Hz), 5.76 (1H, Jꢂ0.8 Hz, H-1); 13C-NMR (CDCl3): d
14.9 (q, C-14), 16.9 (q, C-15), 20.3 (q, C-13), 26.8 (t, C-8), 33.0 (t, C-9),
39.2 (t, C-6), 39.6 (s, C-5), 39.7 (d, C-7), 40.5 (d, C-4), d 42.0 (t, C-3), 68.2
(t, C-12), 74.3 (s, C-11), 124.4 (d, C-1), 171.0 (s, C-10), 199.9 (s, C-2).
Compound 5 (11R) NMR data: see Tables 1 and 2.
Reaction of Compounds 4 and 5 with NaIO4 To a solution of com-
pounds 4 and 5 (10.0 mg) in ethanol (5 ml) was added NaIO4 (40 mg) with
stirring at room temperature. After stirring for 1 h, the mixture was extracted
twice with Et2O (50 ml). The organic phase was washed with brine, dried
(MgSO4), and evaporated to give a residue (13.4 mg). The residue was puri-
fied by a silica gel column chromatography with an n-hexane–AcOEt gradi-
ent to afford diketone (6) (7.9 mg, 78.8%).
Compound 6: Colorless oil, [a]D22 ꢁ163.5° (cꢂ1.74, CHCl3); EI-MS: m/z
220 (Mꢁ; 100%), 205, 177, 150, 135; HR-EI-MS: m/z 220.1458 (Mꢁ),
3—5 and 14—17 of (ꢁ)-valencene (2) did not show an effec- C14H20O2 requires 220.1463; FT-IR (KBr) cmꢀ1: 2942, 1709 (CꢂO), 1667
1
(CꢂO), 1298, 1200; UV (MeOH) lmax nm (log e): 236.5 (4.17); H-NMR
(CDCl3): d 0.99 (3H, d, Jꢂ6.9 Hz, H-14), 1.12 (3H, s, H-15), 2.20 (3H, s,
H-12), 5.79 (1H, s, H-1); 13C-NMR (CDCl3): d 14.9 (q, C-14), 16.7 (q, C-
15), 28.2 (q, C-12), 28.5 (t, C-8), 32.1 (t, C-9), 38.9 (s, C-5), 40.0 (t, C-6),
tive odor.
Experimental
General Experimental Procedures Gas chromatography (GC) was car- 40.2 (d, C-4), 42.1 (t, C-3), 46.8 (d, C-7), 125.3 (d, C-1), 168.5 (s, C-10),
ried out using a Agilent 6890 GC system-5973 Network instrument
equipped with a flame ionization detector on a capillary column (30 mꢄ
250 mmꢄ0.3 mm). The column temperature was programmed from 70 °C to
199.2 (s, C-2), 210.4 (s, C-11).
Reaction of Compounds 4 and 5 with 1,1ꢀ-Thiocarbonyldiimidazole
To a solution of compounds 4 and 5 (30.0 mg) in toluene (7 ml) was added
230 °C. IR spectra were measured on a JASCO FT-IR 500 spectrophotome- 1,1ꢃ-thiocarbonyldiimidazole (45 mg) with stirring at room temperature.
ter. 1H- and 13C-NMR spectra were recorded on a Varian unitiy 600 (1H; After stirring for 7 h, the mixture was purified by silica gel column chro-
600 MHz, 13C; 150 MHz) spectrometer. The solvent used for NMR spectra
matography with an n-hexane–AcOEt gradient to afford thiocarbonates (7,

1428
Vol. 53, No. 11
m/z 234.1612 (Mꢁ), C15H22O2 requires 234.1620; FT-IR (KBr) cmꢀ1: 3359
(OH), 2970, 1652 (CꢂO), 1615 (CꢂC), 1301, 1046; UV (MeOH) lmax nm
(log e): 238 (4.06); NMR data: see Tables 1 and 2.
8) (29.8 mg). The mixture of thiocarbonates was separated by HPLC on sil-
ica gel with n-hexane–EtOAc (1 : 1) to afford 11S-thiocarbonate (7)
(13.1 mg, 37.4%) at Rtꢂ67 min and 11R-thiocarbonate (8) (8.8 mg, 25.1%)
at Rtꢂ70 min.
Biotransformation of (ꢁ)-Nootkatone (2) by B. dothidea An Erlen-
meyer flask (500 ml) containing 200 ml of Czapek-pepton medium was inoc-
ulated with a suspension of B. dothidea (peach PP 8402) and incubated at
30 °C for 7 d in a rotary shaker operating at 100 rpm. (ꢁ)-Nootkatone (2)
(20 mgꢄ5; 100 mg) was added to the culture medium of B. dothidea. The in-
ocubation was then continued for a further 14 d. The culture broth was
treated in the same manner as described above give the crude extract
(424 mg) which was chromatographed on silica gel with an n-hexane–ether
gradient to afford 7a-hydroxynootkatone (13) (22.4 mg, 20.9%) and 11S-
and 11R-nootkatone-11,12-diol (4, 5) (62.7 mg, 54.2%). The ratio of com-
pounds 4 and 5 was determined to be 3 : 2 by HPLC analysis of their thiocar-
bonates (7, 8).
Compound 7: Colorless prisms; mp 182—183 °C; [a]D22 ꢁ167.0°
(cꢂ0.83, CHCl3); EI-MS: m/z 294 (Mꢁ), 278, 217 (100%), 216, 201, 174,
161, 135, 91; HR-EI-MS: m/z 294.1269 (Mꢁ), C16H22O3S requires
294.1290; FT-IR (KBr) cmꢀ1: 2969, 1662 (CꢂO), 1305 (CꢂS), 1197; UV
(MeOH) lmax nm (log e): 237 (4.50); CD (MeOH) lmax nm (De): 319 nm
(ꢀ0.33); 1H-NMR (CDCl3): d 0.97 (3H, d, Jꢂ6.9 Hz, H-14), 1.11 (3H, s, H-
15), 1.55 (3H, s, H-13), 4.28 (1H, d, Jꢂ8.8 Hz, H-12), 4.46 (1H, d, Jꢂ8.8
Hz, H-12), 5.79 (1H, d, Jꢂ1.4 Hz, H-1); 13C-NMR (CDCl3): d 15.0 (q, C-
14), 16.9 (q, C-15), 21.7 (q, C-13), 26.7 (t, C-8), 31.8 (t, C-9), 38.6 (t, C-6),
40.4 (d, C-4), 41.0 (d, C-7), 41.9 (t, C-3), 76.6 (t, C-12), 91.8 (s, C-11),
125.3 (d, C-1), 167.6 (s, C-10), 191.1 (s, CꢂS), 198.9 (s, C-2).
The crystal data for 7 are as follows: Monoclinic, space group P21, aꢂ
8.710 (0) Å, bꢂ8.448 (0) Å, cꢂ10.988 (0) Å, bꢂ102.428, Vꢂ789.599976
(0) Å3, Zꢂ2, Dxꢂ1.530 Mg mꢀ3, Dmꢂ1.500 Mg mꢀ3, m (MoKa)ꢂ2.09
mmꢀ1, Eta; ꢁ1.9. Final R and Rw were 0.044 and 0.074 for 1186 reflections.
The supplementary materials have been deposited at the Cambridge Crystal-
lographic Data Center.
a
7 -Hydroxynootkatone (13): Colorless oil, [a]D ꢁ118.8° (CHCl3, cꢂ
1.0); EI-MS: m/z 234 (Mꢁ, 100), 219, 191, 161, 150, 136, 79, 69; HR-EI-
MS; [Mꢁ] 234.1610, C15H22O2 requires 234.1619; IR (KBr) cmꢀ1: 3445
(OH): 3089, 2941, 1665 (a,b-unsaturated ketone), 1616, 1217; UV (MeOH)
l
max nm (log e): 239.2 nm (4.17); NMR data: see Tables 1 and 2.
Biotransformation of (ꢁ)-Valencene (1) by A. niger A. niger was rota-
Compound 8: Colorless needles; mp 164—165 °C; [a]D22 ꢁ167.0° (cꢂ
0.83, CHCl3); EI-MS: m/z 294 (Mꢁ), 278, 217 (100%), 216, 201, 174, 161,
135, 91; HR-EI-MS: m/z 294.1272 (Mꢁ), C16H22O3S requires 294.1290; FT-
IR (KBr) cmꢀ1: 2941, 1662 (CꢂO), 1307 (CꢂS), 1200; UV (MeOH) lmax
nm (log e): 237 (4.34); CD (MeOH) lmax nm (De): 310 nm (ꢀ0.70); 1H-
NMR (CDCl3): d 1.00 (3H, d, Jꢂ6.9 Hz, H-14), 1.12 (3H, s, H-15), 1.52
(3H, s, H-13), 4.39 (1H, d, Jꢂ8.8 Hz, H-12), 4.46 (1H, d, Jꢂ8.8 Hz, H-12),
5.78 (1H, d, Jꢂ1.4 Hz, H-1); 13C-NMR (CDCl3): d 15.0 (q, C-14), 16.9 (q,
C-15), 21.0 (q, C-13), 27.0 (t, C-8), 31.9 (t, C-9), 38.9 (t, C-6), 40.5 (d, C-4),
41.0 (d, C-7), 42.0 (t, C-3), 76.9 (t, C-12), 91.8 (s, C-11), 125.4 (d, C-1),
167.6 (s, C-10), 191.1 (s, CꢂS), 199.0 (s, C-2).
tory cultivated (100 rpm) in Czapek-pepton medium at 30 °C for 3 d. (ꢁ)-
Valencene (1) (100 mgꢄ15ꢂ1.5 g) was added to the medium and further
cultivated for 5 d. The culture was worked up in a same manner as above.
The EtOAc extract was chromatographed on silica gel (n-hexane–EtOAc
gradient) to give seven metabolites: 3 (14.9 mg; 1.0%), 4 and 5 (202 mg;
13.5%), 14 (16.8 mg; 1.1%), 15 (22.6 mg; 1.5%), 16 (30.2 mg; 2.0%), and
17 (10.7 mg; 0.7%). The ratio of compounds 4 (11S) and 5 (11R) was deter-
mined to be 1 : 3 by HPLC analysis of their thiocarbonates (7, 8).
Compound 14: Colorless oil, [a]D ꢀ71.1° (CHCl3, cꢂ1.0); EI-MS: m/z
236 (Mꢁ), 218, 187 (100), 159 145, 105, 95; HR-EI-MS: [Mꢁ] 236.1749,
C15H24O2 requires 236.1777; IR (KBr) cmꢀ1: 3350 (OH), 3019, 2968, 1460,
1380; UV (MeOH) lmax nm (log e): 235.0 (4.31), 228.0 0(4.29); NMR data:
see Tables 4 and 5.
Compound 15: Colorless oil, [a]D ꢀ50.1° (CHCl3, cꢂ1.0); EI-MS: m/z
250 (Mꢁ), 232, 201, 175 (100), 173, 105, 91, HR-EI-MS: [Mꢁ] 250.1562,
C15H22O3 requires 250.1569; IR (KBr) cmꢀ1: 3417 (OH), 2940, 1666
(CꢂO), 1632, 1046; UV (MeOH) lmax nm (log e): 289.2 (4.31): NMR data:
see Tables 4 and 5.
Compound 16: Colorless oil, [a]D ꢁ17.4° (MeOH, cꢂ1.0); EI-MS: m/z
252 (Mꢁ), 234, 203, 185, 159, 143, 105, 91 (100); HR-EI-MS: [Mꢁ]
252.1708, C15H24O3 requires 252.1726; IR (KBr) cmꢀ1: 3368 (OH), 2969,
2899, 1030, 755; UV (MeOH) lmax nm (log e): 237.0 (4.11); NMR data: see
Tables 4 and 5.
Epoxidation of Nootkatone (1) To a solution of nootkatone (1)
(300.1 mg) in CHCl3 (40 ml) was added mCPBA (687 mg) with stirring at
0 °C. After stirring for 3 h , water was added and the mixture was extracted
with CHCl3. The organic phase was washed with 5% NaHCO3 and brine,
dried (MgSO4), and evaporated to give a residue (314.9 mg). The residue
was purified by silica gel column chromatography with an n-hexane–AcOEt
gradient to afford 11,12-epoxynootkatone (11) (116.1 mg, 82.7%).
Compound 11: Colorless oil, [a]D21 ꢁ173.9° (cꢂ1.01, CHCl3); EI-MS:
m/z 234 (Mꢁ), 216, 206, 176 (100%), 161, 134, 119, 105, 91; HR-EI-MS:
m/z 234.1613 (Mꢁ), C15H22O2 requires 234.1620; FT-IR (KBr) cmꢀ1: 2934,
1
1667 (CꢂO), 1287, 1200; UV (MeOH) lmax nm (log e): 236.5 (4.13); H-
NMR (CDCl3): d 1.06 (3H, s, H-15), 1.08 (3H, d, Jꢂ7.1 Hz, H-14), 1.24,
1.26 (3H, S, H-13), 2.62 (2H, m, H-12), 6.08 (1H, d, Jꢂ1.4 Hz, H-1).
Biotransformation of 11,12-Epoxynootkatone (11) by A. niger A so-
lution of 11,12-epoxynootkatone (11) (20 mg) was added to the culture
medium of A. niger. The incubation was then continued for 1 d at 30 °C. The
culture was filtered in vacuo and the broth was extracted twice with EtOAc
(each 200 ml) with stirring. The EtOAc layers were dried over MgSO4 and
the solvent was evaporated in vacuo to give the crude extract (30.5 mg) as an
oil, which was chromatographed on silica gel with a gradient solvent system
of n-hexane–EtOAc to afford 11S- and 11R-nootkatone-11,12-diol (4 and 5;
ratio 1 : 1) (17.5 mg, 81.4%).
Compound 17: Colorless oil, [a]D ꢁ52.0° (CHCl3, cꢂ1.0); EI-MS; m/z
252 (Mꢁ), 221 (100), 203, 163, 161, 123, 121, 75; HR-EI-MS; [Mꢁ]
252.1736, C15H24O3 requires 252.1726; IR (KBr) cmꢀ1: 3410 (OH), 2941,
1653 (CꢂO), 1045; UV (MeOH) lmax nm (log e): 241.6 (3.94); NMR data:
see Tables 4 and 5.
3,5-Dinitrobenzoylation of Compound 15 To
a solution of 15
(10.5 mg) in pyridine (3 ml) was added 3,5-dinitrobenzoyl chloride (38.4 mg)
and dimethylaminopyridine (10 mg). The mixture was stirred at room tem-
perature overnight. Water was added and the mixture was extracted with
CHCl3. The organic phase was washed with 1 N HCl, 5% NaHCO3, and
brine, and dried (MgSO4) and the solvent was evaporated to give a residue
(15.0 mg) that was purified by a silica gel column chromatography with a n-
hexane–AcOEt gradient to afford 18 (3.9 mg) as colorless prisms; mp 181—
183 °C, [a]D ꢁ34.6° (CHCl3, cꢂ1.0); CI-MS: m/z 445 (MꢁHꢁ, 55%), 233
(16%); HR-CI-MS; [MꢁH]ꢁ found 445.1607, C22H25O8N2 445.1611; IR
(KBr) cmꢀ1: 3451 (OH), 2973, 1733 (CꢂO), 1668 (CꢂO), 1632 (CꢂC),
1280; UV (MeOH) lmax nm (log e): 286.4 (4.10), 230.0 (4.45), 208.0 (4.55);
CD (MeOH) lmax nm (De): 335.8 (ꢁ4.45), 288.7 (ꢀ7.21), 228.7 (ꢀ0.38),
211.7 (ꢁ2.36); 1H-NMR (CDCl3): d 1.03 (3H, s, H-15), 1.12 (3H, d, Jꢂ6.9
Hz, H-14), 1.35 (3H, s, H-13), 4.43 (1H, d, Jꢂ11.5 Hz, H-12), 4.51 (1H, d,
Jꢂ11.5 Hz, H-12), 5.89 (1H, d, Jꢂ9.9 Hz, H-2), 6.01 (1H, m, H-9), 6.89
(1H, d, Jꢂ9.9 Hz, H-1), 9.17 (2H, d, 2.2 Hz, aromatic protons), 9.26 (1H, t,
2.2 Hz, aromatic proton).
Biotransformation of Nootkatone (1) by F. culmorum An Erlenmeyer
flask (500 ml) containing 200 ml of Czapek-pepton medium was inoculated
with a suspension of F. culmorum and incubated at 30 °C for 5 d in a rotary
shaker operating at 100 rpm. After full growth of the microorganisms, a so-
lution of (ꢁ)-nootkatone (1) (20 mg) was added to the culture medium of F.
culmorum. The incubation was then continued for a further 20 d at 30 °C.
The culture was filtered in vacuo and the broth was extracted twice with
EtOAc (each 100 ml) with stirring. The EtOAc layers were dried over
MgSO4 and the solvent was evaporated in vacuo to give the crude extract
(494 mg) which was treated in the same manner as described above to give
9b-hydroxynootkatone (12) (3.2 mg; 14.9%) and (11R)-nootkatone-11,12-
diol (5) (10.9 mg; 47.2%). The purity of compound 5 was determined to be
ca. 95% by HPLC analysis of the thiocarbonate (8).
(11R)-Nootkatone-11,12-diol (5): Colorless oil, [a]D24 ꢁ132.0° (cꢂ0.82,
CHCl3); CI-MS: m/z 253 (MꢁꢁH; 100), 235, 221, 177; HR-CI-MS: m/z
253.1800 (MꢁꢁH), C15H25O3 requires 253.1803; FT-IR (KBr) cmꢀ1: 3359
(OH), 2970, 1652 (CꢂO), 1615 (CꢂC), 1301, 1046; UV (MeOH) lmax nm
(log e): 239 (4.13); NMR data: see Tables 1 and 2.
The crystal data for 18 (Table 3) are as follows: Triclinic, space group P1,
aꢂ6.2170 (3) Å, bꢂ9.0460 (5) Å, cꢂ10.5030 (8) Å, Vꢂ525.56 (6) Å2, aꢂ
68.852 (3) Å, bꢂ87.842 (3) Å, gꢂ73.086 (3) Å, Zꢂ1, Dxꢂ1.360 Mg mꢀ3
,
m (MoKa)ꢂ0.105 mmꢀ1, lꢂ0.71073, final R was 0.0457 for 1883 reflec-
tions. The supplementary materials have been deposited at the Cambridge
Crystallographic Data Center.
9b-Hydroxynootkatone (12): Colorless oil, [a]D24 ꢁ131.3° (cꢂ1.65,
CHCl3); EI-MS: m/z 234 (Mꢁ), 216, 191, 166, 137 (100%), 109; HR-EI-MS:
Reduction of Nootkatone (1) with NaBH4–CeCl3 CeCl3·7H2O

November 2005
1429
(9.24 g) was added to a solution of nootkatone (1) (5.52 g) in EtOH (120 ml)
and stirred at room temperature. NaBH4 (1.45 g) was slowly added over
20 min. The mixture was stirred for 1 h and extracted with Et2O (500 ml) and
1 N HCl (500 ml). The organic phase was washed with brine, dried (MgSO4),
and evaporated to give a residue (6.187 g). The residue was purified by a sil-
ica gel column chromatography with an n-hexane–AcOEt gradient to afford
2a-hydroxyvalencene (19) (4.846 g, 82.7%) as a colorless oil.
Acknowledgments We thank Dr. M. Tanaka, Mr. S. Takaoka, and Miss
Y. Okamoto (TBU) for providing 600-MHz NMR, X-ray crystallographic
analysis, and mass spectra, respectively. We also thank Takasago Interna-
tional Co., Ltd., Japan, for providing valencene and nootkatone.
References
1) Kieslich K., Annual Reports on Fermentation Processes, 1, 267—297
(1977).
2a-Hydroxyvalencene (19): Colorless oil, [a]D24 ꢁ94.2° (cꢂ0.92, CHCl3);
EI-MS: m/z 220 (Mꢁ), 202, 187, 161, 145, 119 (100%), 105; HR-EI-MS:
m/z 220.1818 (Mꢁ), C15H24O requires 220.1827; FT-IR (KBr) cmꢀ1: 3320
(OH), 2928, 1644 (CꢂC), 1024.
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8) Matsumoto T., Hayashi N., Ishida T., Asakawa Y., J. Pharm. Sci., 79,
540—547 (1990).
9) Matsumoto T., Hayashi N., Ishida T., Asakawa Y., Chem. Pharm. Bull.,
40, 1721—1726 (1992).
10) Hashimoto T., Noma Y., Asakawa Y., Heterocycles, 54, 529—559
(2001).
Preparation of Nootkatol (20) by Mitsunobu Reaction of 2a-Hydroxy-
valencene (19) To a solution of p-nitrobenzoic acid (485 mg) and tri-
phenylphosphine (767 mg) in dry toluene (60 ml) at ꢀ30 °C was added 2a-
hydroxyvalencene (19) (500 mg). Diethyl azodicarboxylate-toluene 40% so-
lution (1.14 ml) was added dropwise for 15 min to the mixture. After stirring
for 1 h at ꢀ30 °C, the mixture was extracted with Et2O (300 ml) and a satu-
rated NaHCO3 solution (100 ml). The organic phase was washed with brine,
dried (MgSO4), and evaporated to give a residue (1.295 g). A solution of the
residue in MeOH (30 ml) and H2O (3 ml) was treated with KOH (1.50 g) and
stirred for 12 h at room temperature. The mixture was extracted with Et2O
(200 ml) and brine (100 ml), dried (MgSO4), and evaporated to give a
residue (0.985 g) which was purified by a silica gel column chromatography
with an n-hexane–AcOEt gradient to afford nootkatol (20)19) (208 mg,
41.6%) as a colorless oil.
Nootkatol (20): [a]D25 ꢁ213.3° (cꢂ0.98, CHCl3); EI-MS: m/z 220 (Mꢁ),
202, 187, 161, 145, 119 (100%), 105, 91; HR-EI-MS: m/z 220.1857 (Mꢁ), 11) Haze S., Sakai K., Gozu Y., Jpn. J. Pharmacol., 90, 247—253 (2002).
C15H24O requires 220.1887; FT-IR (KBr) cmꢀ1: 3251 (OH), 2929, 1645 12) Hashimoto T., Asakawa Y., Noma Y., Murakami C., Tanaka M., Kani-
(CꢂC), 1011.
sawa T., Emura M., Jpn. Kokai Tokkyo Koho, 2003-70492A (2003).
Biotransformation of 2a-Hydroxyvalencene (19) by A. niger A. niger 13) Hashimoto T., Asakawa Y., Noma Y., Murakami C., Furusawa M.,
was rotary cultivated (100 rpm) in Czapek-pepton medium at 30 °C for 3 d.
2a-Hydroxyvalencene (19) (100 mg) was added to the medium and further
cultivated for 5 d. The culture was worked up in the same manner as above. 14) Teresa J. de P., Barrero A. F., San Feliciano A., Grande M., Medarde
Kanisawa T., Emura M., Mitsuhashi K., Jpn. Kokai Tokkyo Koho,
2003-250591A (2003).
The EtOAc extract was chromatographed on silica gel (n-hexane–EtOAc
gradient) to give three metabolites: 4 and 5 (7.3 mg; 6.4%), 14 (37.1 mg;
34.6%), and 15 (6.3 mg; 5.5%).
M., Tetrahedron Lett., 43, 4145—4148 (1978).
15) Haines A. H., Jenkins C. S. P., J. Chem. Soc. (C), 1971, 1438—1442
(1971).
Biotransformation of Nootkatol (20) by A. niger A. niger was rotary
cultivated (100 rpm) in Czapek-pepton medium at 30 °C for 3 d. Nootkatol
(20) (100 mg) was added to the medium and further cultivated for 5 d. The
16) Ishida T., Ph. D thesis, Tokushima Bunri University (1987).
17) Krepinsky J., Motl O., Dolejs L., Novotny L., Herout V., Bates R. B.,
Tetrahedron Lett., 29, 3315—3318 (1968).
culture was worked up in the same manner as above. The EtOAc extract was 18) MacLeod W. D., Tetrahedron Lett., 52, 4779—4783 (1965).
chromatographed on silica gel (n-hexane–EtOAc gradient) to give three
metabolites: 4 and 5 (25.0 mg; 21.8%), 15 (6.3 mg; 5.5%) and 16 (11.9 mg;
10.4%).
19) Shoji N., Umeyama A., Asakawa Y., Takemoto T., Nomoto K.,
Ohizumi Y., J. Pharm. Sci., 73, 843—844 (1984).