NPC Natural Product Communications 2018

Vol. 13
No. 8
Biotransformation of Bicyclic Sesqui- and Diterpene 1,2-dials and 923 - 932
Their Derivatives by the Fungus, Aspergillus niger
Yoshinori Asakawa*, Masako Sekita and Toshihiro Hashimoto

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan

asakawa@ph.bunri-u.ac.jp

Received: May 3rd, 2018; Accepted: May 29th, 2018

Microbial biotransformation of naturally occurring pungent sesquiterpene 1,2-dials, polygodial and cinnamodial, and a diterpene 1,2-dial, sacculatal as well as
their tetrahydro derivatives was carried out by using Aspergillus niger. The pungent polygodial and sacculatal are toxic against A. niger not to produce any
metabolites while A. niger biotransformed cinnamodial to the lactonized products in small amount. On the other hands, the dihydroxy derivatives of the
former two dialdehydes were bioconverted by the same fungus to give hydroxy-, oxo-, carboxylic- and epoxy-products. The stereostructures of each metabolite
and their metabolic pathways were described.

Keywords: Sesqui- and diterpene 1,2-dialdehydes, Biotransformation, Aspergillus niger, Fungus, Hydroxylation, Lactonization, Carboxylation.

We are continuing to study on highly efficient production of
functional compounds from plant secondary metabolites and simple
synthetic compounds by using bacteria, microalgae, fungi and
mammals and the direct introduction of oxygen function on
aromatic or cycloaliphatic ring [1-3]. One of the examples of the
industrial scale production of the functional substance is a
sesquiterpene ketone, nootkatone, which is very expensive aroma of
grape fruit and possesses antiobesity activity [4]. Nootkatone was
quantitatively produced from a cheap sesquiterpene hydrocarbon,
valencene obtained from Valencia orange by green microalgae,
Chlorella and fungus Mucor species [4].
Continuing our interest in biotransformation of terpenoids, we
herein report the bioconversion of two natural sesquiterpene 1,2-
dials, polygodial (1) and its synthetic tetrahydro derivative (2), and
9-hydroxy derivative (4) of 2, and cinnamodial (3) and one natural
diterpene 1,2-dial, sacculatal (5) and its tetrahydro derivative (6) by
using the fungus, Aspergillus niger and the structural
characterization and metabolic pathways of each metabolite.
When polygodiol (2, 20 mg) was incubated with A. niger for 3 days,
a hydroxylated product (7) was obtained in 70.5% yield as colorless
needles. The molecular formula, C15H26O3, was established by
HREIMS spectrum, indicating that compound 7 possessed one more
hydroxyl group in the original substrate. This fact was confirmed by
the formation of a triacetate (7a) by the acetylation. The position of
a newly introduced -hydroxy group at C-3 was confirmed by the
coupling patterns of the 1H NMR spectra of 7 and 7a as well as
HMBC and NOESY spectra of 7. In fact, the 1H and 13C NMR
spectra of 7 were completely identical to those of the natural
product, drim-7-ene-3,11,12-triol, isolated from the fungus
Marasmius oreades [12].
Polygodiol (2) (200 mg) was cultured for 4 days as a large scale in
the same condition as described above to afford two more
metabolites (8, 1.6%) and (9, 3.8%) as small amounts, together with
the substrate (2, 8.2%) and the former described metabolite (7,
20.2%). An amount of the substrate may be important to produce
two additional metabolites rather than incubation time.

Polygodial (1) possessing very strong pungent taste, and piscicidal
and antimicrobial activity was isolated from the higher plants,
Polygonum hydropiper, P. minus and P. punctatum and the
liverwort, Porella vernicosa complex [5-8]. Sacculatal isolated
from the liverwort, Trichocoleopsis sacculata and Pellia
endiviifolia also shows very hot taste and antimicrobial and anti-
HIV activity [9-11].
In order to obtain the substrate, polygodial (1), a high amount of the
fresh Polygonum hydropiper collected in Tokushima was extracted
with diethyl ether to afford a green pungent extract, followed by
chromatography on silica gel to furnish (-)-polygodial (1). An
Erlenmeyer flask containing Czapek-peptone was inoculated with a
suspension of A. niger and incubated at 30°C for 3 days in a rotary
shaker. A small amount of 1 (20 mg) was cultured by A. niger for 3
days under the same condition as described above, no metabolites
were obtained. Therefore, polygodial (1) was reduced to polygodiol
(2) by LiAlH4, followed by bioconversion by using the same fungus
for 2-21 days (Figure 1).
The HREIMS of 8 showed the same molecular formula as the
metabolites 7, and its IR spectrum indicated the absorption at 3353
cm-1 ascribe to hydroxy group. In the 1H and 13C-NMR spectra
(Table 1), one methyl group connected to tertiary carbon
disappeared and one oxo methylene proton signals (H 3.01 and
3.31, each doublet; c 71.8) was newly confirmed, indicating that
one of the 1,1-dimethyls at C-4 or methyl group at C-10 was
hydroxylated. The position and the stereochemistry at the -
hydroxylated methyl group at C-15 was confirmed by the 1H-1H
COSY, HMQC, HMBC (Figure 2a) and NOESY spectra (Figure
2b) of 8. In HMBC spectrum, the methylene protons (, 3.01, 3.31)
at C-14 were correlated to C-3, 4, 5 and 13 and NOE was observed
between H-14 and H-5, and H-6 by NOESY. Thus the structure
of 8 was characterized as drim-7-ene-11,12,14-triol.
Compound 9, had the molecular formula, C15H24O3 which was
established by HREIMS. The IR spectrum showed the absorption
bands at 3389 and 1701 cm-1 assignable to a hydroxy, and a ketone
groups which was confirmed by the presence of the signal at c
218.8 in the 13C NMR spectrum. The 1H and 13C NMR of 9 (Table
1) resembled those of 7 except for the presence of a ketone group,
924 Natural Product Communications Vol. 13 (8) 2018
Figure 1: Biotransformation of polygodiol (2) by Aspergillus niger.
Figure 2a: HMBC correlation of 8. Figure 2b: NOE correlation of 8.
indicating that compound 9 was 11,12-dihydroxydrim-7-en-3-one.
This assumption was confirmed by the cross peaks of H-1, 2, 5, 13
and 14 to C-3 in HMBC spectrum.
The same bioconverted products (7, 66-70%) and 6-hydroxy-
polygodiol (7b, 5-10%) were obtained from the metabolites of
polygodiol (2) by the fungus, Mucor plumbeus for 2-6 days [13].
The similar drimane sesquiterpene mono-alcohol drimenol (7c) was
incubated with M. plumbeus to give 3-hydroxy- (7d) and 6-
hydroxydrimenol (7e), along with 3-hydroxy-7,8-epoxy-
drimenol (7f) [13]. Rhizopus arrhizus and A. niger also
bioconverted drimenol (7c) to 3-hydroxydrimenol (7d) [13,14] as
shown in Figure 3, while the present A. niger converted polygodiol
(2) to C-3 oxo- (9) and C-13 hydroxy products (8), together with C-
3-hydroxy product (7, 20-70%) for 3-4 days. From yield of the
metabolites from 2 and 7c, the regio- and stereoselective
introduction of a hydroxy group at C-3 predominantly occurred in
these microorganisms.
Cinnamodial (3) obtained from the Malagasy medicinal plant,
Cinnamosma fragrans shows pepper like pungency, antimicrobial
and insect antifeedant activity [15, 16]. Compound (3, 200 mg) was
incubated with A. niger in the same manner as previously described
to yield three metabolites, (10, 2.2%), (11, 0.05%) and (12, 0.2 %),
respectively (Figure 4).
The molecular formula of 10 was established by a combination of
FABMS, EIMS and HRFABMS as C17H26O5. The presence of an
acetoxy and a hydroxy groups was confirmed by the presence of the
IR absorption bands at 1701 cm-1 and the 1H NMR spectrum at H
2.07 (3H, s) and 3389, and 3389 cm-1 and C 170.4, respectively.
The two aldehyde protons and carbonyl signals present in the
substrate (3) disappeared and a trisubstituted double bond, an
acetate and a hydroxy group on quaternary carbon retained from
the 1H and 13C- NMR spectra (Table 2), instead an oxygenated
Asakawa et al.
Figure 3: Biotransformation of drimenol (7d) by three fungi.
methylene (H: 4.25, 4.61, 2H, each dt;, C 66.5) and a hemiacetal
methine (H: 5.37, 1H, s; C, 98.1) newly appeared in the NMR
spectra. On the basis of the molecular formula and the above
spectral data, compound 10 was suggested to be a tricyclic structure
with a hemiacetal and a tertiary hydroxy group. The conclusive
structure of 10 was established by the connectivity of HMBC
spectrum in which cross-peaks between H-12 and C-7, 8, 9, 11, and
H-11 and C-9 and C-12 were observed, respectively. The
stereochemistry of secondary hydroxy group at C-11 was confirmed
to be by the NOESY in which the correlations between H-11
and H-12, H-1 and H-15, H-2 and H-14 were observed,
respectively. Thus structure of 10 was established as 6-acetoxy-
11,12-epoxy-drim-7-ene-9,11-diol. The metabolite 10 has been
known as the reduction product, 6-O-acethylpereniporin A (10)
from cinnamodial (3) [17]. The partial 1H NMR data of the
synthetic product (10) were reported, however, the assignment of all
of the protons remained to be clarified and its 13C NMR data were
not presented. In Table 2, the assignment of all of both protons and
carbons of the metabolite (10) were presented.
Figure 4: Biotransformation of cinnamodial (3) by A. niger.
Compound 11, possessing the molecular formula, C17H24O5,
confirmed by HRCIMS showed the presence of a tertiary hydroxy
(3405 cm-1), an acetate (1732 cm-1) and a -lactone (1761 cm-1) and
a conjugated double bond (211.4 nm) by IR and UV spectra. The
presence of each functional group was determined by the 1H and
13
C NMR spectra as well as HMBC spectrum in which the
connectivity between H-7 and C-9 and C-12, and H-11 and C-8, 9
and 12 was shown, respectively. On the basis of the above data, the
structure of 11 was deduced to be 6-acetoxy-11,12-epoxy-12-oxo-
drim-7-ene-9-ol. In fact this compound was isolated from
Cinnamosma fragrans and Capsicodendron dinisii belonging to the
same family, Cannelaceae and named cinnamosmolide [15,16, 18].
Microbial biotransformation of terpenoids
Compound 12 was obtained as colorless crystals. Its molecular
formula was determined to be C15H20O3 by HREIMS. The IR, UV
and NMR spectra of 12 showed the presence of a ketone (1718 cm-
1
; C 205.8), a conjugated -lactone (1762 cm-1; 216.6 nm; C
172.4), and a tetrasubstituted double bond (C 121.8, 170.1), a
methylene (H 4.83, 2H, m), and a methylene (C 68.2) on carbon
attached an oxygen function, respectively. The conclusive structure
of 12 was established by its HMBC spectrum. It showed the
correlation between H-5 and C-6, 9, 10, H-7 and C-6, 8, 9, and H-
13 and C-1, 9 and 10, respectively. Thus the structure was deduced
as 11,12-epoxy-6,12-dioxo-drim-8-ene (12) which was known as
fragrolide isolated from Cinnamosma fragrans [16, 19]. All of the
spectral data of 12 were similar to those of the natural product.
Biotransformation of 9-hydroxy-11, 12-diacetoxy-drim-7-ene
(4): Warburganal (13) isolated from Warburgia ugandensis has
been known to show insect antifeedant activity [20]. Warburganal is
9-hydroxypolygodial. In order to obtain some functional
metabolites from 13, the partial synthesis of 13 was performed as
shown in figure 5 [21], however, the reaction mixture did not
contain warburganal, Thus, the intermediate, 9-hydroxy-11,12-
diacetoxydrim-7-ene (4, 50 mg) prepared from 2 by acetylation was
incubated by A. niger in the same manner as previously described
for 2 days to give a hydroxylated metabolite (15, 47.2%) as
colorless oil (Figure 5).
OH
11 11
15
12
2
1
10
9
8 OH
f)
3 5 7
4 6
13
H
14
H H
2 14
f) Ac2O/Py room temp.
g) SeO2/dioxane/120 oC/1hr
h) Aspergillus niger
11
CHO
[(14: 50 mg/2days)]
OH
H H
13
Figure 5: Biotransformation of 9-hydroxy-11,12-diacetoxydrim-7-ene (4) by A. niger.
The molecular formula, C19H30O6, of compound 15 was deduced by
pseudo molecular peak at m/z 377.1917 ([M] + Na)]+ of
HRFABMS. The 1H and 13C NMR spectra of 15 (Table 3) were
very similar to those of the substrate (4) (Table 3) except for the
presence of a hydroxymethine signal [H, 3.31 (1H, d) and C 78.1],
indicating that compound 15 possessed an additional secondary
hydroxy group on one of the two cyclohexane rings. The position of
this functional group at C-3 was established by cross peaks of H-1
to C-3, H-13 and H-14 to C-3 in HMBC spectrum (Figure 6a) and
its configuration was also confirmed to be 3 by the NOE
correlation (Figure 6b) of H-3 to H-1, H-5 and H-14.
Consequently the structure of 15 was accomplished as 11,12-
diacetoxy-drim-7-ene-3,9-diol.
Figure 6a. HMBC correlation of 15. Figure 6b. NOESY correlation of 15
Biotransformation of sacculatal (5): Sacculatal (5, 1.00 g) was
isolated from the fresh liverwort, Pellia endiviifolia. To Czapek
peptone medium including A. niger, sacculatal (5, 20 mg) was
Natural Product Communications Vol. 13 (8) 2018 925
added and incubated for a couple days, however, no metabolites
were obtained. As described in the previous section, two aldehyde
groups were reduced to give sacculatadiol (6, 200 mg) by LiAlH4
which was further biocoverted by the same fungus for 15 days and
the crude metabolites were purified by the same method as
described earlier to obtain (16, 1.6%), (17, 6.7%), (18, 6.1%), and
19 as an acetate (19a) (Figure 7).
Figure 7: Biotransformation of sacculatadiol (6) by A. niger.
OAc OAc
12 OH The HREIMS and EIMS of compound 16 showed the molecular
OAc 9 OAc formula as C17H24O4. The IR absorption bands at a 3400-3100 cm-1
g)
(broad), 1760 and 1706 cm-1 showed the presence of a -lactone
and a carboxyl groups which were confirmed by the 13C NMR
4
signals at C 170.1 and C 178.6 . In the 13C NMR, two olefinic
h) methyls, a methyl carbinol and an olefinic methine disappeared
OAc from that of the substrate (6) and totally 17 carbon signals appeared
12
CHO
OH
including two carbonyl carbon signals, indicating that 16 was
9
OAc
trinorsacculatanolide monocarboxylic acid without an isopropeny
3
HO group attached in sacculatadiol (6). The location of both a -lactone
and a carboxylic acid of 16 were determined by the correlation of
15
H-11 to C-8, 9, 10 and 12, and H-15 and H-16 to C-17 in HMBC
spectrum. Thus, the structure of 16 was elucidated as 11,12-epoxy-
12-oxo-trinorsacculat-7-en-17-oic acid.
The molecular formula of 17 was deduced to be C20H28O5 by
HREIMS and its IR spectrum indicated the presence of a conjugated
carboxylic (3448-2935, 1688 cm-1) and a -lactone (1747 cm-1). The
1
H and 13C NMR spectra of 17 (Table 4) showed the presence of
two carbonyl group (C 169.9, 172.3), a methine (H 3.57, 1H, dd;
C 73.3) and a methylene (H 4.05, 4.39 each t; C 67.1) attached an
oxygen function. The 1H and 13C NMR signals of 17 were very
similar to those of 16, except for the presence of the secondary
hydroxy group, and an olefinic methyl, indicating that 17 possessed
the same sacculatenolide skeleton as that of 16 with a -hydroxy
group at C-3 and a carboxylic group at C-18. This presumption was
supported by the cross-peaks of H-17 to C-15 16, 18, 19 and 20, and
H-3 to C-1, 2, 4, 14 and 15, along with the correlations of H-11 to
C-9, 8, 10 and 12 in the HMBC spectrum (Figure 8a). The
stereochemistry of the secondary hydroxy group at C-3 and
geometry between C-17 and C-18 were established to be  and E
configuration by the NOESY spectrum (Figure 8b) in which
correlations between H-3 and H-1 and H-5, and H-16 and H-19,
respectively. Thus the structure of 17 was characterized as 3-
hydroxy-11,12-epoxy-12-oxo-sacculat-7,17-dien -20-oic acid.
Compound 18, C20H32O5 (m/z 352.32259) established by HREIMS,
displayed the presence of a conjugated carboxylic acid (3500-3100,
926 Natural Product Communications Vol. 13 (8) 2018
Figure 8a: HMBC correlation of 17. Figure 8b: NOESY correlation of 17.
1683 cm-1; C 171.7) and -hydroxy group (3.51, m;C 73.9)
and its 1H and 13C NMR signals (Table 5) are very similar to those
of the original sacculatadiol (6) and except for the absence of one
allylic methyl and the presence of a secondary alcohol in 18,
indicating that the structure 18 was 3, 11, 12-trihydroxy-sacculat-
7,17-dien-20-oic acid. This prediction was further confirmed by
HMBC correlations (Figure 9a) of H-1, 2 and 14to C-3, and H-17 to
C-15, 16, 18, 19 and 20, as seen in the structure of 17.The
stereochemistry of the hydroxy group at C-3 and E-configuration at
C-17 and C-18 double bond were confirmed by NOESY (Figure 9b)
in which correlations of H-3 and H-5, and H-16 and H-19 appeared,
respectively.
Figure 9a: HMBC correlation of 18. Figure 9b: NOESY correlation of 18.
The original metabolite of 19 was difficult to isolate as a pure state,
therefore, it was acetylated with acetic anhydride and pyridine to
afford a triacetate (19a) after the absence of an acetoxy group in the
original metabolite mixture was confirmed by the 1H and 13C NMR
spectra (Table 5). Its molecular formula C26H42O7 was suggested by
HREIMS, the 1H and 13C NMR and HMQC spectra in which 3
acetoxy-, 2 methylenes and one methine attached to oxygen
function, one trisubstituted double bond, a dimethyl carbinol,
together with 5 sp3 methylenes, 2-methyls, 2 sp3 methines and 2
quaternary carbons were observed. The IR spectrum of 19a showed
the presence of a hydroxy (3502 cm-1) and an ester group (1738 cm-
1 7-ene. This presumption was also established by the cross-peaks of
). The 1H and 13C NMR signal patterns of 19a were similar to those
of compound 4, except for the presence of a dimethyl carbinol and
one more acetoxy group in 19a, indicating that the structure of 19a
was suggested to be 11,12,17-triacetoxy-sacculat-7-ene-18-ol. The
above presumption was confirmed by HMBC correlations of H-17
to C-16, C-19 and C-20, as well as an acetoxy carbon at C-17, and
H-19 and H-20 corresponding to the tertiary dimethyl group to C-17
and C-18, and H-11 and H-12 to C- 11 and C-12-acetoxy carbons.
Thus the structure of the original metabolite was sacculat-7-ene-
11,12,17,18-tetraol (19). The absolute configuration at C-17
remained to be clarified.
Two times bigger amount of sacculatadiol (6, 400 mg) was fed by
A. niger in the same medium as described above to afford three
more metabolites, (20, 3.9%), (21, 0.78%) and (22, 0.86%), along
with (17, 44%) and (18, 1.1%) (figure 10).
Compound 20 had a molecular formula, C20H28O5 based on
the HREIMS. The presence of a carboxyl and an ester groups was
suggested by IR absorption bands at 3511-3293 cm-1 and 1743 and
1737 cm-1. Analysis of 1H and 13C NMR spectra (Table 6) showed
that compound 20 possessed the same structure as that of compound
Asakawa et al.
Figure 10: .Biotransformation of sacculatadiol (6) by A. niger.
17. The only difference was the position of the secondary hydroxyl
group. In order to clarify this problem, HMBC and NOESY were
measured. HMBC cross-peaks from H-1 to C-9, 10, 13 and H-2, 3
and H-13 to C-1 established the location of the secondary hydroxy
group at C-1. Furthermore, the presence of a 11,12-olide and a
carboxylic acid at C-18 was confirmed by HMBC cross-peaks from
H-11 to C8, 9, 10 and 12, H-17 to C-15, 16, 19 and 20, and H-19 to
C-17, 18 and 20. The stereochemistry of the hydroxyl group at C-1
was also established as  by NOESY in which the correlations of
H-1 and H-3, H-5 and H-9 were observed. Thus the structure 20
was predicted as 1-hydroxy-11,12-epoxy-12-oxo-sacculat-7,17-
dien-20-oic acid. However, the position of the absorption band at
1737 cm-1 in the IR spectrum of 20 is higher than that of the -
unsaturated carboxylic acid (17), indicating that the proposed
structure was not fit to the proposed structure although all of the
other spectral data were suitable to the proposed structure. This
discrepancy is now under consideration.
The molecular formula of compound 21, C20H30O4, was confirmed
by HREIMS. Analysis of IR, 1H and 13C NMR spectra (Table 6)
showed that 21 contained a hydroxyl group, the same unsaturated -
lactone as shown in 16, 17 and 20 and the NMR spectra resembled
those of the above mentioned sacculatenolides, only difference was
that 21 had a hydroxy group (3450 cm-1; C, 77.1) on quaternary
carbon and two more methyls connected to tertiary carbon attached
on oxygen function. On the basis of the above spectral data, one
remaining oxygen was deduced to be an ether suggesting that the
structure 21 was 5-hydroxy-11,12,17,18-diepoxy-12-oxo-sacculat-
HMBC, which showed correlation of H-3, H-14 to C-5, H-13 to C-
1, 5, 9 and 10, H-11 to C-8 and C-12, H-19 and 20 to C-17 and 18.
The stereochemistry of the tertiary hydroxy group at C-5 was also
established as  by the correlation of NOESY. The cross peaks
were observed between H-13 and H-14, H-1 and H-13 and H-14,
H-11 and H-13, H-6 and H-13, and H-1 and H-9. The
stereochemistry of the epoxide remained to be determined.
The 1H and 13C NMR spectra (Table 7) of the final metabolite 22,
C20H32O4, which was determined by HREIMS, were very similar to
those of the substrate 6. Only difference was that one of two allylic
methyls in 6 was replaced by an -unsaturated carboxylic acid
which was confirmed by the broad (3500-3381 cm-1) and sharp
absorption bands (1684 cm-1) of IR spectrum and the presence of C
171.9 in the 13C NMR spectrum. The both NMR spectra resembled
those of 18. Only difference was that 3-hydroxy group disappeared
in 22. On the basis of the above spectral data, the structure of 22
was easily deduced as 11,12-dihydroxysacculat-7,17-dien-20-oic
acid. This assumption was supported by the presence of the same
HMBC cross-peaks (H-17 to C-16, 18, 19 and 20) as seen in
compound 17, 18 and 20.
Microbial biotransformation of terpenoids
Metabolic pathways of polygodiol (2) and cinnamodial (3), and
sacculatadiol (6): From the metabolites of compound 2, two
metabolic pathways are suggested as shown in Figure 11. The route
(A) shows the stereo-selective direct introduction of the hydroxy
group at C-3 to yield the metabolite 7, followed by oxidation to
produce the 3-oxodrim-7-ene-11,12-diol (9). The route (B) is also
direct hydroxylation at C-15 of 2.
Figure 11: Possible metabolic pathways of polygodiol (2) by A. niger.
Figure 12: Possible metabolic pathways of cinnamodial (3) by A. niger.
Cinnamodial (3) was converted to the hemiacetal and -
unsaturated lactones. It is suggested that there are two metabolic
pathways for the production of the hemiacetals and -lactones from
cinnamodial (3). In the route (A), one of the aldehyde at C-9 was
reduced enzymatically, to give the C-9 primary alcohol, followed
by cyclization to give a C-12-hemiacetal, which is oxidized to give
cinnamosmolide (11), followed by dehydration and migration of the
trisubstituted double bond or hydrogenation, followed by
dehydration to afford the stable -unsaturated -lactone which is
reduced to give 6-hydroxy product, then oxidation to furnish
fragrolide (12). In the second route (B), the aldehyde at C-8 is
reduced to give C-8 primary alcohol, followed by the direct
cyclization to produce compound (10) (Figure 12).
There are two biogenetic pathways of sacculatadiol (6). The first
route (A) is that 6 is converted to C-18 carboxylic acid to produce
compound 22, followed by hydroxylation at C-1 and lactonization
between C-11 and C-12 to furnish compound 20. The same
carboxylic diol (22) is further oxidized to give the triol (18),
followed by lactonization to afford 17. The second route (B)
includes that the formation of a 17,18-epoxide (23) from 6,
followed by hydroxylation at C-5 and lactonization to give 5-
hydroxy-17,18-epoxysacculatenolide (21). The epoxide is further
hydrated to give 17,18-diol, followed by oxidation to give
trinorsacculatenolide-17-oic acid (16) (Figure 13).
Natural Product Communications Vol. 13 (8) 2018 927
Figure 13: Possible metabolic pathways of sacculatadiol (6) by A. niger.
In conclusion, the drimane sesquiterpene dialdehyde, polygodial (1)
and cinnamodial (3) and sacculatane diterpene dialdehyde,
sacculatal (5) which show pungent taste and interesting biological
activities such as antimicrobial, piscicidal, and anti-HIV activity
were incubated by the black fungus, Aspergillus niger. From the
former two dialdehydes, their metabolites were not obtained
because they possess potent antifungal activity [22]. The dihydro
derivative, polygodiol (2) and 11,12-diacetoxy-drim-7-ene-9-ol
(4) were smoothly biotransformed to give 3-hydroxy products (7,
15) in good yield together with 3-oxo- (9) and 15-
hydroxypolygodiol (8). The present method shows the highly
efficient production of 3-hydroxypolygodiol (7) possessing insect
antifeedant activity [23] since a high amount of polygodial (1) is
easily available from the Polygonaceae, Canellaceae and
Winteraceae plants [24]. Cinnamodial (3) was converted to the
hemiacetal and -unsaturated -lactones (10-12), however, 3-
hydroxy and 3-oxo products were not obtained. In case of
sacculatadiol (6), the hydroxylation at C-3, lactonization between
C-11 and C-12, and oxidation at C-17 and C-20 occurred to give the
metabolites (17-22) in small yields. These structures resembled
those of the metabolites obtained from polygodiol (2) and
cinnamodial (3). The biotransformation time of sacculatadiol was
longer than that of polygodiol because the former substrate has
bigger molecule than polygodiol.
Experimental
General: Specific rotations were measured on JASCO DIP-1000 or
JASCO P-1030 at 19-22°C using CHCl3 or MeOH. The UV and IR
spectra were recorded on a Shimadzu UV-1650 and a Shimadzu
FTIR-8400S or a Perkin Elmer FTIR, respectively. For the IR
spectral measurement, the samples were absorbed on a powdered
KBr surface and measured with the diffusion reflection method. The
EI, CI and FAB Mass spectra including high resolution ones were
measured with a JEOL 700 Mstation or JMS AX-500 at 70eV. The
1
H and 13C NMR spectra were recorded on a Varian Unity 600 (1H:
600 13C: 150 MHz), a JNM GX 499 (1H: 400, 13C: 100 MHz) and
928 Natural Product Communications Vol. 13 (8) 2018
Varian Mercury-300 (1H: 300, 13C: 75 MHz) using CDCl3 unless
otherwise stated. Chemical shift values were expressed in 7.26
(ppm) from CDCl3, as a standard (1H NMR) and  77.03 (ppm)
from CDCl3 as standard (13C NMR). TLC: Silica gel plate (Merk
GF254, 0.25mm) was used and developed in glass tank using n-
hexane/EtOAc 4:1) and visualized under UV light (254 nm) and by
spraying with 10% H2SO4 or Godin reagent [25], followed by
heating at 120-130℃. Column chromatography: Silica gel (Merck
silica gel 70-230 and 230-400 mesh), Sephadex LH-20 (Pharmacia
Fine Chemicals) and Cosmosil 75C18-OPN (Nakalai Tesque) were
performed using n-hexane and EtOAc gradient or a mixture of
CHCl3/MeOH (1:1) or CH2Cl2/MeOH (1:1) as the solvents.
Preparative HPLC: Shimadzu LC-10AS/Shimadzu SPD-10A and
RID-10A detectors or JASCO PU-2086 plus/JASCO UV-2070 and
RI-2031 plus were used. COSMOSIL 5SL-II Waters (10 x 250 mm)
and COSMOSIL 5C18-Ar-2 Waters (4.6 x 250 mm and 10 x 250
mm) columns were used for purification of each component.
Microorganisms and medium: Aspergillus niger was isolated from
soil in Osaka prefecture and was identified based on its physiologic
and morphologic features. A Czapek-peptone medium {(1.5%
glucose, 1.5% sucrose, 0.5% polypepton, 0.1% K2HPO4, 0.05%
KCl, 0.05% MgSO4・7H2O, 0.001% FeSO4・7H2O} in distilled
water, pH 7.0)} was used for the bioconversion of each substrate by
A. niger.
Isolation of polygodial (1): The pungent green ether extract (57.2
g) from the fresh Polygonum hydropiper collected in Kuwano
riverside, Anan, Tokushima, Japan in May 2006 was
chromatographed on silica gel (n-hexane/EtOAc gradient) and
Sephadex LH-20 (CHCl3/MeOH 1:1), repeatedly to afford
polygodial (1, 1.36 g) as a white crystal. mp 46-47 oC; []D -37.7 (c
0.60, CHCl3); HREIMS: anal. m/z 234.1597 [M]+ (calcd for
C15H22O2:234.1620). The spectral data (NMR, IR and MS) of 1
were identical to those of the authentic sample and the reference
data [8].
Reduction of polygodial (1): To LiAlH4 (612 mg) suspended in
dried diethyl ether (20 mL) was added polygodial (1) (750 mg) in
dried diethyl ether (13 mL) and stirred for 3 hr at 0.5oC. Ethyl
acetate (20 mL) was added to the reaction mixture at 0oC and stirred
for 10 min, then 1N HCl (10 mL) was added and the reaction
mixture was partitioned between ethyl acetate and water, followed
by evaporation of ethyl acetate to give the residue (711.2 mg) which
was chromatographed on Sephadex LH-20 using CH2Cl2 and
MeOH (1:1) and silica gel using n-hexane-EtOAc gradient to afford
polygodiol (2, 511.5 mg).
Polygodiol (2)
White crystals.
MP: 74-75oC.
[]D: -15.4 (c 1.06, EtOH).
FT/IR: 3617 cm-1.
1
H NMR (300 MHz, CDCl3): d 0.75 (3H, s), 0.86 (3H, s), 0.88 (3H,
s), 3.67 (1H, dd), 3.91 (1H, dd), 3.97 (1H, d), 4.31 (1H, br, d), 5.78
(1H, t).
HREIMS: anal. m/z 238.1931[M]+ (calcd for C15H26O2 234.1620).
EIMS: m/z 238 (M+, 100%), 220 (6), 190 (91), 175 (16), 124 (50),
109 (100), 91 (24), 69 (22), 41 (17).
Biotransformation of polygodiol (2) (20 mg x 1) by Aspergillus
niger: An Erlenmeyer flask (500 mL) containing 200 mL of
Czapek-peptone medium was inoculated with a suspension of A.
niger and incubated at 30 °C for 3 days in a rotary shaker operating
at 100 rpm. After full growth of the microorganisms, a solution of
Asakawa et al.
polygodial (2, 20 mg) was added to the culture medium of A. niger.
The incubation was then continued for a further 3 days at 30°C.
After the completion of the incubation time, the culture was filtered
in vacuo and extracted twice with EtOAc under stirring. The EtOAc
layer was dried over MgSO4 and the solvent was evaporated in
vacuo to give the crude extract (42.2 mg) which was
chromatographed on silica gel with a gradient solvent system of n-
hexane-EtOAc by increasing the amount of EtOAc stepwise in 5%
increments to afford drim-7-ene-3,11,12-triol (7, 15.5 mg).
Drim-7-ene-3,11,12-triol (7)
Colorless needles.
MP: 159-160oC.
[]D: -5.1 (c 0.99, MeOH).
FT/IR: 3274 cm-1.
The 1H and 13C NMR spectra of 7 were identical to those of drim-7-
ene-3,11,12-triol (7) [12].
HREIMS: anal. m/z 254.1887 [M]+ (calcd for C15H26O3 254.1892);
EIMS: m/z 254 (M+, 8 %), 220 (6), 223 (4), 188 (100), 173 (47),
133 (12), 96 (48), 91 (16), 81 (13), 41(10).
Acetylation of 7: Compound (7) was dissolved in dried acetic
anhydride (1.5 mL) and pyridine (1.5) and stirred at room temp. for
1 day. The reaction mixture was treated in usual manner to give
drim-7-ene-3,11,12-yl triacetate (7a, 8.8 mg).
Drim-7-ene-3,11,12-yl triacetate (7a)
[]D +24.1 (c, 0.99, CHCl3).
FT/IR:1735 cm-1.
1
HNMR (300 MHz,CDCl3):  0.85, 0.88, 0.95, 2.02, 2.05, 2.06
(each, 3H, s), 4.11 (1H, dd), 4.25 (1H, dd), , 4.53 (3H, m), 5.91 (1H,
br s).
HRFABMS: anal. m/z 403.2094 [M+Na]+ (calcd for C21H32O6Na
403.2097).
FABMS (m-NBA): m/z 403 [M+Na]+.
Biotransformation of polygodiol (2) (50 mg x 4) by A. niger: The
treatment of 2 as mentioned above gave the metabolites (216.5 mg)
which were subjected to column chromatography on SiO2
(n-hexane/EtOAc gradient) to give 8 fractions. From Fr. 3, the
original substrate (2, 16.5 mg) was recovered. Fr. 8 was further
chromatographed on reverse phase SiO2 (MeOH/H2O 6:4) to afford
6 fractions (8-1 to 8-6). From Fr. 8-6, drim-7-ene-11,12,14-triol
(8, 3.5 mg) was obtained as a pure form. Fr. 8-8 was purified by
prep. HPLC (MeOH/H2O: 6:4) to furnish 11,12-dihydroxydrim-7-
ene-3-one (9, 8.2 mg) and drim-7-ene-3,11,12-triol (7, 43.6 mg).
Drim-7-ene-11,12,14-triol (8)
Colorless oil.
[]D: -16.1 (c, 0.59, MeOH).
FT/IR: 3353 cm-1.
1
H and 13C NMR: (Table 1).
HREIMS: anal. m/z 254.1887 [M]+ calcd. for C15H26O3) 254.1882.
EI/MS: m/z 254 (M+, 10%), 223 (9), 188 67), 175 (70), 144 (22),
109 (100), 105, (44), 81 (27), 55 (19), 41 (17).
11,12-dihydroxydrim-7-en-3-one (9)
Colorless oil.
[]D: -57.4 (c 1.00, MeOH).
FT/IR: 3389, 1701 cm-1.
1
H and 13C NMR: Table 1.
HREIMS: anal. m/z 252.1729 [M]+ C15H24O3 calcd. for 252.1726;
EI/MS: m/z 252 (M+, 6%), 221 (30), 204 (100), 161 (48), 123 (95),
119 (61), 96 (42), 91 835), 41 (24).
Microbial biotransformation of terpenoids Natural Product Communications Vol. 13 (8) 2018 929

Table 1: 1H and 13C NMR data of compounds 8 and 9 (600/150 MHz, CD3OD). Cinnamosmolide (11)
Position
1
8
13 1
9
13
Colorless crystals.
H (J, Hz) C H C
1 1.14 (ddd, 13.2, 13.2, 3.8) 40.3 1.61 (ddd, 14.0, 14.0, 4.1) 39.2
MP: 182-184 oC.
1 2.02 (m) 2.35 (ddd, 14.0, 5.2, 3.8) []D: -211.5 (c, 0.34, CHCl3).
2 1.54 (m) 19.2 2.23 (dt, 14.8, 3.8) 35.5 UV (MeOH) max nm (log): 211.4 (3.65) (c 6.95 x 10-2).
2 1.65 (m) 2.82 (ddd, 14.8, 14.0, 5.2)
FT/IR: 3405, 1761, 1732 cm-1.
3 1.49 (m) 36.6 218.8
3 1.29 (m) The 1H and 13C NMR spectral data of 11 were identical to those of
4 38.6 49.1 cinnamosmolide [16].
5 1.59 (dd, 12.1, 4.7) 43.9 1.67 (dd,, 12.4, 4.7) 52.4 HRCIMS: anal. m/z 309.1692 [M+H]+ (calcd. for C17H25O5
2.02 (m) 24.2 2.06 (m) 24.7
6
1.92 (m) 2;18 (m)
309.1702); CIMS (iso-butane): m/z 309 [M+H]+; EI/MS; m/z 248
6
7 5.77 (t, 2.5) 126.3 5.84 (br, s) 125.9 [(M-60)]+ 39%), 233 (36), 142 (92), 109 (100), 69 (28), 43 883).
8 138.5 138.7
9 2.09 (m) 55.7 2.15 (m) 54.8 Fragrolide (12)
10 36.5 36.6 Colorless crystals.
11 3.62 (dd, 11.3, 7.7) 61.3 3.71 (dd,, 11.3, 6.9) 61.0 MP: 155-157 oC.
3.85 (dd,, 11.3, 2.7) 3.87 (dd,, 11.3, 3.3)
12 3.95 (d, 12.6) 67.0 4.00 (d, 12.9) 66.5 []D: +81.0 (c 0.31, CHCl3).
4.32 (dq, 12.6, 1.1) UV (MeOH) max nm (log): 216.6 (3.70) (c 6.35 x 10-2).
13 0.83 (s) 18.3 1.05 (s) 25.7
14 3.01 (d, 11.0) 71.8 1.12 (s) 22.7
FT/IR: 1762, 1739, 1718 cm-1.
3.31 (d, 11.0) The 1H and 13C NMR spectral data of 12 were identical to those of
15 0.84 (s) 15.6 1.07 (2) 14.5 fragrolide [18].
HREIMS: anal. m/z 248.1922 [M]+ (calcd. for C15H20O3 248.1412);
Table 2: 1H and 13
C NMR data of compounds 10 (600/150 MHz, CDCl3; 300 MHz, CIMS (iso-butane): m/z 309 [M+H]+.
CDCl3 [17]). EIMS; m/z 248 [(M+, 100%), 233 (99), 205 (32), 165 (21), 164 (19),
Position
1
10
13 1
10 (synthetic ) [17] 121 (36), 83 (52), 69 (28), 41 (32).
H (J, Hz) C H
1 1.91 (m) 31.8
1 1.51 (m) Synthesis of 11,12-diacetoxy-drim-7-ene (14) and 11,12-
2 1.52 (m) 17.9 diacetoxy-drim-7-ene-9-ol (4): Polygodiol (2, 443 mg), which
2 1.65 (m)
was obtained by the further reduction of polygodial (455. 4 mg) in
3 1.29 (m) 44.6
3 1.39 (m) the same method as describe earlier, was acetylated with anhydrous
4 33.6 acetic anhydride (5 mL) and pyridine (5 mL). Treatment of the
5 2.03 (d, 4.4) 45.5 2.03 (d,, 4.8) reaction mixture in usual manner gave 11, 12-diacetoxy-drim-7-ene
6 5.61 (m) 67.1 5.61 (m)
7 5.69 (m) 120.0 5.68 (m)
(14, 359.9 mg) after purification using column chromatography
8 141.4 (n-hexane/EtOAc). To compound (14, 245.5 mg) in dioxane was
9 77.7 added SeO2 (20.5 mg) and refluxed at 120oC for 1 hr and the
10 38.3 reaction mixture was filtered to give the residue (290.6 mg), which
11 5.37 (s) 98.1 5.36 (br s)
was further purified on column chromatography (n-hexane/EtOAc
12 4.61 (dt, 13.5, 2.2) 66.5 4.69 (dt, 13.5, 2.3)
12 4.25 (dt, 13.5, 1.6) 4.23 (dt, 13.5, 1.6) gradient) to furnish 11,12-diacetoxydrim-7-ene-9-ol (4, 140 mg).
13 1.00 (s) 32.9 0.99 (s)
14 1.16 (s) 24.5 1.15 (s) 11, 12-Diacetoxy-drim-7-ene (14)
15 1.18 (s) 18.6 1.17( s)
16 170.4 Colorless oil.
17 2.07 (s) 21.7 2.06 (s) []D: +19.8 (c, 0.96, MeOH).
FT/IR: 1734 cm-1.
Biotransformation of cinnamodial (3): Cinnamodial (3) (100 mg HRCIMS: anal. m/z 323.2230 [M+H]+ (calcd. for C19H31O4
x 2) isolated from the Malagasy folk medicinal plant, Cinnamosma 323.2223).
fragrans collected in Madagascar was incubated with A. niger for 4 CIMS (iso-butane): m/z 323 [M+H]+.
days in the same manner as described in the biotransformation of EI/MS; m/z 262 [(M-60)+, 22%), 220 (20), 202 (98), 187 (60), 159
polygodiol (2) to give the metabolites (151.4 mg) which was (49), 133 (51), 109 (100), 105 (36), 43 (89).
chromatographed on reverse SiO2 to divide into two fractions. The 1
H NMR (300 MHz, CDCl3): 0.82, 0.88, 0.90, 2.01, 2.05 (each 3H,
first fraction contained 6-acetoxy-11,12-epoxy-drim-7-ene- s), 4.08 (1H, dd), 4.30 (1H, dd), 4.58 (1H, d), 5.91 (1H, d).
9,11-diol (10, 4.7 mg). The second Fr. was purified by prep. 13
C NMR (75 MHz):  14.4, 18.7, 21.1, 22, 23.6, 33.2, 35.8, 39.2,
HPLC (n-hexane/EtOAc 1:1) to give cinnamosmolide (11, 0.1 mg) 42.0, 49.4, 50.9, 62.9, 67.9, 130.5, 131.6, 170.9, 171.1.
and fragrolide (12, 0.6 mg), respectively.
Diacetoxy-drim-7-ene-9-ol (4)
6-Acetoxy-11,12-epoxy-drim-7-ene-9,11-diol (10) Colorless needles.
Colorless oil. MP: 45-46oC.
[]D: -186.7 (c, 1.00, CHCl3). []D: -53.4 (c 1.00, CHCl3).
FT/IR: 3389, 1701 cm-1. FT/IR: 3525, 1739 cm-1.
HRFABMS: anal. m/z 349.1429 [M+K]+ (calcd. for C17H26O5K 1
H and 13C NMR: Table 3.
349.1417). HRCIMS: anal. m/z 338.2092 [M+H]+ (calcd. for C19H30O5
FABMS (m-NBA+KCl): m/z 349 [M+K]+; EI/MS; m/z 250 [(M- 338.2094).
60)]+ 41%), 221 (31), 204 (81), 189 (65), 161 (49), 126 (78), 109 CIMS (iso-butane): m/z 338 [M+H]+; EI/MS; m/z 320 [(M-18)]+,
(48), 91 (47), 43 (100). 3%), 278 (7), 265 (13), 218 (16), 214 (29), 187 (11), 154 (100), 109
1
H and 13C NMR: Table 2. The 1H NMR spectral data was partially (53), 94 (42), 81 (32), 43(76).
identical to those of the synthetic product (10) named 6-O-
acetylpereniporin A, from cinnamodial (3) [17].
930 Natural Product Communications Vol. 13 (8) 2018 Asakawa et al.

Biotransformation of compound 4: Compound (4, 50 mg) was fractions. The first Fr. was purified by prep. HPLC (n-hexane/
incubated with A. niger for 2 days and the crude metabolites were EtOAc 1:1) to furnish 11,12-Epoxy-12-oxo-trinorsacculat-7-en-17-
treated in the same manner described above to give 11,12- oic acid (16, 3.1 mg). Frs 2 and 3 contained pure 3-hydroxy-11,12-
diacetoxy-drim-7-ene-3,9-diol (15, 29.2 mg). epoxy-12-oxo-saculat-7,17-dien-20-oic acid (17, 15.4 mg) and
3,11,12-trihydroxy-sacculat-7,17-dien-20-oic acid (18, 14.3 mg).
11,12-Diacetoxy-drim-7-ene-3,9-diol (15) The last Fr. was further chromatographed on Sephadex LH-20
Colorless oil. (CHCl3/MeOH 1:1) to give the crude alcohol which was acetylated
[]D: -25.6 (c 0.96, MeOH). with dry acetic anhydride and pyridine (1:1) for overnight, followed
FT/IR: 3516, 3473, 1730 cm-1. by the usual manner to give a triacetate (19a, 13.9 mg).
1
H and 13C NMR: Table 3.
HRFAB/MS: anal. m/z 377.1917[M+Na]+ (calcd. for C19H30O6Na 11,12-Epoxy-12-oxo-trinorsacculat-7-en-17-oic acid (16)
377.1940). Yellow oil.
FABMS (m-NBA): m/z 377 [M+Na]+. []D: -16.7 (c 0.98, CHCl3).
EIMS; m/z 294 [(M-60)+, 12%), 281 (21), 250 (6), 234 (13), 214 UV (MeOH)max nm (log): 223.6 (4.02) (c 1.91 x 10-2).
(36), 203 (20), 154 (98), 123 (51), 112 (44), 94 (39), 43 (100), 187 FT/IR: 3400-3100, 1760, 1706 cm-1.
(60), 159 (49), 133 (51), 109 (100), 105 (36), 43 (89). 1
H and 13C NMR: Table 4.
Table 3: 1H and 13C NMR data of compounds 4 and 15 (600/150 MHz, CDCl3). HREIMS: anal. m/z 292.1664 [M]+ (calcd. for C17H24O4 292.1675).
Position 4 15
EIMS: m/z 292 (M+, 8%), 274 (15), 182 (28, 164 (10), 109 (100),
1
H (J, Hz) 13
C 1
H 13
C 81 (13).
1 1.79 (m) 31.3 2.01 (m) 29.5
1 1.52 (m) 1.57 (m) Table 4: 1H and 13C NMR data of compounds 16 and 17 (600/150 MHz, CDCl3).
2 1.54 (m), 2H 18.4 1.63 (m) 27.0 Position 16 17
1.71 (m) 1
H (J, Hz) 13
C 1
H 13
C
3 1.23 (ddd, 12..6, 12.6, 3.6) 41.4 3.31 (dd, 11.8, 4.4) 78.1 1 1.18 (m) 39.1 1.31 (m) 37.0
3 1.41 (m) 1 1.61 (m) 1.64(m)
4 32.9 38.4 2 1.55 (m) 17.9 1.72 (m), 2H 26.9
5 1.82 (dd, 12.4, 4.7) 41.5 1.67 (dd,, 12.4, 4.7) 52.4 2 1.58 (m)
6 2.16 (dd, 18.7, 4.9) 24.2 2.18 (m) 23.8 3 1.21 (m) 37.4 3.57 (dd, 8.2, 7.7) 73.3
6 1.96 (m) 2.05 (m) 3 1.49 (m)
7 6.06 (d, 5.5, 1.9) 135.2 6.07 (dd, 5.5, 2.2) 135.0 4 34.9 40.9
8 133.1 133.1 5 1.41 (dd, 11.5, 5.2) 47.9 1.51 (dd,, 11.0, 5.8) 43.9
9 74.6 74.7 6 2.39 (m) 24.6 2.26 (m), 2H 24.5
10 40.7 40.5 6 2.12 (m)
11 4.21 (d, 12.1) 65.0 4.22 (d, 12.1) 64.9 7 6.86(d, 6.9, 3.3) 136.0 6.88 (dd, 6.9, 3.3) 135.8
4.29 (d, 12.1) 4.27 (d, 12.1) 8 127.2 127.3
12 4.95 (d, 12.6) 67.4 4.58 (d, 12. 4) 67.1 9 2.83 (m) 50.9 2.83 (m) 50.8
4.63 (d, 12.6) 4.64 (d, 12. 4) 10 34.3 34.0
11 4.38 (t, 9.1) 67.1 4.39 (t, 9.1) 67.1
13 0.91 (s) 33.3 1.03 (s) 28.1
11 4.05 (t, 9.1) 4.05 (t, 9.1)
14 0.94 (s) 22.2 0.92 (s) 15.8
12 170.1 4.58 (d, 12. 4) 169.9
15 0.91 (s) 15.7 0.91 (s) 15.4
4.64 (d, 12. 4)
MeCO2 2.057 (s) 20.9 2.06 (s) 20.9
13 0.85(s) 13.8 0.83 (s) 14.0
2.062 (s) 21.0 2.11 (s) 21.0
14 0.97 (s) 19.5 0.9 4 (s) 16.1
MeCO2 170.7 170.7 15 1.53 (m) 38.3 1.37 (m) 35.5
170.9 170.8 1.69 (m) 1.69 (m)
16 2.29 (dd, 6.9, 1.9) 28.1 2.05 (m) 22.5
Reduction of sacculatal (5): To LiAlH4 (630 mg) in dried diethyl 2.31 (dd, 6.9, 1.9) 2.16 (m)
ether (20 mL) was added sacculatal (5, 1.00 g), which was isolated 17 178.6 6.84 (ddd, 7.4, 7.4,1.4) 144.4
18 127.1
from the Japanese liverwort, Pellia endiviifolia, in dried diethyl 19 . 1.81 (s) 12.1
ether (10 mL). Treatment of the reaction mixture in the same 20 172.3
manner as described above gave sacculatadiol (6, 893.7 mg).
3-Hydroxy-11,12-epoxy-12-oxo-saculat-7,17-dien-20-oic acid
Sacculatadiol (6) (17)
Colorless crystals. Yellow oil.
MP: 90-91oC. []D: +11.2 (c, 1.05, CHCl3).
[]D: +22.8 (c 1.05, CHCl3). UV (MeOH)max nm (log): 218.0 (4.23), 220.8 (4.24) (c 5.26 x
FT/IR: 3305 cm-1. 10-2).
1
H NMR (300 MHz, CDCl3):  0.78, 0.88, 1.59, 1.67 (each 3H, s), FT/IR: 3448-2939, 1747, 1688 cm-1.
3.67 (1H, d), 3.67 (1H, d), 3.89 (1H, dd), 3.98 (1H, d), 4.34 (1H, d), 1
H and 13C NMR: Table 4.
5.05 (1H, d), 5.78 (1H, t). HREIMS: anal. m/z 348.1926 [M]+ (calcd. for C20H28O5 348.1937).
13
C NMR (75 MHz): δ 15.0, 17.6, 18.6, 20.7, 21.8, 23.3, 25.7, 35.3, EIMS: m/z 348 (M+, 65%), 330 (60), 302 (29), 231 863), 218 (53),
35.6, 37.5, 39.1, 44.2, 47.1, 54.6, 61.3, 67.4, 125.1, 127.3, 131.0, 189 (32), 149 (48), 121 (100), 95 (94), 91 (82), 43 (66).
136.8
HREIMS: anal. m/z 306.2557 [M+H]+ (calcd. for C20H34O2 3,11,12-Trihydroxy-sacculat-7,17-dien-20-oic acid (18)
306.2555). Yellow oil.
EIMS: m/z 306 (M+, 9%), 288 (18), 245 (19) %), 215 (23), 175 []D: +44.8 (c 0.99, CHCl3).
(28), 145 (26), 123 (55), 109 (100), 69 (98), 41 (51). UV (MeOH)max nm (log): 217.2 (4.4.08), 220.8 (4.24) (c 5.59 x

10-2).
Biotransformation of 6 by Aspergillus niger for 15 days:
FT/IR: 3448-2939, 1683 cm-1.
Sacculatadiol (6, 50 mg x 4) was incubated with A. niger as 1
H and 13C NMR: Table 5.
described above for 15 days to afford the crude metabolites (207.2
HREIMS: anal. m/z 352.2259 [M]+ (calcd. for C20H32O5 352.2250).
mg) which were fractioned by column chromatography to give 4
Microbial biotransformation of terpenoids Natural Product Communications Vol. 13 (8) 2018 931

1
Table 5: 1H and 13C NMR data of compounds 18 and 19a (600/150 MHz, CDCl3). H NMR and 13C NMR: Table 6.
Position
1
18
13 1
19a
13
EIMS: m/z 348 (M+, 67 %), 330 (38), 302 (33), 235 (34), 217 (66),
H (J, Hz) C H C
1 1.29 (m) 38.9 1.10 (m) 38.9
205 (28), 176 (23), 131 (51), 95 (100), 91 851), 55 (37), 43 (34).
1 2.06 (m) 1.97(m)
2 1.66 (m) 28.1 1.53 (m), 2H 18.3 Table 6: 1H and 13C NMR data of compounds 20 and 21 (600/150 MHz, CDCl3).
2 1.73 (m) Position 20 21
1 13 1 13
3 3.51 (m) 73.9 1.14 (m), 2H 40.4 H (J, Hz) C H C
4 42.1 35.5 1 3.39 (dd, 10.4, 5.2) 79.6 1.66 (m) 33.0
5 1.44 (dd, 11.8, 4.7) 44.9 1.25 (t, 3.9) 47.1 1 1.24(m)
6 2.04 (m) 24.0 2.03 (m) 23.2 2 1.69 (m) 2H 28.0 1.60 (m), 2H 17.7
6 1.99 (m) 1.92 (m) 3 1.56 (m) 35.7 1.57 (m) 32.1
7 5.76 (m) 126.0 5.88 (m) 130.1 3 1.44 (m) 1.36 (m)
8 138.7 131.5 4 35.0 40.3
9 2.08 (m) 55.9 2.22 (m) 50.9 5 1.43 (m) 47.0 77.1
10 36.5 35.8 6 2.35 (m) 24.4 2.35 (m) 32.0
11 3.64 (dd, 11.0, 7.4) 61.2 4.07 (dd, 11.5, 7.7) 62.8 6 2.19 (m) 2.58 (m)
11 3.85 (dd, 11.9, 2.7) 4.30 (dd, 11.5, 7.7) 7 6.86 (m) 135.0 6.77(dd, 6.9, 3.5) 133.2
12 3.97 (d, 12.9) 66.8 4.49 (d, 12.1) 67.7 8 127.7 128.0
4.26 (d, 12.9) 4.55( d, 12.1) 9 2.90 (m) 50.2 3.54 (m) 44.9
13 0.84(s) 15.5 0.84 (s) 14.9 10 40.0 39.2
14 0.89 (s) 17.3 0.89 (s) 20.6 11 4.54 (t, 9.6 ) 69.2 4.42 (t, 9.1) 67.4
15 1.31 (m) 36.6 1.17 (t, 4.1) 37.4 11 4.20 (t, 9.6) 4.10 (t, 9.1)
1.65 (m) 1.41 (m) 12 170.1 170.4
16 2.05 (m) 23.4 1.46 (m) 22.9 13 0.85 (s) 7.8 0.95 (s) 16.7
2.15 (m) 1.58 (m)
14 0.99 (s) 20.0 1.11 (s) 21.3
17 6.76 (ddd, 7.4, 7.4, 1.4) 144.4 4.71 (ddd, 9.6, 9.6, 2.5) 80.8 15 1.28 (m) 42.0 1.30 (m) 35.4
18 128.5 72.5 1.41 (m) 2.03 (m)
19 1.80 (br.s) 12.4 1.20 (s) 25.2 16 2.09 (m) 2H 22.9 1.43 (m) 2H 25.4
20 171.7 1.20 (s) 26.8 17 6.83 (dd, 7.4, 1.4) 144.6 3.30 (m) 79.6
MeCO2 2.01(s) 21.1(x3)
18 127.0 73.2
2.06 (s)
2.12 (s) 19 1.83 (br.s) 12.0 1.17(s)a 23.3a
MeCO2 170.9 20 171.9 1.22 (s)a 26.6a
171.1 a
: Assignments may be interchanged.
171.2
a
: Assignments may be interchanged. Table 7: 1H and 13C NMR data of compound 22 (600/150MHz, CDCl3).
Position 22
EIMS: m/z 352 (M+, 12%), 334 (14), 316 (26), 286 (24), 285 (15), 1
H (J, Hz) 13
C
259 (15), 219 (27), 173 (81), 171 (49), 145 (76), 119 (100), 105 1 1.15 (m) 39.0
1 2.00 (m)
(83), 95 (74), 43 (62).
2 1.56 (m) 18.4
2 1.73 (m)
11,12,17-Triacetoxy-sacculat-7-ene-18-ol (19a) 3 1.26 (m) 37.4
Colorless oil. 3 1.42 (m)
[]D: +19.2 (c 0.98, CHCl3). 4 35.5
5 1.30(m) 46.9
FT/IR: 3502, 1738 cm-1. 6 2.01 (m) 23.3
1
H NMR: Table 4. 6 1.90 (m)
13
C NMR: Table 5. 7 5.79 (m) 127.2
HREIMS: anal. m/z 306.2557 [M+H]+ (calcd. for C20H34O2 8 136.9
9 2.17 (m) 54.5
306.2555). 10 35.6
EIMS: m/z 388 ([M-78]+, 3%), 346 (6), 271 (39), 229 (13), 187 11 3.70 (dd, 11.0, 8.5) 61.4
(91), 146 (71), 133 (95), 109 (54), 71 (36), 43 (100). 11 3.92 (dd, 11.0, 1.6)
12 4.00 (d, 12.4) 67.4
4.37 (d, 12.4)
Biotransformation of 6 by Aspergillus niger for 21 days. 13 0.80(s) 15.0
Sacculatadiol (6) (100 mg x 4) was treated with A. niger for 21 days. 14 0.91 (s) 20.7
The crude metabolites (360.7 mg) was repeatedly chromato-graphed 15 1.30 (m), 2H 42.3
16 2.10 (m) 23.0
on Sephadex LH-20 (CH2Cl2/MeOH 1:1) and SiO2 (n-hexane/ 17 6.85 (ddd, 7.4, 7.4, 1.4) 145.5
EtOAc gradient), and prep. HPLC (MeOH/H2O 4:1) to furnish 1- 18 126.5
hydroxy-11,12-epoxy-12-oxo-sacculat-7,17-dien-20-oic acid (20, 19 1.83 (br. s) 12.0
3.9 mg), 5-hydroxy-11,12,17,18-diepoxy-12-oxo-sacculat-7-ene 20 171.9
(21, 3.4 mg), 11,12-dihydroxysacculat-7,17-dien-20-oic acid (22,
3.8 mg), and the same metabolites, (17, 2.0 mg) and (18, 5.0 mg) 5-hydroxy-11,12,17,18-diepoxy-12-oxo-sacculat-7-ene (21)
which were obtained by the incubation of 6 for 15 days. Yellow oil.
[]D: -8.1 (c, 0.941, CHCl3).
1-Hydroxy-11,12-epoxy-12-oxo-sacculat-7,17-dien-20-oic acid UV (MeOH) max nm (log): 211.6 (2.92), (c 2.98 x 10-3).
(20) FT/IR: 3450, 1752 cm-1.
1
White crystals. H NMR and 13C NMR: Table 6.
MP: 100-102oC. HREIMS: anal. m/z 334.2151 [M]+ (calcd. for C20H30O4 334.2144).
[]D: -22.7 (c 1.01, CHCl3). EIMS: m/z 334 (M+, 33 %), 301 (64), 275 (21), 249 (45), 217 (36),
UV (MeOH)max nm (log): 223.6 (3.03), (c 3.02 x 10-2). 191 (18), 165 (50), 149 (31), 109 (100), 95 (39), 59 (74), 43 (41).
FT/IR: 3511-3293, 1743, 1737 cm-1.
HREIMS: anal. m/z 348.1936 [M+H]+ (calcd. for C20H28O5 11,12-Dihydroxy-sacculat-7,17-dien-20-oic acid (22)
348.1936). White crystals.
932 Natural Product Communications Vol. 13 (8) 2018 Asakawa et al.

MP: 75-77oC. Supplemental materials: HMBC correlations of compounds 9, 11,

[]D : +11.7 (c 1.00, CHCl3). 12, 16, 19a, 20, 21, 22, and NOESY correlations of 10, 20, and 21
FT/IR: 3500-3381, 1684 cm-1. have been deposited as the supplemental charts.
1
H NMR and 13C NMR: Table 7.
HREIMS: anal. m/z 336.2305 [M]+ (calcd. for C20H32O4 336.2301). Acknowledgments - The authors thank Dr. Liva Harinantenaina
EIMS: m/z 336 (M+, 10%), 318 (15), 288 (14), 287 (13), 222 (11), (TBU, now in Ohio State Univ. USA) for his gift of pure
187 (24). 173 (65), 133 (46), 109 (100), 105 (47), 81 (38), 55 (29), cinnamodial. The authors are grateful to Prof. Dr. Y. Noma (TBU)
41 (22). for his valuable suggestion of the medium for Aspergillus niger.

References
[1] Ghani NA, Ismail NH, Noma Y, Asakawa Y. (2017) Microbial transformation of some natural and synthetic aromatic compounds by fungi:
Aspergillus and Neurospora strains. Natural Product Communications, 12, 1237-1240.
[2] (a) Noma Y, Asakawa Y. (2010) Microbial transformation of (-)-nopol benzyl ether: Direct dihydroxylation of benzene ring. Natural Product
Communications, 5, 1339-1341. (b) Asakawa Y, Noma Y. (2010) Biotransformation of sesquiterpenoids. In Comprehensive Natural Products II.
Chemistry and Biology, Mander L, Liu HW (Eds.), Elsevier, Oxford, 3, 856-859. (c) Noma Y, Asakawa Y. (2010) Biotransformation of
monoterpenoids by microorganisms, insects, and mammals. In Handbook of Essential Oils. Baser KHC, Buchbauer G. (Eds.) CRC Press, Boca
Raton, Florida. 585-736. (d) Asakawa Y, Noma Y. (2010) Biotransformation of sesquiterpenoids, ionones, damascones, adamantanes, and
aromatic compounds by green algae, fungi, and mammals. In Handbook of Essential Oils. Baser KHC, Buchbauer G. (Eds.) CRC Press, Boca
Raton, Florida. 737-841.
[3] (a) Noma Y, Asakawa Y. (1994) Chapter XIII: Dunaliella tertiolecta (green microalga): culture and biotransformation of terpenoids and related
compounds. In Biotechnology in Agriculture and Forestry. Medical and Aromatic Plants VII. (Bjaj YPS. (Ed.), Springer, Berlin, 28, 185-202. (b)
Noma Y, Asakawa Y. (1994) Chapter XI. Euglena gracilis Z: Biotransformation of terpenoids and related compounds. In Biotechnology in
Agriculture and Forestry. Medical and Aromatic Plants X. (Bjaj YPS. (Ed.), Springer, Berlin, 41, 194-237. (c) Noma Y, Asakawa Y. (1995)
Chapter V: Aspergillus spp. Biotransformation of terpenoids and related compounds. In Biotechnology in Agriculture and Forestry. Medical and
Aromatic Plants VII. (Bjaj YPS. (Ed.), Springer, Berlin, 33, 62-95..
[4] Furusawa M, Hashimoto T, Noma Y, Asakawa Y. (2005) Highly efficient production of nootkatone, the grape fruit aroma from valencene,
microorganisms. Chemical and Pharmaceutical Bulletin, 53, 1513-1514.
[5] Kubo I, Fujita K, Lee SH. (2001) Antifungal mechanism of polygodial. Journal of Agriculture and Food Chemistry, 49, 1607-1611.
[6] Kubo I, Fujita K, Lee SH, Ha TJ. (2005) Antibacterial activity of polygodial. Phytotherapy Research, 19, 1013-1017.
[7] Asakawa Y, Aratani T. (1976) Sesquiterpenes of Porella vernicosa (Hepaticae). Bulletin de la Societe Chimique de France,1469-1470.
[8] Asakawa Y, Toyota M, Takemoto. (1978) Sesquiterpenes from Porella species. Phytochemistry 17, 457-460.
[9] Asakawa Y, Takemoto T, Toyota M, Aratani T. (1977) Sacculatal and isosacculatal, two new exceptional diterpenedials from the liverwort,
Trichocoleopsis sacculata. Tetrahedron Letters, 18, 1407-1410.
[10] Asakawa Y, Toyota M, Takemoto T (1980) Four new sacculatane-type diterpenoids from Trichocoleopsis sacculata and Pellia endiviifolia.
Phytochemistry, 19, 1799-1803.
[11] Hashimoto T, Okumura Y, Suzuki K, Takaoka S, Kan Y, Tori M, Asakawa Y. (1994) The absolute structures of new 1-hydroxysacculatane-type
diterpenoids with piscicidal activity from the liverwort Pellia endiviifolia. Chemical and Pharmaceutical Bulletin, 42, 1542-1544.
[12] Ayer WA, Craw. (1989) Metabolites of the fairy ring fungus, Marasmius oreades. Part 2. Norsesquiterpenes, further sesquiterpenes, and
agrocybin. Canadian Journal of Chemistry, 67, 1371-1380.
[13] Aranda G, Facon I, Lallemand J-Y, Leclaire M, Azerad M, Cortes M, Lopes J, Ramirez HE. (1992) Microbiological hydroxylation in drimane
series. Tetrahedron Letters, 33, 784-788.
[14] Ramirez HE, Cortes M, Agosin E. (1993) Bioconversion of drimenol into 3-hydroxydrimanes by Aspergillus niger, Effect of culture additives.
Journal of Natural Products, 56, 762-764
[15] Canonica L, Corbella A, Jommi G, Krepinsky J. (1967) The structure of cinnamolide, cinnamosmolide and cinnamodial, sesquiterpenes with
drimane skeleton from Cinnamosma fragrans baillon. Tetrahedron Letters, 8, 2137-2141.
[16] Canonica L, Corbella A, Gariboldi P, Jommi G, Krepinsky J. (1969) Sesquiterpenoids of Cinnamosma fragrans Baillon: Structure of
cinnamolide, cinnamosmolide and cinnamodial. Tetrahedron, 25, 3895-3902,
[17] Garlaschelli L, Mellerio G, Vadari. (1989) Synthetic studies on biologically active natural compounds. Part II: Correlation of cinnamodial with
uvidin a and transformation into (-)-pereniporin A. Tetrahedron, 45, 7379-7386.
[18] Mahmoud I I, Kinghorn GA, Cordell GA, Farnsworth NR. (1990) Potential anticancer agents XVI. Isolation of bicyclofarnesane sesquiterpenoids
from Caspicodendron dinisii. Journal of Natural Products, 43, 365-371.
[19] Canonica L, Corbella A, Gariboldi P, Jommi G, Krepinsky J. (1969) Sesquiterpenoids of Cinnamosma fragrans Baillon: Structure of
bemarivolide, bemadienolide and fragrolide. Tetrahedron, 25, 3903-3908.
[20] Kioy D, Gray AI, Watermann. (1990) A comparative study of the stem-bark drimane sesquiterpenes and leaf volatile oils of Warburgia
ugandensis and W. stuhlmannii. Phytochemistry, 29, 3535-3538.
[21] Urones JG, Marcos IS, Perez BG, Diez D, Lithgow AM, Gomez PM, Basabe P, Garrido NM. (1994) Chemistry of zamoranic acid. Part V.
Homochiral semisyntheses of active drimanes: Pereniporin B, polygodial and warburganal. Tetrahedron, 50, 19995-11012.
[22] Asakawa Y, Ludwiczuk A, Nagashima F. (2013) Chemical constituents of bryophytes. Bio- and chemical diversity, biological activity, and
chemosystematics. In Progress in the Chemistry of Organic Natural Products. Kinghorn AD, Falk H, Kobajashi J. (Eds.) Springer, Vienna. 95,
1-796. Baser KHC, Buchbauer G. (Eds.) CRC Press, Boca Raton, Florida. 737-841.
[23] Justicia J, Olta JE, Barrero AF, Cuadano A, Coloma AG, Curtva JM. (2005) Total synthesis of 3-hydroxydrimanes by titanocene (III)-Evaluation
of their antifeedant activity. European Journal of Organic Chemistry, 712-718.
[24] Asakawa Y, Ludwiczuk A, Harinantenaina L, Toyota M, Nishiki M, Bardon A, Nii K. (2012) Distribution of drimane sesquiterpenoids and
tocopherols in liverworts, ferns and higher plants: Polygonaceae, Canellaceae, and Winteraceae species. Natural Product Communications, 7,
685-692.
[25] Godin P. (1954) A new spray reagent for paper chromatography of polyols and cetones, Nature (London), 174, 134.