Microbial reduction of coumarin, psoralen, and xanthyletin by Glomerella cingulata

Article abstract of DOI:10.1016/j.tet.2010.10.089

Microbial transformation of coumarin, psoralen, and xanthyletin was performed with the fungus Glomerella cingulata. The main reaction pathways involved reduction at α,β-unsaturated δ-lactone ring on coumarin analogue. Coumarin was metabolized by G. cingul

Full text of DOI:10.1016/j.tet.2010.10.089

Tetrahedron 67 (2011) 495e500
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microbial reduction of coumarin, psoralen, and xanthyletin by Glomerella
cingulata
Shinsuke Marumoto, Mitsuo Miyazawa *
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2010
Received in revised form 27 October 2010
Accepted 29 October 2010
Available online 5 November 2010
Microbial transformation of coumarin, psoralen, and xanthyletin was performed with the fungus
Glomerella cingulata. The main reaction pathways involved reduction at a,b-unsaturated d-lactone ring on
coumarin analogue. Coumarin was metabolized by G. cingulata to give the corresponding reduced acid,
hydrocoumaric acid. In the biotransformation of psoralen, two reduced metabolites, 6,7-furano-hydro-
coumaric acid, and 6,7-furano-o-hydrocoumaryl alcohol were isolated from the incubation of psoralen.
Xanthyletin was converted to reduced products 9,9-dimethyl-6,7-pyrano-hydrocoumaric acid and 9,9-
dimethyl-6,7-pyrano-o-hydrocoumaryl alcohol by G. cingulata. The structures of the new compounds
were characterized using spectroscopic techniques. In addition, all of compounds including methyl ester
Keywords:
Biotransformation
Coumarin
derivatives of the metabolites were tested for the b-secretase (BACE1) inhibitory activity in vitro. 6,7-
Psoralen
Xanthyletin
Glomerella cingulata
Furano-hydrocoumaric acid methyl ester was shown to possess BACE1 inhibitory activity, and an IC50
value was 0.84 ꢀ 0.06 mM.
Ó 2010 Published by Elsevier Ltd.
1. Introduction
new production routes for fine chemical, pharmaceutical, and ag-
rochemical compounds. Microorganisms are well known as effi-
7
Coumarins are widely distributed in nature and are found in all
parts of plants.1 These compounds are especially common in
cient and selective catalysts. It is anticipated that the microbial
metabolism of coumarins would produce significant quantities of
metabolites that would be difficult to obtain from chemical syn-
thesis or either animal systems. Moreover, this may provide some
novel metabolites that may serve as starting compounds for semi-
synthesis of other derivative, or as analytical standards for mam-
malian metabolic studies. Previously, we studied the microbial
transformation of furanocoumarins by Glomerella cingulata and
1,2
grasses, orchids, citrus fruits, and legumes. Being so abundant in
nature, coumarins make up an important part of human diet. Based
on chemical structure (Fig. 1), they can be broadly classified as (a)
simple coumarins (e.g., coumarin, 1), (b) furanocoumarins of the
linear (e.g., psoralen, 3) or angular (e.g., angelicine) type, and (c)
pyranocoumarins of the linear (e.g., xanthyletin, 6) or angular (e.g.,
1
seselin) type. Simple coumarins are very widely distributed in the
plant kingdom.1 Interestingly, citrus oils, in particular, contain
3
abundant amounts of both simple as well as furanocoumarins.
Humans are also exposed to furanocoumarins (e.g., bergapten and
xanthotoxin) in umbelliferous vegetables, such as parsnips, celery,
4
and parsley in substantial amounts. A number of furanocoumarins
0
0
act as inhibitors of drug metabolizing enzymes. 6 ,7 -Dihydrox-
ybergamottin and related furanocoumarins dimmers found, e.g., in
grapefruit juice act as highly potent inhibitors of cytochrome P450
5
(
CYP) 3A and other CYP isozymes affecting drug metabolism.
Furthermore, furanocoumarins were the inhibitors of the
b-secre-
tase (BACE1).6
Biotransformation is today considered to be an economically
competitive technology by synthetic organic chemists in search of
*
Corresponding author. Tel.: þ81 6 6721 2332; fax: þ81 6 6727 2024; e-mail
address: miyazawa@apch.kindai.ac.jp (M. Miyazawa).
Fig. 1. Chemical structures of coumarin, psoralen, and xanthyletin derivatives.
0
040-4020/$ e see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.tet.2010.10.089

496
S. Marumoto, M. Miyazawa / Tetrahedron 67 (2011) 495e500
evaluated the transformation products on the BACE1 inhibitory
activity.8 Therefore, it is envisioned that biotransformation of
coumarin (1), psoralen (3), and xanthyletin (6) may provide ana-
logues, which could be tested for new and improved activities. The
goal of the present work was the transformation of coumarins 1, 3,
and 6 by G. cingulata and the evaluation of these compounds as
BACE1 inhibitors (Fig. 1).
HPLC and TLC analysis of the broth extracts of cultures incubated
with paoralen (3) indicated it to be completely metabolized to two
products 4 and 5 at the conversion rate of 47 and 38% yields for 7
days (Fig. 3). To isolate the metabolites, larger scale culture of
G. cingulta was incubated with 3 were performed, the culture was
extracted as described in Experimental section, and metabolites 4
and 5 were obtained.
,9
2
. Results and discussion
Coumarin (1), psoralen (3), and xanthyletin (6) were administered
to fungal cultures of G. cingulata, the cultures were incubated for 7
days. HPLC chromatograms of extracted of cultures incubated with
coumarin (1) indicated that 1 was metabolized to 2 in 30% yield for 7
days (Fig. 2). Subsequently, to obtain sufficient quantities of each
product for broth chemical characterization and bioassay, larger scale
mycelia incubated with 2 were performed, the culture was extracted
as described in Experimental section, and methylated metabolite 2
(
compound 2a) was isolated. Metabolite 2 was obtained by the hy-
drolysis of 2a. The structure of these compounds were determined
1
13
through use of HR-FABMS, H and C NMR (Table 1), HeH COSY,
HSQC, and HMBC spectroscopic analyses, and the metabolite 2 was
10
identified as hydrocoumaric acid.
Fig. 3. Time course in the biotransformation of 3 by G. cingulata: (C) psoralen (3); (-)
metabolite 4; (:) metabolite 5.
þ
HR-FABMS of compound 4 showed [MþH] peaks at m/z
2
11 4
07.0685 (calcd for C11H O , 207.0658), which established a mo-
lecular formula of C11
at 3264 cm and a strong absorption band at 1698 cm in the IR
spectrum suggested the conversion of the original psoralen lactone
into phenol and carboxylic acid functionalities. The C NMR spec-
trum showed 11 resonances distributed as two secondary, four ter-
tiary and five quaternary carbons. The conversion of original two
H
10
O
4
. The presence of a broad absorption band
ꢁ1
ꢁ1
13
olefinic carbons (C-3,
aliphatic secondary carbons (C-3,
indicated the reduction of the C-3,4 double bond. Except for the
d
C
114.7 ppm and C-4,
d
C
144.0 ppm) into two
d
C
34.8 ppm and C-4,
d
C
26.8 ppm)
13
downfield shift of C-2 and C-4a, the C resonances remained the
1
same as those of the starting material (Table 1). Furthermore, the H
NMR spectrum showed the conversion of two doublets (d
H
Fig. 2. Time course in the biotransformation of 1 by G. cingulata: (C) coumarin (1);
-) metabolite 2.
6.38 ppm, d, J¼9.8 Hz, H-3 and
d
H
7.81 ppm, d, J¼9.8 Hz, H-4) into
(
two triplets at
d
H
2.65 (H-3) and 2.98 (H-4). Thus, chemical
d
H
Table 1
The 13C NMR spectroscopic data of compounds 1e8
Position
1a
2b
3c
4a
5c
6b
7c
8c
60.7 (CH
32.3 (CH
24.4 (CH
119.1 (C)
128.0 (CH)
114.7 (C)
152.5 (C)
104.2 (CH)
155.4 (C)
76.1 (C)
2
3
4
160.8 (C)d
116.7 (CH)
143.4 (CH)
118.8 (C)
127.8 (CH)
124.4 (CH)
131.8 (CH)
116.9 (CH)
154.1 (C)
174.7 (C)
34.4 (CH
26.4 (CH
128.1 (C)
130.9 (CH)
120.4 (CH)
128.1 (CH)
116.0 (CH)
156.0 (C)
161.0 (C)
114.7 (CH)
144.0 (CH)
115.4 (C)
119.8 (CH)
124.9 (C)
156.4 (C)
99.9 (CH)
152.0 (C)
146.9 (CH)
106.4 (CH)
174.7 (C)
34.8 (CH
26.8 (CH
124.9 (C)
122.3 (CH)
120.7 (C)
155.5 (C)
98.3 (CH)
154.3 (C)
144.5 (CH)
107.1 (CH)
60.7 (CH
32.6 (CH
25.3 (CH
123.6 (C)
121.6 (CH)
120.9 (C)
154.7 (C)
98.9 (CH)
152.7 (C)
143.9 (CH)
106.0 (CH)
2
)
)
)
161.2 (C)
113.0 (CH)
143.3 (CH)
112.7 (C)
124.7 (CH)
118.5 (C)
155.4 (C)
104.4 (CH)
156.8 (C)
77.7 (C)
174.7 (C)
34.6 (CH
25.8 (CH
120.3 (C)
128.6 (CH)
114.4 (C)
153.4 (C)
104.1 (CH)
156.7 (C)
76.5 (C)
2
2
2
)
)
)
2
)
)
2
)
)
2
2
2
2
)
)
2
2
4
5
6
7
8
8
9
1
1
1
1
a
a
0
1
2
3
131.2 (CH)
120.8 (CH)
28.3 (CH
28.3 (CH
128.0 (CH)
122.8 (CH)
28.1 (CH
28.1 (CH
127.9 (CH)
121.8 (CH)
27.9 (CH
27.9 (CH
3
)
)
3
)
)
3
)
)
3
3
3
a
b
c
Measured in CDCI
Measured in at acetone-d
Measured in CDCI at 125 MHz.
C multiplicities were determined by DEPT 135 .
3
at 100 MHz.
6
125 MHz.
3
d
13
ꢂ

S. Marumoto, M. Miyazawa / Tetrahedron 67 (2011) 495e500
497
structure of metabolite 4 was established as 6,7-furano-hydro-
coumaric acid. Compound 5 was shown to have a molecular formula,
as determined by HR-EIMS. Comparison of this compound
with metabolite 4 revealed significant similarity except for the dis-
appearance of carbonyl group and the presence of oxygenated
methylene in 5. Compound 5 was therefore identified as completely
reduced compound of 4, 6,7-furano-o-coumaryl alcohol.
To clarify the time course of the microbial transformation of
xanthyletin (6) by G. cingulata, a small amount of 6 was incubated
for 7 days. Two metabolites were detected by TLC and HPLC. The
time course of metabolites was measured by HPLC. In this system, 6
was transformed to 7 and 8 at the conversion rate of 57 and 27%
yields for 7 days (Fig. 4). To isolate these metabolites, larger scale
incubation of 6 by G. cingulata was performed for 7 days. After the
biotransformation, the culture was extracted, and metabolites 7
and 8 were isolated.
the main pathway of those metabolism involved reduction at a,b-
unsaturated lactone ring of coumarin skeleton. Compared to the
transformation of psoralen (3) and xanthyletin (6), the bio-
transformation of coumarin (1) in G. cingulata was a much slower
process. The presence of linear-type furano- or pyrano-ring in these
substrates boots the rate of bioconversion. Only linear-type fur-
anocoumarin 3 and pyranocoumarin 6 were converted to com-
pletely reduction to give the corresponding reduced alcohols.
However, previous studies with other linear-type furanocoumarins
were not found to be a completely reduced metabolite. In the me-
tabolism of coumarin (1), the Psudomonas sp., Arthrobacter sp.,
Penicillium sp., Fusarium solani, and Bacillus cereus were known to
11 12 3
C H O
11e14
metabolize 1 by a reductive mechanism,
leading to hydro-
coumaric acid (2), and Colletotrichum capsici completely reduced the
15
lactone moiety of 1 to respective alcohol. This is the first report that
biotransformation completely reduced the lactone moiety of fur-
anocoumarin and pyranocoumarin to respective alcohols. Although
little is known about the enzymatic reduction of coumarins, it is
interesting to speculate on the mechanism of production of the
corresponding reduced acids. Because all coumarins are extremely
stable in the lactone form, it would seem unlikely that the ring is
opened before reduction unless the 3,4-double bond is immediately
isomerized to the trans geometry yielding a stable hydroxy acid. In
the lactone form, the 3,4-double bonds of coumarins are always
more difficult to chemically hydrogenerate than are double bonds in
a side chain or the non-nuclear double bond of a coumarins. If the
fungus has overcome this difficulty, production of the corresponding
reduced acids would follow because hydrolytic opening of the lac-
tone ring of a 3,4-dihydrocoumarin gives a hydroxy acid with little
tendency to lactonize. Therefore, fungal reduction of coumarins
probably proceeds by opening of the lactone ring followed by
isomerization and reduction or by hydrogenation of the 3,4-double
bond followed by, or concomitant with, opening of the lactone ring.
However, intermediate metabolites in each metabolic pathwaywere
not detected in our biotransformation experiments. The data on the
microbial transformed product, metabolite may be used for further
pharmacological evaluation of coumarins.
Fig. 4. Time course in the biotransformation of 6 by G. cingulata: (C) xanthyletin (6);
Subsequently, the b-secretase (BACE1) inhibitory effects of all
(
-) metabolite 7; (:) metabolite 8.
these compounds were evaluated to search for potential novel anti-
Alzheimer agents. Results of evaluation of BACE1 inhibitory activity
established that methyl ester derivatives 4a, and 7a inhibited
54.5ꢀ3.2, and 43.3ꢀ5.1% of the BACE1 activity at a concentration of
1 mM (Fig. 5), and the IC50 value of 4a was 0.84ꢀ0.06 mM. The
þ
HR-FABMS of compound 7 showed [MþH] peaks at m/z
49.2828 (calcd for C14 , 249.2838), which established a mo-
2
17 4
H O
lecular formula of C14
at 3359 cm and a strong absorption band at 1707 cm in the IR
spectrum suggested the conversion of the original xanthyletin lac-
tone into phenol and carboxylic acid functionalities. The C NMR
spectrum showed 14 resonances distributed as two primary, two
secondary, four tertiary and six quaternary carbons. The conversion
H
16
O
4
. The presence of a broad absorption band
ꢁ1
ꢁ1
13
of original two olefinic carbons (C-3,
43.3 ppm) into two aliphatic secondary carbons (C-3,
and C-4, 25.8 ppm) indicated the reduction of the C-3,4 double
d
C
113.0 ppm and C-4,
d
C
1
d
C
34.6 ppm,
d
C
13
bond. Except for the downfield shift of C-2 and C-4a, the C reso-
nances remained the same as those of the starting material (Table 1).
1
Furthermore, the H NMR spectrum showed the conversion of two
doublets (
into two triplets at
chemical structure of metabolite 7 was established as 9,9-dimethyl-
,7-pyrano-hydrocoumaric acid. Compound 8 was shown to have
a molecular formula, C14 as determined by HR-EIMS. Com-
d
H
6.22 ppm, d, J¼9.3 Hz, H-3, and
d
H
7.58, d, J¼9.2 Hz, H-4)
d
H
2.45 ppm (H-3) and
H
d 2.66 ppm (H-4). Thus,
6
18 3
H O
parison of this compound with metabolite 7 revealed significant
similarity except for the disappearance of carbonyl group, and the
presence of oxygenated methylene in 8. Compound 8 was therefore
determined as completely reduced compound of 7, 9,9-dimethyl-
6
,7-pyrano-o-hydrocoumaryl alcohol.
Results of the metabolism of simple coumarin 1 and linear-type
Fig. 5.
b-Secretrase inhibitory activity of coumarin, psoralen, and xanthyletin de-
rivatives. The bar is the mean of ꢀ SD of three experiments. (C) 1; (-) 2; (:) 2a; (;)
furano- or pyranocoumarins 3 and 6 by G. cingulata suggested that
3; (A) 4; (B) 4a; (,) 5; (6) 6; (7) 7; (>) 7a; (þ) 8; (ꢃ) positive control.

498
S. Marumoto, M. Miyazawa / Tetrahedron 67 (2011) 495e500
BACE1 inhibitory effects of methyl ester derivatives 4a and 7a were
stronger than those of corresponding acids, and methyl ester de-
rivative 2a, indicating the presence of methyl ester and furan- and
pyran-systems were important for the inhibitory activity. Psoralen
3.3. Preculture of G. cingulata
Spores of G. cingulata NBRC 5952 (NITE Biological Resource
Center, Japan), which had been preserved on potato dextrose agar
ꢂ
(
2) and its reduced biotransformation product by G. cingulata were
found to significantly lower BACE1 inhibitory activity than did
isoimperatorin, having an additional prenyloxy group at C -posi-
tion, and its biotransformation products. Bergapten, with
a methoxy group at C -position, and its reduced biotransformation
(PDA) at 4 C, were inoculated into 200 mL of sterilized culture
medium (1.5% saccharose, 1.5% glucose, 0.5% polypeptone, 0.05%
5
4 2 2 4 4 2
MgSO $7H O, 0.05%KCl, 0.1% K HPO , and 0.001% FeSO $7H O in
8
distilled H
2
O) in a 500-mL shaking flask, and the flask was shaken
ꢂ
5
(reciprocating shaker, 100 rpm) at 27 C for 3 days.
product were similar inhibitory activity to psoralen and its bio-
9
transformation product, indicating an additional prenyloxy group
3.4. Time course of biotransformation and quantification of
at C
5
-position on furanocoumarin skeleton and its corresponding
metabolite
reduced acid derivative seems to have significant influence.
In conclusion, one metabolite 2 was obtained from the in-
cubation of coumarin (1) with G. cingulata, two new compounds 4
and 5 were obtained from the incubation of psoralen (3), and two
new compounds 7 and 8 were obtained from the incubation of
xanthyletin (6). The results suggested that G. cingulata possess the
Precultured G. cingulata (3 mL) was transferred into two 300-mL
Erlenmeyer flasks containing 100 mL of medium and was stirred
(ca. 100 rpm) for 3 days. After the growth of G. cingulata, coumarins
1, 3, and 6 (46 mmol) in 0.5 mL of dimethyl sulfoxide (DMSO) were
added into the medium, respectively, and cultivated in 7 days. Ev-
ery other day, 2 mL of the culture mediums were extracted with
EtOAc, respectively. These extracts were analyzed by TLC and HPLC.
The mobile phase and detector used were the same as above. The
contents of these compounds were calculated by means of the
absolute calibration curves. The time course of biotransformation of
1, 3, and 6 are shown in Figs. 2e4.
characteristics of reaction of the coumarin skeleton as we previously
reported.8 The reactions involved the reduction of
,9
a
,b
-unsaturated
lactone ring of coumarin skeleton to give the corresponding reduced
acid. In addition, bioconversions of simple linear-types fur-
anocoumarin and pyranocoumarin, those substrates were com-
pletely metabolized in ca. 5 days, and to give both the corresponding
reduced alcohols and acids. These new metabolites will be useful
reference standards for our continuing studies on structure modi-
fication and pharmacological evaluation of coumarins. This in-
vestigation also demonstrates that biotransformation is a powerful
tool for the structural modification of natural products.
3.5. Preparative biotransformation of coumarin (1)
Precultured G. cingulata (5 mL) was transferred into a 500 mL
Erlenmeyer flask containing 300 mL of medium. Cultivation was
ꢂ
carried out at 27 C with stirring (ca. 120 rpm) for 3 days. After the
3
3
. Experimental
growth of G. cingulata, 50 mg of 1 in 1.0 mL of dimethyl sulfoxide
(DMSO) was added into the medium and cultivated for an additional
.1. General experimental details
7 days, together with two controls, which contained either mycelia
with medium or substrate dissolved in DMSO with medium. No
metabolic product was observed in two controls. After the fermen-
tation, the culture medium and mycelia were separated by filtration.
The medium was saturated with NaCl and extracted with EtOAc. The
mycelia were also extracted with EtOAc. Each EtOAc extract was
combined, the solvent was evaporated, and a crude extract (423 mg)
A thin layer chromatography (TLC) was performed on pre-
coated plates (Si gel 60 F254, 0.25 mm, Merck). Compounds were
visualized by spraying plates with 0.5% vanillin in 96% H SO fol-
lowed by brief heating. A Shimazu LC-10A high-performance liquid
chromatography (HPLC) system (Shimazu Co., Ltd., Kyoto, Japan)
was comprised of a quaternary solvent deliver system, an auto-
sampler, a column temperature controller, and photo diode array
2
4
3
was obtained. The extract was distributed between 5% NaHCO aq
and EtOAc, and EtOAc phase was evaporated to give a neutral frac-
tion (159 mg). No metabolite was detected by TLC and HPLC. The
alkali phase was acidified to pH 3 with 1 N HCl and distributed be-
tween water and EtOAc. The EtOAc phase was evaporated, and the
acidic fraction (264 mg) was obtained. Metabolites were detected
from both fractions byTLC and HPLC, respectively. The acidic fraction
(
(
PDA) coupled with analytical works station. A YMC-Pack ODS-AQ
4.6 ꢃ250 mm, 5 m particle size, YMC Co., Ltd., Japan) with a YMC-
m particle size,
m
Pack ODS-AQ guard column (4.6 mmꢃ23 mm, 5
m
YMC Co., Ltd., Japan) were used. The chromatographic parameters
were as follows: solvent A, acetonitrile; solvent B, water; both
modified with 0.1% (v/v) acetic acid. The gradient was set as fol-
lows: 20% A for 10 min at 1.0 mL/min, 20e70% A in 100 min at
2 2
was dissolved in acetone (5 mL), and CH N (1 mL) was added to the
fraction. The solution was evaporated, and the methylation fraction
was obtained. The methylation fraction was subjected to silica-gel
column chromatography (CC) (silica gel 60, 230e400 mesh, Merck)
1.0 mL/min, 70% A for 10 min at 1.0 mL/min. Total runtime was
110 min. The injection volume was 10 L. Electron ionization mass
m
spectrometry (EIMS), high-resolution electron ionization mass
spectrometry (HR-EIMS) fast atom bombardment mass spectrom-
etry (FABMS), and high-resolution fast atom bombardment mass
spectrometry (HR-FABMS) were obtained on a JEOL the Tandem Ms
station JMS-700 TKM. Nuclear magnetic resonance (NMR) spectra
2
with a n-hexaneeEt O gradient (9:1 to 1:4) to yield compound 2a
(25 mg). Compound 2a (13 mg) was dissolved in MeOH (1 mL), 1%
NaOH (2 mL) added tothe solution, and the solutionwas refluxed for
30 min. The solution was acidified to pH 3 with 1 N HCl and dis-
tributed between EtOAc and water. The EtOAc phase was evaporated
1
13
were recorded at 400 or 500 MHz for H and 100 or 125 MHz for
C
to give 2 (9 mg, R
t
¼6.6 min).
on a JEOL AL 400 or ECA-500 spectrometer. IR spectra were de-
termined with a JASCO FT/IR-470 plus Fourier transform infrared
spectrometer. A BACE1 (recombinant human BACE1) assay kit was
purchased from the Pan Vera Co. (United States).
3.5.1. Hydrocoumaric acid (2). White powder; IR (KBr)
n
max 3264,
ꢁ
1 1
1698 cm
6
; H NMR (acetone-d , 500 MHz) d
7.11 (1H, dd, J¼7.5,
1.7 Hz, H-5), 7.01 (1H, ddd, J¼8.0, 7.5, 1.7 Hz, H-7), 6.81 (1H, dd,
J¼8.0,1.2 Hz H-8), 6.73 (1H, dt, J¼7.5, 1.2 Hz, H-6), 2.87 (2H, m, H-4),
13
3
.2. Chemicals
2.59 (2H, m, H-3); C NMR shown as Table 1; HR-FABMS (pos) m/z
1
67.1817 [MþH]þ (calcd for C
9 10 3
H O , 167.1827).
Coumarin (1) and psoralen (3) were purchased from
SigmaeAldrich (Tokyo, Japan). Xanthyletin (6) was synthesized
according to the methods reported previously.
3.5.2. Hydrocoumaric acid methyl ester (2a). White powder; IR
(KBr) nmax 3398, 1714 cm ; H NMR (CDCl , 500 MHz) d 7.10 (2H,
3
16
ꢁ1 1

S. Marumoto, M. Miyazawa / Tetrahedron 67 (2011) 495e500
499
þ
m, H-5, 8), 6.86 (2H, m, H-6, 7), 3.69 (3H, s, COOCH
3
), 2.91 (2H, m,
176.0 (C, C-2),
12 4
(28), 118 (34); HR-EIMS m/z 220.0724 [M] (calcd for C12H O ,
220.0736).
13
H-4), 2.72 (2H, m, H-3); C NMR (CDCl
3
, 125 MHz)
d
1
54.2 (C, C-8a),130.5 (CH, C-5),128.0 (CH, C-7),127.2 (C, C-4a),120.8
CH, C-8), 117.1 (CH, C-6), 52.2 (CH , COOCH ), 34.9 (CH , C-3), 24.6
, C-4); HR-FABMS (pos) m/z 181.2081 [MþH] (calcd for
, 181.2093).
(
(
3
3
2
þ
CH
2
H
3.7. Preparative biotransformation of xanthyletin (6)
C
10
13
O
3
Precultured G. cingulata (5 mL) was transferred into a 500 mL
Erlenmeyer flask containing 300 mL of medium. Cultivation was
ꢂ
3
.6. Preparative biotransformation of psoralen (3)
Precultured G. cingulata (5 mL) was transferred into a 500 mL
carried out at 27 C with stirring (ca. 120 rpm) for 3 days. After the
growth of G. cingulata, 50 mg of 6 in 1.0 mL of dimethyl sulfoxide
(DMSO) was added into the medium and cultivated for an addi-
tional 7 days, together with two controls, which contained either
mycelia with medium or substrate dissolved in DMSO with me-
dium. No metabolic product was observed in two controls. After
the fermentation, the culture medium and mycelia were separated
by filtration. The medium was saturated with NaCl, and extracted
with EtOAc. The mycelia were also extracted with EtOAc. Each
EtOAc extract was combined, the solvent was evaporated, and
a crude extract (391 mg) was obtained. The extract was distributed
Erlenmeyer flask containing 300 mL of medium. Cultivation was
carried out at 27 C with stirring (ca. 120 rpm) for 3 days. After the
growth of G. cingulata, 50 mg of 3 in 1.0 mL of dimethyl sulfoxide
ꢂ
(
DMSO) was added into the medium and cultivated for an addi-
tional 7 days, together with two controls, which contained either
mycelia with medium or substrate dissolved in DMSO with me-
dium. No metabolic product was observed in two controls. After the
fermentation, the culture medium and mycelia were separated by
filtration. The medium was saturated with NaCl, and extracted with
EtOAc. The mycelia were also extracted with EtOAc. Each EtOAc
extract was combined, the solvent was evaporated, and a crude
extract (423 mg) was obtained. The extract was distributed be-
3
between 5% NaHCO aq and EtOAc, and EtOAc phase was evapo-
rated to give a neutral fraction (188 mg). The alkali phase was
acidified to pH 3 with 1 N HCl and distributed between water and
EtOAc. The EtOAc phase was evaporated, and the acidic fraction
(203 mg) was obtained. The acidic fraction was dissolved in ace-
3
tween 5% NaHCO aq and EtOAc, and EtOAc phase was evaporated
to give a neutral fraction (159 mg). The alkali phase was acidified to
pH 3 with 1 N HCl and distributed between water and EtOAc. The
EtOAc phase was evaporated, and the acidic fraction (264 mg) was
obtained. Metabolites were detected from both fractions by TLC and
HPLC, respectively. The acidic fraction was dissolved in acetone
2 2
tone (5 mL), and CH N (1 mL) was added to the fraction. The so-
lution was evaporated, and the methylation fraction was obtained.
The methylation fraction was subjected to CC (silica gel 60,
2
230e400 mesh, Merck) with a n-hexaneeEt O gradient (9:1 to
1:4) to yield compound 7a (42 mg). Compound 7a (18 mg) was
dissolved in MeOH (1 mL), 1% NaOH (2 mL) added to the solution,
and the solution was refluxed for 30 min. The solution was acidi-
fied to pH 3 with 1 N HCl and distributed between EtOAc and
water. The EtOAc phase was evaporated to give 7 (15 mg,
(
2 2
5 mL), and CH N (1 mL) was added to the fraction. The solution
was evaporated, and the methylation fraction was obtained. The
methylation fraction was subjected to silica-gel column chroma-
tography (CC) (silica gel 60, 230e400 mesh, Merck) with a n-hex-
aneeEt
Compound 4a (13 mg) was dissolved in MeOH (1 mL), 1% NaOH
2 mL) added to the solution, and the solution was refluxed for
0 min. The solution was acidified to pH 3 with 1 N HCl and dis-
tributed between EtOAc and water. The EtOAc phase was evapo-
rated to give 4 (9 mg, R
¼28.2 min). The neutral fraction was
subjected to CC using a gradient of n-hexaneeEtOAc (1:0 to 1:9) of
increasing polarity gave to metabolite 5 (11 mg, R
¼27.9 min).
2
O gradient (9:1 to 1:4) to yield compound 4a (25 mg).
R
t
¼45.2 min). The neutral fraction was subjected to CC using
a gradient of n-hexaneeEtOAc (1:0 to 1:9) of increasing polarity
gave to metabolite 8 (8 mg, R
¼44.8 min).
(
3
t
3.7.1. 9,9-Dimethyl-6,7-pyrano-hydrocoumaric acid (7). White
powder; IR (KBr)
500 MHz)
ꢁ1
1
t
n
max 3359, 1707 cm
;
6
H NMR (acetone-d ,
d
6.67 (1H, s, H-5), 6.16 (1H, s, H-8), 6.16 (1H, d, J¼9.8 Hz,
t
H-11), 5.37 (1H, d, J¼9.8 Hz, H-10), 2.66 (2H, t, J¼7.5 Hz, H-4), 2.45
13
(
2H, t, J¼7.5 Hz, H-3), 1.23 (6H, s, H-12, 13); C NMR shown as Table
þ
3
.6.1. 6,7-Furano-hydrocoumaric acid (4). White powder; IR (KBr)
1; HR-FABMS (pos) m/z 249.2828 [MþH] (calcd for C14
17 4
H O ,
ꢁ1 1
n
max 3264, 1698 cm ; H NMR (acetone-d
6
, 500 MHz)
d
7.61 (1H, d,
249.2838).
J¼2.3 Hz, H-9), 7.38 (1H, s, H-5), 6.99 (1H, br s, H-8), 6.73 (1H, dd,
J¼2.3, 1.2 Hz, H-10), 2.98 (2H, t, J¼7.7 Hz, H-4), 2.65 (2H, t, J¼7.7 Hz,
3.7.2. 9,9-Dimethyl-6,7-pyrano-o-hydrocoumaryl alcohol (8). White
powder; IR (KBr) nmax 3319 cm ; H NMR (CDCl , 500 MHz) d 6.70
3
H-3); 13C NMR shown as Table 1; HR-FABMS (pos) m/z 207.0685
ꢁ1 1
þ
[
MþH] (calcd for C11
H
11
O
4
, 207.0658).
(1H, s, H-5), 6.32 (1H, s, H-8), 6.24 (1H, d, J¼9.8 Hz, H-11), 5.45 (1H,
d, J¼9.8 Hz, H-10), 3.64 (2H, t, J¼6.0 Hz, H-2), 2.67 (2H, t, J¼6.6 Hz,
13
3
.6.2. 6,7-Furano-o-hydrocoumaryl alcohol (5). White powder; IR
H-4), 1.83 (2H, m, H-3) 1.40 (6H, s, H-12, 13); C NMR shown as
ꢁ1
1
þ
(
KBr)
n
max 3317 cm
;
H NMR (CDCl
3
, 500 MHz)
d
7.49 (1H, d,
Table 1; EIMS m/z 234 [M] (19), 219 (100), 201 (30), 181 (13), 163
þ
J¼2.3 Hz, H-9), 7.29 (1H, s, H-5), 7.02 (1H, br s, H-8), 6.64 (1H, dd,
(13), 91 (7); HR-EIMS m/z 234.1254 [M] (calcd for C14
18
H O
3
,
J¼2.3, 0.9 Hz, H-10), 3.66 (2H, t, J¼5.7 Hz, H-2), 2.87 (2H, t, J¼6.6 Hz,
234.1256).
13
H-4), 1.92 (2H, m, H-3); C NMR shown as Table 1; EIMS m/z 192
þ
[
(
M] (57), 174 (53), 147 (100), 146 (24), 131 (18), 118 (14), 91 (21), 77
3.7.3. 9,9-Dimethyl-6,7-pyrano-hydrocoumaric acid methyl ester
þ
ꢁ1
1
14); HR-EIMS m/z 192.0782 [M] (calcd for C11
H
12
O
3
, 192.0786).
(7a). White powder; IR (KBr)
CDCl , 500 MHz) 6.67 (1H, s, H-5), 6.33 (1H, s, H-8), 6.22 (1H, d,
J¼9.8 Hz, H-11), 5.45 (1H, d, J¼9.8, 0.9 Hz, H-10), 3.69 (3H, s,
COOCH ), 2.80 (2H, m, H-4), 2.67 (2H, m, H-3) 1.40 (6H, s, H-12, 13);
C NMR (CDCl , 125 MHz) 176.2 (C, C-2) 155.0 (C, C-8a), 152.9 (C,
C-7), 128.1 (CH, C-10), 127.9 (CH, C-5), 121.7 (CH, C-11), 119.3 (C, C-
4a), 114.8 (C, C-6), 105.1 (CH, C-8), 76.1 (C, C-9), 52.2 (CH , COOCH ),
, C-4); EIMS m/z 262
n
max 3397, 1740 cm
;
H NMR
(
3
d
3
.6.3. 6,7-Furano-hydrocoumaric acid methyl ester (4a). White
ꢁ1 1
powder; IR (KBr)
n
max 3398, 1714 cm ; H NMR (CDCl
3
, 500 MHz)
3
13
d
7.48 (1H, d, J¼2.3 Hz, H-9), 7.28 (1H, s, H-5), 7.04 (1H, br s, H-8),
3
d
6
.63 (1H, dd, J¼2.3, 0.9 Hz, H-10), 3.68 (3H, s, COOCH ), 2.99 (2H, t,
3
13
J¼6.5 Hz, H-4), 2.76 (2H, t, J¼6.5 Hz, H-3); C NMR (CDCl
25 MHz) 176.0 (C, C-2), 154.8 (C, C-7), 152.2 (C, C-8a), 144.1 (CH,
C-9), 123.9 (C, C-4a), 121.6 (C, C-5), 121.0 (C, C-6), 106.0 (CH, C-10),
3
,
3
3
1
d
35.2 (CH
2
, C-3), 28.0 (CH
3
, C-12, 13), 23.9 (CH
2
þ
[M] (12), 248 (6), 247 (34), 230 (13), 216 (15), 215 (100), 187 (39),
173 (18); HR-EIMS m/z 262.1203 [M] (calcd for
þ
9
9.8 (CH, C-8), 52.2 (CH
3
, COOCH
3
), 35.5 (CH
2
, C-3), 24.8 (CH
2
, C-4);
C
15
18
H O
4
,
þ
EIMS m/z 220 [M] (31), 188 (100), 160 (82), 147 (48), 146 (75), 131
262.1205).

500
S. Marumoto, M. Miyazawa / Tetrahedron 67 (2011) 495e500
3
.8.
b-Secretase (BACE1) enzyme assay
periments. 6,7-Furano-8a-methoxy-5-prenyloxy hydrocoumaric
acid methyl ester was used as positive control.
8
,9
The assay was carried out according to the supplied manual
6
,8,9
with modifications.
(
Briefly, a mixture of 10
L of BACE1 (1.0 U/mL), 10
the substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM am-
monium bicarbonate), and 10 L of sample dissolved in 30% DMSO
m
l of assay buffer
References and notes
50 mM sodium acetate, pH 4.5), 10
m
ml of
1. Murray, R. D.; Mendez, J.; Brown, S. A. In The Natural Coumarins: Occurrence,
Chemistry and Biochemistry; Murray, R. D. H., Mendez, J., Brown, S. A., Eds.; John
Wiley: New York, NY, 1982.
m
was incubated for 60 min at room temperature in the dark. The
mixture was irradiated at 550 nm and the emission intensity at
2. Robinson, T. In The Organic Constituents of Higher Plants: Their Chemistry and
Interrelationships; Robinson, T., Ed.; Burgess Publishing: Minneapolis, MN, 1967.
3
4
. Stanley, W. L.; Jurd, L. J. Agric. Food Chem. 1971, 19, 1106.
. Ive, G. W.; Holt, D. L.; Ivey, M. C. Science 1981, 213, 909.
5
90 nm was recorded. The inhibition ratio was obtained by the
following equation:
5. Tassaneeyakul, W.; Guo, L.-Q.; Fukuda, K.; Ohta, T.; Yamazoe, Y. Arch. Biochem.
Biophys. 2000, 378, 356.
Inhibitionð%Þ ¼ ½1 ꢁ fðS ꢁ S Þ=ðC ꢁ C Þgꢄ ꢃ 100
6. Marumoto, S.; Miyazawa, M. Phytother. Res. 2010, 24, 510.
0
0
7. Davis, B. G.; Boyer, V. Nat. Prod. Rep. 2001, 18, 618.
8
. Marumoto, S.; Miyazawa, M. Bioorg. Med. Chem. 2010, 18, 455.
Where C was the fluorescence of the control (enzyme, buffer,
and substrate) after 60 min of incubation, C was the fluorescence
of control at zero time, S was the fluorescence of the tested samples
enzyme, sample solution, and substrate) after incubation, and S
was the fluorescence of the tested samples at zero time. To allow for
the quenching effect of the samples, the sample solution was added
to the reaction mixture C, and any reduction in fluorescence by the
sample was then investigated. All data are the mean of three ex-
9. Marumoto, S.; Miyazawa, M. J. Agric. Food Chem. 2010, 58, 7777.
10. Smith, K.; Yang, J. J.; Li, Z.; Weeks, I.; Woodhead, J. S. J. Photochem. Photobiol.,
A 2009, 203, 72.
0
1
1. Shieh, H. S.; Blackwood, A. C. Can. J. Microbiol. 1969, 15, 647.
2. Nakayama, Y.; Nonomura, S.; Tatsumi, C. Agric. Biol. Chem. 1973, 37, 1423.
13. Levy, C. C.; Weinstein, G. D. T. Biochemistry 1964, 3, 1944.
4. Bellis, D. M. Nature 1958, 182, 806.
5. Kumari, G. N. K.; Ganesh, M. R.; Anitha, R.; Aravind, S. Z.; Naturforsch, C. J. Biosci.
004, 59, 405.
16. Kim, S.; Ko, H.; Son, S.; Shin, K. J.; Kim, D. J. Tetrahedron Lett. 2001, 42, 76.
(
0
1
1
1
2