Dimerization of piceatannol by Momordica charantia peroxidase and α-glucosidase inhibitory activity of the biotransformation products

Article abstract of DOI:10.1016/j.bmc.2011.07.032

Stilbenes, especially those oligomers, have great potential to be antihyperglycemic agents. In this study, eight stilbene dimers, including five new ones, were obtained by biotransformation of piceatannol using Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of spectroscopic evidences. These piceatannol dimers displayed potential α-glucosidase inhibitory activities, and trans double bond, tetrahydrofuran ring, and free adjacent phenolic dihydroxyls were found to be important for their activities. Enzymatic biotransformation of stilbenes by M. charantia peroxidase (MCP) was showed to be a prominent way to produce oligomeric stilbenes for antihyperglycemic development.

Full text of DOI:10.1016/j.bmc.2011.07.032

Bioorganic & Medicinal Chemistry 19 (2011) 5085–5092
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dimerization of piceatannol by Momordica charantia peroxidase and
a-glucosidase inhibitory activity of the biotransformation products
⇑
Xiang Wan, Xiao-Bing Wang, Ming-Hua Yang, Jun-Song Wang, Ling-Yi Kong
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Stilbenes, especially those oligomers, have great potential to be antihyperglycemic agents. In this study,
eight stilbene dimers, including five new ones, were obtained by biotransformation of piceatannol using
Momordica charantia peroxidase (MCP) for the first time. Their structures were established on the basis of
Received 20 May 2011
Revised 16 July 2011
Accepted 19 July 2011
Available online 24 July 2011
spectroscopic evidences. These piceatannol dimers displayed potential
a-glucosidase inhibitory activi-
ties, and trans double bond, tetrahydrofuran ring, and free adjacent phenolic dihydroxyls were found
to be important for their activities. Enzymatic biotransformation of stilbenes by M. charantia peroxidase
(MCP) was showed to be a prominent way to produce oligomeric stilbenes for antihyperglycemic
development.
Keywords:
Piceatannol
Momordica charantia peroxidase
Dimerization
Ó 2011 Elsevier Ltd. All rights reserved.
a-Glucosidase inhibition
1. Introduction
Herein, we reported the enzymatic biotransformation process,
the isolation and structural elucidation of these biotransformation
Stilbenes continue to receive substantial interest because of
their varying structures and an array of biological activities. These
compounds and their derivatives are important in drug research
and development due to their apparent activities such as antioxi-
dant, anticancer, and preventive role for Alzheimer’s disease.1–4
Moreover, increasing body of reports have demonstrated that stilb-
enes also possess conspicuous hypoglycemic activities, for exam-
ple, resveratrol analogues from traditional medicinal plants
Pterocarpus marsupium and Rheum undulatum,5,6 and stilbene gly-
cosides from rhubarb and Rumex bucephalophorus.7,8
products (Fig. 1), as well as their in vitro
effects.
a-glucosidase inhibitory
2. Results and discussion
2.1. Synthesis of piceatannol
Piceatannol (1) was synthesized via a key Wittig–Horner reac-
tion using the procedure as shown in Scheme 1.18 The starting
material, 5-(hydroxymethyl)benzene-1,3-diol (10), was benzyl
protected to obtain 11 in 85% yield, and then converted to its
bromide 12 in 35% yield. The bromide 12 was converted into a
phosphorus ylide 13, which reacted with 3,4-bis(benzyloxy)benz-
aldehyde (15), prepared from 3,4-dihydroxybenzaldehyde (14) in
92% yield, to give benzyl protected piceatannol (16) in 40% yield.
Piceatannol (1) was finally obtained by successive deprotection
of the benzyl groups of 16 in a yield of 60%.
Recently, oligomeric stilbenes have been reported to inhibit
amylase and
a
-glucosidase,9,10 with even stronger activity than
their corresponding monomers, making them the fascinating lead
compounds for new antihyperglycemic development.11 Oligomeric
stilbenes are not amply distributed in plant kingdom.1 This fact
and the powerful catalytic ability of Momordica charantia peroxi-
dase (MCP) to transform wider range of monomer into their
oligomers,12–14 prompted us to investigate the enzymatic biotrans-
formation of stilbenes by MCP and their in vitro
a-glucosidase
2.2. Structure elucidation of biotransformation products
inhibitory activities of the biotransformation products. In this
study, the parental compound used is piceatannol, also known as
3-hydroxyresveratrol, that possessed various biological activi-
MCP used in this study is a kind of peroxidase that isolated from
the fruits of M. charantia. With favorable enzyme pH stability (pH
3.8–8.0) and thermostability (20–45 °C), it has found wider appli-
cations than its counterpart peroxidases.12–14 Catalyzed by MCP,
piceatannol in this investigation was successfully transformed to
eight its dimers, including five new ones, the structures of which
were elucidated by analysis of spectroscopic data.
ties,15–17 including -glucosidase inhibitory activity.11 As a result,
a
eight stilbene dimers including five new ones were obtained.
⇑
Corresponding author. Tel./fax: +86 25 83271405.
E-mail address: cpu_lykong@126.com (L.-Y. Kong).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.032

5086
X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
H3CO
3a
HO
4a
OH
3a
2a
1a
2a
1a
7a
8a
OH
4a
HO
HO
OH
3b
OH
3b
4b
A
6a
O 4b
O
A
5a
7a
8a
5a
2b
2b
1b
6a
10a
A'
A'
8b
5b
10a
8b
10b
B'
1b
10b
5b
9b
9b
9a
14a
13a
9a
14a
13a
6b
11b
11b
11a
6b
OH
OH
7b
HO
7b
HO
B
B
11a
12a
B'
14b
14b
12b
12a
12b
13b
13b
OH
OH
HO
HO
HO
OH
HO
1
2
3
OH
OH
4a
4a
OCH3
5a
OAc
5a
3a
2a
3a
2a
A
A
OAc
3a
6a
H
6a
H
1a
4a
1a
HO
13a
OH
11b
12b
14a
B
2a
1a
7a
HO
OH
11b
14a
7a
8a
7a
8a
10b
A
9a
5b
13a
12a
10b
9a
4b
3b
5a
O
O
6b
1b
B
12a
11a
12b
8b
6a
B'
8b
B'
A'
2b
10a 8a
10a
9b
11a
11a
HO
13b
OH
9b
10a
13b
7b
7b
H
6b
5b
7b
14b
H
14b
HO
9a
14a
HO
OH
1b
B
10b
B'
1b
9b
OH
2b
3b
12a
13a
2b
3b
8b
6b
11b
12b
A'
A'
5b
OH
14b
HO
AcO
4b
4b
13b
OH
OH
OH
4
5
6
OH
OCH3
4a
3a
OH
4a
OH
5a
6a
2a
3a
2a
A
OH
3a
A'
5a
4a
HO
HO
7a 1a
8a
1a
2a
1a
OH
13b
HO
6a
14b
OH
14a
14a
B
H
7a
8a
A
5b
9a
HO
13a
10b
9a
8b
4b
3b
11b
12b
13b
5a
O
O
6b
1b
9b
8b
7b
1b
13a
12a
B
7a
8a
B'
6a
12a
11a
12b
B'
A'
2b
10a
11a
10a
9b
7b
10a
10b
7b
11a
H
6b
5b
11b
14b
9a
14a
HO
OH
B
OH
10b
1b
OH
9b
OH
2b
12a
8b
2b
3b
HO
6b
5b
11b
13a
A
A'
B'
3b
OCH3
OH
14b
12b
4b
OH
4b
OH
13b
OH
7
8
9
Figure 1. Chemical structures of compounds 1–9.
HO
OH
PhH2CO
OCH2Ph
PhH2CO
OCH2Ph
c
a
b
CH2OH
CH2Br
CH2OH
11
10
12
PhH2CO
PhH2CO
OCH2Ph
OCH2Ph
d
PhH2CO
e
O
CH
2 P(OEt)2
OCH2Ph
13
16
OH
HO
HO
OCH2Ph
OCH2Ph
OH
OH
OH
f
CHO
CHO
1
15
14
Scheme 1. Synthesis of piceatannol (1). Reagents and conditions: (a) PhCH2Cl, K2CO3, KI, CH3COCH3, rt; (b) PBr3, CH2Cl2, 0 °C; (c) PO(OEt)3, rt; (d) 15, EtONa, 0 °C; (e) BBr3,
CH2Cl2, 0 °C; (f) PhCH2Cl, K2CO3, KI, CH3COCH3, rt.

X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
5087
Compound 2 was obtained as a white powder. Its molecular for-
mula was determined as C28H22O8 by positive HR-ESI-MS, indicat-
ing the formation of a piceatannol dimer. A total of 28 carbon
signals (Table 1) were observed as eight oxygen-bearing and five
carbon-substituted aromatic quaternary carbons, eleven aromatic
and two trans olefinic methines, and two aliphatic methines
according to its 13C NMR and HSQC spectra. Further inspection of
its 1H NMR data (Table 1) with the help of HMBC spectrum re-
vealed the presence of two 1,3,5-tri-substituted aromatic frag-
ments (B, B0) (Fig. 1), one 1,3,4-tri-substituted aromatic fragment
(A), 1,3,4,5-tetra-substituted aromatic fragment (A0), one trans
double bond, and two mutually coupled aliphatic protons.
The HMBC spectrum also enabled the connection of these par-
tial fragments. The HMBC correlations (Fig. 2) from H-7a (dH
5.45, d, 7.8) to C-4b (dC 145.3), C-2a (dC 117.1) and C-6a (dC
121.8), from H-8a (dH 4.45, d, 7.8) to C-5b (dC 130.6) and C-10a
(dC 106.4), suggested the existence of a benzofuran ring among
rings A, A0 and B with a C-1a–C-7a–C-8a–C-9a linkage. The HMBC
cross-peaks of H-7b/C-2b (6b) and H-8b/C-10b (14b) demonstrated
that rings A0 and B0 were connected through the trans double bond
at C-1b and C-9b. The trans orientation of H-7a and H-8a in the fur-
an ring was inferred by the observed large coupling constants
(J = 7.8 Hz) for them and confirmed by correlations (Fig. 2) between
H-7a with H-10a and H-14a, and between H-8a with H-2a and
H-6a in the NOESY spectrum. Compound 2 was thus elucidated
as 4-{trans-3-(3,5-hydroxyphenyl)-5-[(E)-2-(3,5-dihydroxyphenyl)
vivyl]-7-dihydro-2,3-dihydro-1-benzofuran-2-yl]}benzene-1,2-diol.
Compound 3 was obtained as a white powder. Its molecular for-
mula was determined to be C29H24O8 by the observed pseudo
molecular ion at 523.1355 [M+Na]+, 14 mass units more than 2,
in the positive HR-ESI-MS. Its 1H and 13C NMR data resembled
those of 2 except for the presence of an additional methoxyl group
(dH 3.84, 3H, s; dC 56.2) and chemical shift discrepancy in fragment
A (Table 1): the down-field shifted of H-2a signal (
Dd 0.23), and up-
field shifted of H-5a ( d 0.51) and H-6a ( d 0.39) signals. The
D
D
methoxyl group placing at C-3a was determined by its observed
correlation with C-3a in the HMBC spectrum. Therefore, 3 was
determined to be 2-methoxy-4-{trans-3-(3,5-hydroxyphenyl)-5-
[(E)-2-(3,5-dihydroxyphenyl)vivyl]-7-dihydro-2,3-dihydro-1-ben-
zofuran-2-yl]}phenol.
Compound 4 was obtained as a white powder. The molecular
formula was determined to be C30H24O9 by positive HR-ESI-MS.
Its 1H and 13C NMR spectra (Table 1) revealed the existence of
two 1,3,5-tri-substituted aromatic rings (B, B0), two 1,3,4-tri-
substituted aromatic rings (A, A0), one trans double bond, one acet-
yl group, along with two mutually coupled aliphatic protons,
which combined with HMBC cross-peaks of H-7a/C-4b and
H-8a/C-3b (Fig. 3), suggesting the presence of a dioxane ring.
Analysis of its 1D and 2D data allowed the assignments of all
proton and carbon signals of 4 (Table 1). The HMBC correlations
from H-2a (6a) to C-7a and from H-8a to C-10a (14a) suggested
that aromatic rings A and B were joined at C-1a and C-9a, respec-
tively, through C-7a and C-8a of the dioxane ring (Fig. 3). The
HMBC correlations from H-7b to C-2b (6b) and from H-8b to C-
10b (14b) demonstrated that fragments A0 and B0 were connected
by a trans double bond between C-1b and C-9b. Compared with
2, the chemical shifts of H-2a, H-5a and H-6a were down-field
shifted by
Dd 0.37, 0.20 and 0.28, respectively, which combined
with its molecular formula, suggested that 3-OH was acetylated.
The trans orientation of H-7a and H-8a was determined by the ob-
served NOESY cross peaks (Fig. 3) from H-7a to H-10a, H-14a and
from H-8a to H-2a, H-6a. The structure of 4 was thus elucidated
Table 1
1H and 13C NMR data for compounds 2–4
Position
2
3
4
a
b
a
b
a
b
dH
dC
dH
dC
dH
dC
1a
2a
3a
4a
5a
6a
7a
8a
129.7
117.1
146.3
145.9
117.5
121.8
94.3
130.2
117.1
144.5
144.4
117.9
121.1
94.2
130.1
126.3
145.1
144.9
115.7
123.4
81.3
6.82 (d, 2.1)
7.05 (d, 1.8)
7.17 (d, 1.5)
6.79 (d, 8.7)
6.67 (dd, 8.7, 2.1)
5.45 (d, 7.8)
6.28 (d, 8.1)
6.28 (d, 8.1)
5.45 (d, 7.8)
4.54 (d, 7.8)
6.90 (d, 7.8)
6.84 (dd, 7.8, 1.5)
4.85 (d, 8.1)
4.45 (d, 7.8)
57.6
57.4
4.83 (d, 8.1)
81.7
9a
144.2
106.4
158.9
101.7
158.8
106.4
131.1
129.1
145.8
145.3
130.6
127.9
128.3
126.7
139.6
104.8
159
144.2
106.4
158.8
101.6
158.8
106.3
131.0
129.0
145.7
145.2
130.5
127.8
128.2
126.6
139.5
104.7
158.9
102.1
158.9
104.7
56.2
140.2
107.7
159.5
103.8
159.5
107.7
132.4
115.7
145.5
145.1
118.2
121.1
129.0
128.6
140.9
106.1
159.9
103.2
159.9
106.1
10a
11a
12a
13a
14a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
12b
13b
14b
OMe
OAc
C@O
6.06 (d, 1.8)
6.13 (d, 1.8)
6.06 (d, 1.8)
6.80 (d, 2.1)
6.20 (d, 1.5)
6.26 (d, 1.5)
6,20 (d, 1.5)
6.80 (d, 1.8)
6.23 (d, 1.8)
6.24 (t, 1.8)
6.23 (d, 1.8)
7.16 (d, 1.5)
6.93 (d, 8.4)
6.89 (d, 2.1)
6.84 (d, 16.8)
6.95 (d, 16.8)
6.90 (d, 1.8)
6.84 (d, 16.2)
6.92 (d, 16.2)
7.10 (d, 8.4, 1.5)
7.02 (d, 15.6)
6.95 (d, 15.6)
6.39 (d, 1.8)
6.12 (d, 1.8)
6.39 (d, 1.8)
6.39 (d, 1.8)
6.12 (t, 1.8)
6.57 (d, 1.8)
6.27 (d, 1.8)
6.57 (d, 1.8)
2.28 s
102.2
159
104.8
6.39 (d, 1.8)
3.84 s
20.6
168.9
a
Data were measured at 500 MHz.
Data were measured at 125 MHz.
b

5088
X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
HO
4a
3a
H
HO
4a
HO
2a
3a
A
2a
OH
3b
HO
H
OH
3b
5a
1a
4b
H
O
6a
1a
4b
6b
A
O
7a
H
H
2b
1b
8a
5a
7a
A'
2b
6a
8b
10b
B'
A'
5b
H
B
9b
8b
1b
7b
10b
11b
6b
H
OH
9a
14a
13a
9b
7b
11b
11a
OH
14b
HO
9a
11a
B'
12b
14b
HO
B
13b
H
12a
12b
HO
13a
13b
12a
OH
HO
OH
Figure 2. Key HMBC (H?C) and key NOESY (HMH) for compounds 2.
O
OAc
CH3
3a
4a
H
HO
HO
O
3a
A
4a
HO
HO
1a
5a
H
5b
A
H
1a
5b
A'
6a
4b
4b
O
5a
11a
O
7a
6b
1b
H
6b
1b
7a
H
H
A'
H
H
8a
10a
11a
7b
9b
O
3b
8a
O
3b
7b
9a
14a
10b
B'
B
9a
10b
OH
9b
OH
B
12a
13a
8b
12a
13a
11b
12b
11b
12b
H
H
B'
14a
OH
14b
14b
OH
13b
13b
OH
OH
Figure 3. Key HMBC (H?C) and key NOESY (HMH) for compounds 4.
as 5-{trans-3-(3,5-dihydroxyphenyl)-6-[(E)-2-(3,5-dihydroxyphenyl)
vivyl]-2,3-dihydro-1,4-benzodioxin-2-yl}2-hydroxyphenyl aceate.
Compound 5, a white powder, has a molecular formula of
Table 2
1H and 13C NMR data for compounds 5 and 6
Position
5
6
C
32H26O10 as determined by the positive HR-ESI-MS ion at m/z
[M+Na]+ (calcd 593.1424). The 1H and 13C NMR spectra (Table 2)
showed the presence of only 10 protons and 16 carbons suggested
the symmetric nature of 5. In the 1H NMR spectrum, 1,2,3,5-tetra-
substituted aromatic fragment (B), 1,3,4-tri-substituted aromatic
fragment (A0), two aliphatic protons, and one acetoxyl group were
observed. In addition to the acetoxyl group, the 13C NMR spectrum
resolved 14 carbon signals including four oxygen-substituted aro-
matic carbons, three carbon-substituted aromatic carbons, five
aromatic methine carbons, and two aliphatic carbons.
a
b
a
b
dH
dC
dH
dC
1a
2a
3a
4a
5a
6a
7a
128.1
124.4
136.6
145.2
115.3
128.8
54.4
138.1
111.6
147.9
145.2
115.3
120.1
54.4
6.94 (d, 2.1)
6.83 (d, 2.1)
6.70 (d, 8.1)
6.58 (d, 8.1, 2.1)
4.47 s
6.67 (d, 8.1)
6.55 (d, 8.1, 2.1)
4.68 s
8a
3.86 s
60.6
3.86 s
60.6
9a
150.2
123.1
155.2
102.4
159.2
103.3
128.1
124.4
136.6
145.2
115.3
128.8
54.4
150.2
122.9
155.1
102.3
159.1
103.3
128.8
115.1
145.5
143.8
115.8
119.2
54.1
10a
11a
12a
13a
14a
1b
2b
3b
4b
5b
In the HMBC spectrum (Fig. 4), the correlations from H-8a to C-
9a, C-14a, and C-8b (8a), and from H-7b to C-10a and C-8b (8a),
suggested the existence of a benzocyclopentane fragment. The
cross peaks from H-7b to C-2b and C-6b also confirmed the con-
nection of the 1,3,4-tri-substituted aromatic fragment at C-7b.
Moreover, the HMBC correlation from H-3b to the acetyl carbonyl
at dC 168.8, combined with the downshift of H-2b, H-5b and H-6b
6.20 (d, 2.1)
6.64 (d, 2.1)
6.94 (d, 2.1)
6.15 (d, 2.1)
6.57 (d, 2.1)
6.57 (d, 2.1)
6.70 (d, 8.1)
6.58 (d, 8.1, 2.1)
4.47 s
6.67 (d, 8.1)
6.50 (d, 8.1, 2.1)
4.57 s
by
Dd 0.37, 0.20, and 0.28, respectively, suggested the acetylation
6b
7b
of hydroxyl at C-3b. In light of the symmetry of 5 and the methine
nature of C-8a and C-8b, the fusion of the two halves of the whole
structure at C-8a and C-8b was established. The adoption of trans
orientations of H-7a/H-8a and H-7b/H-8b were determined by
the ROESY correlations (Fig. 4) from H-8a to H-2a and H-6a, and
from H-8b to H-2b and H-6b. The ROESY correlation between H-
8a and H-8b demonstrated cis orientations of H-8a and H-8b.
Therefore, the structure of 5 was elucidated as 5,50-(1,3,6,8-tetra-
hydroxy-4b,5,9b,10-tetrahydroindeno[2,1-a]indene-5,10-diyl)-
bis(2-hydroxy-5,1-phenylene) diacetate.
8b
3.86 s
60.6
3.86 s
60.6
9b
150.2
123.1
155.2
102.4
159.2
103.3
20.8
150.2
122.9
155.1
102.3
159.1
103.3
10b
11b
12b
13b
14b
OAc
OAc
C@O
OMe
6.20 (d, 2.1)
6.15 (d, 2.1)
6.57 (d, 2.1)
6.64 (d, 2.1)
2.28 s
2.28 s
20.8
168.8
3.78 br s
56.1
Compound 6, had the molecular formula C29H24O8 as estab-
lished by the positive HR-ESI-MS at 523.1360 [M+Na]+ (calcd for
a
Data were measured at 500 MHz.
Data were measured at 125 MHz.
b
C29H24O8Na, 523.1369). The NMR signals (Table 2) indicated that

X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
5089
enzymatic biotransformation of stilbenes by MCP as a prominent
way to produce oligomeric stilbenes for antihyperglycemic
development.
OH
3a
4a
5a
6a
OH
3a
OAc
4a
A
A
5a
6a
H
2a
OAc
HO
13a
H
1a
H
14a
B
HO
13a
H
4. Experimental section
4.1. General
14a
B
2a
H
H
8a
1a
7a
7a
10b
OH
11b
H
1
2a
OH
9a
7b
8a
8b
9b
12a
10b
11a
HO
9a
7b
11b
12b
10a
H
12b
13b
OH
11a
HO
2b
B'
8b
9b
B'
H
H
14b
1b
H
13b
OH
Optical rotations were carried out on a JASCO P-1020 polarime-
ter. CD spectra were obtained on a JASCO 810 spectropolarimeter.
UV spectra were measured on a Shimadzu UV-2501 PC spectropho-
tometer. 1H- and 13C-NMR, and NOESY spectra were recorded with
Bruker ACF-300 and Bruker ACF-500 (300 MHz and 125 MHz for
1H- and 13C-NMR, respectively) spectrometer in acetone-d6 or
DMSO-d6, chemical shifts were reported in ppm as d value relative
to tetramethylsilane (internal standard). Mass spectra were carried
out on an Agilent Micro Q-TOF mass spectrometer. Column chro-
H
14b
H
1b
A'
2b
6b
5b
H
A'
6b
5b
AcO
3b
AcO
3b
4b
4b
HO
HO
Figure 4. Key HMBC (H?C) and key NOESY (HMH) for compounds 5.
it was an analogue of 5, however, with different substituents: the
absence of two acetoxyl protons and, instead, the appearance of
one methoxyl protons. Since HMBC correlations from methoxyl
protons (dH 3.78, br s) to C-3a (dC 147.9) were observed, the
methoxyl group was assigned at C-3a. The structure of 6 was fur-
ther confirmed by 2D NMR experiments, and thus determined to
matography was carried out with Sephadex LH-20 (20–100
lm,
Pharmacia) and ODS-C18 (100–200 m, Waters). Preparative HPLC
l
was carried out using Agilent 1100 Series with Shim-park RP-C18
column (200 ꢀ 20 mm i.d.) and 1100 Series Multiple Wavelength
Detector. DMSO and
a-glucosidase (E.C.3.2.1.20) were purchased
be
5-(3,4-dihydroxyphenyl)-10-(4-hydroxy-3-methoxyphenyl)-
from Sigma. Other reagents and solvents used were of analytical
4b,5,9b,10-tetrahydroindeno[2,1-a]inden-1,3,6,8-tetrol.
The known compounds maackin (7), cararosinol D (8) and gnet-
ulin (9) were identified by comparison of their physical and spec-
troscopic data with those reported in the literatures.19–21 These
dimers were obtained for the first time by direct biotransformation
from piceatannol, which would be rather difficult by common
organic reactions.
Biotransformation catalyzed by MCP was presumed to be based
on radical reaction (Scheme 2): induced by hydrogen peroxide,
piceatannol was dehydrogenated and rearranged to form different
piceatannol radicals, which, catalyzed by MCP, were combined to
different dimers. Radical reactions were generally not stereo-selec-
tive. As a result, these dimers obtained should be racemes, which
was consistent with the zero values of optical rotations and was
further confirmed by the zero cotton effects in circular dichroism
(CD) spectra of compounds 2, 4, and 6 (Figs. S4, S11, and S18, Sup-
plementary data).
grade and were purchased from reliable commercial sources.
4.2. Synthesis of piceatannol (1)
4.2.1. [3,5-Bis(benzyloxy)phenyl]methanol (11)
To
a solution of 5-(hydroxymethyl)benzene-1,3-diol (10,
0.01 mol) in acetone (5 mL), K2CO3, KI, and (chloromethyl)benzene
were added and stirred at rotation (rt) for 4 h. The reaction solution
was filtrated and evaporated to dryness under reduced pressure,
and washed with 0.5 mol/L NaOH and water, respectively.
The crude product was purified by recrystallisation to afford
[3,5-bis(benzyloxy)phenyl]methanol (11, 0.0085 mol). 1H NMR
(DMSO-d6, 300 MHz): d 7.10–7.43 (10 H), 6.56 (2H, d, J = 2.1 Hz),
6.46 (1H, t), 5.05 (4H, br s), 4.53 (2H, s)]. HPLC analysis 97.7%
(MeOH: H2O = 80: 20 (V/V), tR = 10.58 min).
4.2.2. 1,3-Bis(benzyloxy-5-(bromomethyl)benzene (12)
Phosphorus tribromide was added to the CH2Cl2 solution
(10 mL) of [3,5-bis(benzyloxy)phenyl]methanol (11, 0.008 mol) at
0 °C for 2 h. Ice water was added to reaction solution, and extracted
with diethyl ether. The crude product was evaporated and purified
by silica-gel chromatography using petroleum/ethyl acetate 15:1
as an eluent to give bromide 12 (0.0028 mol, 35%). 1H-NMR
(DMSO-d6, 300 MHz) d: 7.11–7.43 (10H), 6.65 (2H, d, J = 2.1 Hz),
6.55 (1H, s), 5.03 (4H, br s), 4.35 (2H, s). HPLC analysis 96.9%
(MeOH: H2O = 85: 15 (V/V), tR = 8.67 min).
2.3.
a-Glucosidase inhibitory effect in vitro
As shown in Table 3, all the eight dimers inhibited
a-glucosi-
dase to various degrees. The three dimers, 2, 3 and 7, that have a
trans double bond showed stronger activity than the parental com-
pound, piceatannol (1), which suggested the essential role of this
unit for the
sive inhibitory effects of compounds 2 and 3 with IC50 values of
1.13 and 3.04 M, respectively, showed the importance of tetrahy-
a-glucosidase inhibitory ability. Moreover, the impres-
l
drofuran ring for this activity. Activity disparity for dimers with
similar structure, 2/3, 4/7, and 5/6/8, indicated the significance of
free hydroxyls at C-3 and C-4.
4.2.3. 3,4-Bis(benzyloxy)benzaldehyde (15)
To a solution of 3,4-dihydroxybenzaldehyde (14, 0.01 mol) in
ethanol (5 mL), K2CO3, KI, and (chloromethyl)benzene were added
and stirred at rt for 4 h. The reaction solution was filtrated and
evaporated to dryness under reduced pressure, and washed with
0.5 mol/L NaOH and water. The crude product was purified by
recrystallisation to afford 3,4-bis(benzyloxy)benzaldehyde (15).
3. Conclusion
Eight piceatannol dimers including five new ones were obtained
by M. charantia peroxidase (MCP) catalyzed biotransformation of
piceatannol. Raceme nature of these dimers was inferred by the
zero values of their optical rotations and cotton effects in the CD
spectra, suggesting a radical mechanism involved in the MCP cata-
lyzed biotransformation. These piceatannol dimers displayed
4.2.4. 1,2-Bis(benzyloxy)-4-{(E)-2-[3,5-bis(benzyloxy)phenyl]
vinyl}benzene (16)
To a mixture of bromide (12, 0.0026 mol), triethyl phosphite
(5 mL) was added, and stirred at 160 °C for 2 h. The reaction solu-
tion was evaporated at 100 °C under reduced pressure to obtain
potential
a-glucosidase inhibitory activities, and trans double
phosphonate (13).
A solution of phosphonate (13) in DMF
bond, tetrahydrofuran ring, and free hydroxyls at C-3 and C-4 were
found to be important for their activities. Our study favored the
(10 mL) was added dropwise to a suspension of NaOCH3 (3 mL)

5090
X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
O
O
O
O
HO
HO
HO
HO
OH
HO
HO
OH
OH HO
HO
OH
HO
OH
HO
a
c
b
d
O
O
O
O
HO
O
O
HO
OH
HO
OH
HO
HO
HOAc
OH
O
OH
HO
OH
g
h
e
f
HO
O
R
OH
O
OH
O
R
HO
b+d
H
OH
HO
HO
R
OH
R
OH
OH
OH
OH
HO
OH
O
OH
OH
R
HOAc
HO
OH
HO
R
OH
H
d+d
d+h
HO
R
OH
OH
H
HO
HO
R
OH
HO
O
HOAc
HOAc
OH
O
R
HO
O
O
O
HO
O
HO
H
OH
HO
OH
R
R
OH
OH
OH
HO
OH
Scheme 2. Proposed mechanism of the biotransformation of piceatannol.
at 0 °C, and 3,4-bis(benzyloxy)benzaldehyde (15) was added into
the reaction mixture, then stirred at 0 °C for 3 h. Thirty percent
of methanol–water was added to the mixture and stirred. Then
reaction mixture was vacuum filtrated, and the crude product
was recrystallizated to afford 1,2-bis(benzyloxy)-4-{(E)-2-[3,5-
bis(benzyloxy)phenyl]vinyl} benzene (16). 1H NMR (DMSO-d6,
300 MHz) d: 7.11–7.57 (10H), 7.04 (2H, d, J = 15.6 Hz), 6.98 (2H,
br s), 6.85 (1H, d, J = 15.6 Hz), 6.74 (2H, br s), 6.58 (2H, s), 5.07–
5.19 (8H, br s). The compound purity was analyzed by HPLC to
be 97.1% (MeOH: H2O = 88: 12 (V/V), tR = 13.15 min).
4.2.5. Piceatannol (1)
To a solution of compound 16 (0.0012 mol) in CH2Cl2 (20 mL), a
mixture of BBr3 and cyclohexane (5 mL, 1:1) was added, and stirred
at 0 °C for 2 h. Ice water was added to the reaction solution and
solution was extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over anhydrous magnesium
sulfate, and evaporated. The crude product was purified by silica-
gel chromatography using CHCl3/MeOH 10:1 as an eluent to give
piceatannol (1) (0.0098 mol, 79.0%). ESI-MS: [MꢁH]ꢁ 242.9; 1H
NMR (acetone-d6, 300 MHz) d: 7.42 (1H, d, J = 8.1 Hz), 7.04 (1H,

X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
5091
Table 3
4.6.1. 4-{Trans-3-(3,5-hydroxyphenyl)-5-[(E)-2-(3,5-dihydroxy-
phenyl)vivyl]-7-dihydro-2,3-dihydro-1-benzofuran-2-
yl]}benzene-1,2-diol (2)
Inhibitory effect of compounds 1–9 against yeast
glucosidase (IC50
a-
, lM)
White powder, ½a D20
ꢂ
0° (c 0.24, MeOH). HR-ESI-MS m/z 509.1220
Compounds
IC50 (lM)
[M+Na]+ (calcd for C28H22O8Na, 509.1212). UV kmax (MeOH) nm:
322, 280, 218. 1H NMR (acetone-d6, 300 MHz) and 13C NMR
(acetone-d6, 125 MHz) see Table 1.
2
3
4
5
6
7
8
9
1.13
3.04
69.32
131.07
108.58
23.22
79.11
70.16
34.32
20.6
4.6.2. 2-Methoxy-4-{trans-3-(3,5-hydroxyphenyl)-5-[(E)-2-(3,5-
dihydroxyphenyl)vivyl]-7-dihydro-2,3-dihydro-1-benzofuran-
2-yl]}phenol (3)
White powder, ½a D20
ꢂ
0° (c 0.24, MeOH). HR-ESI-MS m/z 523.1355
1 (Piceatannol)
1-Deoxynorjirimycin
[M+Na]+ (calcd for C29H24O8Na, 523.1359). UV kmax (MeOH) nm:
322, 280, 218. See the 1H NMR (acetone-d6, 300 MHz) and 13C
NMR (acetone-d6, 125 MHz) in Table 1.
br s), 6.99–6.82 (3H, m), 6.55 (2H, br s), 6.36 (1H, br s) HPLC anal-
ysis 97.6% (MeOH: H2O = 55: 45 (V/V), tR = 11.33 min).
4.6.3. 5-{Trans-3-(3,5-dihydroxyphenyl)-6-[(E)-2-(3,5-dihy-
droxyphenyl)vivyl]-2,3-dihydro-1,4-benzodioxin-2-yl}
2-hydroxyphenyl aceate (4)
4.3. Plant material
White powder, ½a D20
ꢂ
0° (c 0.24, MeOH). HR-ESI-MS m/z 551.1315
[M+Na]+ (calcd for C30H24O9Na, 551.1318). UV kmax (MeOH) nm:
322, 280, 218. 1H NMR (acetone-d6, 300 MHz) and 13C NMR (ace-
tone-d6, 125 MHz) see Table 1.
Fruits of M. charantia were collected at suburb of Nanjing, China,
and identified by Professor Minjian Qin, Department of Medicinal
Plants, China Pharmaceutical University. A voucher specimen
(No. 000804) was deposited in the Department of Natural Medici-
nal Chemistry, China Pharmaceutical University.
4.6.4. 5,50-(1,3,6,8-Tetrahydroxy-4b,5,9b,10-tetrahydroindeno
[2,1-a]indene-5,10-diyl)bis(2-hydroxy-5,1-phenylene) diacetate
(5)
4.4. Purification of M. charantia peroxidase (MCP)
White powder, ½a D20
ꢂ
0° (c 0.20, MeOH). HR-ESI-MS m/z 593.1414
[M+Na]+ (calcd for C32H26O10Na, 593.1424). UV kmax (MeOH) nm:
272, 254, 216. 1H NMR (acetone-d6, 300 MHz) and 13C NMR (ace-
tone-d6, 125 MHz) see Table 2.
MCP was purified from fruits of M. charantia to electrophoretic
homogeneity by consecutive treatment of ammonium sulfate frac-
tionation, ion exchange chromatography on DEAE-Sepharose FF,
affinity chromatography on Con A Sepharose and gel filtration on
Sephadex G-150 as described in our previous report.12 The purified
MCP exhibited a specific activity of 7757 E.U. of peroxidase per mg
of protein, which was 46-fold higher than that of the crude extract.
4.6.5. 5-(3,4-Dihydroxyphenyl)-10-(4-hydroxy-3-methoxy-
phenyl)-4b,5,9b,10-tetrahydroindeno[2,1-a]inden-1,3,6,8-
tetrol (6)
White powder, ½a D20
ꢂ
0° (c 0.14, MeOH). HR-ESI-MS m/z 523.1360
[M+Na]+ (calcd for C29H24O8Na, 523.1369). UV kmax (MeOH) nm:
254, 216. 1H NMR (acetone-d6, 300 MHz) and 13C NMR (acetone-
d6, 125 MHz) see Table 2.
4.5. Biotransformation of piceatannol in aqueous acetone
A solution of piceatannol (300 mg) in acetone (20 mL) and a
solution of MCP (4 ꢀ 103 U) in buffer (100 mM NaOAC–HOAC, pH
7.3, 50 mL) were mixed and treated with hydrogen peroxide
(0.3%, 10 mL) at 37 °C and stirred for 5 h.
4.6.6. Maackin (7)
White powder, 1H NMR (acetone-d6, 300 MHz) d: 7.16 (1H, d,
J = 1.5 Hz), 7.10 (1H, dd, J = 8.4, 1.5 Hz), 7.02 (1H, d, J = 15.6 Hz),
6.95 (1H, d, J = 15.6 Hz), 6.93 (1H, d, J = 8.4 Hz), 6.80 (1H, d,
J = 1.5 Hz), 6.70 (1H, d, J = 7.8 Hz), 6.57 (2H, d, J = 1.8 Hz), 6.56
(1H, dd, J = 7.8, 1.5 Hz), 6.27 (1H, d, J = 1.8 Hz), 6.24 (1H, t,
J = 1.8 Hz), 6.23 (2H, d, J = 1.8 Hz), 4.85 (1H, d, J = 8.1 Hz), 4.83
(1H, d, J = 8.1 Hz). 13C NMR (acetone-d6, 125 MHz) d: 160.0,
160.0, 159.5, 159.5, 146.6, 146.0, 145.5, 145.1, 140.9, 140.6,
132.4, 129.5, 129.0, 128.6, 121.0, 121.0, 118.2, 116.1, 116.0,
115.7, 107.7, 107.7, 106.0, 106.0, 81.7, 81.3.
4.6. Isolation and identification
The reaction mixture was evaporated to dryness at 40 °C under
reduced pressure and then dissolved in water, partitioned with
ethyl acetate. The ethyl acetate extract was washed with water,
dried with anhydrous Na2SO4 and evaporated to dryness at 40 °C
under reduced pressure to afford a brown powder (310 mg), which
was subjected to RP-C18 column chromatography (
80 g) eluted with MeOH/H2O (40:60 to 60:40, v/v) to afford frac-
tion 1 (52 mg), fraction 2 (55 mg), and fraction 3 (90 mg). Fraction
U
3.0 ꢀ 50,
4.6.7. Cararosinol D (8)
White powder, 1H NMR (acetone-d6, 300 MHz) d: 6.67 (2H, d,
J = 8.1 Hz) , 6.57 (2H, d, J = 2.1 Hz), 6.57 (2H, d, J = 2.1 Hz), 6.50
(2H, dd, J = 8.1, 2.1 Hz), 6.15 (1H, d, J = 2.1 Hz), 4.46 (2H, s), 3.76
(2H, s). 13C NMR (acetone-d6, 125 MHz) d: 159.2, 159.2, 155.3,
155.3, 150.3, 150.3, 145.6, 145.6, 143.8, 143.8, 138.8, 138.8,
123.1, 123.1, 60.6, 60.6, 54.1, 54.1.
1 was subjected to Sephadex LH-20 column chromatography (
to
(35 mg) was submitted to prep-HPLC (MeOH-0.05% TFA in H2O,
U
9
1.5 ꢀ 100, 70 g) eluted with MeOH. Subfractions of
7
35:65, v/v) to give 2 (5 mg) and 7 (13 mg). Fraction 2 was subjected
to Sephadex LH-20 column chromatography (
U
1.5 ꢀ 100, 70 g,
8 mL each) eluted with MeOH. The elution of 12–14 fractions
(37 mg) was submitted to prep-HPLC (MeOH-0.05% TFA in H2O,
47:53, v/v) to give 8 (10 mg) and 9 (6 mg). Fraction 3 was subjected
4.6.8. Gnetulin (9)
Brown powder, 1H NMR (acetone-d6, 300 MHz) d: 7.05 (1H, s),
6.89 (1H, d, J = 1.8 Hz), 6.83 (1H, d, J = 8.1 Hz), 6.78 (1H, d,
J = 2.1 Hz), 6.74 (1H, d, J = 1.8 Hz), 6.69 (1H, d, J = 8.1 Hz), 6.64
(1H, d, J = 8.1 Hz), 6.51 (1H, dd, J = 8.1, 1.8 Hz), 6.34 (2H, d,
J = 1.8 Hz), 6.31 (1H, d, J = 2.1 Hz), 6.20 (1H, t, J = 1.8 Hz), 4.27
to Sephadex LH-20 column chromatography (
U
1.5 ꢀ 100, 70 g
eluted with MeOH. The elution of 10 to 11 fractions (65 mg) was
submitted to prep-HPLC (MeOH-0.05% TFA in H2O, 57:43, v/v) to
give 3 (6 mg), 4 (9 mg), 5 (5 mg), and 6 (5 mg).

5092
X. Wan et al. / Bioorg. Med. Chem. 19 (2011) 5085–5092
(1H, s), 4.19 (1H, s), 3.71 (3H, s), 3.57 (3H, s). 13C NMR (acetone-d6,
125 MHz) d: 159.9, 159.8, 159.8, 155.4, 149.1, 148.1, 148.0, 147.2,
146.6, 145.8, 142.8, 138.4, 130.2, 124.5, 123.8, 123.2, 120.0,
119.5, 117.7, 115.6, 112.2, 106.3, 106.3, 103.8, 101.5, 98.4, 60.7,
57.9, 56.1.
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4.7.
a-Glucosidase inhibition assay
Yeast
a
-glucosidase inhibition assay was performed according
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for 10 min, using 0.01 M p-nitrophenyl
a-D-glucopyranoside
(PNPG) as substrate and 1 U/mL of enzyme, in 0.067 M
a
KH2PO4–Na2HPO4 buffer. Acarbose (98.5%, HPLC) was used as posi-
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Acknowledgment
The research work was supported by the Cultivation Fund of the
Key Scientific and Technical Innovation Project, Ministry of Educa-
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.032. These data
include MOL files and InChiKeys of the most important compounds
described in this article.