Synthesis of glycosyl ferulate derivatives by amine-promoted glycosylation with regioselective hydrolysis using Novozym 435 and evaluation of their antioxidant properties

Article abstract of DOI:10.1016/j.carres.2012.06.019

Various glycosyl ferulates were efficiently synthesized from 2,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (TAGB) with amine by amine-promoted glycosylation without using heavy metal. The resulted acetylated glycosyl ferulates with acetoxyl groups at C-

Full text of DOI:10.1016/j.carres.2012.06.019

Carbohydrate Research 359 (2012) 11–17
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of glycosyl ferulate derivatives by amine-promoted glycosylation
with regioselective hydrolysis using Novozym 435 and evaluation of their
antioxidant properties
Yasutaka Shimotori a, , Kyohei Tsutano b, Kouji Soga b, Yosuke Osawa b, Masakazu Aoyama a,
⇑
Tetsuo Miyakoshi b
a Department of Biotechnology and Environmental Chemistry, National University Corporation Kitami Institute of Technology, 165, Koen-cho, Kitami, Hokkaido 090-8507, Japan
b Department of Applied Chemistry, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki 214-8571, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 April 2012
Received in revised form 28 June 2012
Accepted 29 June 2012
Available online 11 July 2012
Various glycosyl ferulates were efficiently synthesized from 2,4,6-tetra-O-acetyl-a-D-glucopyranosyl
bromide (TAGB) with amine by amine-promoted glycosylation without using heavy metal. The resulted
acetylated glycosyl ferulates with acetoxyl groups at C-2, C-3 and C-4 were regioselectively deacetylated
at C-4 and C-6 positions with Novozym 435. Antioxidant abilities of free ferulic acids and its synthetic
glycosyl ferulates were evaluated by inhibitory effect on autoxidation of bulk methyl linoleate as well
as their radical scavenging activity. The radical scavenging activity on 1,1-diphenyl-2-picrylhydrazyl
(DPPHÅ) decreased in the order ferulic acid > sinapinic acid ꢀ glycosyl sinapinates ꢀ glycosyl feru-
lates > p-coumaric acid > glycosyl p-coumarates. In bulk methyl linoleate, the antioxidant activity order
against autoxidation was very consistent with the scavenging activity order. The results showed that gly-
cosyl ferulates and sinapinates were effective as well as free carboxylic acid forms.
Dedicated to the memory of Dr. Chen-Loung
Chen (North Carolina State University, USA)
Keywords:
Amine-promoted glycosylation
Enzymatic hydrolysis
Regioselectivity
Published by Elsevier Ltd.
Antioxidant property
1. Introduction
procedure of Fischer by way of esterifing hydroxyl groups of
glucose with aglycones, such as alcohols and carboxylic acids. How-
It is well established that various glycosyl ferulates widely oc-
cur in various plants and have various functions in the plant tis-
sues. Feruloyl-D-glycoside and sinapinoyl-D-glycoside which are
ever, in these reactions, the control of stereo-configuration at C-1 of
glucose is difficult. Recently, the procedures using microwave,19
and H2SO4–sillica as catalyst20 are applying for the syntheses of
the glycosyl esters, but in these procedures, the difficulty in control-
ling the stereo-configuration at C-1 of glucose is still not completely
eliminated.
contained in the Citrus fruits have antioxidation activity. These
compounds are reported to have functioned as blood adhesion
molecules.1 It has been reported that p-coumaroyl-
D-glycosyl ester,
one of components in the methanol extract from strawberry has
anti-cancer activity although it was only identified and quantified
in trace amount.2 In addition, the other components in the metha-
nol extract from strawberry, methyl and ethyl cinnamate were
synthesized in vivo through cinnamoyl glucose esters as interme-
diates.3 Although glycosyl ferulates may have similar activities,
their syntheses are very rare.4–6
These lead us to the use of glycosyl halide for the production of
desired glycosyl esters,21 in which glucoses are converted to the
corresponding glycosyl esters with corresponding carboxylic acid
using amine to synthesize glycosyl ferulates without using the
heavy metal compounds as catalysts and with better control of
stereo-configuration at C-1 of glucose. In addition, deacetylation
at C-5 and C-6 positions of the resulting acetylated glycosyl esters
conducted by Novozym 435 to obtain selective control of stereo-
configuration at C-1 of glucose moieties of the corresponding
glycosyl esters (Scheme 1).
In the synthesis of glycosyl esters, catalysts used are mostly
heavy metal compounds such as Ag2CO3,7–9 Ag2O,10–12 Hg
(CN)2,13–15 CdCO3
and SnCl4 as Lewis acids. Therefore, not
16,17
18
only the synthesis of the glycosyl esters is costly, but also the
process is not environmentally friendly. Without using these heavy
metal catalysts, the glycosyl esters can be synthesized using the
Ferulic acid is one of the ubiquitous compounds in nature, espe-
cially rich as an ester form in rice bran pitch, a byproduct of rice oil
production. Application of such industrial waste is important from
the environmental point of view. It has been well-known that
ferulic acid and
c-oryzanol, a mixture of monoesters consisting of
⇑
Corresponding author. Tel.: +81 157 269387; fax: +81 157 247719.
E-mail address: yasu@mail.kitami-it.ac.jp (Y. Shimotori).
ferulic acid and several kinds of triterpene alcohols as
0008-6215/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.carres.2012.06.019

12
Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
Scheme 1. Synthesis of O-glycosyl ester.
cycloartenol and 24-methylenecycloartanol, have an antioxidant
methods. D-Glucose was purchased from Sigma-Aldrich Corp. (St.
22–24
activity.
Such antioxidants are currently expected not only
Louis, MO, USA). trans-Cinnamic acid, p-coumaric acid and sinapi-
nic acid were purchased from Tokyo Chemical Industry Co. Ltd (To-
kyo, Japan). Ferulic acid was purchased from Tsuno Co. Ltd
(Wakayama, Japan). Novozym 435 (immobilized lipase from Can-
dia antarctica) was a gift from Novozymes A/S (Paraná, Brazil). All
other materials were commercially obtained.
to prevent lipid oxidation in food but also to prevent free-radical-
induced diseases such as cancer and atherosclerosis or aging caused
by oxidative tissue degeneration.25 Therefore, there is a possibility
of developing an application of rice bran pitch for human health.
In general, the inhibitory effect of antioxidants on lipid oxida-
tion is influenced by the physicochemical state of the lipid sub-
stance, and several evaluation systems using different physical
conditions are required for better understanding of antioxidant
abilities.26 Although ferulic acid and its glycosyl ester have been
recognized as antioxidants, there are few reports on systematic
evaluation of the antioxidant properties of ferulic acid and its
derivatives in different conditions.
In this paper, we described the antioxidant activities of ferulic
acid and its synthetic glycosyl ferulates, as well as some ferulic acid
related compounds such as p-coumaric acid, sinapinic acid, and
these glycosides. Their antioxidant activity was evaluated on the
basis of their inhibitory effects on the autoxidation of methyl lino-
leate in bulk system and the radical scavenging activity against
1,1-diphenyl-2-picrylhydrazyl (DPPHÅ).
2.2. Glucosylation of ferulic acids
2.2.1. 2,3,4,6-Tetra-O-acetyl-b-
D-glucopyranosyl cinnamate (1a)
A solution of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide
(411 mg, 1.0 mmol), trans-cinnamic acid (296 mg, 2.0 mmol) and i-
Pr2NEt (388 mg, 3.0 mmol) in CH3CN (5 mL) was stirred for 24 h at
room temperature under Ar atmosphere, and 1 g 4 Å molecular
sieves were used for water binding. Progress of the reaction was
monitored by thin layer chromatography. On the completion of
the reaction, the reaction mixture was filtered to remove molecular
sieves, and the solvent was evaporated under vacuum from the
reaction mixture, and EtOAc was added to the residue. The resulting
solution was then neutralized with aqueous NaHCO3, and extracted
with EtOAc, followed by washing with brine. The combined extracts
were dried over anhydrous MgSO4, and the solvent was removed by
evaporating under vacuum. The crude product was purified by flash
column chromatography (hexane–EtOAc, 3:1) to give 1a (244 mg,
2. Experimental
2.1. General
78%) as a white solid. Rf = 0.50 (hexane–EtOAc, 1:1); ½a D20
ꢃ17.7 (c
ꢂ
1.0, THF) [lit. ½a D20
ꢂ
= ꢃ32 (c 0.75, EtOH)27]; IR (KBr): 3011, 1745,
IR spectra were measured on an FT-IR 460plus spectrometer
(JASCO Corp., Tokyo, Japan).1H and 13C NMR spectra were mea-
sured on a JNM-ECA-500 spectrometer (JEOL, Tokyo, Japan) at
500 and 126 MHz, respectively. Deuterated solvents were used as
internal standards. Structural determination of all compounds
was performed by the use of COSY, HMQC, and HMBC NMR
techniques. The high resolution ESI-MS spectra of 2,3,4,6-tetra-O-
1135, 943 cmꢃ1 1H NMR (500 MHz, CDCl3): d = 2.06 (m, 12H,
;
CH3 ꢁ 4 in OAc at C-2, C-3, C-4 and C-6), 3.91 (ddd, J = 9.2, 4.6,
2.3 Hz, 1H, H-5), 4.13 (dd, J = 12.6, 2.3 Hz, 1H, H-6a), 4.32 (dd,
J = 12.6, 4.6 Hz, 1H, H-6b), 5.18 (t, J = 9.2 Hz, 1H, H-4), 5.22 (t,
J = 9.2 Hz, 1H, H-2), 5.28 (t, J = 9.2 Hz, 1H, H-3), 5.86 (d, J = 9.2 Hz,
1H, H-1), 6.42 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 7.41 (m, 3H,
Ph), 7.54 (m, 2H, Ph), 7.76 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–);
13C NMR (126 MHz, CDCl3): d = 20.6 (–CH3 in OAc at C-2 and C-4),
20.7 (–CH3 in OAc at C-3 and C-6), 61.5 (C-6), 67.8 (C-4), 70.3 (C-
2), 72.7 (C-3, C-5), 91.9 (C-1), 116.2 (–C(@O)CH@CH–), 128.4 (Ph),
129.0 (Ph), 130.9 (Ph), 133.8 (Ph), 147.5 (–C(@O)CH@CH–), 164.6
(–C(@O)CH@CH–), 169.3 (–C@O in OAc at C-2), 169.4 (–C@O in
OAc at C-3), 170.0 (–C@O in OAc at C-4) 170.6 (–C@O in OAc at C-6).
acetyl-b-
-glucopyranosyl sinapinate (1d), 2,3-di-O-acetyl-b-
osyl cinnamate (2a), 2,3-di-O-acetyl-b- -glucopyranosyl ferulate
(2c) and 2,3-di-O-acetyl-b- -glucopyranosyl sinapinate (2d) were
measured on a JMS-T100LC mass spectrometer (JEOL, Japan)
equipped with UV detector (254 nm) and Cadenza -C-18
D-glucopyranosyl ferulate (1c), 2,3,4,6-tetra-O-acetyl-b-
D
D
-glucopyran-
D
D
C
D
(4.6 mmID ꢁ 150 mm, Imtakt) column. The LC gradient proceeded
from 70% acetonitrile and 30% water acidified with 0.1% trifluoro-
acetic acid to 20% acetonitrile and 80% acidic water at a flow rate
of 1.0 mL/min. The high resolution APCI-MS spectrum of 2,3-di-
2.2.2. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl p-coumarate
(1b)
This compound was synthesized according to procedure de-
scribed for 1a. Instead of trans-cinnamic acid, p-coumaric acid
(328 mg, 2.0 mmol) was glycosylated with TAGB (411 mg,
1.0 mmol) to give 1b (401 mg, 81%) as a white solid. Rf = 0.45 (hex-
O-acetyl-b-D-glucopyranosyl p-coumarate (2b) was measured on
a JMS-T100LC mass spectrometer (JEOL, Japan) equipped with UV
detector (254 nm) and Synergi MAX-RP 80A (2.0 mmID ꢁ 150
mm, Phenomenex) column. The LC gradient proceeded from 60%
acetonitrile and 40% water acidified with 0.1% trifluoroacetic acid
to 30% acetonitrile and 70% acidic water at a flow rate of 0.2 mL/
min. Absorbance spectra were measured using an UVmini-2400
UV–Vis spectrophotometer (Shimadzu, Kyoto, Japan). Flash column
chromatography was performed using silica gel FL60D (Fuji Silysia
Chemical Ltd, Aichi, Japan). Thin layer chromatography was per-
formed with silica gel F-254 on aluminum plate (Merck Ltd,
Darmstadt, Germany). Solvents were dried following standard
ane–EtOAc, 1:1); ½a D20
ꢂ
ꢃ2.9 (c 1.0, THF) (lit. [
a] Negative specific
rotation, CHCl328); IR (KBr): 3354, 3024, 1756, 1715, 1629, 1603,
1588, 1516, 1231, 1081 cmꢃ1 1H NMR (500 MHz, CDCl3): d = 2.06
;
(m, 12H, CH3 ꢁ 4 in OAc at C-2, C-3, C-4, C-6), 3.92 (ddd, J = 9.2,
4.6, 2.3 Hz, 1H, H-5), 4.16 (dd, J = 12.6, 2.3 Hz, 1H, H-6a), 4.33
(dd, J = 12.6, 4.6 Hz, 1H, H-6b), 5.19 (t, J = 9.2 Hz, 1H, H-4), 5.22
(t, J = 9.2 Hz, 1H, H-2), 5.28 (t, J = 9.2 Hz, 1H, H-3), 5.86 (d,
J = 9.2 Hz, 1H, H-1), 6.23 (d, J = 15.5 Hz, 1H, ꢃC(@O)CH@CH–),

Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
13
6.86 (d, J = 8.6 Hz, 2H, Ph), 7.41 (d, J = 8.6 Hz, 2H, Ph), 7.68 (d,
J = 16.0 Hz, 1H, –C(@O)CH@CH–); 13C NMR (126 MHz, CDCl3):
d = 20.6 (CH3 in OAc at C-2 and C-4), 20.7 (CH3 in OAc at C-3 and
C-6), 61.5 (C-6), 67.8 (C-4), 70.3 (C-2), 72.7 (C-5), 72.8 (C-3), 91.8
(C-1), 113.3 (–C(@O)CH@CH–), 116.0 (Ph), 126.5 (Ph), 130.4 (Ph),
147.3 (–C(@O)CH@CH–), 158.6(Ph), 165.1(–C(@O)CH@CH–),
169.5 (–C@O in OAc at C-2), 169.6 (–C@O in OAc at C-3), 170.2
(–C@O in OAc at C-4), 170.8 (–C@O in OAc at C-6).
NMR (500 MHz, CDCl3): d = 1.92 (s, 3H, CH3 in OAc at C-2), 2.00 (s,
3H, CH3 in OAc at C-3), 2.98 (br, 1H, OH at C-6), 3.53 (m, 1H, H-5),
3.66 (m, 2H, H-4, H-6a), 3.83 (d, J = 10.7 Hz, 1H, H-6b), 3.86 (br,
1H, OH at C-4), 4.98 (t, J = 9.2 Hz, 1H, H-2), 5.13 (t, J = 9.2 Hz, 1H,
H-3), 5.80 (d, J = 9.2 Hz, 1H, H-1), 6.22 (d, J = 16.5 Hz, 1H, –
C(@O)CH@CH–), 6.74 (m, 3H, Ph), 7.39 (d, J = 8.9 Hz, 2H, Ph), 7.60
(d, J = 16.0 Hz, 1H, –C(@O)CH@CH–); 13C NMR (126 MHz, CDCl3):
d = 20.8 (CH3 in OAc at C-2), 21.0 (CH3 in OAc at C-3), 61.8 (C-6),
68.9 (C-4), 70.7 (C-2), 75.7 (C-3), 76.7 (C-5), 92.3 (C-1), 116.4 (–
C(@O)CH@CH–), 128.6 (Ph), 129.1(Ph), 131.1 (Ph), 134.0 (Ph),
147.7 (–C(@O)CH@CH–), 165.2 (–C(@O)CH@CH–), 170.0 (-C@O in
OAc at C-2), 171.5 (-C@O in OAc at C-3). ESI-MS: m/z [M+NH4]+
calcd for C19H26NO9: 412.1602: found: 412.1608.
2.2.3. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl ferulate (1c)
This compound was synthesized according to procedure de-
scribed for 1a. Instead of trans-cinnamic acid, ferulic acid (388 mg,
2.0 mmol) was glycosylated with TAGB (411 mg, 1.0 mmol) to give
1c (388 mg, 74%) as a white solid. Rf = 0.34 (hexane–EtOAc, 1:1);
½
a 2D0
ꢂ
ꢃ52.7 (c 1.0, THF); IR (KBr): 3382, 2969, 1754, 1638, 1588,
2.3.2. 2,3-Di-O-acetyl-b-
This compound was synthesized according to procedure de-
scribed for 2a. 2,3,4,6-tetra-O-acetyl-b- -glucopyranosyl p-couma-
D-glucopyranosyl p-coumarate (2b)
1515, 1372, 1233, 1098 cmꢃ1 1H NMR (500 MHz, CDCl3): d = 2.05
;
(m, 12H, CH3 ꢁ 4 in OAc at C-2, C-3, C-4, C-6), 3.90 (ddd, J = 9.2,
4.6, 2.3 Hz, 1H, H-5), 3.94 (s, 3H, CH3 in OMe at Ph), 4.13 (dd,
J = 12.6, 2.3 Hz, 1H, H-6a), 4.32 (dd, J = 12.6, 4.6 Hz, 1H, H-6b),
5.18 (t, J = 9.2 Hz, 1H, H-4), 5.22 (t, J = 9.2 Hz, 1H, H-2), 5.28 (t,
J = 9.2 Hz, 1H, H-3), 5.86 (d, J = 9.2 Hz, 1H, H-1), 5.91 (s, 1H, OH),
6.26 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 6.93 (d, J = 8.6 Hz, 1H,
Ph), 7.04 (d, J = 1.7 Hz, 1H, Ph), 7.09 (dd, J = 8.3, 2.0 Hz, 1H, Ph),
7.69 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–); 13C NMR (126 MHz,
CDCl3): d = 20.6 (CH3 in OAc), 56.0 (OCH3 at Ph), 61.5 (C-6), 67.9
(C-4), 70.3 (C-2), 72.7 (C-3, C-5), 91.9 (C-1), 109.6 (Ph), 113.4 (–
C(@O)CH@CH–), 114.8 (Ph), 123.9 (Ph), 126.5 (Ph), 146.8 (Ph),
147.6 (–C(@O)CH@CH–), 148.6 (Ph), 164.9 (–C(@O)CH@CH–),
169.4 (2-C@O in OAc at C-3 and C-4), 170.0 (–C@O in OAc at C-6),
170.6 (–C@O in OAc at C-2). ESI-MS: m/z [M+NH4]+ calcd for
D
rate (1b) (468 mg, 1.0 mmol) was hydrolyzed with Novozym 435 to
give 2b (267 mg, 65%) as a white solid. Rf = 0.42 (CHCl3–MeOH,
9:1); ½a 2D0
ꢂ
ꢃ49.7 (c 1.0, THF); IR (KBr): 3351, 2921, 1721, 1604,
1246, 1067, 1036 cmꢃ1
;
1H NMR (500 MHz, CD3OD): d = 1.92 (s,
3H, CH3 in OAc at C-2), 2.00 (s, 3H, CH3 in OAc at C-3), 3.53 (m,
1H, H-5), 3.66 (m, 2H, H-4, H-6a), 3.83 (d, J = 10.7 Hz, 1H, H-6b),
4.75 (t, J = 5.7 Hz, 1H, OH at C-6), 4.98 (t, J = 9.2 Hz, 1H, H-2), 5.13
(t, J = 9.2 Hz, 1H, H-3), 5.56 (d, J = 5.7 Hz, 1H, OH at C-4), 5.80 (d,
J = 9.2 Hz, 1H, H-1), 6.22 (d, J = 16.5 Hz, 1H, –C(@O)CH@CH–), 6.74
(d, J = 7.6 Hz, 2H, Ph), 7.39 (d, J = 8.9 Hz, 2H, Ph), 7.60 (d,
J = 16.0 Hz, 1H, –C(@O)CH@CH–); 13C NMR (126 MHz, CD3OD):
d = 20.6 (CH3 in OAc at C-2), 20.8 (CH3 in OAc at C-3), 61.8 (C-6),
68.9 (C-4), 72.4 (C-2), 76.7 (C-3), 78.5 (C-5), 93.3 (C-1), 113.6 (–
C(@O)CH@CH–), 116.9 (Ph), 126.8 (Ph), 131.5 (Ph), 148.5 (–
C(@O)CH@CH–), 161.6 (Ph), 166.8 (–C(@O)CH@CH–), 171.3(–C@O
in OAc at C-2), 172.1 (–C@O in OAc at C-3). APCI-MS: m/z
[M+Na]+ calcd for C19H22NaO10: 433.1116; found: 433.1111.
C24H32NO13: 542.1873; found: 542.1868.
2.2.4. 2,3,4,6-Tetra-O-acetyl-b- -glucopyranosyl sinapinate (1d)
D
This compound was synthesized according to procedure de-
scribed for 1a. Instead of trans-cinnamic acid, sinapinic acid
(448 mg, 2.0 mmol) was glycosylated with TAGB (411 mg, 1.0 mmol)
to give 1d (405 mg, 73%) as a orange solid. Rf = 0.24 (hexane–EtOAc,
2.3.3. 2,3-Di-O-acetyl-b-
This compound was synthesized according to procedure de-
scribed for 2a. 2,3,4,6-di-O-acetyl-b- -glucopyranosyl ferulate (1c)
D-glucopyranosyl ferulate (2c)
1:1); ½a 2D0
ꢂ
ꢃ6.8 (c 1.0, THF); IR (KBr): 3471, 2943, 1751, 1545, 1223,
D
1070 cmꢃ1; 1H NMR (500 MHz, CDCl3): d = 2.07 (m, 12H, CH3 ꢁ 4 in
OAc), 3.91 (ddd, J = 9.2, 4.6, 2.3 Hz, 1H, H-5), 3.93 (s, 6H, OCH3 at
Ph), 4.14 (dd, J = 12.6, 2.3 Hz, 1H, H-6a), 4.32 (dd, J =12.6, 4.6 Hz,
1H, H-6b), 5.18 (t, J = 9.2 Hz, 1H, H-4), 5.22 (t, J = 9.2 Hz, 1H, H-2),
5.28 (t, J = 9.2 Hz, 1H, H-3), 5.82 (s, 1H, OH), 5.87 (d, J = 9.2 Hz, 1H,
H-1), 6.28 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 6.79 (s, 2H, Ph),
7.67 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–); 13C NMR (126 MHz,
CDCl3): d = 20.5 (CH3 ꢁ 2 in OAc), 20.6 (CH3 in OAc), 56.3 (CH3 in
OCH3 at Ph), 61.5 (C-6), 67.8 (C-4), 70.2 (C-2), 72.6 (C-5), 72.7 (C-3),
91.7 (C-1), 105.4 (Ph), 113.7 (–C(@O)CH@CH–), 125.3 (Ph), 137.8
(Ph), 147.2 (Ph), 147.7 (–C(@O)CH@CH–), 164.8 (–C(@O)CH@CH–),
169.4 (2-C@O in OAc at C-3 and C-4), 170.0 (–C@O in OAc at C-6),
170.6 (–C@O in OAc at C-2). ESI-MS: m/z [M+NH4]+ calcd for
(361 mg, 1.0 mmol) was hydrolyzed with Novozym 435 to give 2c
(361 mg, 82%) as a white solid. Rf = 0.32 (CHCl3–MeOH, 9:1); ½a D20
ꢂ
ꢃ44.8 (c 1.0, THF); IR (KBr) 3464, 3370, 1740, 1248, 1078 cmꢃ1
;
1H NMR (500 MHz, DMSO-d6): d = 1.95 (s, 3H, CH3 in OAc at C-2),
2.01 (s, 3H, CH3 in OAc at C-3), 3.34 (m, 1H, H-5), 3.55 (m, 3H, H-
4, H-5, H-6a), 3.68 (dd, J = 11.2, 3.7 Hz, 1H, H-6b), 3.82 (s, 3H, CH3
in OMe at Ph), 4.66 (s, 1H, OH at C-6), 4.86 (t, J = 9.2 Hz, 1H, H-2),
5.12 (t, J = 9.2 Hz, 1H, H-3), 5.57 (s, 1H, OH at C-4), 5.90 (d,
J = 9.2 Hz, 1H, H-1), 6.46 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 6.80
(d, J = 8.0 Hz, 1H, Ph), 7.14 (dd, J = 8.3, 2.0 Hz, 1H, Ph), 7.35 (s, 1H,
Ph), 7.60 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 9.74 (br, 1H, PhOH);
13C NMR (126 MHz, DMSO-d6): d = 20.4 (CH3 in OAc at C-2), 20.7
(CH3 in OAc at C-3), 55.7 (CH3 in OCH3 at Ph), 60.0 (C-6), 67.9 (C-
5), 70.9 (C-2), 74.9 (C-3), 77.3 (C-4), 91.3 (C-1), 111.3 (Ph), 112.8
(–C(@O)CH@CH–), 115.5 (Ph), 123.9 (Ph), 125.2 (Ph), 147.3 (–
C(@O)CH@CH–), 147.9 (Ph), 150.0 (Ph), 164.8 (–C(@O)CH@CH–),
169.3 (–C@O in OAc at C-2), 169.7 (–C@O in OAc at C-3). ESI-MS:
m/z [M+NH4]+ calcd for C20H28NO11: 458.1662; found: 458.1668.
C25H34NO14: 572.1981; found: 572.1979.
2.3. Regioselective deacetylation with Novozym 435
2.3.1. 2,3-Di-O-acetyl-b-
D
-glucopyranosyl cinnamate (2a)
-glucopyranosyl cinna-
A solution of 2,3,4,6-tetra-O-acetyl-b-
D
mate (1a) (478 mg, 1.0 mmol), MeOH (256 mg, 8.0 mmol) and Nov-
ozym 435 (0.8 g) in tert-butyl methyl ether (10 mL) was stirred for
24 h at 50 °C. The progress of the reaction was monitored with thin
layer chromatography. Upon completion of the reaction, Novozym
435 was filtered off the reaction mixture, and the solvent was then
removed under vacuum. The residue was purified with flash chro-
matography (CHCl3–MeOH, 20:1) to give 2a (386 mg, 98%) as a
2.3.4. 2,3-Di-O-acetyl-b-
This compound was synthesized according to procedure de-
scribed for 2a. 2,3,4,6-di-O-acetyl-b- -glucopyranosyl sinapate
D-glucopyranosyl sinapinate (2d)
D
(1d) (528 mg, 1.0 mmol) was hydrolyzed with Novozym 435 to give
3d (423 mg, 90%) as a white solid. Rf = 0.43 (CHCl3–MeOH, 9:1);
½
a 2D0
ꢂ
ꢃ39.6 (c 1.0, THF); IR (KBr): 3446, 2997, 1740, 1412, 1135,
942 cmꢃ1 1H NMR (500 MHz, DMSO-d6): d = 1.95 (s, 3H, CH3 in
;
white solid. Rf = 0.42 (CHCl3–MeOH, 9:1); ½a D20
ꢂ
ꢃ49.4 (c 1.0, THF);
OAc at C-2), 2.00 (s, 3H, CH3 in OAc at C-3), 3.54 (m, 3H, H-4, H-5,
H-6a) 3.68(m, 1H, H-6b), 3.80 (s, 6H, CH3 ꢁ 2 in OMe at Ph), 4.75
IR (KBr): 3446, 2935, 1751, 1635, 1244, 1074, 1034 cmꢃ1
;
1H

14
Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
(t, J = 5.7 Hz, 1H, OH at C-6), 4.86 (t, J = 9.2 Hz, 1H, H-2), 5.11 (t,
J = 9.2 Hz, 1H, H-3), 5.56 (d, J = 5.7 Hz, 1H, OH at C-4), 5.91 (d,
J = 9.2 Hz, 1H, H-1), 6.52 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 7.06
(s, 2H, Ph), 7.61 (d, J = 16.0 Hz, 1H, –C(@O)CH@CH–), 9.08 (br, 1H,
PhOH); 13C NMR (126 MHz, DMSO-d6): d = 20.2(CH3 in OAc at C-
2), 20.6 (CH3 in OAc at C-3), 56.1 (CH3 in OCH3 at Ph), 60.0 (C-6),
67.1 (C-4), 70.9 (C-2), 75.0 (C-3), 77.3 (C-5), 96.1 (C-1), 106.7 (Ph),
113.2 (–C(@O)CH@CH–), 124.0 (Ph), 138.9 (Ph), 147.4 (–
C(@O)CH@CH–), 148.0 (Ph), 164.7(–C(@O)CH@CH–), 169.0 (–C@O
in OAc at C-2), 169.6(–C@O in OAc at C-3). ESI-MS: m/z [M+NH4]+
calcd for C21H30NO12: 488.1777; found: 488.1768.
acetoxyl group at C-1 was also unexpectedly undergoing hydroly-
sis. Therefore, we had focused attention on lipase that has the
capability for regioselective deacetylation. Among lipase, Novozym
435 has wide substrate specificity, and well established properties
in stereo- and regioselective activities in hydrolysis of ester groups.
Therefore, it could hydrolyze acetoxyl groups at C-2, C-3, C-4, and
C-6 of the tetra-O-acetyl glycosyl esters without hydrolyzing the
ester group at C-1. In hydrolysis of esters with Novozym 435, ethyl
ether is in general used as solvent. However, in hydrolysis of the
tetra-O-acetyl glycosyl esters with Novozym 435, t-BuOMe was
used as solvent. In addition, the optimal reaction temperature is
50 °C where Novozym 435 has the maximum activity. When the
tetra-O-acetyl glycosyl esters 1b and 1c were used as substrates,
they were not completely soluble in t-BuOMe, as a consequence
a mixture of t-BuOMe/benzene (3:1) was used in hydrolysis of
the substrates. As a result, the 24 h reaction proceeded normally
for all substrates. However, all of the products were glycosyl esters
2, in which only acetoxyl groups at position C-4 and C-6 were
hydrolyzed (Table 2). In addition, the reaction time was extended
to 24 h, and the reaction did not proceed any more to yield the
products. From these results, it has been identified that the Nov-
ozym 435 has selectively hydrolyzed acetoxyl groups at position
C-4 and C-6 in glycosyl esters 1. Although glycosyl cinnamate 1a
was consumed completely during the enzymatic hydrolysis, sub-
strates 1b and 1d were detected in the reaction mixture. Thus,
the regioselectivity of Novozym 435 for substrate specificity, the
glycosyl ester 1a is the highest (Table 2, entry 1), and the enzyme
showed least affinity to 1b (Table 2, entry 2). The active site of Can-
dida antarctica lipase B (Novozym 435) was reported.30 Active site
of lipase, catalytic triad, is generally formed by three amino acids.
These are serine, which is the active center, histidine and aspartate.
The active site of Candida antarctica lipase B is composed by
Ser105, His187 and Asp224. The reaction mechanism of lipase-cat-
alyzed hydrolysis of esters was reported.31 The reaction of hydro-
lysis starts with the formation of tetrahedral intermediate with
alcohol, where ester as a substrate is covalently linked to side-
chain oxygen of catalytic serine. The crucial hydrogen bond from
H of catalytic histidine to serine O and the oxygen of alcohol moi-
ety are formed. Transfer of H to the oxygen of the alcohol moiety
splits away the alcohol. It assumed that the hydrolyzed glycosyl es-
ters (2a–d) are produced by this mechanism. Steric hindrance of
the acetoxyl group at C-6 was smaller than those of other acetoxyl
groups and feruloyloxyl group because of the primary alcohol at
glucose C-6 position. Steric hindrance of the acetoxyl group at C-
4 was also smaller compared with those at C-2 and C-3 for the
methylene group at C-6. For this reason, Novozym 435 preferen-
tially incorporated the acetoxyl group at C-4 and C-6 into its active
site, and hydrolyzed these acetoxyl groups into glycosyl esters 2.
Naoshima et al. reported that the C–O distance between the oxy-
gen of serine and the carbonyl carbon of substrate ester affected li-
pase selectivity.32 Lipase preferentially hydrolyzed substrate esters
which had short C–O distance. It seemed that the small steric hin-
drance had come easily close to the oxygen of serine at active site,
and the acetoxyl groups at C-4 and C-6 were hydrolyzed. The pro-
gress of Novozym 435-catalyzed hydrolysis was monitored by TLC,
2.4. Evaluation of scavenging effect on DPPHÅ radicals
The antioxidant potential of glycosyl ferulates was estimated by
measuring their free radical scavenging activity using 2,2-diphe-
nyl-1-picrylhydrazyl (DPPHÅ) as free radical according to modifica-
tion of the method of Silva et al.29 The reaction mixture (10 mL)
comprised of freshly made 0.15 mM DPPHÅ in ethanol (7000
l
L),
different concentrations of each glycosyl ferulates (1, 5, and
10 mol) in 300 L DMSO, and Tris–HCl buffer (pH 7.4, 100 mM).
l
l
The reaction mixture was kept for 30 min in a water bath at
25 °C under dark and optical density was measured at 517 nm.
DPPHÅ has an unpaired electron, which gives purple color, and
when this electron is balanced, the color is lost. The compounds
which can give an electron to the DPPHÅ can bleach the color.
The scavenging activity of tested compound was measured as the
decrease in absorbance of the DPPHÅ solution without test com-
pounds. All analyses were carried out in triplicate.
2.5. Evaluation of the inhibitory effect on autoxidation of
methyl linoleate
To 1 g of methyl linoleate, 25
compound was mixed in a 50 mL vial, and the mixture was agitated
under ultrasonic wave for 30 s. A 25 L aliquot of acetone without
lL of the acetone solution of the test
l
sample was added for control. After purging the acetone with nitro-
gen, the mixture was placed in an oven at 40 °C in dark. Final concen-
tration of each sample was 0.05 lmol/g oil. An aliquot of oil sample
was measured at 234 nm with a UVmini-2400 UV–Vis spectropho-
tometer every 24 h at 20 °C. All tests were run in triplicate.
3. Results and discussion
3.1. Glucosylation of ferulic acids
Glucosylated carboxyl acids derived from cinnamic acid, such as
trans-cinnamic acid, p-coumaric acid, ferulic acid and sinapinic
acid, were achieved from 2,3,4,6-tetra-O-acetyl-
syl bromide (TAGB) via SN2 reaction of the bromide leaving group
to the corresponding 2,3,4,6-tetra-O-acetyl- -glycopyranosyl
a-D-glucopyrano-
a-D
carboxylate with high yield (more that 70%, except for 1b), respec-
tively. i-Pr2NEt and Et3N were used as a Brønsted base to deproto-
nate the carboxylic acid making it more nucleophilic. In the case of
1b using i-Pr2NEt as a base, the yield is lower than that of other
glycosyl esters (see Table 1, entry 2). Precipitate was observed only
in the preparation of 1b. It seemed that carboxylic acid and i-
Pr2NEt gave the salt and this lead to the low yield. Therefore,
Et3N was used instead of i-Pr2NEt, and the salt was not observed.
The yield of 1b increased to as high as 81%.
Table 1
Amine-promoted glycosylation of carboxylic acid with amine
Entry
Amine
Product
Yield (%)
1
2
3
4
5
i-Pr2NEt
i-Pr2NEt
Et3N
i-Pr2NEt
i-Pr2NEt
1a
1b
72
55
81
74
73
3.2. Regioselective deacetylation with Novozym 435
1c
1d
In order to deacetylate acetoxyl groups at C-2, C-3, C-4, and C-6,
inorganic bases were first used in hydrolysis. However, the

Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
15
Table 2
potency of sinapinic acid and its glycosyl esters compared to other
ferulic acids. An increase in concentration of antioxidant in the
presence of ferulic acid led to no change in its DPPHÅ radical scav-
enging activity, while the activity of glycosyl ferulates (1b and 2b)
increased with concentration. A possible explanation for the con-
centration dependence might be a slight difference in the solubility
of the test solvent. Glycosyl ferulates possessed acetoxyl and/or
hydroxyl groups, and these high polar groups raise the solubility
in the tested medium. It seemed that the lower polarity of free
ferulic acid within ferulic acid derivatives might result in higher
effectiveness on DPPHÅ radical scavenging effect in good agreement
with ‘polar paradox’, according to which polar antioxidants are
more efficient in an apolar medium, and conversely, apolar antiox-
idants are more efficient in a polar medium.41 Free p-coumaric acid
and its glycosyl esters (1b and 2b) showed lower scavenging activ-
ity compared with ferulic and sinapinic acid derivatives. The scav-
enging ability of hydroxycinnamic acids against DPPHÅ radical was
dependent on the number of hydroxyl groups on the benzene ring
and ortho substitution with the electron donor methoxy group
which increases the stability of the phenoxy radical.39,42 Therefore,
the ability of ferulic and sinapinic acid derivatives was higher than
that of p-coumaric acid derivatives. In p-coumaric acid derivarives,
glycosyl p-coumarates (1b and 2b) exhibited lower ability com-
pared with free p-coumaric acid, and there was no great difference
among glycosyl p-coumarates. Pekkarinen et al. explained the low
antioxidant activity of 2,3-dihydroxybenzoic acid.40 2,3-Dihy-
droxybenzoic acid forms an intramolecular hydrogen bond be-
tween carbonyl oxygen and hydroxyl group hydrogen atom,
which would explain a low activity in DPPHÅ radical scavenging
activity. From this fact, it assumed that the formation of intramo-
lecular hydrogen bonding between carbonyl oxygen at acetoxyl
group and 4-hydroxy group decreased the activity of glycosyl
p-coumarates. Additionally, DPPHÅ radical scavenging activity of
Novozym 435-catalyzed deacetylation
Entry
Solvent
Product
Yield (%)
1
2
3
4
t-BuOMe
t-BuOMea
t-BuOMea
t-BuOMe
2a
2b
2c
2d
98
65
82
90
a
t-BuOMe:PhH = 3:1
and only hydrolyzed glycosyl esters 2 except unreacted 1 were ob-
served. This showed that Novozym 435 specifically hydrolyzed the
acetoxyl groups at C-4 and C-6. Kazlauskas et al. suggested the
relationship between the structure of active site and substrate to
enantioselectivity.33 An empirical rule, which is based only on
the presence of steric factors in substrate molecules, was proposed
to predict the enantioselectivity toward secondary alcohols and
their esters displayed by lipase. Recently, the relationship between
structure of active site and substrate and enantioselectivity of li-
pase is reported.34–37 Lemke et al. investigated particularly about
the enantioselectivity of Pseudomonas cepacia lipase using 69 kinds
of substrate.38 The kinetic resolution of 3-(aryloxy)propan-2-ol
derivatives by transesterification with vinyl acetate in organic sol-
vents in the presence of Pseudomonas cepacia lipase was measured.
This lipase showed high E value (>100) to the substrates which
have acyloxy groups such as n-pentanoate, n-nonanoate, and n-
pentadecanoate. In contrast, it expressed low E value, about 30,
compared to a similar substrate that possessed n-hexanoate, n-
octanoate, and n-heptadecanoate. The slight difference of chain
length has a great effect on the enantioselectivity of lipase. Fur-
thermore, Lemke et al. concluded that the enantioselectivity of li-
pase was determined not by volume of active site but by shape.
In this paper, Novozym 435 exhibited not enantioselectivity but
regioselectivity for hydrolysis of acetylated glycosyl esters. How-
ever, it seemed that in this case in which lipase recognized not
the volume but the shape of substrate applied to Novozym 435-
catalyzed regioselective hydrolysis. Although all acetoxyl groups
at C-2, C-3, C-4, and C-6 were of the same structure and shape, each
steric hindrance around every acetoxyl group was different. It as-
sumed that Novozym 435 recognized the shape of substrate, and
specifically hydrolyzed only acetoxyl groups at C-4 and C-6.
a
-tocopherol decreased with the increasing concentration. It
seemed that the solubility of -tocopherol for the tested medium
had huge effect on the radical scavenging activity. The
0.1 mol/mL solution of -tocopherol showed up clear and color-
less. In contrast, the 0.5 and 1.0 mol/mL solution was slightly sus-
pended. This caused the low radical scavenging activity of
a
a
l
a
l
0.1
1.0
l
l
mol/mL
mol/mL.
a-tocopherol solution compared with 0.5 and
3.4. Inhibitory effect on autoxidation of methyl linoleate
3.3. Radical scavenging of ferulic acids and its glycosyl esters
Figure 2 shows the inhibitory effect on autoxidation of bulk
methyl linoleate using ferulic acids and glycosyl ferulate deriva-
tives. Tested ferulic acid derivatives except p-coumaric acid and
its glycosyl esters (1b and 2b) showed higher antioxidant activity
The present study demonstrates the antioxidant properties of
ferulic acids and glycosyl ferulate derivatives synthesized by eval-
uation of the DPPHÅ radical scavenging activity and the inhibitory
effect on autoxidation of methyl linoleate in bulk phase. Figure 1
shows the DPPHÅ radical scavenging activity of ferulic acids and
the corresponding glycosyl ferulates. Scavenging ability of all
tested compounds increased with concentration in the range of
compared with a-tocopherol. The antioxidation activity decreased
in the order sinapinic acid > glycosyl sinapinates (1d > 2d) > ferulic
acid ꢀ glycosyl ferulates (1c ꢀ 2c) > p-coumaric acid ꢀ glycosyl p-
coumarates (1b ꢀ 2b). The antioxidant activity order against
autoxidation of bulk methyl linoleate at 40 °C was largely consis-
tent with the scavenging activity order, suggesting that their anti-
oxidant efficiencies result in their electron-donating ability. The
results were in accordance with those reported by Pekkarinen
et al. who measured the inhibition effects of some phenolic acids,
including hydroxycinnamic acids, on the formation of hydroperox-
ides in bulk methyl linoleate at 40 °C.40 Cuvelier et al. evaluated
the antioxidant activity of some phenolic acids based on measuring
the disappearance of methyl linoleate in dodecane under heating
(110 °C). They reported that the activity order was caffeic
acid > sinapinic acid > ferulic acid > p-coumaric acid, and the activ-
ity depended on their stability of aryloxy radicals.43 Marinova and
Yanishlieva also obtained the same activity order by determination
of hydroperoxides, the primary oxidative products at 100 °C.44
0.1–1.0 lmol/mL. Ferulic acid had the best radical scavenging
activity compared with other tested compounds, with the ability
about 90% in all concentrations. The activity decreased in the order
ferulic acid > sinapinic acid ꢀ glycosyl sinapinates (1d ꢀ 2d) > gly-
cosyl ferulates (1c < 2c) > p-coumaric acid > glycosyl p-coumarates
(1b < 2b). The activity of sinapinic acid was somewhat lower than
that of ferulic acid. These data are in disagreement with results re-
ported by Pekkarinen et al. and Kylli et al., who measured the
DPPHÅ radical scavenging of ferulic acids.39,40 These discrepancies
may be due to experimental differences, such as antioxidant con-
centration and a composition of medium. Tested glycosyl ferurates
except glycosyl sinapinates (1d and 2d) showed lower activity
compared with free ferulic acids. With sinapinic acids no great dif-
ference was seen. This may be explained by the great antioxidant

16
Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
0.1 µmol/mL
0.5 µmol/mL
1.0 µmol/mL
Figure 1. Radical scavenging activity (DPPH test) of free ferulic acids and its glycosyl esters.
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Control
α-Tocopherol
Quercetin
p-coumaric acid
Glycosyl p-coumarate 1b
Glycosyl p-coumarate 2b
Ferulic acid
Glycosyl ferulate 1c
Glycosyl ferulate 2c
Sinapinic acid
Glycosyl sinapinate 1d
Glycosyl sinapinate 2d
0
5
10
15
20
25
30
Oxidation time [days]
Figure 2. Inhibition of the formation of hydroperoxides in bulk methyl linoleate by ferulic acids and its glycosyl esters.
From these results, there was no temperature dependent substance
in the antioxidant activity of free ferulic acids against autoxidation
of bulk methyl linoleate. The antioxidant activity between free
ferulic acid and glycosyl ferulates showed a little difference. The
esterification to glycosyl esters did not decrease the antioxidant
activity of ferulic acid. The activity between free sinapinic acid
and its glycosyl esters (1d and 2d) was different. The antioxidant
activity of free sinapinic acid and quercetin showed to be almost
same. Free sinapinic acid exhibited higher antioxidant activity
compared with glycosyl sinapinates (1d and 2d). In glycosyl sinap-
inates (1d and 2d), the antioxidant activity of 1d was higher than
that of 2d. Kylli et al. evaluated the antioxidant activity of free sina-
pinic acid and its glycosyl ester and reported the difference.39 It
was reported that the affinities of antioxidants toward the air–oil
interfaces in bulk oil affect antioxidant activity. These are in dis-
agreement with our results. The activity strongly depended on
the applied medium and reflected polar or apolar nature of the
compound. It was stated, that polar antioxidant acts more effec-
tively in apolar medium and apolar antioxidant in medium with
polar solvent. Compound 1d possessed four acetoxyl groups, and
2d possessed two acetoxyl and two hydroxyl groups. Therefore,
the molecular polarity of glycosyl sinapinates (1d and 2d) was dif-
ferent from free sinapinic acid. It seemed that this caused the dif-
ference of affinity toward the air–oil interfaces in bulk oil and
affected the inhibitory effect on autoxidation of methyl linoleate.
In the case of p-coumaric acid and its glycosyl esters, glycosyl p-
coumarates (1b and 2b) showed stronger activity than free p-cou-
maric acid at 40 °C. Glycosyl p-coumarates (1b and 2b) possessed
hydroxyl group and/or acetoxyl group, and these groups increased
molecular polarity. These results were in good agreement with the
‘polar paradox’.
4. Conclusion
The synthesis of 2,3-di-O-acetyl-b-
carboxylic acids from 2,3,4,6-tetra-O-acetyl-
bromide has been achieved via amine-promoted glycosylation pro-
cedure in which the -glucose derivatives were converted to the
D-glucopyranosyl esters with
a-D-glucopyranosyl
a-D
corresponding glycosyl esters using amine without using the heavy
metal compounds as catalysts. The resulting 2,3,4,6-tetra-O-acetyl-
b-
D-glucopyranosyl esters were regioselectively deacetylated with
Novozym 435. In the glycosyl esters 1a, 1b, 1c, and 1d, acetoxyl

Y. Shimotori et al. / Carbohydrate Research 359 (2012) 11–17
17
9. Xie, W.; Tang, J.; Gu, X.; Luo, C.; Wang, G. J. Anal. Appl. Pyrolysis. 2007, 78, 180–
184.
groups at C-4 and C-6 were regioselectively deacetylated. The reg-
ioselective deacetylation of only acetoxyl groups at C-4 and C-6 in
these compounds with Novozym 435 is primarily interesting. Thus,
the syntheses of 2,3-O-acetylglycosyl esters of the corresponding
ferulic acids were accomplished without using the heavy metal
catalysts, which are not only cost-effective but also an environ-
mentally friendly method. The antioxidant properties of ferulic
acids and its glycosyl esters were investigated. The antioxidant
activities of ferulic acids and its glycosyl esters (1c and 2c), as well
as those of the other tested ferulic acids, were almost in agreement
with their radical scavenging activities when measured using a
bulk oil at 40 °C. Sinapinic acid and its glycosyl esters (1d and
2d) had the most effective antioxidant activity among tested feru-
lic acids against autoxidation of methyl linoleate. The esterification
to glycosyl esters and subsequent regioselective deacetylation with
lipase did not decrease the antioxidant activity of ferulic acid in
oxidation of methyl linoleate and sinapinic acid in scavenging ef-
fect on DPPHÅ radicals.
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Acknowledgements
We thank Masaaki Kenmotsu, Mitsuru Kaminaga and Masashi
Shoji for supporting this work. We are also grateful to the Novozymes
A/S for the generous gift of Novozym 435. Our works were partly sup-
ported by the Academic Frontier Project for Private Universities:
matching fund subsidy for MEXT (2007–2011), and the Research Pro-
ject Grant B by the Institute of Science and Technology, Meiji Univer-
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