Establishment of Camellia sinensis cell culture with high peroxidase activity and oxidative coupling reaction of dibenzylbutanolides

Article abstract of DOI:10.1016/S0040-4039(02)01631-3

We succeeded in establishment of Camellia sinensis cell culture with high peroxidase activity. When dibenzylbutanolides (1a-c) were reacted with C. sinensis cell culture, peroxidase-catalyzed oxidative coupling of 1a-c to cyclic products (2a-c) proceeded

Full text of DOI:10.1016/S0040-4039(02)01631-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6915–6917
Establishment of Camellia sinensis cell culture with high
peroxidase activity and oxidative coupling reaction of
dibenzylbutanolides
Masumi Takemoto,a,* Youichi Aoshima,b Nikolay Stoynovc and James Peter Kutneyc
aSchool of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
bShizuoka Agricultural Experiment Station, 678-1 Tomigaoka Toyoda-cho, Iwata-gun, Shizuoka 438-0803, Japan
cDepartment of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada V6T 1Z1
Received 17 July 2002; accepted 2 August 2002
Abstract—We succeeded in establishment of Camellia sinensis cell culture with high peroxidase activity. When dibenzylbutanolides
(1a–c) were reacted with C. sinensis cell culture, peroxidase-catalyzed oxidative coupling of 1a–c to cyclic products (2a–c)
proceeded quantitatively in the absence of foreign hydrogen peroxide as cofactor. © 2002 Elsevier Science Ltd. All rights reserved.
derived from cotyledons of ‘Sayamakaori’ seeds
expressed the highest POD activity per g calli. When
the calli were cultured on MS medium3 with 10 mg/L
2,4-dichlorophenoxyacetic acid (2,4-D), POD activity
was assayed to be raised by 2ꢀ3 times. Next, we
assayed POD activities of C. sinensis, Daucus carota,
Catharanthus roseus and Nicotiana tabacum suspension
cell cultures with purpurogallin assay.4 The POD activ-
ity was determined by combining 5% pyrogallol (2 mL),
freshly-prepared 0.5% hydrogen peroxide (1 mL), water
(14 mL), 0.1 M pH 6.4 phosphate buffer (2 mL) and
enzyme sample (1 mL), recording absorbance at 420 nm
and calculating the number of units present from the
standard curve for purpurogallin. POD activities of
various plant cell cultures are shown in Table 1. The
highest level of POD activity was found in 12-day-old
C. sinensis cell culture (entry 1). The cells of C. sinensis
cell culture excreted POD into the broth during growth
(entry 2).
The development of enzymes for oxidation reaction
aimed at green chemistry is very important.
Horseradish peroxidase (HRP) is a commercially avail-
able metalloporphyrin enzyme and has been established
as an effective biocatalyst for organic and inorganic
oxidation reactions by using hydrogen peroxide or
organic hydroperoxides.1 Kutney et al. reported HRP-
catalyzed carbonꢀcarbon bond formation with phenolic
systems: enzyme-catalyzed ring closured reaction of
dibenzylbutanolides suitable for biotransformation to
lignans as potential intermediates for the synthesis of
etoposide which is now used clinically as an antitumor
agent.2 However, the addition of H2O2 to the reaction
mixture brought about red–brown darkening of the
solvent to decrease chemical yield. From these points of
view, we have investigated plant cell cultures in which
cell wall peroxidases rapidly metabolize a huge amount
of H2O2 produced by the addition of foreign substrates.
Recently, we have found Camellia sinensis cell culture,
an efficient source of peroxidase (POD) enzymes. In
this work, we would like to report establishment of C.
sinensis cell culture with high POD activity and the
oxidative coupling of dibenzylbutanolides (1a–c) by its
culture.
Table 1. The POD activity in the plant cell culture
(9S.D.)
Entry
Plant cell culture
POD U/mL
1
2
3
4
5
6
7
8
C. sinensis
C. sinensis
D. carota
D. carota
N. tabacum
N. tabacum
C. roseus
Cell
Broth
Cell
Broth
Cell
Broth
Cell
15.590.32
12.490.50
8.090.20
1.690.15
9.690.32
7.990.26
0.590.10
0.190.05
We carried out an examination to establish C. sinensis
callus culture providing us high POD activity. The calli
Keywords: oxidation; coupling reaction; catalysts; enzymes and
enzyme reaction.
* Corresponding author. Fax: +81-54-264-5740; e-mail: takemoto@
ys2.u-shizuoka-ken.ac.jp
C. roseus
Broth
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01631-3

6916
M. Takemoto et al. / Tetrahedron Letters 43 (2002) 6915–6917
When the previously synthesized racemic dibenzyl-
butanolides 1a–c5 were subjected to C. sinensis plant
cell culture, racemic cyclic products 2a–c5 were given
quantitatively in the absence of foreign hydrogen per-
oxide as cofactor as shown in Table 2. The oxidative
coupling reaction was performed by two methods, that
is: [A] with freely suspended plant cell culture in the
stationary phase after 12 days of incubation, [B] with
immobilized plant cell culture. In the case of C. sinensis,
1a–c were quantitatively cyclized to the desired (trans
relationship of the lactone ring) ring-closed product
(2a–c) not only with method [A], but also with [B] in
the absence of foreign hydrogen peroxide as cofactor
(entries 2, 3, 10, 14). Next, we attempted the repetitive
use of immobilized C. sinensis cell culture (ICSC). After
three consecutive reuses, the biocatalyst still maintained
the POD activities to afford 2a–c quantitatively (entries
4, 5, 11, 12, 15, 16). To our surprise, we succeeded in
the biotransformation of 1a–c to 2a–c with ICSC in
hexane containing no water (entries 6, 13, 17). In this
case, all of the substrates were incorporated in the
immobilized cell immediately on adding. This method
has the advantage of extracting with a small amount of
an organic solvent and is a very good system for
water-insoluble substrates. Studies are now in progress
to establish enantioselective oxidation with C. sinensis
cell culture.
our previous papers.6–16 ICSC were prepared according
to the following procedure. Freely suspended C. sinensis
(4.6 g cells and 20 mL broth) and 60 mL B5 medium17
in the stationary phase after 8 days of incubation was
mixed with 5% sodium alginate solution (80 mL). The
resultant mixture was dropped into a 0.6% CaCl2 solu-
tion (1000 mL) and rinsed with water to give ICSC.
ICSC (including 7 g cells and 30 mL broth) was added
to freshly prepared B5 medium (80 mL per flask) and
was shaken on a rotary shaker (110 rpm) in the dark at
25°C. A substrate (35 mg) was added to the freely
suspended plant cell cultures (19 g cells and 80 mL
broth), ICSC cells (including 7 g cells and 30 mL broth)
in 80 mL freshly B5 medium or ICSC cells (including 7
g cells and 30 mL broth) in 20 mL hexane. After
regular intervals of incubation, the incubation mixture
was filtered, the filtered cells or immobilized cells were
washed with CH2Cl2, and the filtrates were combined.
The combined mixture was extracted with CH2Cl2. The
organic layer was dried over anhydrous MgSO4. In the
case of reaction in hexane, homogenated cells were
extracted with CH2Cl2. Butanolides 1a and 1b were
synthesized by the synthetic route of Kutney.18,19
Butanolide 1c was synthesized by the same synthetic
route of Kutney.19
Acknowledgements
For a typical experiment, we used suspension-cultured
cells which had originally been isolated from C. sinen-
sis, N. tabacum, D. carota and C. roseus as described in
This work was supported in part by the Sumitomo
Foundation.
Table 2. Biotransformation of dibenzylbutanolide (1a–c) to cyclic product (2a–c) by peroxidase enzymes
R3
R3
R1
R2
R1
R2
a; R1=OH, R2=OCH(CH3)2, R3=OH
b; R1=OH, R2=OMe, R3=H
O
O
O
O
Enzyme
c; R1=OMe, R2=OH, R3=H
H3CO
OCH3
H3CO
OCH3
OH
OH
( )-2a-c
_
+
_
+
( )-1a-c
Entry
Substr.
Enzyme
Method
Time (h)
Solvent
Product 2a–c (%)
Recovery 1a–c (%)
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1c
1c
1c
1c
HRP(H2O2)
C. sinensis
C. sinensis
(Second reuse)
(Third reuse)
C. sinensis
C. roseus
N. tabacum
D. carota
0.5
6
18
15
0
0
0
0
24
23
66
83
0
0
0
26
0
0
A
B
B
B
B
A
A
A
B
B
B
B
B
B
B
B
B5 medium
B5 medium
B5 medium
B5 medium
Hexane
B5 medium
MS medium
MS medium
B5 medium
B5 medium
B5 medium
Hexane
Quant.
Quant.
Quant.
Quant.
52
37
14
17
Quant.
Quant.
Quant.
48
Quant.
Quant.
Quant.
52
24
24
24
24
168
168
168
24
24
24
24
1
9
10
11
12
13
14
15
16
17
C. sinensis
(Second reuse)
(Third reuse)
C. sinensis
C. sinensis
B5 medium
B5 medium
B5 medium
Hexane
(Second reuse)
(Third reuse)
C. sinensis
1
1
1
0
17

M. Takemoto et al. / Tetrahedron Letters 43 (2002) 6915–6917
6917
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