Synthesis of styrenes through the biocatalytic decarboxylation of trans-cinnamic acids by plant cell cultures

Article abstract of DOI:10.1248/cpb.49.639

A novel method for producing styrenes from trans-cinnamic acids was developed. When trans-cinnamic acid was incubated with plant cell cultures at room temperature, styrene was obtained. 4-Hydroxy-3-methoxystyrene (2a), 3-nitrostyrene (2f) and furan (2g) were synthesized quantitatively.

Full text of DOI:10.1248/cpb.49.639

May 2001
Notes
Chem. Pharm. Bull. 49(5) 639—641 (2001)
639
Synthesis of Styrenes through the Biocatalytic Decarboxylation of
trans-Cinnamic Acids by Plant Cell Cultures
*
Masumi TAKEMOTO and Kazuo ACHIWA
School of Pharmaceutical Sciences, University of Shizuoka, 52–1 Yada, Shizuoka 422–8526, Japan.
Received November 24, 2000; accepted January 15, 2001
A novel method for producing styrenes from trans-cinnamic acids was developed. When trans-cinnamic acid
was incubated with plant cell cultures at room temperature, styrene was obtained. 4-Hydroxy-3-methoxystyrene
(2a), 3-nitrostyrene (2f) and furan (2g) were synthesized quantitatively.
Key words decarboxylation; styrene; trans-cinnamic acid; plant cell culture; enzyme
The chemical methodologies for obtaining styrenes are
Very recently, we developed a novel method for the decar-
mainly i) the dehydrogenation of ethylbenzene, ii) the chlori- boxylation of trans-cinnamic acids (1a—i) by plant cell cul-
nation of ethylbenzene and subsequent removal of hydrogen tures to the corresponding styrenes or furan (2a—i), as
halide, and iii) the decarboxylation of trans-cinnamic acids. shown in Fig. 1.20) In this paper, we would like to report our
Of these methodologies, the decarboxylation of trans-cin- use of plant cell cultures in detail.
namic acids is the most widely used chemical method for
In this work, we used suspension-cultured cells which had
preparing styrenes or stilbenes. Typical decarboxylation is originally been isolated from Nicotiana (N.) tabacum “Bright
carried out by heating under reflux at 200—300 °C for 4— Yellow-2”, Daucus (D.) carota, Camellia (C.) sinensis and
5 h in quinoline in the presence of a Cu powder (Y. Ͼ50%). Catharanthus (C.) roseus. These cell cultures (N. tabacum,
Quinoline is useful as a solvent for the decarboxylation of D. carota, C. roseus) were prepared as described in our pre-
unsaturated acids because it is basic enough to form the re- vious papers.10,11,15) C. sinensis callus tissues were obtained
quired carboxylate anion and also because it boils at a tem- from the shoot tips after a 4- to 6-week induction
perature favorable for decarboxylation. This method, how- period when Murashige and Skoog’s (MS) medium21) was
ever, needs a high temperature.
used. The callus was inoculated into liquid Gamborg’s
The biocatalytic decarboxylation of trans-cinnamic acid is B5 medium22) containing 1.25 mg/l of 2,4-dichlorophenoxy
the most promising method, because it takes advantage of the acetic acid (2,4-D) as an auxin, 1 mg/l of benzyladenine as a
mild reaction conditions for preparing styrenes. The known cytokinin and 5% sucrose. Subculturing was performed
decarboxylative enzymes are mainly as follows: i) pyruvate every 15 d by transferring 10 ml of 2-week-old culture into
decarboxylase,1) ii) oxalate decarboxylase,2) iii) glutamate 80 ml of fresh Gamborg’s B5 medium. Incubation was done
decarboxylase,3) iv) benzoylformate decarboxylase,4) v) on a rotary shaker (110 rpm) at 25 °C in the dark.
aconitate decarboxylase,5) and vi) aspartate 4-decarboxy-
A biodecarboxylative reaction was performed by the fol-
lowing four methods to optimize conditions: A) with freely
lase.6)
In the case of trans-cinnamic acids, b-phenylacrylic acid suspended plant cells in the stationary phase after 10 d of in-
was decarboxylated by Aspergillus niger to give styrene.7) cubation (10 g of cells in 20 ml of a medium); B) with ho-
Aerobacter has been found to decarboxylate trans-4-hydroxy- mogenized plant cell culture (10 g) in 0.1 M phosphate buffer
cinnamic acid to the corresponding 4-hydroxystyrene.8) solution (pH 6.0, 20 ml); C) with homogenized plant cell cul-
However, only a few attempts to decarboxylate other trans- ture (10 g) in 0.1 M phosphate buffer solution (pH 6.4, 20 ml);
cinnamic acids by a decarboxylase have been reported.
and D) with homogenized plant cell culture (10g) in 0.1 M
In recent years, much attention has been paid to the ability phosphate buffer solution (pH 7.0, 20 ml). In the case of
of cultured plant cells to enantioselectively transform not method A, a substrate (50 mg) was added to the freely sus-
only secondary metabolites, but also organic foreign sub- pended C. roseus (B-5 medium, pH 5.5), N. tabacum “Bright
strates.9—14) The biotransformation of the exogenous sub- Yellow-2” (MS medium, pH 5.8), D. carota (MS medium,
strates by plant cell cultures can be summarized according to pH 5.8), and C. sinensis (B-5 medium, pH 5.8). The mixture
classes of chemical reactions as follows: 1) hydroxylation, 2) was shaken at 25 °C in a rotary shaker (110 rpm) in the dark.
oxido-reduction between alcohols and ketones, 3) reduction In the case of methods B, C and D, 10 g of plant cells were
of the carbon–carbon double bond, 4) glycosyl conjugation, homogenized in 20 ml 0.1 M phosphate buffer {B (pH 6.0), C
5) hydrolysis, and 6) miscellaneous reactions.
(pH 6.4), D (pH 7.0)}. A substrate (50 mg) was added to the
We have reported synthetic methods by plant cell cul- homogenate. The subsequent procedure was the same as for
tures as follows: 1) the asymmetric reduction of benzoyl
pyridines,10,11,15) 2) asymmetric hydrolysis of (a-acetoxyben-
zyl)pyridines,10,11,15) 3) deracemization of racemic alcohols,
i.e., 100% conversion of racemic alcohols to the correspond-
ing chiral alcohols,16—18) 4) dediastereomerization of diben-
zylbutanolides, i.e. reactions allowing the transformation of
two diastereomers into one diastereomer in quantitative
yield.19)
Fig. 1
To whom correspondence should be addressed. e-mail: takemoto@ys2.u-shizuoka-ken.ac.jp
© 2001 Pharmaceutical Society of Japan

640
Vol. 49, No. 5
Table 2. Decarboxylation of Cinnamic Acid Derivatives (1a—i) with
Table 1. Decarboxylation of trans-Ferulic Acid (1a) with Plant Cell Cul-
Plant Cell Cultures
tures
Plant cell
cultures
Product 2
C.Y. (%)
Recovery 1
C.Y. (%)
Entry
Substrate
Plant cell
culture
Time
(d)
Product 2a Recovery 1a
Entry
Method
C.Y. (%)
C.Y. (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
1a
C. roseus
N. tabacum
D. carota
C. roseus
N. tabacum
D. carota
C. roseus
N. tabacum
D. carota
C. sinensis
D. carota
D. carota
C. sinensis
C. sinensis
C. sinensis
D. carota
C. sinensis
D. carota
C. sinensis
Quant.
Quant.
30
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
C. roseus
C. roseus
C. roseus
C. roseus
N. tabacum
N. tabacum
N. tabacum
N. tabacum
D. carota
D. carota
D. carota
D. carota
C. sinensis
C. sinensis
C. sinensis
C. sinensis
A
B
C
D
A
B
C
D
A
B
C
D
A
B
C
D
5
3
3
3
5
5
5
5
5
5
5
5
5
5
5
5
Quant.
Quant.
0
0
65
80
86
85
63
84
55
83
86
85
88
0
1b
1c
10
Quant.
0
Trace
Trace
30
5
32
Trace
Trace
Trace
Trace
Quant.
Quant.
Trace
10
65
Trace
Quant.
20
84
0
Quant.
0
50
0
25
30
0
0
0
0
0
25
100
65
65
100
100
100
100
100
1d
1e
1f
1g
1h
0
87
76
78
82
1i
10
Trace
method A.
When trans-ferulic acid (1a) was subjected to plant cell
culture in a medium, 4-hydroxy-3-methoxystyrene (2a) was
given quantitatively as shown in Table 1. In the case of C.
roseus, 1a was quantitatively decarboxylated to 2a not only
with method A, but also with B and C (entries 1, 2, 3). But,
in the cases of N. tabacum, the decarboxylation proceeded
quantitatively with only method B and C (entries 6, 7). In the
case of D. carota, the decarboxylation proceeded with only
method B and C in 25—30% yield (entries 10, 11). In this
biodecarboxylation, C. sinensis had no ability to convert 1a
to 2a (entries 13—16).
Next, it was reported that E-a-phenylcinnamic acid (1j)
was decarboxylated in the presence of 2CuO·Cr2O3 in quino-
line at 230 °C to afford Z-stilbene in 75% yield.26) Then, we
tried the decarboxylation of 1j with plant cell cultures. But
C. roseus, N. tabacum, D. carota and C. sinencis cell cultures
had no ability to decarboxylate 1j.
Thus, we have developed a novel method for producing
styrenes from trans-cinnamic acids through the biocatalytic
decarboxylation by plant cell cultures. Studies are now in
progress to shorten the reaction time.
In these experiments, we have succeeded in the biodecar-
boxylation of 1a with homogenized plant cell culture in 0.1 M
phosphate buffer solution (pH 6.4) [method C].
Next, we attempted the decarboxylation of other trans-cin-
namic acids [trans-cinnamic acid (1b), 4-hydroxycinnamic
Experimental
Thin layer chromatography (TLC) was performed on silica gel (Kieselgel
60F254 on aluminum sheets, Merck). All compounds were located by spray-
ing the TLC plate with a 10% solution of phosphomolybdic acid in ethanol
and heating it on a hot plate. Preparative TLC was performed on preparative
layer chromatography plates (Kieselgel 60F254 2 and 0.5 mm, Merck). Col-
acid (1c), 3-hydroxycinnamic acid (1d), 2-hydroxycinnamic umn chromatography was performed on silica gel (Kieselgel 60, 70—230
mesh, Merck).
acid (1e), 3-nitrocinnamic acid (1f), 2-furancarboxylic acid
(1g), 4-methoxycinnamic acid (1h), and 4-chlorocinnamic
acid (1i)] using method [C], as shown in Table 2. The decar-
Cultivation of N. tabacum “Bright Yellow-2” Cells N. tabacum
“Bright Yellow-2” was subcultured every 7 d by transferring 1.3 ml of a 1-
week culture into 80 ml of MS medium containing 2,4-D (0.2 mg/l) and 3%
boxylation of 1f and 1g with C. sinensis gave 3-nitrostyrene
(2f) and furan (2g) quantitatively (entries 14, 15). In the case
of 1c, 4-hydroxystyrene (2c) was given in 30—32% yield by
C. roseus or D. carota (entries 7, 9). But, in the case of 1d
and 1e, decarboxylated products, 3-hydroxystyrene (2d) and
2-hydroxystyrene (2e), were obtained in trace quantities by
D. carota. (entries 11, 12). In the case of 1b, 1h and 1i, the
corresponding products, styrene (2b), 4-methoxystyrene (2h)
and 4-chlorostyrene (2i), were given in low chemical yields
(entries 4, 17, 18). These styrenes (2b,23) 2f,24) 2h24) and 2i24)
and furan 2g25) were chemically synthesized by the decar-
boxylation of trans-cinnamic acids (1b, 1f—i) in the pres-
ence of a copper powder in quinoline at 185—195 °C for 2—
4 h (Y. Ͼ50%). A major advantage of our method is that the
decarboxylation with plant cell culture proceeds mildly at
room temperature.
sucrose (pH 5.8) on a rotary shaker (110 rpm) at 25 °C in the dark.
Cultivation of C. roseus Cells Suspension cells of C. roseus were sub-
cultured every 7 d by transferring a 1-week culture (8 ml) into B5 medium
(80 ml) containing 2,4-D (1mg/l) and 2% sucrose (pH 5.5) on a rotary
shaker (110 rpm) at 25°C in the dark.
Cultivation of D. carota Cells Suspension cells of D. carota were sub-
cultured every 7 d by transferring a 1-week culture (8 ml) into MS medium
(80 ml) containing 2,4-D (2 mg/l) and 3% sucrose (pH 5.8) on a rotary
shaker (110 rpm) at 25 °C in the dark.
Preparation of C. sinensis Callus Callus tissues were prepared from
seedlings of C. sinensis. The seedlings were rinsed with EtOH (30 s) and
NaOCl (2% aqueous solution), followed by washing with sterile distilled
H2O (ϫ5), and were inoculated onto MS medium containing 2,4-D (10 mg/l)
as an auxin and 3% sucrose with 0.8% agar at 25 °C. The shoot tips after a
4- to 6-week induction period were transferred into 10 ml of fresh B5
medium (10 ml) containing 2,4-D (1.25 mg/l) and 5% sucrose. Incubation
was done in a rotary shaker (110 rpm) at 25 °C in the dark. The first subcul-
tures took place at 2 to 3 weeks. To this culture, fresh B5 medium (70 ml)
containing 2,4-D (1.25 mg/l) and 5% sucrose was added. Subculturing was

May 2001
641
performed every 14 d by transferring 10 ml of 1-week-old culture into fresh
B5 medium (80 ml) containing 2,4-D (1.25 mg/l) and 5% sucrose.
Biotransformation of Substrates with Method (A) A substrate (1a—
j) (50 mg) was added to the freely suspended C. roseus (B-5 medium pH
5.5), N. tabacum ‘Bright Yellow-2’ (MS medium, pH 5.8), D. carota (MS
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