Oxidative cleavage of the C-C bond of 3,6-dialkylcyclohexane-1,2-diones by cell suspension cultures of Marchantia polymorpha

Article abstract of DOI:10.1016/S0031-9422(02)00373-4

Biotransformation of 3,6-dialkylcyclohexane-1,2-diones by cell suspension cultures of Marchantia polymorpha involves regio-selective oxidative cleavage of the C-C bond to give the corresponding oxocarboxylic acids shortened by one carbon unit. In the case of cyclohexane-1,2-dione, adipic acid was obtained.

Full text of DOI:10.1016/S0031-9422(02)00373-4

Phytochemistry 61 (2002) 669–673
www.elsevier.com/locate/phytochem
Oxidative cleavage of the C–C bond of 3,6-dialkylcyclohexane-1,2-
diones by cell suspension cultures of Marchantia polymorpha
a
a
a
a
b
Yoshitomo Matsuura , Wen Chai , Etsuko Endoh , Jyumpei Suhara , Hiroki Hamada ,
a,
C. Akira Horiuchi *
aDepartment of Chemistry, Rikkyo (St. Paul’s) University, Nishi-Ikebukuro, Toshima-Ku, Tokyo 171-8501, Japan
bDepartment of Applied Science, Okayama University of Science, 1-1 Ridai-Cho, Okayama 700-0005, Japan
Received 24 January 2002; received in revised form8 August 2002
Abstract
Biotransformation of 3,6-dialkylcyclohexane-1,2-diones by cell suspension cultures of Marchantia polymorpha involves regio-
selective oxidative cleavage of the C–C bond to give the corresponding oxocarboxylic acids shortened by one carbon unit. In the
case of cyclohexane-1,2-dione, adipic acid was obtained.
#
2002 Elsevier Science Ltd. All rights reserved.
Keywords: Marchantia polymorpha; Leguminosae; Biotransformation; Oxidative cleavage of the C–C bond; Oxocarboxylic acids
1
. Introduction
2. Results and discussion
Previous synthetic studies (He et al., 1999; He and
2.1. Products of biotransformation
Horiuchi, 1999; Ji and Horiuchi, 2000) for producing
oxocarboxylic acids and esters involve the use of metal-
lic oxidizing agents to catalyze the regiospecific oxida-
tive ring-opening of cyclohexanones. Some of these
methods unfortunately are associated with the use of
toxic reagents and heavy metals and the reaction pro-
ducts are racemic.
There has recently been growing interest in the ability
of plant cultured cells to performstereo- and regios-
elective biotransformations.This is in order to facilitate
production of compounds that are useful in synthetic
organic chemistry. Previous studies in our laboratory
have successfully shown how cell cultures could be used,
fromthe viewpoint of green che mi stry, in catalyzing
such biotransformations (Hirata et al., 1989; Hamada et
al., 1991, 1993, 1997; Gotoh et al., 1994). The present
study focuses on the use of M. polymorpha cell cultures
to produce oxocarboxylic acids that are important
intermediates in organic synthesis.
The biotransformation of 3,6-dialkylcyclohexane-1,2-
diones (1a–1e) by suspension cells of M. polymorpha is
shown in Table 1 and Scheme 1. Compounds 1a–1d gave
oxocarboxylic acids 2a–2d, respectively, whose structures
were identified by HRMS, CIMS, EIMS, IR and NMR
spectroscopic analysis as follows. The IR spectrumof 2a
showed a characteristic absorption of a carboxylic acid
ꢀ
1
1
(1708 cm ). The H NMR spectra showed the presence of
a methyl group at ꢀ=1.21 (3H, d) and of an isopropyl
13
group at ꢀ=1.10 (6H, d); and the C NMR spectrumhad
signals at ꢀ=214.3 and 182.4, which were assigned to the
carbonyl and carboxylic acid groups, respectively. There-
fore, compound 2a was presumed to be 2,6-dimethyl-5-
oxoheptanoic acid. Moreover, this structure was supported
+
by the fragmentation patterns (m/z 154[M–H O] , 129[M–
2
+
+
C H ] , and 101[M–C H O] ) of its GC–MS spectrum .
3
7
4
7
Compound 2b had absorption at 1720 (C¼O) and
ꢀ
1
1
1708 cm (COOH) in the IR spectrumand the
NMR spectrumdisplayed a singlet at ꢀ=2.16 (3H) due
H
to the CH C¼O and a doublet ꢀ=1.20 (3H) due to the
3
1
3
CH CH functionality; the C NMR spectrumalso
3
*
Corresponding author. Tel.: +81-3-3895-2397; fax: +81-3-5994-
434.
E-mail address: horiuchi@rikkyo.ac.jp (C.A. Horiuchi).
exhibited signals at ꢀ=208.4 and 182.1 assigned to the
carbonyl group and carboxylic acid, respectively.
3
0
031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00373-4

670
Y. Matsuura et al. / Phytochemistry 61 (2002) 669–673
ꢀ
1
1
Accordingly, 2b was presumed to be 2-methyl-5-oxo-
1
ester carbonyl groups (1736 cm ). The H NMR spectra
exhibited the presence of methyl ester groups at ꢀ=3.68
hexanoic acid. The H NMR spectra of compounds 2c
and 2d both showed a triplet (3H, J=7.3 Hz) at ꢀ=1.05
(3H, s, COOCH ) for 3a and ꢀ=3.67 (3H, s) for 3b, and the
3
C NMR spectra displayed signals at ꢀ=214.0 and 176.7
13
due to the CH – for 2c; and triplet (2H, 7.3 Hz) and
3
(
2H, J=7.1 Hz) at ꢀ=2.35 and ꢀ=2.38, due to the
for 3a and at ꢀ=208.1 and 176.6 for 3b which were
assigned as the carbonyl carbon and ester carbonyl carbon
functionalities, respectively. The structures of 3a and 3b
were also supported by the MS analysis of the fragmenta-
tion pattern. Lastly, in the case of cyclohexane-1,2-dione
(1e), adipic acid was obtained as the product (Table 1),
whose structure was confirmed by the IR and H and 13C
NMR spectra with an authentic sample.
CH COOH, respectively. The C¼O stretching band
2
ꢀ
1
appeared at 1720 and 1712 cm for 2c and 1712 and
ꢀ
1
1
698 cm for 2d, respectively. Fromthese results, the
structures of the oxo carboxylic acids were assigned to be
-oxoheptanoic acid (2c) and 5-cyclohexyl-5-oxopenta-
5
1
noic acid (2d), respectively. These structures were also
supported by analysis of the 13C NMR spectra. Further,
2
a and 2b were converted into methyl oxocarboxylates
3a,3b) using diazomethane. The IR spectra of com-
In the biotransformation of 1a, 1-p-mentene-2,4-diol-
3-one (4) was also isolated as an intermediate. Its struc-
ture was established as follows: the IR spectrumshowed
characteristic absorptions for hydroxyl group (3456
(
pounds 3a and 3b showed the characteristic absorption of
ꢀ ꢀ1
1
Table 1
Biotransformation
M. polymorphia
cm ), carbonyl (1674 cm ) and C–C double bond
of
3,6-dialkylcyclohexane-1,2-diones
using
1646 cm ) functionalities. The 1H NMR spectra
revealed the presence of two hydroxyl groups at
=3.17 [(C-4)–OH] and 5.73 [(C-2)–OH], and the 13C
ꢀ1
(
ꢀ
NMR spectra had resonances corresponding to a car-
bonyl group at ꢀ=197.9 and a C–C double bond at
ꢀ=131.3 (C-4) and 141.4 (C-2). Mass spectral analysis
gave a m/z 184, presumably due to a molecular ion
peak of C H O . It is thus assumed that oxidative
0
1
16
3
cleavage of the C–C bond involves a related decarboxy-
lation step.
Run
Substrate (mg)
Product
Yield (%)a
2
.2. Possible reaction mechanism
1
2
3
4
5
6
1a (80)
1a (20)
1b (80)
1c (80)
1d (80)
1e (80)
2a
2a
60
92
32
52
58
10
2b
2c
The reaction mechanism of diosphenol (1a) metabo-
lism(Sche me 2) described by Nishi mu ra and Mihara
(1986), suggested that 2-(1-methylethyl)-5-oxohexanoic
acid (2a ) could be produced. However, the bio-
2d
Adipic acid
0
a
0
Isolated yield.
transformation of 1a produced 2a rather than 2a . It is
Scheme 1. Substrates and biotransformation products.

Y. Matsuura et al. / Phytochemistry 61 (2002) 669–673
671
thus considered that a hydroperoxy radical attacks at
the C -position of 1a–1d and then the C -peroxy ions of
spectra were measured on a Jeol GSX 400 spectrometer.
Samples were dissolved in CDCl3 with TMS as the
internal standard. GC–MS (EI) analyses were per-
formed on a Shimadzu GCMS-QP5050 with an ionizing
energy of 70 eV. HRMS (EI) analyses were performed
on a JMS-GC mateII/HP-6890 with an ionizing energy
of 70 eV. CIMS (iso-butane reagent gas) were recorded
on a Shimadzu GCMS-QP5050 with an ionizing energy
of 300 eV. 13C NMR spectroscopic assignments are
reported in Table 2.
4
4
3
tinuously, the peroxy ion attacks at the C -carbonyl
,6-dialkylcyclo-hexane-1,2-dione are formed. Con-
1
group and cleavage of the C –C bond occurs according
1
2
to Scheme 2, and the CO group is eliminated. This
reaction mechanism is supported by the fact that 1-p-
menthene-2,4-diol-3-one (4) is obtained as an inter-
mediate. Moreover, the transformation of diosphenol
(1a) under oxygen atmosphere without cell suspension
cultures of M. polymorpha resulted in the recovery of
the starting material. This result rules out the assump-
tion that the cleavage of the C –C bond is effected
Compounds 1a–1d were prepared according to Utaka
et al. (1980). Compound 1e (extra pure grade) of Tokyo
Chemical Co. Ltd. was used without purification.
1
2
solely by oxygen.
3.2. Incubation of substrates 1a–1e
3
. Experimental
Cultured cells of M. polymorpha (60 g) were trans-
planted to MS (Murashige Skoog) medium (200 ml)
containing 1 ppmof 2,4-dichlorophenoxyacetic acid and
the suspension cells were then incubated under shaking
3
.1. General
ꢁ
Melting points were determined on a Yanaco micro
melting point apparatus. IR spectra were recorded on a
Jasco FT-IR 230 spectrometer. 1H and 13C NMR
(120 rpm) at 25 C in the light (2000 lÂ) for 8 days.
Using diosphenol (1a) (80 mg) as an example it was next
added to suspension cells, which were incubated for 8
days. Cultured cells were removed by filtration, and the
filtrate was extracted with EtOAc–Et O (1:1). The
2
organic layer was washed with a satd. solution of NaCl,
dried over anhydr. Na SO and filtered. After the solvent
was removed, the oxocarboxylic acid 2a was obtained as
a yellow oil. Then the oil was subjected on silica gel.
Chromatography, elution of which with hexane–EtOAc
Table 2
13C NMR chemical shift assignments for products (2a–2d, 3a, 3b)
2
4
2a
2b
2c
2d
3a
3b
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
182.4
38.7
27.2
37.6
214.3
40.9
18.2
17.1
18.2
182.1
38.5
30.0
41.0
208.4
27.1
17.0
179.0
36.0
18.8
41.1
211.1
33.4
7.8
179.4
33.1
10.3
39.2
213.6
50.9
25.6
17.2
28.5
176.7
38.7
27.5
37.6
214.0
40.9
18.3
176.6
38.6
27.4
41.1
208.1
29.7
17.1
(
7:3) gave pure oxocarboxylic acid 2a (see Table 1 for
yields).
The experimental procedure followed was similar to
that reported in the literature (Fales et al., 1973). Oxo-
carboxylic acids (2a–2d) were treated with diazo-
methane in dry Et O. The ethereal solution was washed
2
25.8
18.2
with a satd. soln. of NaCl, dried with anhydr. Na SO
4
2
and the solvent was removed in vacuo. The corre-
0
Scheme 2. Proposed reaction mechanism of biotransformation of diosphenol (1a) leading to two different products 2a and 2a . (Mechanismfor
formation of 2a is adapted fromNishimura and Mihara, 1986).

672
Y. Matsuura et al. / Phytochemistry 61 (2002) 669–673
sponding methyl ester was obtained as colorless oil.
In the case of substrate 1a, 1-p-menthene- 2,4-diol-3-
one (4) was also isolated as an intermediate. Then,
cyclohexane-1,2-dione (1e) was transformed to adipic
acid.
3.7. 5-Oxoheptanoic acid (2c)
+
Oil; HRMS (EI): m/z 144.0784 [M] (0.3), 126.0679
+
+
[M–H O] (100), 98 [M–H O–CO] (20.1); CIMS (m/
2
2
+
+
z): 145 [M+H] (71), 127 [(M+H)–H O] (100);
2
+
+
EIMS m/z 126 [M–H O] (2), 98 [M–H O–CO] (5),
2
2
+
ꢀ1
3
.3. 2,6-Dimethyl-5-oxoheptanoic acid (2a)
74 [M-H O-CO-C H ] (5); IR (NaCl): nmax cm
2
2
4
1
36.0, 33.4, 18.8, 7.8; H NMR (CDCl ): ꢀ 1.05 (t, 3H,
720, 1712; 13C NMR (CDCl ): ꢀ 211.1, 179.0, 41.1,
3
+
1
Oil; HRMS (EI): m/z 172.1099 [M] (20.2), 154.1017
+
3
+
[
[
M–H O] (100); CIMS m/z 173 [M+H] (100), 155
2
J=7.3 Hz, H-7), 1.89 (q, 2H, J=7.3 Hz, H-3), 2.35 (t,
2H, J=7.3 Hz, H-2), 2.43 (q, 2H, J=7.3 Hz, H-6), 2.49
(t, 2H, J=7.3 Hz, H-4), 7.8 (s, 1H, H-1).
+
+
(M+H)- H O] (96); EIMS m/z 154 [M–H O] (2),
2 2
+
+
1
ꢁmax cm
29 [M-43] (15), 101 [M–C H O] (32); IR (NaCl):
4 7
ꢀ1
1718, 1708; 13C NMR (CDCl ): ꢀ 214.3,
3
1
1
82.4, 40.9, 38.7, 37.6, 27.2, 18.2, 17.1; H NMR
3.8. 5-Cyclohexyl-5-oxopentanoic acid (2d)
(
w/2=7.0 Hz, H-2), 1.21 (d, 3H, J=7.0 Hz, H-8), 1.10
CDCl ): ꢀ 2.61 (m, 1H, w/2=7.3 Hz, H-6), 2.54 (m, 1H,
3
ꢁ
Needles fromEtOH; mp 56.9–58.1
C; HRMS (EI):
26
ꢁ
m/z 198.1260 [M]+ (100), 180.1141 [M–H O]+ (21.2);
CIMS (m/z): 199 [M+H]+ (100), 181 [(M+H)–H O]
(
1
d, 6H, J=7.0 Hz, H-7 and H-9); [a] ½aꢂ ꢀ13.5 (c
D
2
+
9.4,CHCl ).
3
2
+
(37); EIMS m/z 115 [M–C H ]
(CH ) COOH]
(28), 111 [M–
+
6
11
+
3
.4. Methyl 2,6-dimethyl-5-oxoheptanoate (3a)
(13), 87 [M–C H CO]
6
(26), 83
2
3
11
+
+
[
C H ] (81), 60 [M–C H COCH CH ] (12); IR
6 11 6 11 2 3
(KBr): ꢁmax cm 1712, 1698; 13C NMR (CDCl ): ꢀ
213.6, 179.4, 50.9, 39.2, 33.1, 28.5, 25.8, 25.6, 10.3; H
NMR (CDCl ): 1.89 (m, 2H, w/2=7.1 Hz, H-3), 2.32
(m, 1H, w/2=7.1 Hz, H-6), 2.38 (t, 2H, J=7.1 Hz, H-2),
ꢀ 2.54 (t, 2H, J=7.1 Hz, H-4).
+
ꢀ1
Oil; CIMS m/z 187 [M+H] (100); EIMS m/z 143
+
3
+
1
[
M–(CH ) CH] , 15 [M–(CH ) CHCO] ; IR (NaCl):
2
3
3 2
ꢀ
1
1736, 1720, 1164; 13C NMR (CDCl ): ꢀ
14.0, 176.7, 51.6, 40.9, 38.7, 37.6, 27.5, 18.3, 18.2, 17.2;
nmax cm
3
3
2
1
H NMR (CDCl ): ꢀ 3.68 (s, 3H, H-10), 2.60 (m, 1H, w/
3
2
=7.0 Hz, H-6), 2.49 (m, 1H, w/2=7.0 Hz, H-2), 2.47
m, 2H, w/2=7.0 Hz, H-4), 2.81 (m, 2H, w/2=7.0 Hz,
(
H-3), 1.16 (d, 3H, J=7.0 Hz, H-8), 1.08 (d, 6H, J=7.0
Hz, H-7 and H-9).
3.9. 1-p-Menthene-2,4-diol-3-one (4)
ꢁ
Needles fromEtOH; mp 70–76 C; CIMS (m/z): 185
+
+
[
M+H] (100); EIMS m/z 184 [M] (8), 166 [M–
+
+
3
.5. 2-Methyl-5-oxohexanoic acid (2b)
H O] (4), 141 [M–(CH ) CH] (34), 113 (54), 95 (16),
2 3 2
7
NMR (CDCl ): ꢀ 197.9, 141.4, 131.3, 76.4, 32.1, 30.6,
1 (34); IR (KBr): nmax cm 3456, 1674, 1646; 13
C
ꢀ
1
+
Oil; HRMS (EI): m/z 144.0784 [M] (6.5), 126.0676
3
M–H O] (19.1), 115.0403 [M–CHO]+ (100); CIMS
+
1
[
27.3, 16.9, 16.0; H NMR (CDCl ): ꢀ 0.75 (d, 3H, J=7.0
3
2
m/z): 145 [M+H]+ (100), 127 [(M+H)– H O] (60);
+
Hz, H-9), 1.02 (d, 3H, J=7.0 Hz, H-10), 1.90 (s, 3H, H-
7), 1.97 (m, 2H, H-5a/b and H-8), 2.27 (m, 2H, H-5a/b
and H-6a/b), 2.47 (m, 1H, H-6a/b).
(
EIMS m/z 144 [M] (1), 126 [M–H O] (5), 115 [M–
2
+
+
2
CHO]+ (21), 87 [M–CH COCH ]
+
(27), 57 [M–
ꢀ1
3
2
+
CH (CH )CHCOOH] (100); IR (NaCl): nmax cm
2
3
1
720, 1708; 13C NMR (CDCl ): ꢀ 208.4, 182.1, 41.0,
3
1
References
38.5, 30.0, 27.1, 17.0 ; H NMR (CDCl ):ꢀ 2.53 (m, 1H,
w/2=6.6 Hz, H-2), 2.16 (s, 3H, H-6), 1.20 (d, 3H, J=7.0
Hz, H-7); ½ꢂꢂD +5.7 (c 2.47, CHCl ).
3
2
6
ꢁ
Fales, H.M., Jaouni, T.M., Babashak, J.F., 1973. Simple device for
preparing ethereal diazomethane without resorting to codistillation.
Analytical Chemistry 45, 2302–2303.
3
3
.6. Methyl 2-methyl-5-oxohexanoate (3b)
Gotoh, S., Aoki, M., Iwaeda, T., Izumi, S., Hirata, T., 1994. Asym-
metric hydrolyses of 1,2-diacetoxycyclohexanes by the cultured sus-
pension cells of Marchantia polymorpha. Chemistry Letters 1519–
1520.
+
Oil;CIMS (m/z): 159 [M+H] (100); EIMS (m/z):
+
+
1
CH CO] (16), 101 [M–CH COCH ] (51), 98 [M–
HCOOCH3] (96), 88 [M–CH COCH CH ] (100); IR
2
43 [M–CH ] (2), 126 [M–CH OH] (64), 115 [M–
3 3
Hamada, H., Yasumune, H., Fuchikami, Y., Hirata, T., Sattler, I.,
Williams, H.J., Scott, A.I., 1997. Biotransformation of geraniol,
nerol and (+)- and (-)-carvone by suspension cultured cells of
Catharanthus roseus. Phytochemistry 44, 615–621.
+
+
3
3
2
+
+
3
2
ꢀ
1
1736, 1720, 1164; 13C NMR
(
(
1
NaCl): ꢁmax cm
CDCl ): ꢀ 208.1, 176.6, 50.8, 41.1, 38.6, 29.7, 27.4, 17.1;
H NMR (CDCl ): ꢀ 3.67 (s, 3H, H-8), 2.46 (m, 1H, w/
3
Hamada, H., Konishi, H., Williams, H.J., Scott, A.I., 1991. Bio-
transformation of testosterone isomers by a green cell suspension
culture of Marchantia polymorpha. Phytochemistry 30, 2269–2270.
Hamada, H., Naka, S., Kurban, H., 1993. Stereoselective reduction in
the biotransformation of androstane derivatives by cell suspension
cultures of Marchantia polymorpha. Chemistry Letters 2111–2112.
3
2
(
3
=7.0 Hz, H-2), 2.31 (m, 2H, w/2=7.0 Hz, H-4), 2.14
s, 3H, H-6), 2.01 (m, 2H, w/2=7.0 Hz, H-3), 1.17 (d,
H, J=7.2 Hz, H-7).

Y. Matsuura et al. / Phytochemistry 61 (2002) 669–673
673
He, L., Kanamori, M., Horiuchi, C.A., 1999. Oxidation of 2-alkylcy-
cloalkanones with iodine-cerium(IV) salts in alcohols. Journal of
Chemical Research (S) 122–123.
Ji, S.-J., Horiuchi, C.A., 2000. Photo-cleavage of carbon-carbon bond
of a-iodocycloalkanones giving o,o-dialkoxyalkanoic ester in alco-
hol. Bulletin of the Chemical Society of Japan 73, 1645–1652.
Nishimura, O., Mihara, S., 1986. Aroma constituents of blackcurrant
buds (Ribes Nigrum L.). Flavors and Fragrances: a world perspec-
tive. Proceedings of the 10th International Congress of Essential
Oils, Fragrances and Flavors, Washington, DC, USA, 16–20
November 1986, pp. 375–386.
He, L., Horiuchi, C.A., 1999. Oxidation of 2-substituted cycloalk-
anones with cerium(IV) sulfate tetrahydrate in alcohols and acetic
acid. Bulletin of the Chemical Society of Japan 72, 2515–2521.
Hirata, T., Tang, Y., Okano, K., Suga, T., 1989. Stereochemistry of
reduction of the endocyclic double bond of (ꢀ)-carvone with the
enzyme preparation from cultured cells of Nicotiana tabacum.
Phytochemistry 28, 3331–3333.
Utaka, M., Matsushita, S., Takeda, A., 1980. Convenient preparation
of 1,2-cyclohexanediones. Chemistry Letters 779–780.