Asymmetric hydrogenation of the C-C double bond of 1- and 1,2-methylated maleimides with cultured suspension cells of Marchantia polymorpha

Article abstract of DOI:10.1016/j.tetasy.2006.06.030

Suspension cultured cells of Marchantia polymorpha have the potential to hydrogenate the C-C double bonds of 2-methyl- and 2,3-dimethylmaleimide derivatives to give enantiomerically pure (2R)-2-methyl- and (2R,3R)-2,3-dimethylsuccinimide derivatives, resp

Full text of DOI:10.1016/j.tetasy.2006.06.030

Tetrahedron: Asymmetry 17 (2006) 1859–1862
Asymmetric hydrogenation of the C–C double bond
of 1- and 1,2-methylated maleimides with cultured
suspension cells of Marchantia polymorpha
Mohamed-Elamir F. Hegazy,a Kozo Shishidob and Toshifumi Hirataa,*
aDepartment of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima 739-8526, Japan
bGraduate School of Pharmaceutical Science, The University of Tokushima, 1-78 Shomachi, Tokushima 770-8505, Japan
Received 22 May 2006; accepted 20 June 2006
Available online 21 July 2006
Abstract—Suspension cultured cells of Marchantia polymorpha have the potential to hydrogenate the C–C double bonds of 2-methyl-
and 2,3-dimethylmaleimide derivatives to give enantiomerically pure (2R)-2-methyl- and (2R,3R)-2,3-dimethylsuccinimide derivatives,
respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tured cells of M. polymorpha have a high potential for
the hydrogenation of the C–C double bond of maleimides.6
Therefore, we selected the cultured suspension cells of
M. polymorpha as a biocatalyst and carried out the
biotransformation of N-substituted 2-methylmaleimides
(1 and 2), 2,3-dimethylmaleimides 3, and N-phenylitaconi-
mide 5. It was found that the endocyclic C–C double bond
of 1–4 was hydrogenated to give the corresponding succin-
imides 6–9, respectively, as shown in Table 1. Substrates 2
and 3 were completely transformed to the succinimides 7
and 8, respectively, by incubation for 5 days. However,
the substrate 5 having an exocyclic C–C double bond was
not hydrogenated by the cultured cells. In the biotransfor-
mation of the dimethylmaleimides 3 and 4, only trans-di-
methylsuccinimides 8 and 9 were produced, respectively,
but cis-dimethylsuccinimides 10 and 11 were not found as
a product, in spite of careful GLC analyses of the reaction
mixtures. This indicates that anti-addition of the hydrogen
atoms occurs in hydrogenation of the C–C double bond of
the maleimides.
It is well known that transformation using biocatalysts al-
lows structural modification to provide useful chiral sub-
stances under mild and ecologically compatible
conditions. We have previously found the ability of cul-
tured plant cells to hydrogenate the C–C double bond of
enones with discrimination of its enantiotopic faces to the
corresponding optically active ketones.1–4 In the course of
the development of new chiral hydrogenation reactions,
we recently found that the hydrogenation of the C–C dou-
ble bond of N-phenyl-2-methylmaleimide by cultured plant
cells was highly enantioselective.5,6 In continuation of this
work, we have now studied the reactivity and enantioselec-
tivity in the reduction of 2-methylated and 2,3-dimethyl-
ated maleimide derivatives with cultured cells of
Marchantia polymorpha.
2. Results and discussion
In our previous work, we studied the biotransformation of
N-substituted maleimides with several plant cells, such as
Nicotiana tabacum, Catharanthus roseus, Parthenocissus
tricuspidata, and M. polymorpha, and found that the cul-
To clarify the effect of the N-substituted groups, such as
phenyl, benzyl, and p-methoxyphenyl, for reactivity in the
reduction of C–C double bond with cultured cells of
M. polymorpha, the time courses in the biotransformation
of N-substituted maleimides, 1–4 and 12, were followed.
As shown in Figure 1, it was found that the presence of a
p-methoxyphenyl group decreases the reactivity slightly.
*
Corresponding author. E-mail: thirata@sci.hiroshima-u.ac.jp
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.06.030

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M.-E. F. Hegazy et al. / Tetrahedron: Asymmetry 17 (2006) 1859–1862
CH3
H3C
urations of the products, 6 and 7, should be R. The peak
analyses of the 1H NMR spectra with a chiral shift reagent8
and the GLC with a chiral column showed the enantiomeric
purities of 6 and 7 to be 99% ee.
H3C
H2C
O
O
O
O
O
O
N
R
N
R
N
R
On the other hand, the optical rotations of 2,3-dimethyl-
succinimides, 8 and 9, which were obtained by the biotrans-
formation of N-substituted 2,3-dimethylmaleimides, 3 and
4, were dextro-rotatory. This indicated that N-phenyl-2,3-
1: R= p-Methoxy-Ph
2: R= -CH2Ph
12: R=Ph
3: R= Ph
4: R= p-Methoxy-Ph
5
dimethylsuccinimide
8 and N-p-methoxyphenyl-2,3-di-
CH3
H3C
O
CH3
O
H3C
H3C
methylsuccinimide 9, had 2R,3R-configuration, because
the optical rotation of (2R,3R)-2,3-dimethylsuccinimide
was reported to be dextro-rotatory.9 The peak analyses
of the 1H NMR spectrum with a chiral shift reagent
showed the enantiomeric purities of 8 and 9 to be 99% ee.
O
O
O
O
N
R
N
N
R
R
10: R=Ph
11: R= p-Methoxy-Ph
8: R=Ph
9: R= p-Methoxy-Ph
6: R= p-Methoxy-Ph
7: R= -CH2Ph
13: R=Ph
Thus, it was found that the cultured suspension cells of
M. polymorpha have an ability to hydrogenate enantioselec-
tively the C–C double bond of 2- and 2,3-methylated male-
imides to afford highly enantiomerically pure succinimide
derivatives. Recently, Oishi et al. reported the utilization
of a chiral 2,3-dimethylsuccinimide derivative as a model
compound for the development of a polymer having chiral
recognition ability.10 It is fascinating to note that the enan-
tioface selective hydrogenation of the 2- and 2,3-alkylated
maleimide derivatives with cultured plant cells as a biocata-
lyst is one of the useful methods for the generation of chiral-
ity. In addition, the optically active succinimides, obtained
by these biotransformations, could be useful chiral building
blocks for the synthesis of biologically active natural prod-
ucts, for example, pyrrolizidine and indolizidine alkaloids.
Table 1. Biotransformation of N-substituted maleimides, 1–4, by cultured
cells of M. polymorpha
Substrate Product Incubation Conversion ee
Configuration
time (d)
(%)
(%)
1
2
3
4
6
7
8
9
5
5
5
5
89
99
99
77
99
>99
99
R
R
R,R
R,R
>99
100
80
60
40
20
0
13
3. Experimental
7
6
8
9
3.1. General experimental procedures
Analytical and preparative TLCs were carried out on glass
sheets coated with silica gel (0.25 mm or 0.5 mm; Merck sil-
ica gel 60 GF254). The NMR spectra were measured with a
JEOL LA-500 (500 MHz, 1H; 125 MHz, 13C) spectrometer
with TMS as an internal standard. Optical rotations were
measured with a JASCO DIP-370 digital polarimeter.
GLCs with FID were carried out on a capillary column
(0.25 mm · 30 m) coated with 0.25 lm ZB-5 (Zebron)
using N2 as carrier gas (60 cm3 minꢀ1) at column tempera-
ture programmed from 180 to 250 ꢁC at 5 ꢁC/min; injector
temperature 250 ꢁC and on a chiral capillary column
(0.25 mm · 20 m) coated with 0.25 lm CHIRALDEX G-
0
1
2
3
4
Incubation time / day
Figure 1. Time-course experiments in the biotransformation of maleimide
substrates (1–4 and 12) into succinimide derivatives with cultured cells of
M. polymorpha.
Although clear evidences for the difference have not been
obtained, it would presumably be a consequence of the
electronic effect of the p-OMe substituent.
TA (Tokyokasei) using N2 as carrier gas (60 cm3 minꢀ1
)
at column temperature programmed from 150 to 180 ꢁC
at 5 ꢁC/min; injector temperature 180 ꢁC. HPLCs were car-
ried out on Puresil C18 column (Waters) using CH3CN/
H2O = 2:3 (v/v) as the eluent.
Absolute configuration and enantiomeric purity of the prod-
ucts were determined by their specific rotations, the peak
analysis of the 1H NMR with chiral shift reagent, and peak
intensity of GLC with a chiral column. Optical rotations of
N-p-methoxyphenyl-2-methylsuccinimide 6 and N-benzyl-
2-methylsuccinimide 7, obtained by the biotransformation
of N-substituted 2-methylmaleimides, were dextro-rotatory.
Since the optical rotation of (R)-2-methylsuccinimide deriv-
ative was reported to be dextro-rotatory,6,7 absolute config-
3.2. Substrates
N-p-Methoxyphenyl-2-methylmaleimide 1, N-benzyl-2-
methylmaleimide 2, N-phenyl-2,3-dimethylmaleimide 3,
N-p-methoxyphenyl-2,3-dimethylmaleimide
N-phenylitaconimide 5 were prepared by use of a combina-
tion of acid anhydrides such as 2,3-dimethylmalonic anhy-
4,
and

M.-E. F. Hegazy et al. / Tetrahedron: Asymmetry 17 (2006) 1859–1862
1861
dride, citraconic anhydride, and itaconic anhydride and
amines such as p-methoxy aniline and benzyl amine,
according to the reported procedure.11
100 ml of MSK-II medium with 2% glucose, 0.1% inositol,
10 mM of 2,4-D, and cultured on a rotary shaker (110 rpm)
for 10 days at 25 ꢁC under illumination (4000 lx).
An amine and acid anhydride were mixed in high pressure
vessel, which was capped with a teflon screw. The vessel
was submerged in oil bath at 120 ꢁC overnight. The reac-
tion mixture (yellow solid) was dissolved with ethyl acetate
and filtrated through a short silica gel column. The ethyl
acetate solution was concentrated after addition of 10%
NaOH solution. The residue was purified by column chro-
matography on silica gel eluted with ethyl acetate and
n-hexane (1:9) to give the substrates, 1, 2, 3, 4, and 5.
3.4. Biotransformation of maleimides 1–5 with cultured plant
cells of M. polymorpha
To the flask containing the suspended cells (about 20 g) of
M. polymorpha in MSK-II medium (100 ml), each substrate
1–5 (20 mg) in DMSO (0.2 ml) was administered, and the
cultures were incubated at 25 ꢁC on a rotary shaker
(110 rpm) under illumination (4000 lx). After the incuba-
tion, the cell and medium were separated by filtration with
suction. The filtrated medium was extracted with diethyl
ether and the extract was concentrated by rotary evapora-
tor to give crude extracts. These crude extracts were subject
to preparative TLC with ether and n-hexane (3:1) to give
products 6–9 in a pure form. The conversion yields of the
products were determined by GLC analysis of the crude ex-
tracts, as shown in Table 1. The structure of each product
was identified by NMR and MS analyses.
3.2.1.
N-p-Methoxyphenyl-2-methylmaleimide
1. 1H
NMR (CDCl3) d 2.18 (3H, d, J = 1.5 Hz, 2-Me), 3.84
(3H, s, OMe), 6.48 (1H, q, J = 1.5 Hz, 3-H), 6.98 (2H, d,
J = 8.5 Hz, 20- and 60-H), 7.25 (2H, d, J = 8.5 Hz, 30- and
50-H); 13C NMR (CDCl3) d 11.1 (2-Me), 55.5 (OMe),
114.4 (C-20 and C-60), 124.2 (C-10), 127.3 (C-3), 127.5 (C-
30 and C-50), 145.7 (C-2), 158.9 (C-40), 169.9 (C-1), 170.3
(C-4).
25
3.4.1. N-p-Methoxyphenyl-2-methylsuccinimide 6. ½aꢁD
¼
25
3.2.2. N-Benzyl-2-methylmaleimide 2. 1H NMR (CDCl3)
d 2.01 (3H, d, J = 1.5 Hz, 2-Me), 6.48 (1H, q, J = 1.5 Hz,
3-H), 4.60 (2H, s, 70-H2), 7.20 (2H, d, J = 8.5 Hz, 20- and
60-H), 7.26 (3H, m, 30-, 40-, and 50-H); 13C NMR (CDCl3)
d 10.8 (Me), 41.3 (C-70), 127.3 (C-20 and C-60), 127.6 (C-30
and C-50), 128.2 (C-40), 128.8 (C-3), 136.4 (C-10), 145.6 (C-
2), 170.0 (C-1), 171.3 (C-4).
þ4:0 ꢂ 0:3 (c 0.13, CHCl3) {lit.7 ½aꢁD ¼ þ8 ꢂ 0:4 (c 1.2,
CHCl3) for (R)-N-phenyl-2-methylsuccinimide}; IR
(CHCl3) 1718 cmꢀ1 (CO); H NMR (CDCl3) d 1.48 (3H,
1
d, J = 7.0 Hz, 2-Me), 3.06 (1H, ddq, J = 11.5, 3.5, and
7.0 Hz, 2-H), 2.53 (1H, dd, J = 13.5 and 4.0 Hz, 3-Ha),
3.12 (1H, dd, J = 13.5 and 11.5 Hz, 3-Hb), 3.86 (3H, s,
OMe), 7.01 (2H, d, J = 8.5 Hz, 30- and 50-H), 7.23 (2H,
d, J = 8.5 Hz, 20- and 60-H); 13C NMR (CDCl3) d 16.9
(2-Me), 34.8 (C-3), 36.4 (C-2), 55.5 (OMe), 114.4 (C-20
and C-60), 124.6 (C-10), 127.1 (C-30 and C-50), 176.0 (C-
4), 179.7 (C-1). The enantiomeric purity of the product
was determined on the basis of the peak analysis of the
3.2.3. N-Phenyl-2,3-dimethylmaleimide 3. 1H NMR
(CDCl3) d 2.03 (6H, s, 2- and 3-Me), 7.25–7.35 (3H, m,
20-, 40- and 60-H), 7.45 (2H, t, J = 8.5 Hz, 30- and 50-H);
13C NMR (CDCl3) d 8.8 (2- and 3-Me), 125.7 (C-20 and
C-60), 127.4 (C-10), 128.9 (C-30 and C-50), 131.9 (C-40),
137.4 (C-2 and C-3) 170.4 (C-1 and C-4).
1
methyl proton signals of the H NMR with chiral shift re-
agent, Eu(hfc)3 (Sigma Ltd.).8 The methyl proton signals of
racemic N-p-methoxyphenyl-2-methylsuccinimide were
observed at d 2.15 (d, J = 7.5 Hz; relative integral
value = 100) and 2.22 (d, J = 7.5 Hz; integral value = 100)
in the CDCl3 solution of the sample and Eu(hfc)3 (1:1 mol
ratio). In the NMR spectrum of the product 6 with
Eu(hfc)3, the ratio of relative integral value of the methyl
proton signals at d 2.15 (d, J = 7.5 Hz) and 2.22 (d,
J = 7.5 Hz) was about 99.5–0.5.
3.2.4. N-p-Methoxyphenyl-2,3-dimethylmaleimide 4. 1H
NMR (CDCl3) d 2.07 (6H, s, 2- and 3-Me), 3.84 (3H, s,
OMe), 6.98 (2H, d, J = 9.0 Hz, 20- and 60-H), 7.25 (2H,
d, J = 9.0 Hz, 30- and 50-H); 13C NMR (CDCl3) d 8.8 (2-
and 3-Me), 55.5 (OMe) 114.3 (C-20 and C-60), 124.6 (C-
10), 127.3 (C-30 and C-50), 137.3 (C-2 and C-3), 158.7 (C-
40), 171.2 (C-1 and C-4).
25
3.2.5. N-Phenylitaconimide 5. 1H NMR (CDCl3) d 3.43
(2H, t, J = 2.3 Hz, 3-H), 5.67 (1H, t, J = 2.1 Hz, 5-Ha),
6.37 (1H, t, J = 2.3 Hz, 5-Hb), 7.23 (2H, d, J = 7.5 Hz,
20- and 60-H), 7.32 (2H, t, J = 7.5 Hz, 40-H), 7.40 (2H, t,
J = 7.5 Hz, 30- and 50-H); 13C NMR (CDCl3) 41.0 (C-3),
123.7 (C-20 and C-60), 124.1 (C-5), 125.2 (C-40), 127.5
(C-30 and C-50), 131.5 (C-10), 179.1 (C-1) and 187.6 (C-4).
3.4.2.
N-Benzyl-2-methylsuccinimide
7. ½aꢁD ¼
þ14:0 ꢂ 0:7 (c 0.18, CHCl3); IR (CHCl3) 1703 cmꢀ1
1
(C@O); H NMR (CDCl3) d 1.27 (3H, d, J = 6.5 Hz, 2-
Me), 2.76 (1H, ddq, J = 11.5, 3.5 and 6.5 Hz, 2-H), 2.25
(1H, dd, J = 12.5 and 3.5 Hz, 3-Ha), 2.78 (1H, dd,
J = 12.5 and 11.5 Hz, 3-Hb), 4.58 (2H, s, 70-H), 7.25 (3H,
m, 30-, 40- and 50-H), 7.32 (2H, d, J = 6.5 Hz, 20- and 60-
H); 13C NMR (CDCl3) d 16.6 (2-Me), 34.7 (C-3), 36.4
(C-2), 42.3 (C-5), 127.8 (C-40), 128.6 (C-20 and C-60),
128.7 (C-30 and C-50), 135.8 (C-10), 176.0 (C-4), 180.2
(C-1). The methyl proton signals of racemic N-benzyl-2-
methylsuccinimide were observed at d 2.07 (d, J = 6.5 Hz;
relative integral value = 100) and 2.31 (d, J = 6.5 Hz;
integral value = 100) in the CDCl3 solution of the sample
and Eu(hfc)3 (1:1 mol ratio). The relative intensity of the
methyl proton signal in the 1H NMR of the product 7 with
3.3. Plant material
The cells of M. polymorpha12 have been subcultured every
3 weeks using MSK-II medium13 containing 2% glucose,
0.1% inosoitol, 10 mM of 2,4-dichrolophenoxyacetic acid
(2,4-D) for more than 10 years in our laboratory. Prior
to use for biotransformation experiments, the cultured cells
were transplanted to a 300 ml conical flask containing

1862
M.-E. F. Hegazy et al. / Tetrahedron: Asymmetry 17 (2006) 1859–1862
Eu(hfc)3 was over 99.5% in the signal at d 2.07 (d,
J = 6.5 Hz).
taken out and filtrated. The filtrate was extracted with
diethyl ether. The conversion yield of the product was
determined on the basis of the peak ratio in the GLC
and HPLC analyses of the ether extract.
25
3.4.3.
N-Phenyl-2,3-dimethylsuccinimide
8. ½aꢁD ¼
20
þ21:0 ꢂ 0:8 (c 0.03, CHCl3) (lit.9 ½aꢁD ¼ þ53:6 ꢂ 0:4 (c
1.4, CHCl3) for (2R,3R)-2,3-dimethylsuccinimide); IR
(CHCl3) 1709 cmꢀ1 (C@O); 1H NMR (CDCl3) d 1.42
(6H, d, J = 7.0 Hz, 2- and 3-Me), 2.35 (2H, dq, J = 12.5
and 6.0 Hz, 2- and 3-H), 7.28 (2H, d, J = 7.5 Hz, 20- and
60-H), 7.35 (2H, t, J = 7.5 Hz, 40-H), 7.45 (2H, t,
J = 7.5 Hz, 30- and 50-H); 13C NMR (CDCl3) d 15.2 (2-
and 3-Me), 43.2 (C-2 and C-3), 126.3 (C-20 and C-60),
128.4 (C-10), 129.0 (C-30 and C-50), 132.0 (C-40), 178.4
(C-1 and C-4). The methyl proton signals of racemic N-
phenyl-2,3-dimethylsuccinimide were observed at d 2.36
(d, J = 5.5 Hz; relative integral value = 100) and 2.49 (d,
J = 5.5 Hz; integral value = 100) in the CDCl3 solution
of the sample and Eu(hfc)3 (1:1 mol ratio). The relative
3.6. Preparation of racemic N-substituted trans-2,3-
dimethylsuccinimides
N-Substituted trans-2,3-dimethylsuccinimides were pre-
pared by the isomerization of N-substituted cis-2,3-dimeth-
ylsuccinimides, according to the reported procedures.10 To
a solution of N-phenyl-cis-2,3-dimethylsuccinimide 10
(15 mg, 0.073 mmol) in DMSO (0.4 ml), three drops of
Sp [L-(ꢀ)-sparteine] (Aldrich) were added and the mixture
was heated at 60 ꢁC for 8 days in oil bath. The reaction
mixture was subjected to silica gel column chromatography
(hexane/ethyl acetate = 1/1, v/v) to give racemic N-phenyl-
trans-2,3-dimethylsuccinimide. The conversion of the cis-
2,3-dimethylsuccinimide to the trans-isomer was 85%, on
the basis of the peak ratio of the GLC analysis of the crude
product.
1
intensity of the methyl proton signal in the H NMR of
the product 8 with Eu(hfc)3 was over 99.5% in the signal
at d 2.36 (d, J = 5.5 Hz). The enantiomeric purity of the
product was confirmed on the basis of peak analysis using
GLC on chiral column: retention times in the GLC were
12.60 for (2R,3R)-8 and 12.37 for (2S,3S)-8, and the peak
ratio indicated that the enantiomeric excess of (2R,3R)-8
is about 99%.
Racemic
N-p-methoxyphenyl-trans-2,3-dimethylsuccini-
mide was also prepared from N-p-methoxyphenyl-cis-2,
3-dimethylsuccinimide 11 in a yield of 83% by a similar
procedure as above.
3.4.4.
N-p-Methoxyphenyl-2,3-dimethylsuccinimide
25
References
9. ½aꢁD ¼ þ15:0 ꢂ 0:4 (c 0.08, CHCl3); IR (CHCl3)
1709 cmꢀ1 (C@O); 1H NMR (CDCl3) d 1.41 (6H, d,
J = 6.0 Hz, 2- and 3-Me), 2.55 (2H, dq, J = 12.5 and
6.0 Hz, 2- and 3-H), 3.80 (3H, s, OMe), 6.94 (2H, d,
J = 8.5 Hz, 20- and 60-H), 7.17 (2H, d, J = 8.5 Hz, 30- and
50-H); 13C NMR (CDCl3) d 15.1 (2- and 3-Me), 43.2 (C-2
and C-3), 55.4 (OMe) 114.4 (C-20 and C-60), 124.6 (C-10),
127.6 (C-30 and C-5), 158.0 (C-40), 178.4 (C-1 and C-4).
The methyl proton signals of racemic N-p-methoxyphen-
yl-2,3-dimethylsuccinimide were observed at d 2.07 (d,
J = 6.0 Hz; relative integral value = 100) and 2.16 (d,
J = 6.0 Hz; integral value = 100) in the CDCl3 solution
of the sample and Eu(hfc)3 (1:1 mol ratio). The relative
intensity of the methyl proton signal in the 1H NMR of
the product 9 with Eu(hfc)3 was over 99.5% in the signal
at d 2.07 (d, J = 6.0 Hz).
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3.5. Time-course experiments
Cultured cells of M. polymorpha (about 20 g) were trans-
ferred to several 300 ml Erlenmeyer flasks containing
100 ml MSK-II medium and each (20 mg) of the substrates,
1–4 and 12, was added to the suspension cultures and incu-
bated at 25 ꢁC in rotary shaker (110 rpm) under illumina-
tion. At a regular time interval, one of the flasks was