Highly enantioselective synthesis of α,α-disubstituted malonamic acids through asymmetric hydrolysis of dinitriles with Rhodococcus sp. CGMCC 0497

Article abstract of DOI:10.1039/b210743k

Highly enantioselective hydrolysis of α,α-disubstituted malononitriles by the strain Rhodococcus sp. CGMCC 0497 expressing both nitrile hydratase and amidase activity to give (R)-α,α-disubstituted malonamic acids which could be converted to valuable (R)-

Full text of DOI:10.1039/b210743k

Highly enantioselective synthesis of a,a-disubstituted malonamic acids
through asymmetric hydrolysis of dinitriles with Rhodococcus sp.
CGMCC 0497†
Zhong-Liu Wu and Zu-Yi Li*
State Key Laboratory of Bio-organic & Natural Products Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China.
E-mail: lizy@pub.sioc.ac.cn; Fax: (+86)021-64166128; Tel: (+86)021-64163300-1406
Received (in Cambridge, UK) 31st October 2002, Accepted 3rd January 2003
First published as an Advance Article on the web 14th January 2003
Highly enantioselective hydrolysis of a,a-disubstituted mal-
ononitriles by the strain Rhodococcus sp. CGMCC 0497
expressing both nitrile hydratase and amidase activity to
give (R)-a,a-disubstituted malonamic acids which could be
converted to valuable (R)- or (S)-a-alkylated amino acids are
reported and the yields of the products are improved
remarkably at a lower reaction temperature.
Fig. 1 Hydrolysis products of a-benzyl-a-methylmalononitrile 1a.
(52%, 95% ee) and 3 (44%, 1.2% ee). The high enantiomeric
excess of 5a encouraged us to better explore reaction conditions
to improve its chemical yield.
Nitrile-converting enzymes have been known for several
decades1 and demonstrated great potential in organic synthesis.
Moreover, organonitriles are versatile intermediates in organic
synthesis and can be readily prepared by a number of methods.
However, the substrates studied for nitrile-converting enzymes
are still very limited2 and the ability of the enzymes to catalyze
stereoselective conversion remains largely unexploited. So far,
most studies focus on the enantioselective conversion of
racemic nitriles, such as a-alkyl nitriles, a-hydroxy nitriles, a-
amino nitriles and b-acetoxy nitriles,3 while only a few on
prochiral nitriles4,5 especially malononitrile derivatives.6
The products of asymmetric hydrolysis of a,a-disubstituted
malononitriles catalyzed by nitrile-converting enzymes could
serve as precursors of a-alkylated a-amino acids. This class of
non-proteinogenic amino acids play an important role in the
design of conformationally modified bioactive peptides and in
the inhibition of enzyme activities.7 Their extensive use is only
limited by the availability of enantiopure compounds in large
scale.8 As a result, the synthesis of optically pure a-alkylated a-
amino acids has attracted considerable attention in recent
years.
It is well known that enzyme-catalyzed hydrolysis of nitriles
proceeds by two routes: by nitrilase or by a combination of
nitrile hydratase (NHase) and amidase through an intermediate
amide.10 Rhodococcus sp. CGMCC 0497 acts mainly by the
latter route.9b The possible pathway of the hydrolysis of 1a was
illustrated as Scheme 1. It is clear that a-benzyl-a-methylmalo-
namic acid 5a may derive from two ways: one involved
2-cyano-2-methyl-3-phenylpropionic acid 3 and the other
involved a-benzyl-a-methylmalonamide 4.
Scheme 1 Possible procedure of the hydrolysis of 1a.
We have screened and optimized the culture condition of the
strain Rhodococcus sp. CGMCC 0497, which was isolated by
our group and proved to have high nitrile-converting activity
and enantioselectivity.9 We report here a highly efficient
enantioselective hydrolysis of a,a-disubstituted malononitriles
using Rhodococcus sp. CGMCC 0497 to afford (R)-a,a-
disubstituted malonamic acids. We found that the results were
improved remarkably when the reaction temperature decreased
from 30 to 20 °C. A number of malonamic acid derivatives were
obtained with excellent enantiomeric excesses and high
yields.
Further experiments were carried out using racemic 3 and 4
as substrates respectively (Scheme 2). The transformation of
racemic 3 did not occur at all after one day and 3 was recovered
quantitatively, while the diamide 4 was converted to 5a in 94%
ee and > 99% yield, which demonstrated that 5a derived mostly
from diamide 4 and the ratio of products 5a to 3 depends on the
value of k3/k2.
The reactions were initially carried out at 30 °C, the
conventional incubation temperature in asymmetric hydrolysis
catalyzed by nitrile-converting enzymes. In accordance with the
literature,6 a-butyl-a-methylmalononitrile can be converted to
(R)-a-butyl-a-methylmalonamic acid neatly by the strain
Rhodococcus sp. CGMCC 0497. However, when a-benzyl-a-
methylmalononitrile 1a was used as substrate, most probably
due to the steric hindrance, the product isolated was a complex
mixture of hydrolysis intermediates. After 24 h, the reaction
gave a mixture of (S)-2 (70%, 48% ee), (R)-3 (19%, 72% ee) and
4 (8%). After 90 h, the reaction gave a mixture of (S)-2 (22%,
99% ee), (R)-3 (35%, 6% ee) and 5a (41%, 88% ee)(Fig. 1). By
prolonging reaction time to 112 h, the mixture converted to 5a
Scheme 2 Hydrolysis of racemic 3 and 4.
In the successful application of NHase to the industrial
production of acryamide, the reaction is performed at an
especially low temperature (2–4 °C),11,12 which, as M.
Kobayashi et al. explained, reduces the amidase activity and
exerts little effect on the NHase activity.11 This phenomenon
promoted us to explore the possibility of enhancing the value of
k3/k2 by decreasing reaction temperature. Expectedly, decreas-
† Electronic supplementary information (ESI) available: full experimental
details. See http://www.rsc.org/suppdata/cc/b2/b210743k/
386
CHEM. COMMUN., 2003, 386–387
This journal is © The Royal Society of Chemistry 2003

ing the reaction temperature to 20 °C increased the yield of 5a
to 84% and the enantiomeric excess was 96% ee.
The results of enantioselective hydrolysis of various a,a-
disubstituted malononitriles by Rhodococcus sp. CGMCC 0497
(Scheme 3) at 20 °C or 30 °C are summarized in Table 1.‡ As
shown, in all cases, the chemical yields of a,a-disubstituted
malonamic acids 5 were greatly improved at 20 °C compared to
that at 30 °C and all the products were achieved in excellent
enantioselectivity. The strain tolerates aromatic ring sub-
stituents in the ortho-, meta-, and para-positions and all para-
substituted substrates gave slightly high enantiomeric excesses
than ortho- and meta- ones. 2-Phenylethyl-2-methylmalononi-
trile 1i gave product 2-phenylethyl-2-methylmalonamic acid 5i
with 96% yield and > 99% ee as the exclusive product at 20
°C.
Scheme 4 Synthesis of either (R)- or (S)-a-alkylated amino acid: (a) DMF,
EtBr, K2CO3, rt; (b )DMF, Hg(OAc)2, NBS, EtOH, rt; (c) 20% HCl, reflux;
(d) P2O5, toluene; (e) 3 N NaOH, THF, rt; (f) SOCl2, NaN3, MeOH.
Professor Lijun Xia and Miss Zuoding Ding for performing the
HPLC analysis.
Notes and references
Scheme 3 Asymmetric hydrolysis of a,a-disubstituted malononitriles.
‡ A suspension of 10 g washed wet cells and 80 ml 0.1 mM potassium
phosphate buffer (pH = 7.0) was incubated at 30 or 20 °C for 30 min with
continuously magnetic stirring before the addition of 1 (100 mg in 100 ml
acetone). The reaction was quenched by centrifugation. The resulting
supernatant was acidified and extracted with ethyl acetate and dried over
Na2SO4. After concentration, the residue was purified by flash chromatog-
raphy on silica gel (elute+petroleum ether–EtOAc–AcOH 150+100+1).
Table 1 Enantioselective hydrolysis of various a,a-disubstituted malononi-
triles by Rhodococcus sp. CGMCC 0497
Entrya
Substrate
X
T/°C
Yield (%) ee (%)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1b
1b
1c
1c
1d
1d
1e
1e
1f
p-CH3
p-CH3
p-F
30
20
30
20
30
20
30
20
30
20
30
20
30
20
30
20
31
58
43
80
42
83
40
81
30
58
40
85
34
65
90
96
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99
> 99c
> 99c
97
1 K. V. Thimann and S. Mahadervan, Arch. Biochem. Biophys, 1964, 105,
133; Y. Asano, K. Fujishiro, Y. Tani and H. Yamada, Agri. Biol. Chem.,
1982, 46, 1165.
2 J. Crosby, J. Moilliet, J. S. Parratt and and N. J. Turner, J. Chem. Soc.,
Perkin Trans. 1, 1994, 1679; M. Wieser and T. Nagasawa, in
Stereoselective Biocatalysis, ed. R. N. Patel, Dekker, New York, 2000,
pp. 461–486.
p-F
p-Cl
p-Cl
p-Br
p-Br
p-MeO
p-MeO
m-Cl
m-Cl
o-Cl
o-Cl
H
3 M.-X. Wang, G. Lu, G.-J. Ji, Z.-T. Huang, O. Meth-Cohn and J. Colby,
Tetrahedron: Asymmetry, 2000, 11, 1123; S. Payne, S. Wu, R. D.
Fallon, G. Tudor, B. Stieglitz, J. M. Turner and M. J. Nelson,
Biochemistry, 1997, 36, 5447; K. Yamamoto, K. Oishi, I. Fujimatsu and
K. I. Komatus, Appl. Environ. Microbiol., 1991, 57, 3028; N. Layh, A.
Stolz, S. Forster, F. Effenberger and H.-J. Knackmuss, Arch. Microbiol.,
1992, 158, 405; A. M. Macadam and C. J. Knowles, Biotechnol. Lett.,
1985, 7, 865; M.-X. Wang and S.-J. Lin, J. Org. Chem., 2002, 67, 6542;
H. Idogaki, N. Kasai, M. Takeuchi, M. Hatada and T. Suzuki,
Tetrahedron: Asymmetry, 2001, 12, 369.
4 G. DeSantis, Z. Zhu, W. A. Greenberg, K. Wong, J. Chaplin, S. R.
Hanson, B. Farwell, L. W. Nicholson, C. L. Rand, D. P. Weiner, D. E.
Robertson and M. J. Burk, J. Am. Chem. Soc., 2002, 124, 9024.
5 F. Effenberger and S. Osswald, Synthesis, 2001, 12, 1866; T. Beard, M.
A. Cohen, J. S. Parratt, N. J. Turner, J. Crosby and J. Moilliet,
Tetrahedron: Asymmetry, 1993, 4, 1085; H. Kakeya, N. Sakai, A. Sano,
M. Yokoyama, T. Sugai and H. Ohta, Chem. Lett., 1991, 1823.
6 M. Yokoyama, T. Sugai and H. Ohta, Tetrahedron: Asymmetry, 1993, 4,
1081.
7 B. M. Trost and X. Ariza, Angew. Chem., Int. Ed. Engl., 1997, 36, 2635
and references therein; D. Ma and K. Ding, Org. Lett., 2000, 2, 2515; J.
S. Clark and M. D. Middleton, Org. Lett., 2002, 4, 765; T. Ooi, M.
Takeuchi, M. Kameda and K. Maruoka, J. Am. Chem. Soc., 2000, 122,
5228.
8 C. Cativiela and M. D. Diaz-de-Villegas, Tetrahedron: Asymmetry,
1998, 9, 3517 and references therein.
9 (a) Z.-L. Wu and Z.-Y. Li, Tetrahedron: Asymmetry, 2001, 12, 3305; (b)
Z.-L. Wu and Z.-Y. Li, Biotechnol. Appl. Biochem., 2002, 35, 61.
10 K. Faber, Biotransformations in organic chemistry: a textbook, 4th ed.,
Spring-Verlag, Berlin, 2000.
1f
1g
1g
1h
1h
1i
98
98c
99c
99d
Ii
H
> 99e
a All the reactions were carried out for 6 days at 30 °C or 7 days at 20 °C
unless stated otherwise. b Determined by HPLC on a Chiralpak AS column
with hexane–propan-2-ol mixtures unless stated otherwise. c Determined by
HPLC on a Chiralcel OJ column. d The reactions were carried out for 90 h.
e The reactions were carried out for 98 h.
The products of the enantioselective hydrolysis of a,a-
disubstituted malononitriles, (R)-a,a-disubstituted malonamic
acids 5, could afford either (R)- or (S)-a-alkylated amino acids
after routine conversion. For example, (R)-a-benzyl-a-me-
thylmalonamic acid 5a was transferred to (R)-9 or (S)-9 in a
yield of 81% or 89%. (Scheme 4). a-Methylphenylalanine 9 is
an efficient b-turn and helix former, much stronger than its non-
methylated parent compound phenylalanine.8
In conclusion, we have demonstrated a successful application
of the strain Rhodococcus sp. CGMCC 0497 in the asymmetric
hydrolysis of a,a-disubstituted malononitriles to afford en-
antiopure (R)-a,a-disubstituted malonamic acids, which can be
converted to either (R)- or (S)-a-alkylated amino acids. The new
strategy to carry out the reaction at a lower temperature greatly
improved the efficacy of the reaction.
11 M. Kobayashi, T. Nagasawa and H. Yamada, TIBTECH, 1992, 10,
402.
12 M. Kobayashi and S. Shimizu, Curr. Opin. Chem. Biol., 2000, 4, 95.
We are grateful to the National Science Foundation of China
(Grant No. 20032020) for financial support. We also thank
CHEM. COMMUN., 2003, 386–387
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