Enantioselective biotransformation of α,α-disubstituted dinitriles to the corresponding 2-cyanoacetamides using Rhodococcus sp. CGMCC 0497

Article abstract of DOI:10.1016/S0957-4166(03)00456-7

A new application of nitrile-converting enzymes in the synthesis of optically active α,α-disubstituted-α-cyanoacetamides from α,α-disubstituted-malononitriles with whole cells of Rhodococcus sp. CGMCC 0497 is described. The products were obtained with enantiomeric excesses of up to >99%, and yields of up to 53%. They are very useful chiral intermediates especially for the synthesis of chiral α,α-disubstituted amino acids but have never been synthesized directly by chemical or enzymatic methods.

Full text of DOI:10.1016/S0957-4166(03)00456-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2133–2142
Enantioselective biotransformation of a,a-disubstituted dinitriles
to the corresponding 2-cyanoacetamides using Rhodococcus sp.
CGMCC 0497
Zhong-Liu Wu and Zu-Yi Li*
State Key Laboratory of Bioorganic & Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
Received 5 April 2003; accepted 4 June 2003
Abstract—A new application of nitrile-converting enzymes in the synthesis of optically active a,a-disubstituted-a-cyanoacetamides
from a,a-disubstituted-malononitriles with whole cells of Rhodococcus sp. CGMCC 0497 is described. The products were obtained
with enantiomeric excesses of up to >99%, and yields of up to 53%. They are very useful chiral intermediates especially for the
synthesis of chiral a,a-disubstituted amino acids but have never been synthesized directly by chemical or enzymatic methods.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
metric hydrolysis of prochiral a,a-disubstituted dini-
triles, which, after asymmetric desymmetrization, could
afford important precursors of many physiologically
active compounds and chiral building blocks, such as
b-lactams12 and substituted succinic acids,13 especially
enantiopure a,a-dialkylated a-amino acids. This group
of amino acids induces dramatic conformational
changes when incorporated into peptides. They can act
as enzyme inhibitors and also as chiral building blocks
for the synthesis of pharmaceuticals and other biologi-
cal agents.14,15
The enantioselective biotransformation of nitriles has
attracted increasing interest in recent years due to the
easy availability of nitrile compounds and the versatile
utility of the optically active products, amides and
acids.1 So far, the kinetic resolutions of several classes
of racemic nitriles using nitrile-converting enzymes have
been studied in detail, such as a-alkyl nitriles,2 a-
hydroxy nitriles,3 a-acyloxy nitriles,4 a-amino nitriles,5
and b-acetoxy nitriles.6 There are also several reports
on the desymmetrization of 3-substituted glutaroni-
triles.3b,7 However, the hydrolysis of prochiral mal-
ononitriles has been somewhat neglected except that
Sugai and Ohta et al. have reported the successful
hydrolysis of a-butyl-a-methylmalononitrile to a-butyl-
a-methylmalonamic acid.8
We have made some progress in the production of
optically active malonamic acids by hydrolysis of a,a-
disubstituted dinitriles using Rhodococcus sp. CGMCC
0497,16 but the reaction time is very long, which
prompted us to try to find an alternative way. Clearly
the most important thing in the reaction is to enantiose-
lectively distinguish the two cyano groups by enzymes,
no matter that the products are cyanoamides,
cyanoacids or amideacids, because all can be converted
to amino acids. If cyanoamides or cyanoacids with high
enantiomeric excesses could be achieved instead of
amideacids, the reaction may be terminated earlier and
be more practical. Herein we report in detail the
hydrolysis of a-benzyl-a-methylmalononitrile 1a and
the synthesis of a variety of (S)-a,a-disubstituted-a-
cyanoacetamides via asymmetric hydrolysis of a,a-di-
substituted malononitriles catalyzed by the strain
Our previous study has demonstrated that Rhodococcus
sp. CGMCC 0497, a strain isolated from soil by our
group,9 was able to transform a variety of a-substituted
arylacetonitriles into optically active a-substituted aryl-
acetamides and a-substituted arylacetic acids with
excellent enantiomeric excesses,10 and b-hydroxy nitriles
into b-hydroxy amides and b-hydroxy acids with high
activity and moderate enantioselectivity.11 Quite
recently, we have focused our attention on the asym-
* Corresponding author. Tel.: +86-021-64163300-1406; fax: +86-021-
64166128; e-mail: lizy@pub.sioc.ac.cn
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00456-7

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Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
Rhodococcus sp. CGMCC 0497. The products were
achieved with enantiomeric excesses of up to >99%.
and enantiomeric excess of 3a seemed less regular, while
the absolute configuration of 2a was consistently S and
its enantiomeric excess evidently increased with pro-
longed reaction time (entries 3–6). The cyanoamide
(S)-2a could be obtained with an excellent enantiomeric
excess of >99% in a yield of 52% within 72 h (entry 5).
Considering the easy removal of acids 3a by basifica-
tion, the isolation of (S)-2a can be achieved
conveniently.
2. Results and discussion
In order to gain reproducible results, the fresh microor-
ganism cultivated under optimal conditions was used.
The enantiomeric excesses of cyanoamides 2 were deter-
mined directly or after conversion to the corresponding
ethyl esters by HPLC with chiral stationary phase. The
enantiomeric excesses of cyanoacids 3 and amideacids 5
were determined by means of HPLC with chiral sta-
tionary phase after conversion to the corresponding
methyl or ethyl esters. The absolute configurations of
3a17and 5a13 were obtained by comparing the direction
of specific rotations with those in literature, and that of
2a was assigned by comparing the retention time of the
corresponding ethyl ester with the samples derived from
3a using HPLC with chiral stationary phase. The abso-
lute configurations of other cyanoamide compounds 2
were assumed by the analogy with known enzyme-cata-
lyzed reaction. The cyanoacids 3 were converted to the
corresponding cyanoamides and their retention time on
chiral HPLC or direction of specific rotations were
Though it is well known that biotransformations of
nitriles to the corresponding acids proceed by two
distinct routes: by nitrilase or by a combination of
nitrile hydratase and amidase,1 the detailed information
on the pathway of the hydrolysis of 1a to the four
products 2a, 3a, 4a and 5a remains still largely unex-
plored. Our early work has demonstrated that product
5a was mostly derived from 4a by (R)-selective ami-
dase, rather than from 3a by nitrile hydratase.16 To
shed more light on the multi-step biotransformation
procedure,
(rac)-2-cyano-2-methyl-3-phenyl-
propanamide 2a was synthesized and subjected to the
reaction catalyzed by Rhodococcus sp. CGMCC 0497.
The results were illustrated in Scheme 2. It is clear that
cyanoacid 3a came from 2a by an (R)-selective ami-
dase. Calculation according to the yields and enan-
tiomeric excesses of 2a and 3a, as well as the rules of
kinetic resolution, demonstrated that more (S)-2a con-
verted to 4a and 5a than (R)-2a did. Thus, it can be
inferred that diamide 4a came from 2a by an (S)-selec-
tive nitrile hydratase and the total effect of the two
steps, 2a to 3a and 2a to 4a, was (R)-selective, because
the recovered 2a was of (S)-configuration. That is
consistent with the results obtained in Table 1, entries
3–5. The enantiomeric excess of (S)-2a increased as the
reaction continued, for (R)-2a was preferred in the
following steps and the (S)-2a was retained. Prolonging
the reaction time to 90 h, the enantiomeric excess of
compared with
configurations.
2
to determine the absolute
2.1. Hydrolysis of a-benzyl-a-methylmalononitrile
To investigate the possibility of transforming dinitriles
to chiral cyanoamides or cyanoacids by Rhodococcus
sp. CGMCC 0497, the hydrolysis of a-benzyl-a-methyl-
malononitrile 1a at different temperatures and pH was
studied in detail (Scheme 1). As shown in Table 1, in all
cases, the substrate was hydrolyzed completely, but the
products isolated were a complex mixture of hydrolysis
intermediates in most cases. The absolute configuration
Scheme 1.
Table 1. Hydrolysis of a-benzyl-a-methyl malononitrile 1a by Rhodococcus sp. CGMCC
Entry
pH
T (°C)
Time (h)
(S)-2a
3a
4a
Yield (%)
Ee (%)
Yield (%)
Ee (%)
Yield (%)
1
7.0
7.0
7.0
7.0
7.0
7.0
8.0
8.9
10
20
30
30
30
30
30
30
24
24
24
66
72
90
72
72
66
21
70
56
52
22
49
45
45
46
48
9
16 S
78 R
72 R
52 R
49 R
6 R
17
52
8
8
7
–
–
–
2a
3
12
19
33
35
35
46
50
4
93
5
>99
>99
87
6b
7
40 R
30 R
8
81
a 5a obtained in 13% yield.
b (R)-5a obtained in 41% yield and 88% ee.

Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
2135
Scheme 2.
(S)-2a remains >99%, while the yield decreased (Table
1, entry 6). In this situation, (R)-2a has been exhausted
and (S)-2a was consequently converted to 4a and 4a to
5a, or to 3a to make the enantiomeric excess of (S)-3a
decrease dramatically. We can further deduced from the
>50% yield and >99% enantiomeric excess of (S)-2a
(Table 1, entry 5) that the process of 1a to 2a was
catalyzed by an (S)-selective nitrile hydratase. The
enantiomeric excess of 2a was thus enhanced by the
processes that produce it (1a to 2a) and consume it (2a
to 3a and 4a), which exhibit the selectivity of the
opposite direction.
formations of 4 and 5 were not observed as that in the
hydrolysis of 1a.
Substrates 1b to 1h are most similar to 1a in structure
with a methyl and a benzyl group at the a position of
the malononitrile, but the benzyl is substituted by
groups such as methyl-, methoxy-, chloro-, bromo-, etc.
These substitutions especially relatively large p-methyl,
p-methoxy and o-chloro groups, seem to exert distinct
influences on the reaction. As show in Table 1, 1a could
be converted to amides and acids completely within 24
h, while 1b–h could not be converted even after 75 h
(Table 2, entries 1–9); hydrolysis of 1a could afford
(S)-2a successfully in 52% yield and >99% ee, while
hydrolysis of 1b–h only afforded 2b–h in yields of
20–42% and ee of 2–81%.
2.2. Hydrolysis of various a,a-disubstituted
malononitriles
Based on the above results, a variety of a,a-disubsti-
tuted malononitriles 1 were subjected to the enzymatic
transformation. The reactions were monitored by TLC
to produce chiral cyanoamides 2 with both good yield
and ee (Scheme 3).
It seemed that the substitutions increased steric hin-
drance, which made the conversion of dinitriles 1 to
cyanoamides 2 much slower. The slowness of the for-
mation of compound 2 led to the hydrolysis of (S)-2,
which had been expected to remain, as well as (R)-2 to
cyanoacid 3. At the same time, hydration of the
remaining dinitrile 1 to afford 2 was continuing with
low to medium enantioselectivity, which led to the low
ee value of product 2. On the contrary, a-benzyl-a-
methyl malononitrile 1a was converted completely to
To our disappointment, the reactions were generally
unsuccessful with the conversion of substrates incom-
plete and the enantiomeric excesses of products low to
medium. During the reaction time of 75–120 h, com-
pounds 2 and 3 were obtained in most cases, while the
Scheme 3.
Table 2. Hydrolysis of various a,a-disubstituted malononitriles 1 by Rhodococcus sp. CGMCC at 30°C
Entry
Substrate 1
X
R
Time (h)
Recovered 1 (%)
(S)-2
3
Yield (%)
Ee (%)
Yield (%)
Ee (%)
1
3
4
5
6
8
9
11
12
13
1b
1c
1d
1e
1f
1g
1h
1i
p-Me
p-F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH3
CH2CH3
Allyl
75
75
75
75
75
75
75
90
72
24
10
11
14
33
16
34
–
30
42
39
39
20
35
26
92
37
47
64
78
42
78
2
81
44
65
64
60a
30
40
39
17
33
41
29
–
14 S
16 R
ND S
NDa
30 S
ND R
NDa
–
p-Cl
p-Br
p-MeO
m-Cl
o-Cl
H
1j
1k
H
H
44
46
–
–
–
–
120
a Absolute configuration undetermined.

2136
Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
cyanoamide 2a first by Rhodococcus sp. CGMCC
(Table 1, entry 3), and 2a to 3a or 4a in succession.
Though the first step was less selective, the enan-
tiomeric excess of 2a was enhanced by the next steps.
was observed that in the process of the hydrolysis of 1c,
1d and 1f, there was very little 2 all along and the
reaction mixture consisted mainly of cyanoacids 3,
amideacids 5 and unreacted dinitriles 1. It seemed that
on this reaction condition, the conversion of 2 to the
diamide 4 accelerated as well as the conversion of 1 to
2, which made the accumulation of product 2 hard to
achieve and the amideacids 5 formed instead.
The products cyanoacids 3 were not our concern, but
their unexpected absolute configuration caught our
attention. Unlike any other situations in our enzymatic
hydrolysis of nitriles using Rhodococcus sp. CGMCC,
which gave products with concordant absolute configu-
ration, the hydrolysis of dinitriles gave cyanoacids 3
with absolute configuration of both R and S. We
speculated that this phenomenon may not be caused by
the difference of selectivity of the amidase towards
different substrates, for there are both (R)- and (S)-
cyanoacids for the same substrate (Table 1), but caused
by the complexity of the multi-step reaction. The ami-
dase is most probably (R)-selective consistently. The
transformation of (S)-2 following (R)-2 because of the
slow conversion of 1 to 2 may result in the formation of
(S)-cyanoacids 3 as well as the low ee of (S)-
cyanoamides 2.
While under the same condition, the hydrolysis of 1b,
1f and 1h, the relatively bulky substrates, gave products
(S)-2 with good to excellent enantiomeric excesses
(Table 3). The enantiomeric excess of 2b increased from
64% (Table 2, entry 1) to 88% (Table 3, entry 1); that of
2f increased from 2% (Table 2, entry 6) to 96% (Table
3, entry 2); that of 2h increased from 44% (Table 2,
entry 9) to >99% (Table 3, entry 3); and the yields of
these three cyanoamides increased as well. In these
cases, decreased temperatures enhanced the conversion
of dinitriles 1 to cyanoamides 2 more than 2 to
diamides 4, thus (S)-2 were successfully accumulated.
Interestingly, the absolute configurations of cyanoacids
3b and 3f are R, opposite to that got at 30°C. The
absolute configuration of 3 seems in close relation to
the enantiomeric excess of 2. The accelerated conver-
sion of 1 to 2, the enantioselective hydrolysis of (R)-2
and the retaining of (S)-2 resulted in both the forma-
tion of (R)-3 and the excellent enantiomeric excess of 2.
Compounds 1i–k are substrates bearing a benzyl or
phenylethyl group and a substitution of ethyl or allyl,
larger than methyl group of the substrates 1a–h, on the
a position of the malononitriles. It seems that amidase
in Rhodococcus sp. CGMCC 0497 is very sensitive to
the steric hindrance, for no acid was formed in the
three reactions. As observed in our early study, it is
difficult to hydrolyze this kind of amides with two
substituents both larger than methyl by the amidase in
Rhodococcus sp. CGMCC 0497.18 Though the hydroly-
sis of 1i to 2i proceeded smoothly and completely with
medium enantioselectivity, the ineffectiveness of ami-
dase towards 2i made the enantiomeric excess of 2i
remain as it formed. Prolonged reaction time took no
effect. 1j and 1k were recovered nearly half of the
feeding with 2j and 2k as the sole product.
2.3. Synthesis of enantiopure amino acids
The products of the enantioselective hydrolysis of a,a-
disubstituted
malononitriles,
(S)-a,a-disubstituted
cyanoacetamides 2, could be conveniently converted to
either (R)- or (S)-a-alkylated amino acids using routine
rearrangement reactions and hydrolysis reactions. For
example, enantiopure (S)-2-cyano-2-methyl-3-phenyl-
propanamide 2a was transferred to (R)-9 or (S)-9 in a
yield of 89 or 93% (Scheme 4). This non-proteinogenic
amino acid is an efficient b-turn and helix former, much
stronger than its non-methylated parent compound
phenylalanine.14a
To improve the yields and enantiomeric excesses of
(S)-cyanoamides 2, we carried out reactions at 20°C
instead of 30°C. We have observed that lower reaction
temperatures favor the conversion of the substrates in
the hydrolysis b-hydroxy nitriles by Rhodococcus sp.
CGMCC 0497.11 It was expected that the reactions at
20°C may result in the faster transformation of dini-
triles 1 to cyanoamides 2, then more (S)-2 could be
retained in the sequent amidase-catalyzed reaction.
3. Conclusion
We have shown a new application of nitrile-converting
enzymes to the synthesis of optically active a,a-disubsti-
Substrates 1b–h were subjected to the hydrolysis reac-
tion at 20°C. The reactions were monitored by TLC. It
tuted-a-cyanoacetamides
from
a,a-disubstituted-
malononitriles with whole cells of Rhodococcus sp.
Table 3. Hydrolysis of a,a-disubstituted malononitrile 1 by Rhodococcus sp. CGMCC 0497 at 20°C
Entry
Substrate 1
X
R
Time (h)
Recovered 1 (%)
(S)-2
3
Yield (%)
Ee (%)
Yield (%)
Ee (%)
1
2
3
1b
1f
1h
p-Me
p-MeO
o-Cl
CH3
CH3
CH3
85
90
95
7
11
6
49
51
53
88
96
>99
27
26
27
51 R
21 R
NDa
a Absolute configuration undetermined.

Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
2137
Scheme 4. Reagents and conditions: (i) DMF, Hg(OAc)2, NBS, MeOH, rt; (ii) 20% HCl, reflux; (iii) EtOH, 98% H2SO4, reflux;
(iv) EtOH, 30% H2O2, 10N NaOH, rt; (v) DMF, Hg(OAc)2, NBS, EtOH, rt.
CGMCC 0497. The products were obtained with enan-
tiomeric excesses of up to >99%, and yields of up to
53%. It was found that reaction temperature had a
great effect on the yields and enantiomeric excesses of
the products. As far as we know, homochiral a,a-disub-
stituted-a-cyanoacetamides have never been synthesized
directly by chemical or enzymatic methods in spite that
they are very useful homochiral intermediates. In this
paper, the enzymatic method, combined with the conve-
nient conversion of the products to amino acids, pro-
vides a new way to the synthesis of enantiopure
a,a-disubstituted amino acids.
temperature for 3–10 h. The reaction was quenched by
aqueous NH4Cl solution, extracted with ethyl acetate,
dried on MgSO4 and purified by flash chromatography.
The product was dissolved in acetone (50 ml). Methyl
iodide, or ethyl bromide, or allyl bromide (20 mmol)
and solid K2CO3 (25 mmol) were added. The mixture
was stirred overnight at room temperature. After filtra-
tion and concentration, the residue was purified by
flash chromatography to yield a,a-disubstituted
malononitriles.
Method B (for 1j and 1k): To a solution of malononi-
trile (1.32 g, 20 mmol) and phenylethyl bromide (10
mmol) in THF (40 ml) cooled by cryohydrate, was
added solid tert-BuOK (10 mmol). The mixture was
stirred overnight at room temperature. The following
procedure was as described in Method A.
4. Experimental
4.1. Materials and methods
4.2.1. 2-Benzyl-2-methylmalononitrile, 1a. Mp 94.3–
95.3°C, Lit.19 94.5–95.5°C; 1H NMR (300 MHz,
CDCl3): l 7.35–7.45 (m, 5H, Ar-H), 3.22 (s, 2H, CH2),
1.81 (s, 3H, CH3); IR (KBr): 2251 (CN); MS m/z (%):
170 (M+, 1), 91 (100).
The commercially available reagents were used without
further purification. Melting points were determined on
1
a Mettler FP62 and are uncorrected. H NMR spectra
were recorded on a Bruker AMX-300 (300 MHz) spec-
trometer at room temperature with TMS as internal
standard. Chemical shifts in ppm were positive for
upfield shifts. IR spectra were recorded neat or in KBr
and measured in cm−1, using a Shimadzu IR-440 IR
spectrophotometer. EI-MS spectra were recorded on an
HP 5989A. High-resolution mass spectra were obtained
on a Finnigan MAT8430. Microanalyses were carried
out on an Italian Carlo-Erba 1106. Polarimetry was
carried out using an optical activity Perkin–Elmer
241ML polarimeter and the measurements were made
at the sodium D-line with a 10 cm pathlength cell.
Concentrations (c) are given in g/100 ml. Enantiomeric
excesses: Chiral HPLC was conducted with a PE NEL-
SON NCI900 using Chiralpak AS, AD or Chiralcel OJ,
OD column at a flow rate of 0.7 ml/min with 2-
propanol/hexane as the mobile phase.
4.2.2. 2-(4%-Methylbenzyl)-2-methylmalononitrile, 1b. Mp
1
88.6–89.6°C; H NMR (300 MHz, CDCl3): l 7.25, 7.20
(AB, 4H, J=8.1 Hz, ArH), 3.17 (s, 2H, CH2), 2.37 (s,
3H, CH3), 1.79 (s, 3H, CH3); IR (KBr): 2253 (CN); MS
m/z (%): 184 (M+, 5), 105 (100). Anal. calcd for
C12H12N2: C, 78.23; H, 6.57; N, 15.20. Found: C, 78.36;
H, 6.50; N, 15.33.
4.2.3. 2-(4%-Florobenzyl)-2-methylmalononitrile, 1c. Mp
1
126.1–127.1°C; H NMR (300 MHz, CDCl3): l 7.38–
7.33 (m, 2H, ArH), 7.14–7.08 (m, 2H, ArH), 3.19 (s,
2H, CH2), 1.82 (s, 3H, CH3); IR (KBr): 2253 (CN); MS
m/z (%): 188 (M+, 2), 173 (1), 109 (100). Anal. calcd for
C11H19FN2: C, 70.20; H, 4.82; F, 10.09; N, 14.88.
Found: C, 70.22; H, 4.98; F, 10.05; N, 14.99.
4.2. General procedure for dinitriles synthesis
4.2.4. 2-(4%-Chrolobenzyl)-2-methylmalononitrile, 1d. Mp
1
Method A (for 1a–i): To a suspension of NaH (60%)
400 mg (10 mmol) in 40 ml THF and 4 ml DMF, was
added dropwise a solution of malononitrile (1.32 g, 20
mmol) in 5 ml THF at room temperature. After 2 h,
benzyl bromide or chloride (10 mmol) in THF (10 ml)
was added within 2 h. Stirring was continued at room
88.9–89.9°C; H NMR (300 MHz, CHCl3): l 7.40, 7.31
(AB, 4H, J=8.4 Hz, ArH), 3.18 (s, 2H, CH2), 1.82 (s,
3H, CH3); IR (KBr): 2254 (CN); MS m/z (%): 206
(M++2, 1), 204 (M+, 4), 127 (33), 125 (100). Anal. calcd
for C11H9ClN2: C, 64.56; H, 4.43; Cl, 17.32; N, 13.69.
Found: C, 64.43; H, 4.60; Cl, 17.39; N, 13.68.

2138
Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
4.2.5. 2-(4%-Bromobenzyl)-2-methylmalononitrile, 1e. Mp
containing 20 ml of a medium consisting of 0.5 g of
polypepton, 0.5 g of beef extract and 1 g of glucose per
100 ml of tap water, pH 7.0. Then the subculture was
inoculated into a 5 l shaking flask containing 1 l of the
rich medium consisting of 1 g of glucose, 0.5 g of beef
1
86.3–87.3°C; H NMR (300 MHz, CHCl3): l 7.55, 7.25
(AB, 4H, J=8.4 Hz, ArH), 3.17 (s, 2H, CH2), 1.82 (s,
3H, CH3); IR (KBr): 2247 (CN); MS m/z (%): 250
(M++2, 5), 248 (M+, 5), 171 (94), 169 (100). Anal. calcd
for C11H9BrN2: C, 53.04; H, 3.64; Br, 32.08; N, 11.25.
Found: C, 53.05; H, 3.48; Br, 32.20; N, 11.06.
extract, 0.25
g of methacrylamide, 100 mg of
K2HPO4·3H2O, 75 mg of KH2PO4, 10 mg of NaCl, 0.1
ml of mineral medium per 100 ml of tap water with
methacrylamide added 24 h later. The pH of each
medium was adjusted to around 7.0–7.2 by addition of
2N NaOH or 3N HCl. After incubation at 30°C with
reciprocal shaking for 48 h, the organism was harvested
by centrifugation using an HIMAC centrifuge CR20B2
(Hitachi, Japan) with a RPR9-2 rotor (6800g, 30 min
10°C). Cells were washed with 100 mM potassium
phosphate buffer (pH 7.0) and centrifugated.
4.2.6. 2-(4%-Methoxybenzyl)-2-methylmalononitrile, 1f.
Mp 68–69°C; 1H NMR (300 MHz, CDCl3): l 7.28, 6.93
(AB, 4H, J=9.0 Hz, ArH), 3.82 (s, 3H, OMe), 3.16 (s,
2H, CH2), 1.79 (s, 3H, CH3); IR (KBr): 2253 (CN); MS
m/z (%): 200 (M+, 4), 121 (100). Anal. calcd for
C12H12N2O: C, 71.98; H, 6.04; N, 13.99. Found: C,
71.87; H, 6.05; N, 13.80.
4.2.7. 2-(3%-Chrolobenzyl)-2-methylmalononitrile, 1g. Mp
1
81.3–82.3°C; H NMR (300 MHz, CDCl3): l 7.39–7.46
4.4. General procedure with whole cells and determina-
tion of enantiomeric excess
(m, 4H, ArH), 3.19 (s, 2H, CH2), 1.84 (s, 3H, CH3); IR
(KBr): 2250 (CN); MS m/z (%): 206 (M++2, 4), 204
(M+, 12), 127 (34), 125 (100). Anal. calcd for
C11H9ClN2: C, 64.56; H, 4.43; Cl, 17.32; N, 13.69.
Found: C, 64.39; H, 4.24; Cl, 17.15; N, 13.72.
A suspension of 10 g washed wet cells and 80 ml 0.1
mM potassium phosphate buffer (pH 7.0) was incu-
bated at 30 or 20°C for 30 min with continuous mag-
netic stirring before the addition of the substrate, a
solution of 100 mg of a,a-disubstituted malononitrile
dissolved in 100 ml of acetone. The reaction was
quenched by centrifugation. The resulting supernatant
was made alkaline with 2N NaOH to pH 12 and
extracted with diethyl ether. The organic solutions,
after drying (MgSO4) and concentration, gave the
amide 2 and 4, and unreacted dinitrile 1. Separation
was effected by column chromatography. The aqueous
solution was then acidified using 3N HCl to pH 2 and
extracted with diethyl ether. After concentration, the
residue was purified by flash chromatography on silica
gel (elute: petroleum ether/EtOAc/AcOH 150:100:1) to
afford the acids 3 and 5.
4.2.8. 2-(2%-Chrolobenzyl)-2-methylmalononitrile, 1h. Mp
1
61.9–62.9°C; H NMR (300 MHz, CHCl3): l 7.55–7.51
(m, 1H, ArH), 7.51–7.46 (m, 1H, ArH), 7.36–7.26 (m,
2H, ArH), 3.48 (s, 2H, CH2), 1.85 (s, 3H, CH3); IR
(KBr): 2253 (CN); MS m/z (%): 206 (M++2, 2), 204
(M+, 6), 127 (34), 125 (100). Anal. calcd for C11H9ClN2:
C, 64.56; H, 4.43; Cl, 17.32; N, 13.69. Found: C, 64.79;
H, 4.65; Cl, 17.29; N, 13.69.
4.2.9. 2-Benzyl-2-ethylmalononitrile, 1i. White solid, mp
59.4–60.4°C, Lit.20 mp. 60°C; 1H NMR (300 MHz,
CHCl3): l 7.42–7.36 (m, 5H, ArH), 3.21 (s, 2H, CH2),
2.02 (q, 2H, J=7.2 Hz, CH2), 1.32 (t, 3H, J=7.5 Hz,
CH3); IR (KBr): 2247 (CN); MS m/z (%): 184 (M+, 2),
155 (M+−Et, 2), 91 (100).
To a solution of a,a-disubstituted cyanoacetic acid 3
(0.1 mmol) in DMF (0.1 ml) was added bromoethane
or iodomethane (2 mmol) and anhydrous K2CO3 (2
mmol). The reaction was carried out at room tempera-
ture for one day to achieve the esters and the esters
were subjected to chiral HPLC.
4.2.10. 2-Phenylethyl-2-ethylmalononitrile, 1j. Oil, 1H
NMR (300 MHz, CDCl3): l 7.38–7.33 (m, 2H, ArH),
7.30–7.23 (m, 3H, ArH), 3.04–2.98 (m, 2H, CH2), 2.24–
2.19 (m, 2H, CH2), 2.06 (q, 2H, J=7.5 Hz, CH2), 1.32
(t, 3H, J=7.5 Hz, CH3); IR (film): 2248 (CN); MS m/z
(%): 198 (M+, 21), 105 (46), 91 (100); HRMS calcd for
(C13H14N2)+: 198.11570. Found: 198.11300.
Amides 2 were subjected to chiral HPLC directly, or
dissolved in ethanol gently refluxed at the presence of
catalytic concentrated sulfuric acid for 3 h to yield the
corresponding esters.
4.2.11. 2-Phenylethyl-2-allylmalononitrile, 1k. Oil, 1H
NMR (300 MHz, CDCl3): l 7.37–7.31 (m, 2H, ArH),
7.29–7.21 (m, 3H, ArH), 5.98–5.84 (m, 1H, CHꢀC),
5.46–5.37 (m, 2H, CH2ꢀC), 3.02–2.96 (m, 2H, CH2),
2.74 (d, 2H, J=7.5 Hz, CH2), 2.23–2.17 (m, 2H, CH2);
IR (film): 2249 (CN); MS m/z (%): 211 (M++1, 4), 210
(M+, 27), 91 (100); HRMS calcd for (C14H14N2)+:
210.11570. Found: 210.11255.
4.4.1. Enzymatic hydrolysis of 2-benzyl-2-methyl-
malononitrile,
1a.
(S)-2-Cyano-2-methyl-3-phenyl-
propanamide 2a. Mp 96.5–97.5°C; [h]1D7=+46.85 (c
0.98, CHCl3), >99% ee; enantiomeric excess was deter-
mined by HPLC on a Chiralcel OD column with hex-
ane/2-propanol mixtures 8:2 and the retention time for
the (R)- and (S)-enantiomers was 13.8 and 16.8 min,
1
4.3. Microorganism and cultivation
respectively; H NMR (300 MHz, CDCl3): l 7.40–7.20
(m, 5H, Ar-H), 6.04 (s, br, 1H, NH), 5.52 (s, br, 1H,
NH), 3.26 (d, 1H, J=13.5 Hz, CH), 2.96 (d, 1H,
J=13.5 Hz, CH), 1.66 (s, 3H, CH3); IR (KBr): w 3378,
3320 (NH), 2244 (CN), 1666 (CꢀO); MS m/z (%): 189
(M++1, 3), 188 (M+, 21), 173 (M+−CH3, 4), 144 (M+−
The strain Rhodococcus sp. CGMCC 0497 is available
in CGMCC (China General Microbiological Culture
Collection Center). Rhodococcus sp. CGMCC 0497 was
subcultured at 30°C for 24 h in a 100 ml shaking flask

Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
2139
CONH2, 19), 91 (100). Anal. calcd for C11H12N2O: C,
70.19; H, 6.43; N, 14.88. Found: C, 69.98; H, 6.51; N,
14.81. (R)- or (S)-2-Cyano-2-methyl-3-phenylpropanoic
acid 3a. Mp 94.6–95.6°C, Lit.17 84°C; [h]D25=−15.1 (c
0.53, CHCl31), 52% ee, R, {Lit.17 [h]D=27.2 (c 2,
CHCl3), S}; H NMR (300 MHz, CDCl3): l 9.83 (br s,
1H, OH), 7.37–7.28 (m, 5H, Ar-H), 3.27 (d, 1H, J=
13.5 Hz, CH), 3.07 (d, 1H, J=13.8 Hz), 1.65 (s, 3H,
CH3); IR (KBr): w 3074 (br OH), 2263 (CN), 1747
(CꢀO); MS m/z (%): 189 (M+, 2), 174 (M+−CH3, 3), 144
(M+−COOH, 3), 91 (100). 2-Benzyl-2-methylmalon-
amide 4a. White solid, mp 195.4–196.4°C, Lit.21 202–
CONH2, 8), 109 (100). Anal. calcd for C11H11FN2O: C,
64.07; H, 5.38; F, 9.21; N, 13.58. Found: C, 64.05; H,
5.39; F, 9.05; N, 13.62. (R)-2-Cyano-2-methyl-3-(4%-
florophenyl)propanoic acid 3c. [h]2D1=−3.8 (c 0.177,
1
CHCl3), 16% ee; H NMR (300 MHz, CDCl3): l 8.58
(br s, 1H, OH), 7.31–7.27 (m, 2H, ArH), 7.05–6.99 (m,
2H, ArH), 3.25 (d, 1H, J=13.8 Hz, CH), 3.02 (d, 1H,
J=13.5 Hz, CH), 1.64 (s, 3H, CH3); IR (film): 2950–
3200 (br OH), 2254 (CN), 1733 (CꢀO); MS m/z (%):
207 (M+, 2), 162 (6), 161 (7), 109 (100); HRMS calcd
for (C11H10FNO2)+: 207.06960. Found: 207.07429.
1
203°C; H NMR (300 MHz, DMSO): l 7.22–7.12 (m,
4.4.4. Enzymatic hydrolysis of 2-(4%-chrolobenzyl)-2-
methylmalononitrile, 1d. (S)-2-Cyano-2-methyl-3-(4%-
chlorophenyl)propanamide 2d. Mp 118.8–119.8°C;
[h]2D6=+15.7 (c 0.57, CHCl3), 42% ee; enantiomeric
excess was determined by HPLC on a Chiralpak AD
column with hexane/2-propanol mixtures 9:1 and the
retention time for (R)- and (S)-enantiomer was 17.8
and 15.5 min, respectively; 1H NMR (300 MHz,
CDCl3): l 7.47, 7.18 (AB, 4H, J=8.1 Hz, ArH), 6.06
(br s, 1H, NH), 5.57 (br s, 1H, NH), 3.23 (d, 1H,
J=13.5 Hz, CH), 2.90 (d, 1H, J=13.2 Hz, CH), 1.66 (s,
3H, CH3); IR (KBr): 3456, 3311, 3196, 2249 (CN), 1664
(CꢀO); MS m/z (%): 224 (M++2, 3), 222 (M+, 7), 127
(33), 125 (100). Anal. calcd for C11H12ClNO3: C, 59.33;
H, 4.98; Cl, 15.92; N, 12.58. Found: C, 59.46; H, 4.98;
Cl, 15.90; N, 12.64. (S)-2-Cyano-2-methyl-3-(4%-
chlorophenyl)propanoic acid 3d. [h]2D1=+8.0 (c 1.46,
9H, ArH, 2NH2), 3.06 (s, 2H, CH2), 1.10 (s, 3H, CH3);
IR (KBr): w 3389, 3205 (NH), 1692, 1663 (CꢀO); MS
m/z (%): 206 (M+, 7), 189 (10), 162 (M+−CONH2, 100),
160 (41). (R)-2-Benzyl-2-methylmalonamic acid 5a.
White solid, mp 117.9–118.9°C, Lit.13 120–121°C;
[h]1D5=−15.0 (c 1.07, MeOH), 94% ee; {Lit.13 [h]D=−4.4
1
(c 0.5, MeOH), R}; H NMR (300 MHz, acetone-d6): l
7.29–7.19 (m, 5H, ArH), 3.61 (br s, 3H, NH2, OH), 3.25
(d, 1H, J=13.8 Hz), 3.20 (d, 1H, J=13.5 Hz), 1.37 (s,
3H, CH3); IR (KBr): w 3423, 3204 (NH), 3034 (br OH),
1745, 1659 (CꢀO); MS m/z (%): 207 (M+, 2), 91 (100).
4.4.2. Enzymatic hydrolysis of 2-(4%-methylbenzyl)-2-
methylmalononitrile, 1b. (S)-2-Cyano-2-methyl-3-(4%-
methylphenyl)propanamide 2b. Mp 114.1–115.1°C;
[h]D26=+12.0 (c 0.25, CHCl3), 64% ee; [h]2D5=+16.7 (c
0.76, CHCl3), 88% ee; enantiomeric excess was deter-
mined by HPLC on a Chiralpak AD column with
hexane/2-propanol mixtures 9:1 and the retention time
for R and S enantiomer was 17.2 and 11.2 min, respec-
1
CHCl3); H NMR (300 MHz, CDCl3): l 7.6 (br s, 1H,
OH), 7.32, 7.25 (AB, 4H, J=8.4 Hz, ArH), 3.25 (d, 1H,
J=14.1 Hz, CH), 3.02 (d, 1H, J=13.8 Hz, CH), 1.65 (s,
3H, CH3); IR (film): 2800–3200 (br OH), 2254 (CN),
1731 (CꢀO); MS m/z (%): 225 (M++2, 3), 223 (M+, 9),
127 (33), 125 (100); HRMS calcd for (C11H10ClNO2)+:
223.04001. Found: 223.04123.
1
tively; H NMR (300 MHz, CDCl3): l 7.19, 7.14 (AB,
4H, J=8.4 Hz, ArH), 6.04 (br s, 1H, NH), 5.59 (br s,
1H, NH), 3.22 (d, 1H, J=13.5 Hz, CH), 2.92 (d, 1H,
J=13.5 Hz, CH), 2.34 (s, 3H, CH3), 1.64 (s, 3H, CH3);
IR (KBr): 3443, 3420, 3189 (br NH), 2237 (CN), 1695
(CꢀO); MS m/z (%): 202 (M+, 6), 105 (100). Anal. calcd
for C12H14N2O: C, 71.26; H, 6.98; N, 13.85. Found: C,
71.32; H, 7.04; N, 13.78. (R) or (S)-2-Cyano-2-methyl-
3-(4%-methylphenyl)propanoic acid 3b. [h]2D4=+3.5 (c
4.4.5. Enzymatic hydrolysis of 2-(4%-bromobenzyl)-2-
methylmalononitrile, 1e. (S)-2-Cyano-2-methyl-3-(4%-
bromophenyl)propanamide 2e. Mp 119.5–120.5°C;
[h]2D6=+23.0 (c 0.33, CHCl3), 78% ee; enantiomeric
excess was determined by HPLC on a Chiralpak AD
column with hexane/2-propanol mixtures 9:1 and the
retention time for (R)- and (S)-enantiomer was 17.3
and 14.8 min, respectively; 1H NMR (300 MHz,
CDCl3): l 7.32, 7.24 (AB, 4H, J=8.4 Hz, ArH), 6.06
(br s, 1H, NH), 5.57 (br s, 1H, NH), 3.24 (d, 1H,
J=13.5 Hz, CH), 2.92 (d, 1H, J=13.2 Hz, CH), 1.66 (s,
3H, CH3); IR (KBr): 3389, 3312, 3205, 2248 (CN), 1669
(CꢀO); MS m/z (%): 268 (M++2, 9), 266 (M+, 8), 224
(4), 222 (4), 171 (91), 169 (100). Anal. calcd for
C11H11BrN2O: C, 49.46; H, 4.15; Br, 29.91; N, 10.49.
Found: C, 49.59; H, 4.16; Br, 30.05; N, 10.55. 2-Cyano-
2-methyl-3-(4%-bromophenyl)propanoic acid 3e. [h]1D8=
1
1.05, CHCl3), 14% ee, S; H NMR (300 MHz, CDCl3):
l 7.19, 7.14 (AB, 4H, J=8.4 Hz, ArH), 6.45 (br s, 1H,
OH), 3.23 (d, 1H, J=13.5 Hz, CH), 3.01 (d, 1H,
J=13.5 Hz, CH), 2.33 (s, 3H, CH3), 1.65 (s, 3H, CH3);
IR (film): 2850–3250 (br OH), 2251 (CN), 1739 (CꢀO);
MS m/z (%): 203 (M+, 4), 105 (100); HRMS calcd for
(C12H13NO2)+: 203.09463. Found: 203.09419.
4.4.3. Enzymatic hydrolysis of 2-(4%-florobenzyl)-2-
methylmalononitrile, 1c. (S)-2-Cyano-2-methyl-3-(4%-
florophenyl)propanamide 2c. Mp 138.4–139.4°C;
[h]2D2=+26.4 (c 0.582, CHCl3). 78% ee; enantiomeric
excess was determined by HPLC on a Chiralpak AD
column with hexane/2-propanol mixtures 9:1 and the
retention time for (R)- and (S)-enantiomer was 17.3
and 15.0 min, respectively; 1H NMR (300 MHz,
CDCl3): l 7.31–7.26 (m, 2H, ArH), 7.06–7.01 (m, 2H,
ArH), 6.05 (br s, 1H, NH), 5.55 (br s, 1H, NH), 3.25 (d,
1H, J=13.5 Hz, CH), 2.92 (d, 1H, J=13.5 Hz, CH),
1.65 (s, 3H, CH3); IR (KBr): 3472, 3316, 2249 (CN),
1664 (CꢀO); MS m/z (%): 206 (M+, 5), 162 (M+−
1
+8.75 (c 1.37, CHCl3); H NMR (300 MHz, CDCl3): l
7.48, 7.24 (AB, 4H, J=8.7 Hz, ArH), 6.99 (br s, 1H,
OH), 3.23 (d, 1H, J=13.8 Hz, CH), 3.01 (d, 1H,
J=13.5 Hz, CH), 1.65 (s, 3H, CH3); IR (film): 2800–
3200 (br OH), 2254 (CN), 1731 (CꢀO); MS m/z (%):
269 (M++2, 9), 267 (M+, 9), 171 (96), 169 (100), 115
(17), 90 (38); HRMS calcd for (C11H10BrNO2)+:
266.98949. Found: 266.98855.

2140
Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
4.4.6. Enzymatic hydrolysis of 2-(4%-methoxybenzyl)-2-
methylmalononitrile, 1f. (S)-2-Cyano-2-methyl-3-(4%-
methoxyphenyl)propanamide 2f. Mp 95.9–96.9°C;
[h]2D8=+41.0 (c 1.60, CHCl3), 96% ee; enantiomeric
excess was determined by HPLC on a Chiralpak AS
column with hexane/2-propanol mixtures 8:2 and the
retention time for (R)- and (S)-enantiomer was 19.3
and 16.1 min, respectively; 1H NMR (300 MHz,
CDCl3): l 7.22, 6.86 (AB, 4H, J=8.4 Hz, ArH), 6.04
(br s, 1H, NH), 5.61 (br s, 1H, NH), 3.80 (s, 3H, OMe),
3.19 (d, 1H, J=13.5 Hz, CH), 2.90 (d, 1H, J=13.5 Hz,
CH), 1.63 (s, 3H, CH3); IR (KBr): 3427, 3307, 3201 (br
NH), 2247 (CN), 1670 (CꢀO); MS m/z (%): 218 (M+,
6), 174 (M+−CONH2, 1), 121 (100). Anal. calcd for
C12H14N2O2: C, 66.04; H, 6.47; N, 12.84. Found: C,
66.00; H, 6.23; N, 12.97. (R) or (S)-2-Cyano-2-methyl-
3-(4%-methoxyphenyl)propanoic acid 3f. [h]1D9=+7.5 (c
IR (KBr): 3408, 3199, 3140 (NH), 2244 (CN), 1702
(CꢀO); MS m/z (%): 225 (M++3, 1), 223 (M++1, 2), 187
(M+−Cl, 77), 127 (34), 125 (100). Anal. calcd for
C11H12ClNO3: C, 59.33; H, 4.98; Cl, 15.92; N, 12.58.
Found: C, 59.28; H, 4.96; Cl, 16.18; N, 12.60. 2-Cyano-
2-methyl-3-(2%-chlorophenyl)propanoic acid 3h. 1H
NMR (300 MHz, CDCl3): l 7.48–7.40 (m, 2H, ArH),
7.30–7.26 (m, 2H, ArH), 6.05 (br s, 1H, OH), 3.45 (d,
1H, J=14.1 Hz, CH), 3.37 (d, 1H, J=14.4 Hz, CH),
1.69 (s, 3H, CH3); IR (film): w 3483 (br OH), 2253
(CN), 1731 (CꢀO); MS m/z (%): 225 (M++2, 3), 223
(M+, 10), 127 (37), 125 (100); HRMS calcd for
(C11H10ClNO2)+: 223.04001. Found: 223.03989.
4.4.9. Enzymatic hydrolysis of 2-benzyl-2-ethylmalononi-
trile, 1i. (S)-2-Cyano-2-ethyl-3-phenylpropanamide 2i.
Mp 115.2–116.2°C; [h]1D9=+38.9 (c 0.941, CHCl3), 65%
1
1
1.0, CHCl3), 30% ee, S; H NMR (300 MHz, CDCl3):
ee; H NMR (300 MHz, CDCl3): l 7.36–7.28 (m, 5H,
l 7.22, 6.87 (AB, 4H, J=8.4 Hz, ArH), 6.58 (br s, 1H,
OH), 3.80 (s, 3H, OMe), 3.22 (d, 1H, J=13.5 Hz, CH),
3.00 (d, 1H, J=13.8 Hz, CH), 1.62 (s, 3H, CH3); IR
(film): 2850–3250 (br OH), 2253 (CN), 1747 (CꢀO); MS
m/z (%): 219 (M+, 2), 121 (100); HRMS calcd for
ArH), 6.05 (br s, 1H, NH), 5.79 (br s, 1H, NH), 3.23 (d,
1H, J=13.5 Hz, CH), 2.96 (d, 1H, J=13.5 Hz), 2.13
(m, 1H, CH), 1.83 (m, 1H, CH), 1.11 (t, 3H, J=7.2 Hz,
CH3); IR (KBr): 3390, 3185 (br NH), 2245 (CN), 1695
(CꢀO); MS m/z (%): 202 (M+, 15), 187 (1), 91 (100).
Anal. calcd for C12H14N2O: C, 71.26; H, 6.98; N, 13.85.
Found: C, 71.22; H, 6.95; N, 13.87.
(C11H13NO)+:
175.09795.
[(M+−CH3)]
175.09971.
Found:
4.4.7. Enzymatic hydrolysis of 2-(3%-chrolobenzyl)-2-
methylmalononitrile, 1g. (S)-2-Cyano-2-methyl-3-(3%-
chlorophenyl)propanamide 2g. Mp 137.0–138.0°C;
[h]2D5=+33.7 (c 0.895, CHCl3), 81% ee; enantiomeric
excess was determined by HPLC on a Chiralpak AD
column with hexane/2-propanol mixtures 9:1 and the
retention time for (R)- and (S)-enantiomer was 18.3
and 13.6 min, respectively; 1H NMR (300 MHz,
CDCl3): l 7.29–7.19 (m, 4H, ArH), 6.15 (br s, 1H,
NH), 5.95 (br s, 1H, NH), 3.24 (d, 1H, J=13.5 Hz,
CH), 2.91 (d, 1H, J=13.5 Hz, CH), 1.65 (s, 3H, CH3);
IR (KBr): 3461, 3325, 3190, 2245 (CN), 1668 (CꢀO),
1626, 1406, 1243, 792; MS m/z (%): 224 (M++2, 9), 222
(M+, 23), 180 (13), 178 (36), 127 (33), 125 (100). Anal.
calcd for C11H11ClN2O: C, 59.33; H, 4.98; Cl, 15.92; N,
12.58. Found: C, 59.39; H, 5.01; Cl, 15.82; N, 12.67.
(R)-2-Cyano-2-methyl-3-(3%-chlorophenyl)propanoic
acid 3g. Mp 101.0–102.0°C; 1H NMR (300 MHz,
CDCl3): l 7.32–7.21 (m, 4H, ArH), 6.22 (br s, OH),
3.25 (d, 1H, J=13.8 Hz, CH), 3.03 (d, 1H, J=13.5 Hz,
CH), 1.66 (s, 3H, CH3); IR (film): 3477 (br OH), 2253
(CN), 1741 (CꢀO); MS m/z (%): 225 (M++2, 2), 223
(M+, 7), 127 (32.8), 125 (100); HRMS calcd for
(C11H10ClNO2)+: 223.04001. Found: 223.03706.
4.4.10. Enzymatic hydrolysis of 2-phenylethyl-2-ethyl-
malononitrile,
1j.
(S)-2-Cyano-2-ethyl-4-phenylbu-
1
tamide 2j. 64% ee, [h]9D=+11.1 (c 1.076, CHCl3). H
NMR (300 MHz, CDCl3): l 7.32–7.29 (m, 2H, ArH),
7.23–7.18 (m, 3H, ArH), 6.42 (s, br, 1H, NH), 6.14 (s,
br, 1H, NH), 2.84 (td, 1H, J1=12.9 Hz, J2=4.8 Hz,
CH), 2.70 (td, 1H, J1=12.9 Hz, J2=5.0 Hz, CH), 2.25
(td, 1H, J1=12.9 Hz, J2=5.2 Hz, CH), 2.06–1.98 (m,
2H), 1.81 (dt, J1=21.3 Hz, J2=7.2 Hz, CH), 1.11 (t,
3H, J=7.2 Hz, CH3); IR (KBr): 3383, 3182 (NH), 2245
(CN), 1694 (CꢀO); MS m/z (%): 217 (M++1, 4), 200 (2),
112 (100), 105 (25); HRMS calcd for (C13H16N2O)+:
216.12626. Found: 216.12489.
4.4.11. Enzymatic hydrolysis of 2-phenylethyl-2-allyl-
malononitrile,
1k.
(S)-2-Cyano-2-allyl-4-phenylbu-
tamide 2k. Mp 99.8–108.8°C; [h]1D7=+5.5 (c 0.86,
1
CHCl3), 60% ee; H NMR (300 MHz, CDCl3): l 7.32–
7.14 (m, 5H, ArH), 6.34 (s, br, 1H, NH), 5.95 (s, br,
1H, NH), 5.91–5.76 (m, 1H, ꢀCH), 5.30 (d, 1H, J=6.3
Hz, ꢀCH), 5.25 (s, 1H, ꢀCH), 2.89–2.79 (m, 1H), 2.75–
2.63 (m, 2H), 2.53–2.45 (m, 1H), 2.32–2.17 (m, 1H),
2.05–1.93 (m, 1H); IR (KBr): 3388, 3178 (NH), 2246
(CN), 1695 (CꢀO); MS m/z (%): 229 (M++1, 1), 124
(100), 91 (49); HRMS calcd for (C14H16N2O)+:
228.12626. Found: 228.12724.
4.4.8. Enzymatic hydrolysis of 2-(2%-chrolobenzyl)-2-
methylmalononitrile, 1h. (S)-2-Cyano-2-methyl-3-(2%-
chlorophenyl)propanamide 2h. Mp 138.9–139.9°C;
[h]D25=+25.4 (c 1.14, CHCl3), 44% ee; [h]2D5=+64.9 (c
1.54, CHCl3), >99% ee; enantiomeric excess was deter-
mined by HPLC on a Chiralpak AD column with
hexane/2-propanol mixtures 9:1 and the retention time
for R and S enantiomer was 18.3 and 15.1 min, respec-
4.4.12. Enzymatic hydrolysis of (rac)-2-cyano-2-methyl-
3-phenylpropanamide, 2a. A suspension of 7 g of
washed wet cells and 56 ml of 0.1 mM potassium
phosphate buffer (pH 7.0) was incubated at 30°C for 30
min with continuously magnetic stirring, then a solu-
tion of 70 mg (rac)-2-cyano-2-methyl-3-phenyl-
propanamide 2a dissolved in 70 ml of acetone was
added. The reaction was continued for 24 h and
quenched by centrifugation. The post treatment was
carried out as described in General procedure. The
1
tively; H NMR (300 MHz, CDCl3): l 7.44–7.37 (m,
2H, ArH), 7.28–7.24 (m, 2H, ArH), 6.22 (br s, 1H,
NH), 5.78 (br s, 1H, NH), 3.41 (d, 1H, J=14.1 Hz,
CH), 3.33 (d, 1H, J=14.1 Hz, CH), 1.68 (s, 3H, CH3);

Z.-L. Wu, Z.-Y. Li / Tetrahedron: Asymmetry 14 (2003) 2133–2142
2141
resulting product was purified by flash chromatography
to afford mg (S)-2-cyano-2-methyl-3-phenyl-
4.5.3.
(R)-2-Ethoxycarbonylamino-2-methyl-3-phenyl-
9
propanoic acid ethyl ester, 8. The product 7 was dis-
solved in dry DMF (1.5 ml). Hg(OAc)2 (61 mg, 0.19
mmol), dry ethanol (220 mg, 5 mmol) and NBS (37 mg,
0.21 mmol) were added at room temperature. After 16
h, the resulting mixture was poured into water and
extracted with ether, washed and dried on Na2SO4. The
solvent was removed under reduced pressure and the
resulting product was purified by flash chromatography
to afford (R)-8 (98% yield). Oil; enantiomeric excess
was determined by HPLC on a Chiralcel OD column
with hexane/2-propanol mixtures 8:2 and the retention
time for (R)- and (S)-enantiomer was 10.4 and 17.8
propanamide 2a (13% yield, 61% ee), 17 mg (R)-2-
cyano-2-methyl-3-phenylpropanoic acid 3a (24% yield,
96% ee), 12 mg 2-benzyl-2-methylmalonamide 4a (16%
yield) and 35 mg (R)-2-benzyl-2-methylmalonamic acid
5a (45% yield, 97% ee). The physical data of the
products are the same as those described in Section
4.4.1.
4.5. Synthesis of amino acids
4.5.1. (S)-2-Methoxycarbonylamino-2-methyl-3-phenyl-
propanonitrile, 6. The product 2a (0.16 mmol) was
dissolved in dry DMF (1.5 ml). Hg(OAc)2 (61 mg, 0.19
mmol), dry methanol (5 mmol) and NBS (37 mg, 0.21
mmol) were added at room temperature. After 16 h, the
resulting mixture was poured into water and extracted
with ether, washed and dried on Na2SO4. The solvent
was removed under reduced pressure and the resulting
product was purified by flash chromatography to afford
(S)-6 (96% yield). Lit.17 mp 81°C; [h]2D2=−46.6 (c 0.798,
CHCl3), {Lit.17 [h]D=−46.1 (c 2, CHCl3), S}; enan-
tiomeric excess was determined by HPLC on a Chiral-
cel OJ column with hexane/2-propanol mixtures 9:1
and the retention time for (R)- and (S)-enantiomer was
1
min, respectively; H NMR (300 MHz, CDCl3): l 7.30–
7.23 (m, 3H, Ar-H), 7.08–7.05 (m, 2H, Ar-H), 5.37 (br
s, 1H, NH), 4.27–4.10 (m, 4H, 2OCH2), 3.43 (d, 1H,
J=13.8 Hz, CH), 3.18 (d, 1H, J=13.2 Hz, CH), 1.64 (s,
3H, CH3), 1.33–1.23 (m, 6H, 2CH3); IR (film): w 3358
(NH), 1721 (CꢀO), 704; MS m/z (%): 279 (M+, 1), 206
(M+−COOEt, 43), 188 (100); HRMS calcd for
(C15H21NO4)+: 279.14706. Found: 279.15017.
4.5.4. (S)- and (R)-a-Methylphenylalanine, 9. (S)-2-
Methoxycarbonylamino-2-methyl-3-phenylpropanoni-
trile
7
or (R)-2-ethoxycarbonylamino-2-methyl-3-
1
36.6 and 40.5 min, respectively; H NMR (300 MHz,
phenylpropanoic acid ethyl ester 8 (0.13 mmol) was
hydrolyzed by refluxing for 3 h with 20% aqueous
hydrochloric acid (3 ml). The solution was evaporated
under vacuum. To the residue was added distilled water
(3 ml) and evaporated again. The residue was purified
by Dowex 50×2-400 ion-exchange resin (Acros), yield
95%. [h]1D7=−22.0 (c 0.61, H2O), S; [h]1D7=+21.8 (c 0.73,
CDCl3): l 7.40–7.34 (m, 3H, ArH), 7.30–7.27 (m, 2H,
ArH), 4.94 (br s, 1H, NH), 3.74 (s, 3H, OMe), 3.29 (d,
1H, J=13.8 Hz, CH), 3.19 (d, 1H, J=13.5 Hz, CH),
1.66 (s, 3H, CH3); IR (KBr): w 3325 (NH), 2240 (CN),
1699 (CꢀO); MS m/z (%): 219 (M++1, 8), 218 (M+, 15),
192 (M+−CN, 99), 91 (100).
H2O), R; {Lit.22 [h]D=−22 (c 1, H2O), S}; H NMR
1
4.5.2. (S)-2-Benzyl-2-methylmalonamic acid ethyl ester,
7. (S)-2a (28 mg, 1.5 mmol) was dissolved in 2 ml
anhydrous ethanol. Three drops of concentrated sulfate
acid was added and the mixture was refluxed gently.
The resulting mixture was poured into cool NaHCO3
aqueous solution and extracted with ethyl acetate,
washed and dried on MgSO4. The solvent was removed
under reduced pressure and the resulting product was
purified by flash chromatography (98%). The product
was dissolved in 1 ml ethanol. 30% H2O2 (2 ml) and 10
M NaOH (0.05 ml) were added at 0°C. After 3 h at
room temperature, the resulting mixture was extracted
with ethyl acetate, washed and dried on MgSO4. The
solvent was removed under reduced pressure and the
resulting product was purified by flash chromatography
to give 34 mg (S)-7 (98%). Mp 90.9–91.9°C; [h]1D6=+3.3
(c 2.6, CHCl3); enantiomeric excess was determined by
HPLC on a Chiralcel AS column with hexane/2-
propanol mixtures 85: 15 and the retention time for R
and S enantiomer was 21.0 and 16.4 min, respectively;
1H NMR (300 MHz, CDCl3): l 7.30–7.23 (m, 3H,
Ar-H), 7.17–7.14 (m, 2H, Ar-H), 6.98 (s, br, 1H, NH),
5.79 (s, br, 1H, NH), 4.19 (q, 2H, J=7.2 Hz), 3.37 (d,
1H, J=13.5 Hz), 3.12 (d, 1H, J=13.5 Hz, CH), 1.45 (s,
3H, CH3), 1.27 (t, 3H, J=7.2 Hz, CH3); IR (KBr): w
3395, 3327 (NH), 1722 (CꢀO), 1694, 1667 (CꢀO); MS
m/z (%): 235 (M+, 5), 218 (8), 190 (16), 162 (22), 91
(100). Anal. calcd for C13H17NO3: C, 66.36; H, 7.28; N,
5.95. Found: C, 66.35; H, 7.07; N, 5.88.
(300 MHz, D2O): l 7.22–7.19 (m, 3H, ArH), 7.10–7.07
(m, 2H, ArH), 3.11 (d, 1H, J=14.4 Hz, CH), 2.79 (d,
1H, J=14.4 Hz, CH), 1.37 (s, 3H, CH3); IR (KBr):
2500–3300, 1650 (CꢀO); MS (ESI): 202 ([M+Na]+), 180
([M+H]+).
Acknowledgements
We are grateful to the National Science Foundation of
China (Grant No. 20032020) for financial support. We
also thank Professor Lijun Xia and Miss Zuoding Ding
for performing the HPLC analysis.
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