Synthesis of optically active α-methylamino acids and amides through biocatalytic kinetic resolution of amides

Article abstract of DOI:10.1016/j.tetasy.2005.06.023

Catalyzed by Rhodococcus sp. AJ270, a nitrile hydratase and amidase containing microbial whole-cell catalyst, under very mild conditions, a number of racemic α-methylamino amides were resolved into the corresponding optically active (S)-(+)-α-methylamino acids and (R)-(-)-α- methylamino amides. The steric requirement of the amidase against α-amino phenylacetamides bearing methyl group(s) at α-amino nitrogen and/or α-carbon was also studied. Coupled with the chemical hydrolysis of amide, the biotransformation process provided a direct synthesis of α-methylamino acids in both enantiomeric forms from readily available racemic amides.

Full text of DOI:10.1016/j.tetasy.2005.06.023

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2409–2416
Synthesis of optically active a-methylamino acids and
amides through biocatalytic kinetic resolution of amides
Mei-Xiang Wang,* Jun Liu, De-Xian Wang and Qi-Yu Zheng
Laboratory for Chemical Biology, Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100080, China
Received 25 March 2005; revised 19 June 2005; accepted 20 June 2005
Abstract—Catalyzed by Rhodococcus sp. AJ270, a nitrile hydratase and amidase containing microbial whole-cell catalyst, under very
mild conditions, a number of racemic a-methylamino amides were resolved into the corresponding optically active (S)-(+)-a-meth-
ylamino acids and (R)-(ꢀ)-a-methylamino amides. The steric requirement of the amidase against a-amino phenylacetamides bearing
methyl group(s) at a-amino nitrogen and/or a-carbon was also studied. Coupled with the chemical hydrolysis of amide, the biotrans-
formation process provided a direct synthesis of a-methylamino acids in both enantiomeric forms from readily available racemic
amides.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
catalyzed by the amidase,10 are effective and environ-
mentally friendly methods for the production of carbox-
ylic acids and their amide derivatives. The microbial
hydration of acrylonitrile to acrylamide, for instance,
is one of the largest industrial biotransformations in
the world.11 Recent studies12,13 have demonstrated that
biotransformations of nitriles complement the existing
asymmetric chemical and enzymatic synthetic methods
for carboxylic acids and their derivatives. The distinct
features of enzymatic transformations of nitriles are
the formation of enantiopure carboxylic acids, and the
straightforward generation of enantiopure amides,
which are valuable organonitrogen compounds in syn-
thetic chemistry. Very recently, it was found14 that the
combination of the nitrile hydratase and the amidase
in Rhodococcus sp. AJ270 microbial cells15 is a powerful
biocatalytic system to transform a variety of racemic
a-amino nitriles into highly enantiopure D-amino acid
amides and L-amino acids. The amidase is also able to
resolve a-alkylarylglycine amides to yield optically
active (S)-a-alkylarylglycines and (R)-a-alkylarylglycine
amides.16 Our interests13,14,16 in the understanding of
the scope and mechanism of the nitrile hydratase and
the amidase, and in the synthesis of optically active
N-methyl a-amino acids have led us to the current study.
We report herein a general and convenient synthesis of
optically active (S)-a-methylamino acids and (R)-a-
methylamino amides from the kinetic resolution of
amides catalyzed by the amidase within Rhodococcus
There has been an increasing interest in the synthesis of
optically active a-methylamino acids because of their
significant biological properties.1,2 A number of syn-
thetic methods reported to date mainly involve the
methylation of N-protected a-amino acid derivatives,3
methylation of a-azido carboxylic acids,4 reductive ami-
nation5 and reduction and deprotection of oxazolidi-
nones, which are derived from the cyclocondensation
of N-protected a-amino acids and paraformaldehyde.6
In addition, the resolution of racemic a-methylamino
acids using chiral acids and bases7 and the enzymatic
resolution of N-acylated N-methyl a-amino acids8 have
also been reported. However, most of the methods
reported suffer from at least one drawback such as low
chemical yields, narrow substrate spectrum, long syn-
thetic steps including tedious protection and deprotec-
tion, harsh reaction conditions and the problems of
racemization.
Biotransformations of nitriles, either through direct con-
version from a nitrile to a carboxylic acid catalyzed by a
nitrilase9 or through the nitrile hydratase-catalyzed
hydration of a nitrile followed by the amide hydrolysis
*
Corresponding author. Tel.: +86 10 62565610; fax: +86 10
62564723; e-mail: mxwang@iccas.ac.cn
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.06.023

2410
M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
sp. AJ270 cells. Using structurally diversified a-amino
amide substrates as probes, we are also able to define
the steric requirements of the amidase action.
decreased (entries 1–6), clearly indicating a kinetic reso-
lution process. Under the optimal conditions, such as in
a neutral buffer solution (pH 7.0) for 60 h, racemic
( )-1a was resolved effectively into (R)-(ꢀ)-1a and
(S)-(+)-2a in excellent chemical yields with excellent
enantioselectivities (entry 7).
2. Results and discussion
We first tested the Rhodococcus sp. AJ270 cell-catalyzed
reaction of racemic a-methylamino phenylacetonitrile.
Unfortunately, in stark contrast to the efficient and
highly enantioselective reaction of racemic a-amino phen-
ylacetonitrile,14 the a-methylamino analogue did not
undergo biocatalytic hydrolysis. Efforts to improve the
conversion of the nitrile, such as by varying the pH of
the buffered solution, reaction temperature and concen-
tration of the substrate were made, but all met with fail-
ure. Under the various reaction conditions including the
control experiments in the absence of biocatalyst, the
substrate was found to undergo a very rapid and spon-
taneous decomposition. The inefficient hydration of the
nitrile in this case is most probably due to the instability
of the substrate and the inhibition of the nitrile hydra-
tase, a believed cobalt- or iron-containing enzyme,10b
by the cyanide ion generated from decomposition of
a-methylamino phenylacetonitrile.
Encouraged by these results, we then extended this reac-
tion to other racemic a-methylamino amide substrates.
In order to examine the influence of the structure of
the substrates on the biocatalytic kinetic resolution,
both the number of a-methylamino arylacetamides
( )-1b–j bearing a different substituent at different
substitution positions on the benzene ring and a-methyl-
amino alkanamides ( )-1k–m were prepared and sub-
jected to incubation with Rhodococcus sp. AJ270
(Scheme 2 and Table 2). The results summarized in
Table 2 shows that the amidase accepted all the racemic
a-methylamino amides tested as substrates. The conver-
sion of the amide, however, depended strongly on the
nature of the substituent and the substitution pattern
on the arene ring of the substrates. For example, while
N-methylamino 4-fluorophenylacetamide ( )-1b (entry
2) and N-methylamino 3-chlorophenylacetamide ( )-1d
(entry 4) underwent the equally efficient biohydrolysis
as the parent amide ( )-1a (entry 1), the introduction
of other substituents into either the 3- or 4-position of
the benzene ring generally resulted in the slow hydroly-
sis, and the time for ca. 50% conversion increased from
1–2 days to 3–4.5 days. The presence of an ortho substi-
tuent such as in ( )-1e (entry 5) and ( )-1i (entry 9), led
to even slower reaction. It is of interest to note that all
methyl-substituted substrates ( )-1g–i (entries 7–9)
underwent slower hydrolysis than their chloro-substi-
tuted counterparts (entries 3–5). In the case of a-methyl-
amino-b-phenylpropionamide ( )-1k (entry 11) and a-
methylamino-b-methylbutyramide ( )-1l (entry 12), bio-
catalytic hydrolysis proceeded very rapidly in 2–4 h to
give more than 50% conversion. The conversion of
cyclohexyl-substituted methylaminoacetamide ( )-1m
(entry 13) was comparable to that of a-methylamino
4-chlorophenylacetamide ( )-1c (entry 3). The influence
of the steric effect of the substituent appeared obvious;
the smaller the steric bulkiness around the stereogenic
centre that is adjacent to amide functional group, the
faster the biocatalytic hydrolysis.
Having considered the high enantioselectivity of the
amidase within Rhodococcus sp. AJ270 and the stability
of a-methylamino amides, we then concentrated on the
kinetic resolution of racemic a-methylamino phenylacet-
amide ( )-1a (Scheme 1 and Table 1). In a phosphate
buffer with pH ranging from 7.0 to 7.6, Rhodococcus
sp. AJ270 readily transformed racemic ( )-1a into the
optically active (R)-(ꢀ)-amide (ꢀ)-1a and (S)-(+)-acid
(+)-2a. With the progress of reaction, the enantiomeric
excess value of (ꢀ)-1a increased while that of (+)-2a
NHMe
CONH2
(R)-(-)-1a
NHMe
CONH2
(+/-)-1a
NHMe
CO2H
(S)-(+)-2a
i
+
Ph
Ph
Ph
Scheme 1. Biocatalytic kinetic resolution of racemic a-methylamino
phenylacetamide ( )-1a. Reagents and condition: (i) Rhodococcus sp.
AJ270, phosphate buffer, 30 ꢁC.
On the other hand, the scrutiny of the results tabulated
in Table 2 revealed a varied (S)-enantioselectivity of the
amidase depending on the structure of the substrates.
For example, while the biohydrolysis of parent a-methyl-
amino phenylacetamide ( )-1a (entry 1) furnished excel-
lent enantioselectivity (E17 = 94), its 4-fluoro ( )-1b
Table 1. Biocatalytic kinetic resolution of racemic a-methylamino
phenylacetamide ( )-1a
Entry pH Time (R)-(ꢀ)-1a (R)-(ꢀ)-1a (S)-(+)-2a (S)-(+)-2a
(h)a (%)b
(% ee)c
(%)b
(% ee)c
1
2
3
4
5
6
7
7.0 48
56
58
57
41
43
43
48
73
64
43
40
41
58
56
56
47
96
93
92
75
78
75
93
7.25 48
7.6 48
7.0 72
7.25 72
7.6 72
7.0 60
64
>99.5
>99.5
>99.5
93
NHMe
CONH2
(R)-(-)-1a-m
NHMe
CONH2
(+/-)-1a-m
NHMe
CO2H
(S)-(+)-2a-m
i
+
R
R
R
a Substrate 1a (2 mmol) was incubated with Rhodococcus sp. AJ270
whole cells (2 g wet weight) in a phosphate buffer (0.1 M) at 30 ꢁC.
b Isolated yield.
Scheme 2. Amidase-catalyzed kinetic resolution of racemic a-meth-
ylamino amides ( )-1. Reagents and condition: (i) Rhodococcus sp.
AJ270, phosphate buffer, 30 ꢁC.
c Enantiomeric excess values were determined by chiral HPLC analysis.

M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
Table 2. Amidase-catalyzed kinetic resolution of racemic a-methylamino amides ( )-1
2411
Entry
1
R
Time (d)a
(ꢀ)-1 (%)b
(ꢀ)-1 (% ee)c
93
93
57
5
(+)-2 (%)b
(+)-2 (% ee)c
E
1
1a
1b
1c
1d
1e
1f
C6H5
2.5d
1
48
45
57
48
68
47
43
53
72
47
38
49
55
47
53
37
46
31
47
56
46
23
51
60
51
42
93
83
85
15
33
16
57
81
13
76
56
13
96
94
36
21
2
4-FC6H4
4-ClC6H4
3-ClC6H4
2-ClC6H4
4-BrC6H4
4-MeC6H4
3-MeC6H4
2-MeC6H4
4-MeOC6H4
C6H4CH2
Me2CH
3
2
4
1
1.4
2
5
7
<3
6
6
4.5
7
1.5
9.4
7
1g
1h
1i
85
67
4
8
3
19
1.3
26
9
7e
4
10
11
12
13
1j
95
73
21
39
1k
1l
2 hd
4 h
2.5
7.5
1.6
1m
c-C6H11
71
a The substrate 1 (1 mmol) was incubated with Rhodococcus sp. AJ270 whole cells (2 g wet weight) in phosphate buffer (pH 7.0, 0.1 M, 50 ml) at
30 ꢁC.
b Isolated yield.
c Enantiomeric excess values were determined by chiral HPLC analysis.
d 2 mmol of the substrate were used.
e 0.5 mmol of the substrate were used.
R1 NR2R3
Ph CONH2
(+/-)-1a, n-q
R1 NR2R3
Ph CONH2
(R)-(-)-1a, n-q
(entry 2), 4-chloro ( )-1c (entry 3), 4-methyl ( )-1g (en-
try 7) and 4-methoxy ( )-1j (entry 10) substituted amide
analogues gave modest enantiocontrol, with E values
ranging roughly from 10 to 36. Very low enantioselec-
tion was obtained for a-methylamino 4-bromophenyl-
acetamide ( )-1f (entry 6). Variation of the substitution
pattern from the para- to the meta- or ortho-position
on the benzene ring resulted in a dramatic decrease in
enantioselectivity (entries 3–5, 7 and 9) with the excep-
tion of a-methylamino 3-methylphenylacetamide ( )-
1h, which afforded modest selection (E = 19) (entry 8).
Low enantioselectivity was observed for alkanamides
( )-1k and ( )-1l, whereas a-cyclohexyl methylamino-
acetamide ( )-1m showed a comparable enantioselectivity
(E = 71) as that of aromatic analogues ( )-1a and ( )-
1b. The aforementioned stereoselectivities suggested that
the enantioselection of the amidase-catalyzed kinetic
resolution of a-methylamino amides was determined
by both electronic and steric factors of the substrate,
with the latter being a pronounced one.
R1 NR2R3
i
+
Ph
CO2H
(S)-(+)-2a, n-q
Scheme 3. Biocatalytic hydrolysis of substituted a-amino phenylace-
tamides. Reagents and condition: (i) Rhodococcus sp. AJ270, phos-
phate buffer pH 7.0, 30 ꢁC.
steric encumbrance of the amino group from NH2
[( )-1n]14 to NHMe ( )-1a or to NMe2 ( )-1o led to a
dramatic decrease of conversion. This was shown by
the inefficient hydrolysis of a-dimethylamino phenylacet-
amide ( )-1o in a prolonged incubation time with
whole-cell catalyst (entry 3). Introduction of an addi-
tional methyl group to the stereogenic centre caused
the biocatalytic hydrolysis of the corresponding quater-
nary carbon-centred substrates ( )-1p16 and ( )-1q more
sluggishly. Only under the conditions using a lower sub-
strate concentration with a long period of interaction
with Rhodococcus sp. AJ270, did the reaction give 50%
conversion of the starting amide (entries 4 and 5). More
surprisingly, the amidase displayed a clear-cut enantio-
selection power depending on the presence of one more
methyl group either at the amino nitrogen or the stereo-
genic carbon centre. This was shown by the observation
To shed further light on the steric requirement of the
amidase involved in Rhodococcus sp. AJ270, more a-
amino amide substrates were employed to probe the
reactivity and enantioselectivity of the enzyme (Scheme
3 and Table 3). In the case of phenylglycine amides bear-
ing a tertiary carbon stereogenic centre, increasing the
Table 3. Biocatalytic hydrolysis of substituted a-amino phenylacetamides
Entry
Subs.
Substituents
Conditionsa
(ꢀ)-1 (%)b
(ꢀ)-1 (% ee)c
(+)-2 (%)b
(+)-2 (% ee)c
E
1
2
3
4
5
1n14
1a
R1 = H, R2 = H, R3 = H
R1 = H, R2 = H, R3 = Me
R1 = H, R2 = Me, R3 = Me
R1 = Me, R2 = H, R3 = H
R1 = Me, R2 = H, R3 = Me
2 mmol, 2 h
41
48
91
46
46
84
93
14
95
17
51
47
6
85
93
6
32
94
—
42
2 mmol, 60 h
1 mmol, 168 h
1 mmol, 17.5 h
0.5 mmol, 168 h
1o
1p16
1q
50
48
84
32
2.3
a The substrate was incubated with Rhodococcus sp. AJ270 whole cells (2 g wet weight) in a phosphate buffer (0.1 M) at 30 ꢁC.
b Isolated yield.
c Enantiomeric excesses were determined by chiral HPLC analysis.

2412
M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
NHMe
CO2H
4. Experimental
NHMe
CONH2
HCl (6M),60-80oC
94%
Ph
Ph
4.1. General
(R)-(-)-3a
(R)-(-)-1a
e.e. 87%
The configurations of optically active a-substituted a-
amino acids were determined by the comparison of
their direction of optical rotation with that of authentic
samples, while the configurations of amides were ob-
tained by correlating optical rotation of their chemi-
cally hydrolyzed amino acid products with that of
amino acids of known configurations. For unknown
optically active amino acids and amides obtained from
biotransformation, their configurations were tentatively
assigned by the comparison of both their direction of
optical rotation and the retention times under identical
HPLC analysis conditions using the same chiral
stationary phase with that of (S)-(+)-methylamino
phenylacetic acid 2a and (R)-(ꢀ)-a-methylamino
phenylacetamide 1a, respectively, briefly assuming that
the introduction of a substituent on the benzene ring
did not change the direction of the optical rotation
and the order of chromatogram. The enantiomeric
excesses of all compounds were obtained with a Shima-
dzu LC-10AVP HPLC system. A Chirex(D)-Penicill-
amine column with a mixture (1:9) of 2-propanol and
aqueous CuSO4 solution (2 nM) as the mobile phase
at a flow rate of 0.5–0.9 ml/min was employed for the
analysis of all acids. For amides, a Cyclobond ITM
2000DMP column with a mixture (5:95) of methanol
and acetonitrile buffered with acetic acid and triethyl-
amine (100:0.35:0.1) as the mobile phase at a flow rate
of 0.3–1.0 ml/min was used.
e.e. > 99.5%
Scheme 4. Preparation of optically active (R)-(ꢀ)-a-methylamino
phenylacetic acid.
of high enantiomeric excess values for (R)-amides 1a,
1n14 and 1p16 and (S)-acids 3a (E = 94), 2n (E = 32)14
and 2p (E = 42)16 and very low ones for products 1o,
1q, 2o and 2q (E = 2.3). Though it is difficult at this stage
to rationalize the origin of the enantioselectivity of the
amidase, it is most likely that the synergetic effects of
electronic and steric features of the substrates play a
subtle but significant role to allow the amidase to show
varied enantio-differentiating power. In other words, to
achieve an efficient and highly enantioselective biotrans-
formation of a-amino amides, it is advantageous to con-
sider both electronic and steric effects of a substituent
when engineering the substrate.
To demonstrate the practical utility of the biocatalytic
synthesis of a-methyamino acids, a gram-scale biotrans-
formation of amide was attempted. Thus, to two flasks
each containing a suspension of Rhodococcus sp.
AJ270 whole cells (4 g wet weight) in phosphate buffer
(pH 7.0, 0.1 M, 100 ml) was added ( )-1a (24 mmol)
portion-wise over 5 days. After incubation for another
4 days, highly enantiopure (S)-(+)-acid 2a (1.17 g, 30%
yield, ee 96%) was yielded. Chemical hydrolysis of
enantiopure amide (R)-(ꢀ)-1a in hydrochloric acid
(6 M) afforded acid (R)-(ꢀ)-3a with partial racemization
(Scheme 4).
4.2. General procedure for the biocatalytic kinetic
resolution of racemic amides
To an Erlenmeyer flask (100 ml) with a screw cap were
added Rhodococcus sp. AJ270 cells15 (2 g wet weight)
and potassium phosphate buffer (0.1 M, pH 7.0, 50 ml)
and the resting cells activated at 30 ꢁC for 30 min with
orbital shaking. Racemic amino acid amides 1 (Tables
1–3) were added in one portion to the flask and the
mixture incubated at 30 ꢁC using an orbital shaker
(200 rpm). The reaction, monitored by TLC, was
quenched after a specified period of time (see Tables
1–3 and Schemes 1–3) by removing the biomass through
a Celite pad filtration. The resulting aqueous solution
was concentrated to about 10 ml and then acidified to
pH 3–4 with 2 M HCl. After the precipitate was
removed by filtration, the solution was concentrated
again to 5 ml. The products were isolated and purified
by chromatography using consecutively a cationic
exchange resin column (Dowex, 50 · 8), a reverse phase
silica gel column (35–70 lm) and a Sephadex G-25 col-
umn. All products were characterized by their spectral
data and comparison of the melting points and optical
rotary power with that of the known compounds, which
are listed as follows, or by full characterization.
3. Conclusion
In conclusion, we have shown a general and convenient
method for the synthesis of optically active (R)-(ꢀ)-a-
methylamino amides and (S)-(+)-a-methylamino acids
from the kinetic resolution of racemic amides catalyzed
by the amidase within Rhodococcus sp. AJ270 cells
under very mild conditions. The reaction results are
heavily dependent upon both the nature of the substitu-
ent and its substitution pattern on the substrate. The
results obtained from the current and previous stud-
ies13,14,16 have indicated that the amidase involved in
the Rhodococcus sp. AJ270 cells exhibit a predictive
(S)-enantioselectivity against both a-mono-substituted-
and a,a-disubstituted a-amino arylacetamides irrespec-
tive of the nature of the substituents attached on the
benzene ring. Coupled with the chemical hydrolysis of
amide, this biotransformation process provided a useful
approach to a-methylamino acids in both enantiomeric
forms from the readily available racemic amino amides.
The various highly enantiopure a-amino acids and their
amide derivatives obtained from biotransformation
should serve as useful chemical entities in organic
synthesis and bioorganic chemistry, which is being
actively pursued in this laboratory.
4.2.1. Enzymatic hydrolysis of racemic a-methylamino
phenylacetamide 1a. (R)-(ꢀ)-a-Methylamino phenylacet-
25
amide 1a: mp 176–177 ꢁC; ½aꢁ ¼ ꢀ98.4 (c 0.5, MeOH);
D
ee 99% (HPLC, tR = 8.9 min, tS = 11.5 min, flow rate

M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
2413
0.8 ml/min); IR (KBr) m 3286, 1690 cmꢀ1
;
1H NMR
135.7, 128.6, 125.8, 62.6, 29.1; MS (ESI) m/z (%) 202
(33), 200 [M+1]+ (100). Anal. Calcd for [C9H12ClNO2]+:
200.0473. Found: 200.0474.
(300 MHz, CDCl3) d 7.43–7.32 (m, 5H), 6.93 (s, 1H),
5.44 (s, 1H), 4.06 (s, 1H), 2.47 (s, 3H); 13C NMR (CDCl3)
d 174.9, 139.1, 128.9, 128.2, 127.3, 69.8, 35.4; MS (ESI) m/z
(%) 165 [M+1]+ (100). Anal. Calcd for C9H12N2O: C,
65.83; H, 7.36; N, 17.06. Found: C, 65.80; H, 7.32; N,
17.06.
4.2.4. Enzymatic hydrolysis of racemic a-methylamino
3-chlorophenylacetamide 1d. (R)-(ꢀ)-a-Methylamino
25
D
3-chlorophenylacetamide 1d: mp 120–121 ꢁC; ½aꢁ
¼
ꢀ0.8 (c 1.5, MeOH); ee 5% (HPLC analysis of the cor-
(S)-(+)-a-Methylamino phenylacetic acid 2a: mp 207 ꢁC
responding acid, tR = 65.9 min, tS = 44.9 min, flow rate
25
25
D
(dec); ½aꢁ ¼ þ116.4 (c, 0.5, 1 M HCl) {lit.7 ½aꢁ ¼ þ130
0.7 ml/min); IR (KBr) m 3285, 3057, 1691, 1668 cmꢀ1
;
D
(c, 0.5, 1 M HCl)}; ee 96% (HPLC, tR = 12.8 min,
tS = 9.4 min, flow rate 0.8 ml/min); IR (KBr) m 2310–
1H NMR (300 MHz, CDCl3) d 7.41–7.27 (m, 4H), 6.91
(s, 1H), 5.44 (s, 1H), 4.04 (s, 1H), 2.47 (s, 3H); 13C
NMR (CDCl3) d 174.1, 140.9, 134.7, 130.1, 128.5,
127.4, 125.6, 69.3, 35.3; MS (ESI) m/z (%) 201
[M+2+1]+ (39), 199 [M+1]+ (100). Anal. Calcd for
C9H11ClN2O: C, 54.42; H, 5.58; N, 14.10. Found: C,
54.35; H, 5.71; N, 13.79.
3061, 1628, 1593 cmꢀ1
;
1H NMR (300 MHz,
D2O + CF3CO2D) d 7.34–7.25 (m, 5H), 4.42 (s, 1H),
2.42 (s, 3H); 13C NMR (D2O + CF3CO2D) d 170.4,
130.0, 127.8, 127.4, 126.4, 64.7, 28.9; MS (ESI) m/z
166 [M+1]+ (100). Anal. Calcd for [C9H12NO2]+:
166.0862. Found: 166.0863.
(S)-(+)-a-Methylamino 3-chlorophenylacetic acid 2d:
mp 198 ꢁC (dec); ½aꢁ ¼ þ14.9 (c 1.5, 1 M HCl); ee
;
0.7 ml/min); IR (KBr) m 2415–3061, 1590 cmꢀ1 1H
NMR (300 MHz, D2O + CF3CO2D) d 7.38–7.19 (m,
4H), 4.43 (s, 1H), 2.44 (s, 3H); 13C NMR (D2O + CF3-
CO2D) d 168.4, 134.2, 130.3, 130.0, 129.7, 127.7,
125.9, 62.8, 29.7; MS (ESI) m/z (%) 202 (68), 200
[M+1]+ (100). Anal. Calcd for [C9H11ClNO2]+:
200.0473. Found: 200.0474.
25
D
15% (HPLC, tR = 65.9 min, tS = 44.9 min, flow rate
4.2.2. Enzymatic hydrolysis of racemic a-methylamino
4-fluorophenylacetamide 1b. (R)-(ꢀ)-a-Methylamino 4-
25
D
fluorophenylacetamide 1b: mp 135–137 ꢁC; ½aꢁ
¼
ꢀ82.0 (c 0.5, MeOH); ee 93% (HPLC, tR = 8.2 min,
tS = 9.8 min, flow rate 0.8 ml/min); IR (KBr) m 3285,
3033, 1697 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.41–
1
7.36 (m, 2H), 7.01 (t, J = 4.7 Hz, 2H), 6.94 (s, 1H),
5.54 (s, 1H), 4.04 (s, 1H), 2.46 (s, 3H); 13C NMR
(DMSO-d6) d 173.9, 163.0, 159.8, 136.4, 129.1, 114.9,
114.6, 67.2, 34.2; MS (ESI) m/z (%) 183 [M+1]+ (100).
Anal. Calcd for C9H11FN2O: C, 59.33; H, 6.09; N,
15.38. Found: C, 59.36; H, 6.08; N, 15.35.
4.2.5. Enzymatic hydrolysis of racemic a-methylamino
2-chlorophenylacetamide 1e. (R)-(ꢀ)-a-Methylamino
25
D
2-chlorophenylacetamide 1e: mp 118–120 ꢁC; ½aꢁ
¼
(S)-(+)-a-Methylamino 4-fluorophenylacetic acid 2b:
ꢀ2.4 (c 3.0, MeOH); ee <3% (HPLC, tR = 18.6 min,
tS = 22.9 min, flow rate 0.3 ml/min); IR (KBr) m 3219,
25
mp 211 ꢁC (dec); ½aꢁ ¼ þ78.4 (c 0.5, 1 M HCl); ee
D
83% (HPLC, tR = 18.8 min, tS = 12.8 min, flow rate
3038, 1701 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.40–
1
0.7 ml/min); IR (KBr) m 2319–3055, 1605, 1590 cmꢀ1
;
7.23 (m, 4H), 7.04 (s, 1H), 6.39 (s, 1H), 4.50 (s, 1H),
2.43 (s, 3H); 13C NMR (CDCl3) d 174.4, 136.7, 133.9,
130.0, 129.4, 129.3, 127.3, 65.7, 35.3; MS (ESI) m/z
(%) 201 [M+2+1]+ (41), 199 [M+1]+ (100). Anal. Calcd
for C9H11ClN2O: C, 54.42; H, 5.58; N, 14.10. Found:
C, 54.26; H, 5.64; N, 13.73.
1H NMR (300 MHz, D2O + CF3CO2D) d 7.31–7.26
(m, 2H), 7.05 (t, J = 8.8 Hz, 2H), 4.44 (s, 1H), 2.42 (s,
3H); 13C NMR (D2O + CF3CO2D) d 170.1, 165.9,
162.6, 131.3, 125.2, 117.5, 117.1, 63.9, 30.8; MS (ESI)
m/z (%) 184 [M+1]+ (100). Anal. Calcd for
[C9H10FNO2]+: 184.0768. Found: 184.0768.
(S)-(+)-a-Methylamino 2-chlorophenylacetic acid 2e:
mp 224 ꢁC (dec); ½aꢁ ¼ þ46.1 (c 0.5, 1 M HCl); ee
;
0.8 ml/min); IR (KBr) m 2312–3056, 1632 cmꢀ1 1H
NMR (300 MHz, D2O + CF3CO2D) d 7.43–7.26 (m,
4H), 4.89 (s, 1H), 2.48 (s, 3H); 13C NMR (D2O + CF3-
CO2D) d 171.7, 134.2, 131.5, 130.5, 130.3, 129.9,
128.1, 63.6, 31.4; MS (ESI) m/z (%) 202 (33), 200
[M+1]+ (100). Anal. Calcd for [C9H11ClNO2]+:
200.0473. Found: 200.0474.
25
D
33% (HPLC, tR = 33.6 min, tS = 22.4 min, flow rate
4.2.3. Enzymatic hydrolysis of racemic a-methylamino
4-chlorophenylacetamide 1c. (R)-(ꢀ)-a-Methylamino
25
D
4-chlorophenylacetamide 1c: mp 150–152 ꢁC; ½aꢁ
¼
ꢀ21.0 (c 0.5, MeOH); ee 57% (HPLC, tR = 8.0 min,
tS = 9.4 min, flow rate 0.8 ml/min); IR (KBr) m 3290,
3021, 1697 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.37–
1
7.30 (m, 4H), 6.92 (s, 1H), 5.53 (s, 1H), 4.04 (s, 1H),
2.46 (s, 3H); 13C NMR (CDCl3) d 173.9, 136.9, 133.6,
128.5, 128.1, 68.6, 34.8; MS (ESI) m/z (%) 201
[M+2+1]+ (36), 199 [M+1]+ (100). Anal. Calcd for
C9H11ClN2O: C, 54.42; H, 5.58; N, 14.10. Found: C,
54.25; H, 5.63; N, 13.75.
4.2.6. Enzymatic hydrolysis of racemic a-methylamino
4-bromophenylacetamide 1f. (R)-(ꢀ)-a-Methylamino
25
D
4-bromophenylacetamide 1f: mp 164–166 ꢁC; ½aꢁ
¼
(S)-(+)-a-Methyl 4-chlorophenylacetic acid 2c: mp
ꢀ7.8 (c 2.5, MeOH); ee 6% (HPLC, tR = 8.1 min,
tS = 9.2 min, flow rate 0.8 ml/min); IR (KBr) m 3420,
25
D
(HPLC, tR = 59.5 min, tS = 36.1 min, flow rate 0.9 ml/
219 ꢁC (dec); ½aꢁ ¼ þ67.3 (c 0.5, 1 M HCl); ee 85%
3288, 1669 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.48
1
min); IR (KBr) m 2662–3061, 1637, 1585 cmꢀ1
;
1H
7.34 (d,
(d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 6.91 (s,
1H), 5.42 (s, 1H), 4.03 (s, 1H), 2.46 (s, 3H); 13C NMR
(CDCl3) d 173.6, 139.6, 130.9, 129.4, 120.2, 67.3, 34.1;
MS (ESI) m/z (%) 245 [M+2+1]+ (97), 243 [M+1]+
NMR (300 MHz, D2O + CF3CO2D)
J = 6.5 Hz, 2H), 7.25 (d, J = 6.7 Hz, 2H), 4.44 (s, 1H),
d
2.43 (s, 3H); 13C NMR (D2O + CF3CO2D) d 168.2,

2414
M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
(100). Anal. Calcd for C9H11BrN2O: C, 44.47; H, 4.56;
N, 11.52. Found: C, 44.19; H, 4.53; N, 11.39.
4.2.9. Enzymatic hydrolysis of racemic a-methylamino
2-methylphenylacetamide 1i. (R)-(ꢀ)-a-Methylamino
25
D
2-methylphenylacetamide 1i: mp 118–119 ꢁC; ½aꢁ
¼
(S)-(+)-a-Methylamino 4-bromophenylacetic acid 2f:
ꢀ0.3 (c 3.0, MeOH); ee 4% (HPLC, tR = 15.6
min, tS = 17.4 min, flow rate 0.3 ml/min); IR (KBr) m
25
D
16% (HPLC, tR = 88.4 min, tS = 51.4, flow rate 0.8 ml/
mp 215 ꢁC (dec); ½aꢁ ¼ þ19.4 (c 1.0, 1 M HCl); ee
3418, 3319, 3147, 1682 cmꢀ1 1H NMR (300 MHz,
;
min); IR (KBr) m 2250–3078, 1586 cmꢀ1
;
1H NMR
CDCl3) d 7.37–7.16 (m, 4H), 7.02 (s, 1H), 5.57 (s, 1H),
4.30 (s, 1H), 2.47 (d, J = 6.7 Hz, 3H); 13C NMR
(CDCl3) d 175.0, 137.7, 136.6, 130.8, 127.9, 126.7,
126.5, 65.9, 35.8, 19.7; MS (ESI) m/z (%) 179 [M+1]+
(100). Anal. Calcd for C10H14N2O: C, 67.39; H, 7.92;
N, 15.72. Found: C, 67.52; H, 7.93; N, 15.65.
(300 MHz, D2O + CF3CO2D) d 7.50 (d, J = 8.4 Hz,
2H), 7.18 (d, J = 8.4 Hz, 2H), 4.43 (s, 1H), 2.42 (s,
3H); 13C NMR (D2O + CF3CO2D) d 168.4, 131.8,
129.1, 126.6, 123.8, 62.7, 29.3; MS (ESI) m/z (%) 246
[M+2+1]+ (96), 244 [M+1]+ (100). Anal. Calcd for
C9H10BrNO2: C, 44.26; H, 4.10; N, 5.74. Found: C,
44.21; H, 4.27; N, 5.93.
(S)-(+)-a-Methylamino 2-methylphenylacetic acid 2i:
25
D
13% (HPLC, tR = 21.6 min, tS = 15.4 min, flow rate
mp 235 ꢁC (dec); ½aꢁ ¼ þ12.9 (c 1.5, 1 M HCl); ee
4.2.7. Enzymatic hydrolysis of racemic a-methylamino
4-methylphenylacetamide 1g. (R)-(ꢀ)-a-Methylamino
0.8 ml/min); IR (KBr)
m 2342–3057, 1630, 1613,
25
4-methylacetamide 1g: mp 166–167 ꢁC; ½aꢁ ¼ ꢀ81.6 (c
1
D
1569 cmꢀ1; H NMR (300 MHz, D2O + CF3CO2D) d
7.14 (s, 4H), 4.37 (s, 1H), 2.39 (s, 3H), 2.17 (s, 3H);
13C NMR (D2O + CF3CO2D) d 169.4, 137.2, 130.8,
129.7, 126.4, 125.4, 59.5, 29.5, 17.3; MS (ESI) m/z 180
[M+1]+ (100). Anal. Calcd for [C10H14NO2]+:
180.1019. Found: 180.1019.
0.5, MeOH); ee 85% (HPLC, tR = 9.7 min, tS =
11.7 min, flow rate 0.8 ml/min); IR (KBr) m 3291, 3025,
1697 cmꢀ1 1H NMR (300 MHz, CDCl3) d 7.34–7.14
;
(m, 4H), 6.91 (s, 1H), 5.42 (s, 1H), 4.02 (s, 1H), 2.46
(s, 3H), 2.33 (s, 3H); 13C NMR (DMSO-d6) d 174.2,
137.2, 136.2, 128.5, 127.1, 67.8, 34.2, 20.6; MS (ESI)
m/z (%) 179 [M+1]+ (100). Anal. Calcd for
C10H14N2O: C, 67.39; H, 7.92; N, 15.72. Found: C,
67.66; H, 8.01; N, 15.61.
4.2.10. Enzymatic hydrolysis of racemic a-methylamino
4-methoxyphenylacetamide 1j. (R)-(ꢀ)-a-Methylamino
25
4-methoxyphenylacetamide
1j:
mp
172–173 ꢁC;
½aꢁ ¼ ꢀ75.9 (c 0.5, MeOH); ee 95% (HPLC,
D
tR = 11.3 min, tS = 13.4 min, flow rate 0.8 ml/min); IR
(S)-(+)-a-Methylamino 4-methylphenylacetic acid 2g:
25
D
57% (HPLC, tR = 23.2 min, tS = 46.5 min, flow rate
mp 196 ꢁC (dec); ½aꢁ ¼ þ50.1 (c 1.0, 1 M HCl); ee
(KBr) m 3286, 3255, 1695, 1666 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) d 7.32 (d, J = 8.7 Hz, 2H), 6.89 (d,
J = 9.0 Hz, 2H), 6.85 (s, 1H), 5.36 (s, 1H), 4.01 (s,
1H), 3.80 (s, 3H), 2.45 (s, 3H); 13C NMR (DMSO-d6)
d 174.3, 158.4, 132.2, 128.3, 113.4, 67.4, 55.0, 34.2; MS
(ESI) m/z (%) 195 [M+1]+ (100). Anal. Calcd for
C10H14N2O2: C, 61.84; H, 7.27; N, 14.41. Found: C,
61.47; H, 7.24; N, 14.31.
0.7 ml/min); IR (KBr) m 2319–3052, 1616, 1597 cmꢀ1
;
1H NMR (300 MHz, D2O + CF3CO2D) d 7.19–7.13
(m, 4H), 4.70 (s, 1H), 2.44 (s, 3H), 2.26 (s, 3H); 13C
NMR (D2O + CF3CO2D) d 169.4, 140.8, 129.5, 127.6,
124.9, 63.3, 29.6, 19.2; MS (ESI) m/z 180 [M+1]+
(100). Anal. Calcd for [C10H14NO2]+: 180.1019. Found:
180.1020.
(S)-(+)-a-Methylamino 4-methoxyphenylacetic acid 2j:
mp 177 ꢁC (dec); ½aꢁ ¼ þ131.8 (c 0.5, 1 M HCl); ee
4.2.8. Enzymatic hydrolysis of racemic a-methylamino
25
D
76% (HPLC, tR = 17.5 min, tS = 11.8 min, flow rate
3-methylphenylacetamide 1h. (R)-(+)-a-Methylamino
25
D
3-methylphenylacetamide 1h: mp 131–132 ꢁC; ½aꢁ
¼
0.8 ml/min); IR (KBr) m 2243–3059, 1615, 1584 cmꢀ1
;
ꢀ68.0 (c 0.5, MeOH); ee 76% (HPLC, tR = 4.84
1H NMR (300 MHz, D2O + CF3CO2D) d 7.19 (d,
J = 6.7 Hz, 2H), 6.88 (d, J = 6.7 Hz, 2H), 4.33 (s, 1H),
3.67 (s, 3H), 2.37 (s, 3H); 13C NMR d 173.4, 159.9,
130.1, 125.3, 114.9, 66.5, 55.5, 31.1; MS (ESI) m/z 196
[M+1]+ (100). Anal. Calcd for C10H13NO3: C, 61.53;
H, 6.71; N, 7.18. Found: C, 61.52; H, 6.69; N, 7.58.
min, tS = 7.32 min, flow rate 1.0 ml/min); IR (KBr)
3290, 3065, 1688, 1668 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 7.26–7.10 (m, 4H), 6.95 (s, 1H), 5.50 (s, 1H),
4.01 (s, 1H), 2.46 (s, 3H), 2.35 (s, 3H); 13C NMR
(CDCl3) d 175.3, 139.0, 138.5, 129.0, 128.7, 128.0,
124.2, 69.8, 35.5, 21.4; MS (ESI) m/z (%) 179
[M+1]+ (100). Anal. Calcd for C10H14N2O: C, 67.39;
H, 7.92; N, 15.72. Found: C, 67.66; H, 8.01; N,
15.83.
4.2.11. Enzymatic hydrolysis of racemic a-methyl-b-
phenylpropionamide 1k. (ꢀ)-a-3-Methylamino-b-phen-
25
ylpropionamide 1k: mp 133–134 ꢁC; ½aꢁ ¼ ꢀ66.3 (c
D
0.5, MeOH); ee 73% (HPLC analysis of the correspond-
(S)-(+)-a-Methylamino 3-methylphenylacetic acid 2h:
25
D
81% (HPLC, tR = 33.1 min, tS = 21.4 min, flow rate
ing acid, t(ꢀ) = 33.3 min, t(+) = 28.5 min, flow rate
mp 200 ꢁC (dec); ½aꢁ ¼ þ83.2 (c 0.5, 1 M HCl); ee
1
0.8 ml/min); IR (KBr) 3279, 3228, 1683, 1662 cmꢀ1; H
0.8 ml/min); IR (KBr) m 2313–3061, 1606, 1584 cmꢀ1
;
NMR (300 MHz, CDCl3) d 7.36–7.22 (m, 5H), 7.01
(s, 1H), 5.33 (s, 1H), 3.25–3.19 (m, 2H), 2.73 (dd, J =
14.6, 10.4 Hz, 1H), 2.31 (s, 3H); 13C NMR (CDCl3) d
176.7, 137.5, 129.1, 128.8, 127.0, 66.1, 39.2, 35.5; MS
(ESI) m/z (%) 179 [M+1]+ (100). Anal. Calcd for
C10H14N2O: C, 67.39; H, 7.92; N, 15.72. Found: C,
67.39; H, 7.95; N, 15.81.
1H NMR (300 MHz, D2O + CF3CO2D) d 7.23–7.03
(m, 4H), 4.35 (s, 1H), 2.39 (s, 3H), 2.17 (s, 3H); 13C
NMR (D2O + CF3CO2D) d 172.6, 139.8, 132.0, 130.4,
129.3, 129.0, 125.4, 66.7, 30.8, 20.3; MS (ESI) m/z (%)
180 [M+1]+ (100). Anal. Calcd for [C10H14NO2]+:
180.1019. Found: 180.1019.

M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
2415
(+)-a-Methylamino-b-phenylpropionic acid 2k: mp
1655 cmꢀ1 1H NMR (300 MHz, CDCl3) d 7.37–7.28
;
25
D
192 ꢁC (dec); ½aꢁ ¼ þ27.8 (c 1.5, 1 M HCl); ee 56%
(m 5H), 6.95 (s, 1H), 5.72 (s, 1H), 3.73 (s, 1H), 2.23 (s,
6H); 13C NMR (CDCl3) d 174.5, 136.2, 128.8, 128.5,
128.2, 43.9; MS (ESI) m/z (%) 179 [M+1]+ (100). Anal.
Calcd for C10H14N2O: C, 67.39; H, 7.92; N, 15.72.
Found: C, 67.62; H, 7.84; N, 15.57.
(HPLC, t(ꢀ) = 33.1 min, t(+) = 28.5 min, flow rate 0.8
1
ml/min); IR (KBr) m 2310–3060, 1588 cmꢀ1; H NMR
(300 MHz, D2O + CF3CO2D) d 7.24–7.10 (m, 5H),
3.66 (t, J = 6.2 Hz, 1H), 3.03 (d, J = 6.2 Hz, 2H), 2.49
(s, 3H); 13C NMR d 169.2, 132.1, 128.2, 127.2, 61.1,
33.7, 30.8; MS (ESI) m/z (%)180 [M+1]+ (100). Anal.
Calcd for [C10H15NO2]+: 180.1019. Found: 180.1020.
(S)-(+)-a-Dimethylamino phenylacetic acid 2o: mp 236
25
ꢁC (dec); ½aꢁ ¼ þ5.5 (c 0.5, 1 M HCl) {lit.7 (R)-(ꢀ)-a-
D
25
D
H2O)}; ee 6% (HPLC, tR = 16.5 min, tS = 7.8 min, flow
dimethylamino-phenylacetic acid: ½aꢁ ¼ ꢀ122 (c, 1.0,
4.2.12. Enzymatic hydrolysis of racemic a-methylamino-
b-methylbutyramide 1l. (ꢀ)-a-Methylamino-b-methyl-
rate 0.8 ml/min); IR (KBr) m 2320–3066, 1628 cmꢀ1
;
25
butyramide 1l: mp 83–84 ꢁC; ½aꢁ ¼ ꢀ0.5 (c 1.5,
1H NMR (300 MHz, D2O + CF3CO2D) d 6.70–6.67
(m, 5H), 4.23 (s, 1H), 2.21 (s, 3H), 1.78 (s, 3H); 13C
NMR (D2O + CF3CO2D) d 167.6, 129.9, 128.3, 127.6,
125.7, 70.5, 41.6, 38.6; MS (ESI) m/z (%) 180 [M+1]+
(100). Anal. Calcd for [C10H14NO2]+: 180.1019. Found:
180.1019.
D
MeOH); ee 21% (HPLC analysis of the corresponding
acid, t(ꢀ) = 20.7 min, t(+) = 17.5 min, flow rate 0.5 ml/
min); IR (KBr) m 3283, 3084, 1698, 1664 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 6.96 (s, 1H), 5.41 (s, 1H),
2.77 (d, J = 4.7 Hz, 1H), 2.41 (s, 3H), 2.11–2.00 (m,
1H), 1.01 (d, J = 7.0 Hz, 3H), 0.94 (d, J = 7.0 Hz, 3H);
13C NMR (CDCl3) d 177.0, 70.9, 36.1, 31.3, 19.5, 18.0;
MS (ESI) m/z (%) 131 [M+1]+ (100). Anal. Calcd for
C6H14N2O: C, 55.35; H, 10.84; N, 21.52. Found: C,
55.15; H, 10.65; N, 21.53.
4.2.15. Enzymatic hydrolysis of racemic a-methylamino-
a-phenylpropionamide 1q. (ꢀ)-a-Methylamino-a-phen-
25
ylpropionamide 1q: mp 90–91 ꢁC; ½aꢁ ¼ ꢀ3.8 (c
D
0.5, MeOH); ee 17% (HPLC analysis of the corres-
ponding acid, t(ꢀ) = 28.5 min, t(+) = 25.0 min, flow rate
(+)-a-Methylamino-b-methylbutyric acid 2l: mp 219 ꢁC
0.5 ml/min); IR (KBr) m 3419, 3212, 1643 cmꢀ1 1H
;
25
(dec); ½aꢁ ¼ þ8.8 (c 0.5, 1 M HCl); ee 76% (HPLC,
NMR (300 MHz, CDCl3) d 7.61–7.25 (m, 5H), 6.92 (s,
1H), 5.53 (s, 1H), 2.30 (s, 3H), 1.73 (s, 3H); 13C NMR
(CDCl3) d 173.9, 136.9, 133.6, 128.5, 128.1, 68.6, 34.8;
MS (ESI) m/z (%) 199 [M+1]+ (100). Anal. Calcd for
C10H14N2O: C, 67.39; H, 7.92; N, 15.72. Found: C,
67.62; H, 7.84; N, 15.57.
D
t(ꢀ) = 20.7 min, t(+) = 17.5 min, flow rate 0.5 ml/min);
IR (KBr)
m
2309–3050, 1580 cmꢀ1
;
1H NMR
(300 MHz, D2O + CF3CO2D) d 3.59–3.54 (m, 1H),
2.64–2.60 (m, 3H), 2.18–2.14 (m, 1H), 0.96–0.84 (m,
6H); 13C NMR (D2O + CF3CO2D) d 168.8, 66.0, 31.3,
27.7, 16.1, 14.9; MS (ESI) m/z (%) 132 [M+1]+ (100).
Anal. Calcd for C6H13NO2: C, 54.94; H, 9.99; N,
10.68. Found: C, 54.44; H, 9.86; N, 10.61.
(+)-a-Methylamino-a-phenylpropionic acid 2q: mp
25
D
208 ꢁC (dec); ½aꢁ ¼ þ30.3 (c 0.5, 1 M HCl); ee 32%
(HPLC, t(ꢀ) = 28.5 min, t(+) = 25.9 min, flow rate 0.5
ml/min); IR (KBr) m 3476, 3186, 2494–3039, 1631,
4.2.13. Enzymatic hydrolysis of racemic a-methylamino-a-
1
cyclohexylacetamide 1m. (ꢀ)-a-Methylamino-a-cyclo-
1616 cmꢀ1; H NMR (300 MHz, D2O + CF3CO2D) d
25
hexylacetamide 1m: mp 160–161.5 ꢁC; ½aꢁ ¼ ꢀ2.6 (c
7.32 (s, 5H), 2.21 (s, 3H), 1.71 (s, 3H); 13C NMR
(D2O + CF3CO2D) d 171.9, 130.7, 129.4, 128.5, 125.2,
65.8, 25.9, 15.8; MS (ESI) m/z (%) 180 [M+1]+ (100).
Anal. Calcd for [C10H14NO2]+: 180.1019. Found:
180.1020.
D
2.5, MeOH); ee 39% (HPLC analysis of the corresponding
acid, t(ꢀ) = 64.3 min, t(+) = 53.2 min, flow rate 0.5 ml/
min); IR (KBr) m 3312, 3144, 1670, 1618 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 6.97 (s, 1H), 6.14 (s, 1H),
2.76 (d, J = 4.3 Hz, 1H), 2.40 (m, 3H), 1.73–1.05 (m,
11H); 13C NMR (CDCl3) d 176.9, 70.8, 41.3, 36.1, 30.1,
28.5, 26.33, 26.26, 26.22; MS (ESI) m/z (%) 171 [M+1]+
(100). Anal. Calcd for C9H18N2O: C, 63.49; H, 10.66;
N, 16.45. Found: C, 63.40; H, 10.58; N, 16.50.
4.3. Chemical hydrolysis of (R)-(ꢀ)-a-methylamino
phenylacetamide 1a
A mixture of (R)-(ꢀ)-a-methylamino phenylacetamide
(ꢀ)-1a (100 mg, ee >99.5%) in 6 M HCl (10 ml) was
stirred at 60 ꢁC for 5 h and then was kept at 80 ꢁC for
another 5 h. The acid product was purified using a
cationic exchange resin column (Dowex, 50 · 8) and a
Sephadex G-25 column to give (R)-(ꢀ)-a-methylamino
phenylacetic acid (+)-2a: 94.1 mg (94%); mp 213 ꢁC;
(+)-a-Methylamino-a-cyclohexylacetic acid 2m: mp
25
D
245 ꢁC (dec); ½aꢁ ¼ þ46.2 (c 1.0, 1 M HCl); ee 96%
(HPLC, t(ꢀ) = 64.3 min, t(+) = 53.2 min, flow rate 0.5
1
ml/min); IR (KBr) m 2310–3061, 1581 cmꢀ1; H NMR
(300 MHz, D2O + CF3CO2D) d 3.51–3.50 (m, 1H),
2.63–2.50 (m, 3H), 1.80–0.90 (m, 11H); 13C NMR
(D2O + CF3CO2D) d 169.1, 65.9, 37.5, 31.6, 27.4, 26.6,
24.3, 24.1, 23.9; MS (ESI) m/z (%) 172 [M+1]+ (100).
Anal. Calcd for [C9H18NO2]+: 172.1331. Found: 172.1333.
25
25
½aꢁ ¼ ꢀ101.7 (c 0.5, 1 M HCl) {lit.7 ½aꢁ ¼ ꢀ171 (c,
D
D
0.5, 1 M HCl)}, ee 87% (HPLC, tR = 12.8 min,
tS = 9.4 min, flow rate 0.8 ml/min). The spectral data
are identical with those of (S)-(+)-3a.
4.2.14. Enzymatic hydrolysis of racemic a-dimethylamino
phenylacetamide 1o. (R)-(ꢀ)-a-Dimethylamino pheny-
Acknowledgements
25
lacetamide 1o: mp 160–161.5 ꢁC; ½aꢁ ꢀ 7.5 (c
D
0.5, MeOH); ee 14% (HPLC, tR = 6.5 min, tS =
13.4 min, flow rate 0.75 ml/min); IR (KBr) 3349, 3177,
We thank the State Major Fundamental Research
Program (2003CB716005, G2000077506), the Ministry

2416
M.-X. Wang et al. / Tetrahedron: Asymmetry 16 (2005) 2409–2416
7. Shiraiwa, T.; Baba, Y.; Miyazaki, H.; Sakata, S.; Kawam-
ura, S.; Uehara, M.; Kurokawa, H. Bull. Chem. Soc. Jpn.
1993, 66, 1430, and references cited therein.
8. Groeger, U.; Drauz, K.; Klenk, H. Angew. Chem., Int. Ed.
Engl. 1992, 31, 195.
of Science and Technology (Grant No. 2002CCA03100),
the National Science Foundation of China, and the
Chinese Academy of Sciences for financial support.
M.-X. Wang also thanks O. Meth-Cohn, J. Colby and
C. OÕReilly for discussion.
9. Kobayashi, M.; Shimizu, S. FEMS Microbiol. Lett. 1994,
120, 217, and references cited therein.
10. (a) Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin
Trans. 1 1997, 1099–1104; (b) Meth-Cohn, O.; Wang,
M.-X. J. Chem. Soc., Perkin Trans. 1 1997, 3197, and
references cited therein.
11. Nagasawa, T.; Schimizu, H.; Yamada, H. Appl. Microbiol.
Biotechnol. 1993, 40, 189.
12. For a recent review, see: Sugai, T.; Yamazaki, T.;
Yokoyama, M.; Ohta, H. Biosci. Biotechnol. Biochem.
1997, 61, 1419, and references cited therein.
13. (a) Wang, M.-X.; Lin, S.-J.; Liu, C.-S.; Zheng, Q.-Y.; Li,
J.-S. J. Org. Chem. 2003, 68, 4570; (b) Wang, M.-X.; Feng,
G.-Q. J. Org. Chem. 2003, 68, 621; (c) Wang, M.-X.; Wu,
Y. Org. Biomol. Chem. 2003, 535; (d) Wang, M.-X.; Feng,
G.-Q.; Zheng, Q.-Y. Adv. Synth. Catal. 2003, 345, 695; (e)
Wang, M.-X.; Zhao, S.-M. Tetrahedron: Asymmetry 2002,
13, 1695; (f) Wang, M.-X.; Li, J.-J.; Ji, G.-J.; Li, J.-S.
J. Mol. Catal. B: Enzyme 2001, 14, 77; (g) Wang, M.-X.;
Liu, C.-S.; Li, J.-S. Tetrahedron: Asymmetry 2001, 12,
3367; (h) Wang, M.-X.; Lu, G.; Ji, G.-J.; Huang, Z.-T.;
Meth-Cohn, O.; Colby, J. Tetrahedron: Asymmetry 2000,
11, 1123; (i) Wang, M.-X.; Feng, G.-Q. New J. Chem.
2002, 1575; (j) Wang, M.-X.; Feng, G.-Q.; Zheng, Q.-Y.
Tetrahedron: Asymmetry 2004, 15, 347.
References
1. Fairlie, D. P.; Abbenante, G.; March, D. R. Curr. Med.
Chem. 1995, 2, 654.
2. (a) Cody, W. L.; He, J. X.; Reily, M. D.; Haleen, S. J.;
Walker, D. M.; Reyner, E. L.; Stewart, B. H.; Doherty, A.
M. J. Med. Chem. 1997, 40, 2228; (b) Haviv, F.;
Fitzpatrick, T. D.; Swenson, R. E.; Nicholas, C. J.; Mort,
N. A.; Bush, E. U.; Diaz, G.; Barmmert, G.; Nguyen, A.;
Nellans, H. N.; Hoffman, D. J.; Johnson, E. S.; Greer, J.
J. Med. Chem. 1993, 36, 363; (c) Vitoux, B.; Aubry, A.;
Cung, M. T.; Marraud, M. Int. J. Pept. Protein Res. 1986,
26, 617.
3. (a) Olsen, R. K. J. Org. Chem. 1970, 35, 1912; (b) Coggins,
J. R.; Benoiton, N. L. Can. J. Chem. 1971, 49, 1968; (c)
McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973,
51, 2562, and references cited therein; (d) Kitagawa, I.;
Lee, N. K.; Kobayashi, M.; Shibuya, H. Tetrahedron 1991,
47, 2169; (e) Prashad, M.; Har, D.; Hu, B.; Kim, H.-Y.;
Repic, O.; Blacklock, T. J. Org. Lett. 2003, 5, 125.
4. Dorow, R. L.; Gingrich, D. E. J. Org. Chem. 1995, 60,
4986.
5. (a) Chu, K. S.; Negrete, G. R.; Konopelski, J. P. J. Org.
Chem. 1991, 56, 5196; (b) Luke, R. W. A.; Boyce, P. G. T.;
Dorling, E. K. Tetrahedron Lett. 1996, 37, 263.
6. (a) Freidinger, R. M.; Hinkle, J. S.; Perlow, D. S.; Arison,
B. H. J. Org. Chem. 1983, 48, 77; (b) Aurelio, L.;
Brownlee, R. T. C.; Hughes, A. B. Aust. J. Chem. 2000,
53, 425; (c) Aurelio, L.; Box, J. S.; Brownlee, R. T. C.;
Hughes, A. B.; Sleebs, M. M. J. Org. Chem. 2003, 68,
2652.
14. Wang, M.-X.; Lin, S.-J. J. Org. Chem. 2002, 67, 6542.
15. Blakey, A. J.; Colby, J.; Williams, E.; OÕReilly, C. FEMS
Microbiol. Lett. 1995, 129, 57.
16. Wang, M.-X.; Lin, S.-J.; Liu, J.; Zheng, Q.-Y. Adv. Synth.
Catal. 2004, 346, 439.
17. (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J.
J. Am. Chem. Soc. 1982, 104, 7294; (b) Program ÔSelec-
tivityÕ by Faber, K.; Hoenig, H. http://www.cis.TUGraz.
at/orgc/.