Practical and convenient enzymatic synthesis of enantiopure α-amino acids and amides

Article abstract of DOI:10.1021/jo0256282

Catalyzed by the nitrile hydratase and the amidease in Rhodococcus sp. AJ270 cells under very mild conditions, a number of α-aryl- and α-alkyl-substituted DL-glycine nitriles 1 rapidly underwent a highly enantioselective hydrolysis to afford D-(-)-α-amino acid amides 2 and L-(+)-α-amino acids 3 in high yields with excellent enantiomeric excesses in most cases. The overall enantioselectivity of the biotransformations of nitriles originated from the combined effects of a high L-enantioselective amidase and a low enantioselective nitrile hydratase. The influence of the substrates on both reaction efficiency and enantioselectivity was also discussed in terms of steric and electronic effects. Coupled with chemical hydrolysis of D-(-)-α-phenylglycine amide, biotransformation of DL-phenylglycine nitrile was applied in practical scale to produce both D- and L-phenylglycines in high optical purity.

Full text of DOI:10.1021/jo0256282

standing reaction scope and applications of both nitrile
hydratase and amidase involved in Rhodococcus AJ 270
and in synthesizing enantiopure amino acid and amide
derivatives9 led us to undertake the study of biotrans-
formation of R-amino nitriles.10
P r a ctica l a n d Con ven ien t En zym a tic
Syn th esis of En a n tiop u r e r-Am in o Acid s
a n d Am id es
Mei-Xiang Wang* and Shuang-J un Lin
Center for Molecular Science, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100080, China
At the outset of our project, few examples of enantio-
selective biotransformations of R-amino nitriles had been
reported. The nitrilase of Rhodococcus rhodochrous PA-
3411 and a nitrilase-containing Acinetobacter sp. culture12
had the ability to convert several aliphatic DL-R-amino
nitriles into L-amino acids in moderate to high enantio-
meric excesses, while the nitrilase-catalyzed hydrolysis
of DL-phenylglycine nitrile with Aspergillus furmigatus13
had been shown to form L-phenylglycine in 80% ee. Quite
recently, Sheldon and co-workers14 screened a microbial
cell able to catalyze the enantioselective hydrolysis of DL-
phenylglycine nitrile to give L-acid and D-amide with high
ee.
Considering the optimal working pH for nitrile hy-
dratase of Rhodococcus sp. AJ 270 and to prevent the
decomposition of R-amino nitriles in aqueous solution, the
biotransformation was conducted in a phosphate buffer
with pH 7.62. As described in Table 1, Rhodococcus sp.
AJ 270 catalyzed effectively the biotransformations of a
number of racemic arylglycine nitriles 1a -h and alkyl-
glycine nitriles 1i-k to give the corresponding optically
active D-(-)-glycine amide 2 and L-(+)-glycine 3 deriva-
tives (Scheme 1). The reaction rate and enantiomeric
excesses, however, were dependent upon both the elec-
tronic and steric effects of the substituent. In the case of
aromatic R-amino nitriles, the hydrolysis proceeded
rapidly (within 4 h) under identical conditions except
2-methyl-substituted substrate 1e took 12 h to transform
its cyano function group into the amide 2e and then into
the acid 3e (Entry 7). Good to excellent enantiocontrol
was obtained for almost all aromatic R-amino nitriles and
therefore some enantiopure D-(-)-glycine amides such as
2b-d ,f and L-(+)-glycines such as 3d ,e,g were produced
in very high yield. Attempts were made to improve the
enantioselectivity of the biotransformation of phenyl-
glycine nitrile 1a by altering the buffer pH from 7.62 to
mxwang@infoc3.icas.ac.cn
Received February 19, 2002
Abstr a ct: Catalyzed by the nitrile hydratase and the
amidease in Rhodococcus sp. AJ 270 cells under very mild
conditions, a number of R-aryl- and R-alkyl-substituted DL-
glycine nitriles 1 rapidly underwent a highly enantioselec-
tive hydrolysis to afford D-(-)-R-amino acid amides 2 and
L-(+)-R-amino acids 3 in high yields with excellent enantio-
meric excesses in most cases. The overall enantioselectivity
of the biotransformations of nitriles originated from the
combined effects of a high L-enantioselective amidase and a
low enantioselective nitrile hydratase. The influence of the
substrates on both reaction efficiency and enantioselectivity
was also discussed in terms of steric and electronic effects.
Coupled with chemical hydrolysis of D-(-)-R-phenylglycine
amide, biotransformation of DL-phenylglycine nitrile was
applied in practical scale to produce both D- and L-phenyl-
glycines in high optical purity.
Biotransformations of nitriles,1 through either a direct
nitrilase-catalyzed conversion of a nitrile to a carboxylic
acid2 or a nitrile hydratase-catalyzed hydration of a
nitrile followed by the hydrolysis of amide to acid by the
action of the amidase,3 have been demonstrated as being
unique and environmentally benign methods for the
synthesis of chiral carboxylic acids and their amide
derivatives because of the excellent selectivity and very
mild reaction conditions. Our earlier work has shown that
Rhodococcus sp. AJ 270, a robust nitrile hydratase/
amidase-containing microorganism,4 was able to hydro-
lyze a wide range of structurally diverse mono-5 and
dinitriles6 with excellent chemo- and regioselectivieties.
Very recently, we have found that Rhodococcus sp. AJ 270
could efficiently and enantioselectively catalyze the hy-
drolysis of a number of racemic R-substituted phenylac-
etonitriles7 and 2-arylcyclopropanecarbonitriles8 to pro-
duce the corresponding enantiopure carboxylic acids and
amides in high yields. Our continuous interest in under-
(7) (a) Wang, M.-X.; Lu, G.; J i, G. J .; Huang, Z.-T.; Meth-Cohn, O.;
Colby, J . Tetrahedron: Asymmetry 2000, 11, 1123. (b) Wang, M.-X.;
Li, J .-J .; J i, G.-J .; Li, J .-S. J . Mol. Catal. B: Enzym. 2001, 14, 77.
(8) Wang, M.-X.; Feng, G.-Q. Tetrahedron Lett. 2000, 41, 6501.
(9) For reviews of chemical and enzymatic syntheses, see: (a)
Kochetkov K.; Belikov, V. Russ. Chem. Rev. 1987, 56, 1045. (b)
Williams, R. M. Synthesis of Optically Active R-Amino Acids; Perga-
mon: Oxford, UK, 1989. (c) Williams, R. M.; Hexdrix, J . A. Chem. Rev.
1992, 92, 889. (d) Duthaler, R. Tetrahedron, 1994, 50, 1539. (e) Calms,
M.; Daunis, J . Amino Acids 1999, 16, 215. (f) Miyazawa, T. Amino Acids
1999, 16, 191. (g) Enzyme Catalyst in Organic Synthesis; Drauz, K.,
Waldmann, H., Eds.; VCH: Weinheim, Germany, 1995. (h) Wong, C.-
H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry;
Pergamon: New York, 1994. (i) Preparative Biotransformation: Whole
Cell and Isolated Enzymes in Organic Chemistry; Roberts, S. M., Ed.;
Wiley: New York, 1993. (j) Stereoselective Biocatalysis, Patel, R. N.,
Ed.; Marcel Dekker: New York, 2000.
(1) For recent reviews, see: (a) Sugai, T.; Yamazaki, T.; Yokoyama,
M.; Ohta, H. Biosci. Biotechnol. Biochem. 1997, 61, 1419. (b) Crosby,
J .; Moiliet, J .; Parratt, J . S.; Turner, N. J . J . Chem. Soc., Perkin Trans.
1 1994, 1679.
(2) (a) Harper, D. B. Biochem. Soc. Trans. 1976, 4, 502. (b) Harper,
D. B. Biochem. J . 1977, 165, 309. (c) Kobayashi, M.; Shimizu, S. FEMS
Microbiol. Lett. 1994, 120, 217.
(3) (a) Asano, Y.; Tani, Y.; Yamada, H. Agric. Biol. Chem. 1980, 44,
2251. (b) Asano, Y.; Fujishiro, K.; Tani, Y.; Yamada, H. Agric. Biol.
Chem. 1982, 46, 1165. (c) Asano, Y.; Tachibana, M.; Tani, Y.; Yamada,
H. Agric. Biol. Chem. 1982, 46, 1175.
(4) Blakey, A. J .; Colby, J .; Williams, E.; O’Reilly, C. FEMS
Microbiol. Lett. 1995, 129, 57.
(5) (a) Meth-Cohn, O.; Wang, M.-X. Tetrahedron Lett. 1995, 36, 9561.
(b) Meth-Cohn, O.; Wang, M.-X. J . Chem. Soc., Perkin Trans. 1 1997,
1099.
(6) (a) Meth-Cohn, O.; Wang, M.-X. Chem. Commun. 1997, 1041.
(b) Meth-Cohn, O.; Wang, M.-X. J . Chem. Soc., Perkin Trans. 1 1997,
3197.
(10) Part of this work has appeared as a preliminary communica-
tion: Wang, M.-X.; Lin, S.-J Tetrahedron Lett. 2001, 42, 6925.
(11) Bhalla, T. C.; Miura, A.; Wakamoto, A.; Furuhashi, K. Appl.
Microbiol. Biotechnol. 1992, 37, 184.
(12) Macdam, A. M.; Knowles, C. J . Biotechnol. Lett. 1985, 7, 865.
(13) Choi, S. Y.; Goo, Y. M. Arch. Pharm. Res. 1986, 9, 45.
(14) Wegman, M. A.; Heinemann, U.; van Rantuijk, F.; Stolz, A.;
Sheldon, R. A. J . Mol. Catal. B: Enzym. 2001, 11, 249.
10.1021/jo0256282 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/30/2002
6542
J . Org. Chem. 2002, 67, 6542-6545

TABLE 1. En a n tioselective Biotr a n sfor m a tion s of DL-r-Am in o Nitr iles 1
amide 2
yieldb (%)
acid 3
yieldb (%)
entry
substrate 1
R
pH; time (h)a
eec (%)
eec (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1a
1a
1a
1b
1c
1d
1e
1f
1g
1h
1i
C6H5
C6H5
C6H5
4-ClC6H4
3-ClC6H4
4-MeC6H4
2-MeC6H4
4-MeOC6H4
3-MeOC6H4
3,4-OCH2OC6H3
cyclohexyl
C6H5CH2
pH 7.62; 2.5
pH 7.13; 8
49
43
74
>99
47
52
93
52
50
50
42
51
46
48
47
81
42
47
93
>99
<5
pH 7.13; 25d
pH 7.62; 4
42
48
42
45
45
47
39
45
17
48
48
>99
>99
86
80
>99
77
>99
88f
96
97
pH 7.62; 2.7
pH 7.62; 3
95
>99
>99
96
pH 7.62; 12
pH 7.62; 1.5
pH 7.62; 1.5
pH 7.62; 2.2
pH 7.62; 21e
pH 7.62; 0.6d
pH 7.62; 0.7d,e
pH 7.62; 1.2e
>99
99
>99f
13
1j
1j
1k
C6H5CH2
(CH3)2CH
77
51
22f
34f
a
Substrate (2 mmol) was incubated at 30 °C with Rhodococcus sp. AJ 270 cells (2 g wet weight) in phosphate buffer (0.1 M, 50 mL). The
reaction conditions were not optimized. Isolated yield. c Determined by HPLC analysis.
1 mmol of the substrate was used.
b
d
e Biotransformations were performed at 20 °C. f Determined by HPLC analysis of the corresponding N-benzoylated derivatives.
TABLE 2. Kin etic Resolu tion of DL-r-a m in o Acid Am id es 2
amide 2
yieldb (%)
acid 3
yieldb (%)
entry
substrate 2
R
pH; time (h)a
eec (%)
eec (%)
1
2
3
4
5
2a
2e
2f
2g
2j
C6H5
pH 7.0; 2
pH 7.0; 9
pH 7.0; 2
pH 7.0; 2
pH 7.0; 2
41
45
46
51
43
84
61
>99
94
51
53
53
47
56
85
60
90
95
60
2-MeC6H4
4-MeOC6H4
3-MeOC6H4
C6H5CH2
87
a
Substrate (2 mmol) was incubated at 30 °C with Rhodococcus sp. AJ 270 cells (2 g wet weight) in phosphate buffer (0.1 M, 50 mL). The
reaction conditions were not optimized. Isolated yield. c Determined by HPLC analysis.
b
SCHEME 1
SCHEME 2
7.13. Although the reaction proceeded in 8 h, much slower
than that at pH 7.62, no detrimental effect such as
decomposition of the substrate was observed. The enan-
tioselectivity of the biotransformation was enhanced and
both amide 2a and acid 3a were obtained in enantiopure
forms (entry 2). It should be noted that when a pH of
7.13 was applied to the rest of the nitriles tested, the
biotransformation appeared very slow and the nitrile
substrates underwent a notorious decomposition to form
cyanide which further inhibited the nitrile hydratase.
Under the same reaction conditions, Rhodococcus sp.
AJ270 displayed much higher enzymatic activities against
R-amino nitriles 1i-k derived from alkyl aldehydes, and
a very small amount of amide product was isolated from
the reaction. This is in sharp contrast to the biotrans-
formation of aromatic R-amino nitrile such as 1a that
took more than 1 day to complete its conversion to acid
(entry 3). Only at a lower temperature such as 20 °C did
the biotransformation afford an appreciable amount of
amide. The enantiocontrol of the reaction of cyclohexyl-
glycine nitrile 1i was excellent, being comparable to that
from the reaction of arylglycine nitriles. Nitriles 1j and
1k , however, led to low to moderate enantioselectivity
(entries 13 and 14).
AJ 270. The incubation was terminated at 50% conversion
of the substrate to give optically active D-(-)-amides 2
and L-(+)-acids 3 with enantiomeric excesses ranging
from 60% to >99% (Scheme 2, Table 2), indicating that
a high L-enantioselective amidase16 was involved in the
cells. In general, however, the enantiocontrol of the
kinetic resolution of R-amino acid amides 2 appeared less
effective than that of the biotransformation of nitriles 1.
To prepare enantiopure L-(+)-R-amino acids and their
D-(-)-amides, it is apparently advantageous to apply the
biotransformation of nitriles rather than of amides in
terms of reaction efficiency, enantioselectivity, and easy
availability of the starting materials.
The results of the biotransformation of nitriles and of
amides showed that the amidase involved in Rhodococcus
sp. AJ 270 is L- or S-enantioselective, while nitrile hy-
dratase probably also exhibits L- or S-enantiocontrol but
to a lower degree. The enantioselectivity of the overall
hydrolysis of a nitrile results from the combined effects
of the nitrile hydratase and the amidase, with the latter
being a dominant factor. The presence of an aromatic
nucleus or of a cyclohexyl moiety adjacent to the cyano
or amido function gave excellent enantioselectivity of the
To shed further light on the biotransformation of
nitriles, a selected set of racemic R-amino acid amides,
prepared from the chemical hydration of the nitriles with
concentrated H2SO4 (98%),15 was fed to Rhodococcus sp.
(15) Hassan, N. A.; Bayer, E.; J ochims, J . C. J . Chem. Soc., Perkin
Trans. 1 1998, 3747.
(16) A D-enantioselective aminopeptidase from Ochrobactrun an-
thropi was reported. Asano, Y.; Nakazawa, A.; Kato, Y.; Kondo, K. J .
Biol. Chem. 1989, 264, 14233.
J . Org. Chem, Vol. 67, No. 18, 2002 6543

wet weight) and potassium phosphate buffer (0.1 M, 50 mL) and
the resting cells were activated at 30 or 20 °C for 30 min with
orbital shaking. DL-Amino nitrile hydrochlorides 1 (2 mmol) or
DL-amino acid amides 2 (2 mmol) were added in one portion to
the flask and the mixture was incubated at 30 or 20 oC with
use of an orbital shaker (200 rpm). The reaction, monitored by
TLC, was quenched after a specified period of time (see Tables
1 and 2) by removing the biomass through a Celite pad filtration.
The resulting aqueous solution was concentrated. The products
were isolated and purified by chromatography with consecutive
use of a cationic exchange resin column (Dowex, 50 × 8), a
Sephadex G-25 column, and a reverse phase silica gel column
(35-70 µm). All products were characterized by their spectra
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.
SCHEME 3
reaction. The further increase of steric bulkiness of the
aromatic group by introducing an o-methyl group, how-
ever, slowed the reaction rate and lowed the enantiose-
lectivity. These results indicated clearly the dependence
of effective enantioselection of the amidase and nitrile
hydratase upon the steric feature of the substrates.
To test the practical use of biotransformation of nitrile
in the preparation of optically active R-amino acids in
both enantiomeric forms, we finally conducted a multi-
gram scale reaction employing DL-phenylglycine nitrile
1a as the substrate. By adding the substrate in aliquot
to Rhodococcus sp. AJ 270 culture in one flask during a
15 h time span, up to 19 mmol of 1a was transformed
into the D-(-)-phenylglycine amide 2a (yield 46%) and
L-(+)-phenylglycine 3a (yield 53%) with enantiomeric
excesses being >99% and 91%, respectively. In a well-
controlled reaction, the enantiomeric excess of acid would
reach >99%. In refluxing hydrochloric acid (6 N), enan-
tiopure D-(-)-phenylglycine amide 2a was hydrolyzed to
give D-(-)-phenylglycine in almost quantitative yield
without racemization (Scheme 3).
In conclusion, we have shown a very convenient and
practical method for the syntheses of D-(-)-R-amino acid
amides and L-(+)-R-amino acids with high optical purity
from enantioselective biotransformations of DL-R-amino
nitriles with Rhodococcus sp. AJ 270 cells under very mild
conditions. The overall enantioselection of the biotrans-
formations was determined by the combined effects of a
dominantly high L-enantioselective amidase and a low
L-enantioselective nitrile hydratase involved in the cell.
Coupled with the chemical hydrolysis of amide, this
biotransformation process provided efficient syntheses of
optically pure arylglycines in both enantiomeric forms
from readily available Strecker nitriles. It can also be
developed into a valuable synthetic alternative to other
nonproteinogenic and nonnaturally occurring amino ac-
ids, which is being actively investigated in this labora-
tory.
En zym a tic Hyd r olysis of DL-P h en ylglycin e Nitr ile (1a ).
D-(-)-Phenylglycine amide (2a ) as a white solid: 8 h (43%); [R]25
D
-108 (c 0.5, MeOH); ee >99% (HPLC); mp 126-127 °C; IR (KBr)
3378, 3339, 3269, 1679 cm-1; 1H NMR (DMSO-d6) δ 7.49 (s, 1H,
CONHH), 7.20-7.45 (m, 5H, ArH), 7.04 (s, 1H, CONHH), 4.29
(s, 1H, CH); MS (EI) m/z 106 ([M - CONH2]+, 100%). Anal. Calcd
for C8H10N2O: C, 63.98; H, 6.71; N, 18.65. Found: C, 63.90; H,
6.45; N, 18.30.
L-(+)-Phenylglycine 3a as white powder: 8 h (52%); [R]25
D
+141 (c 0.95, 1 N HCl) (lit19 [R]25 -155 (c 1.0, 1 N HCl, D-(-)-
D
phenylglycine); ee >99% (HPLC); mp 252 °C (lit.20 mp 246 °C);
IR (KBr) 3130-2611, 1610 cm-1 1H NMR (D2O) δ 7.24-7.28
;
(m, 5H, ArH), 4.96 (s, 1H, CH).
In a multigram scale biotransformation reaction, DL-phenyl-
glycine nitrile 1a (3.2 g, 19 mmol) was added in 10 portions (∼2
mmol each) to the suspension of Rhodococcus sp. AJ 270 (2 g wet
weight) in phosphate buffer (pH 7.70, 40 mL) during a time span
of 12 h. Another 30 mL of the culture suspension containing 1
g of cells were also added to the reaction flask at 4.5 and 9 h
time intervals. After a total of 15 h of incubation at 30 °C, and
workup, the reaction afforded D-(-)-phenylglycine amide 2a (1.31
g, 46%) with ee >99% and L-(+)-phenylglycine 3a (1.52 g, 53%)
with ee 91%.
En zym a tic Hyd r olysis of DL-4-Ch lor op h en ylglycin e Ni-
tr ile (1b). D-(-)-4-Chlorophenylglycine amide (2b) as white
powder: 4 h (42%); [R]25D -87 (c 0.55, MeOH); ee >99% (HPLC);
mp 94 °C; IR (KBr) 3360, 3312, 3290, 1666 cm-1 1H NMR
;
(DMSO-d6) δ 7.49 (s, 1H, CONHH), 7.40 (d, J ) 8.6 Hz, 2H,
ArH), 7.35 (d, J ) 8.6 Hz, 2H, ArH), 7.06 (s, 1H, CONHH), 4.29
(s, 1H, CH); MS (EI) m/z 142 (32), 140 ([M - CONH2]+, 100%).
Aanl. Calcd for C8H9ClN2O: C, 52.04; H, 5.91; N, 15.17. Found:
C, 52.15; H, 5.01; N, 15.20.
L-(+)-4-Chlorophenylglycine (3b) as white powder: 4 h (52%);
[R]25D +129.4 (c 0.34, 1 N HCl) [lit.21 -142 (c 0.5, 1 N HCl), D-(-)-
4-chlorophenylglycine]; ee 97% (HPLC); mp 229 °C (lit.20 mp 238
1
°C); IR (KBr) 2978-2654, 1633 cm-1; H NMR (D2O) δ 7.26 (d,
Exp er im en ta l Section 17
J ) 6.9, 2H, ArH), 7.20 (d, J ) 6.9, 2H, ArH), 4.96 (s, 1H, CH).
En zym a tic Hyd r olysis of DL-3-Ch lor op h en ylglycin en i-
tr ile (1c). D-(-)-3-Chlorophenyglycine amide 2c as white pow-
All starting DL-amino nitriles were synthesized following a
modified Strecker reaction from the corresponding aldehydes
according to the literature.18 Racemic amino amides and amino
acids as standards were prepared by chemical hydrolysis of DL-
amino nitriles following the literature methods.15,19 The con-
figurations of optically active L-amino acids 3 were determined
by the comparison of their direction of optical rotation with that
of authentic samples, while the configurations of D-amino amides
2 were obtained by correlating optical rotation of their chemically
hydrolyzed amino acid products with that of amino acids of
known configurations. All enantiomeric excess values were
obtained from HPLC analysis with use of Crownpark CR(+) and
Chiralcel OD and OJ columns (see Supporting Information).
Gen er al P r ocedu r e for th e Biotr an sfor m ation s of Am in o
Nitr iles a n d Am in o Am id es. To an Erlenmeyer flask (100 mL)
with a screw cap was added Rhodococcus sp. AJ 270 cells6 (2 g
der: 2.7 h (48%); [R]25 -80 (c 0.5, MeOH); ee >99% (HPLC);
D
mp 140 °C; IR (KBr) 3392, 3352, 1685, 1669 cm-1
;
1H NMR
(DMSO-d6) δ 7.52 (s, 1H, CONHH), 7.46 (s, 1H, ArH), 7.31-
7.36 (m, 3H, ArH), 7.08 (s, 1H, CONHH), 4.30 (s, 1H, CH); MS
(EI) m/z 142 (32), 140 ([M - CONH2]+, 100%). Anal. Calcd for
C8H9ClN2O: C, 52.04; H, 5.91; N, 15.17. Found: C, 52.20; H,
4.96; N, 14.76.
L-(+)-3-Chlorophenylglycine 3c as white powder: 2.7 h (50%);
[R]25 +137 (c 0.35, 1 N HCl) [lit.22 -118 (c 1.0, 1 N NH4OH),
D
D-(-)3-chlorophenylglycine]; ee 95% (HPLC); mp 232 °C (lit.20
mp 238 °C); IR (KBr) 2628-3058, 1615, 1602 cm-1
(D2O) δ 6.86-7.02 (M, 4H, ArH), 4.69 (s, 1H, CH).
;
1H NMR
En zym a tic Hyd r olysis of DL-4-Meth yp h en ylglycin e Ni-
tr ile 1d . D-(-)-4-Methyphenylglycine amide (2d ) as white
powder: 3 h (42%); [R]25 -100 (c 0.5, MeOH); ee 86% (HPLC);
D
(17) General experimental information was given in ref 6.
(18) Hanafusa, T.; Ichihara, I.; Ashida, T. Chem. Lett. 1987, 687.
(19) Yokozeki, K.; Mitsugi, K.; Iwagami, H. German Patent, 285245,
1978.
(20) Zhang, Y.-M.; Li, W.-X. Synth. Commun. 1988, 18, 1685.
(21) Vermier, J . M.; Hegedus, L. S.; Miller, D. B. J . Org. Chem. 1992,
57, 6914.
(22) Garcia, M. J .; Azerad, R. Tetrahedron: Asymmetry 1997, 8, 85.
6544 J . Org. Chem., Vol. 67, No. 18, 2002

mp 104-105 °C; IR (KBr) 3370, 3309, 1661, 1654 cm-1; 1H NMR
(DMSO-d6) δ 7.40 (s, 1H, CONHH), 7.24 (d, J ) 7.9 Hz, 2H,
ArH), 7.08 (d, J ) 7.9 Hz, 2H, ArH), 6.91 (s, 1H, CONHH), 4.21
(s, 1H, CH), 2.25 (s, 3H, CH3); MS (EI) m/z 120 ([M - CONH2]+,
100%). Anal. Calcd for C9H12N2O: C, 65.83; H, 7.37; N, 17.06.
Found: C, 65.50; H, 7.67; N, 17.36.
En zym a tic Hyd r olysis of DL-Cycloh exylglycin e Nitr ile
(1i). D-(-)-Cyclohexylglycine amide 2i as a white solid: 21 h
(45%); [R]25 -20 (c 0.5, MeOH); ee 88% (HPLC analysis of the
D
corresponding N-benzoylated compound); mp 145-146 °C; IR
(KBr) 3349, 3258 (sh) 3195 cm-1; 1H NMR (DMSO-d6) δ 7.23 (s,
1H, CONHH), 6.88 (s, 1H, CONHH), 2.83 (d, J ) 5.1 Hz, 1H,
CH), 1.43-1.67 (m, 6H), 0.91-1.18 (m, 5H); 13C NMR (DMSO-
d6) δ 177.2, 59.8, 41.8, 29.8, 27.5, 26.24, 26.20, 26.0; MS (FAB)
m/z 156 (M+), 155 ([M - 1]+). Anal. Calcd for C8H16N2O: C,
61.51; H, 10.32; N, 17.93. Found: C, 61.18; H, 10.30; N, 17.74.
L-(+)-4-Methyphenylglycine (3d ) as white powder: 3 h (50%);
[R]25 +175 (c 0.9, 1 N HCl)) [lit.15 [R]23 +151 (c 1.0, 1N HCl);
D
D
ee >99% (HPLC); mp 247 °C (lit.20 mp 244 °C); H NMR (D2O)
δ 7.14 (s, 4H, ArH), 4.95 (s, 1H, CH), 2.14 (s, 3H, CH3).
En zym a tic Hyd r olysis of DL-2-Meth yp h en ylglycin e Ni-
tr ile (1e). D-(-)-2-Methyphenylglycine amide 2e as white
1
L-(+)-Cyclohexylglycine 3i as a white solid: 21 h (47%); [R]25
D
+28 (c 0.5, 1 N HCl) [lit.23 [R]25D +31.3 (c 1.0 1N HCl)]; ee >99%
(HPLC analysis of the corresponding N-benzoylated compound);
mp 255 °C dec (lit.23 mp >300 °C); IR (KBr) 2611-3020, 1595
(sh), 1584 cm-1; 1H NMR (D2O) δ 3.32 (d, J ) 5.2, 1H, CH), 0.52-
1.16 (m, 11H); MS (FAB) m/z 157 (M+), 156 ([M - 1]+). Anal.
Calcd for C8H15NO2: C, 61.12; H, 9.62; N, 8.91. Found: C, 60.90;
H, 9.71; N, 8.91.
powder: 12 h (45%); [R]25 -72 (c 1.0, MeOH); ee 80% (HPLC);
D
mp 117-118 °C; IR (KBr) 3351, 3274, 1661, 1618 cm-1; 1H NMR
(DMSO-d6) δ 7.39 (s, 1H, CONHH), 7.09-7.28 (m, 5H, ArH and
CONHH), 4.42 (s, 1H, CH), 2.34 (s, 3H, CH3); MS (EI) m/z 120
([M - CONH2]+, 100%). Anal. Calcd for C9H12N2O: C, 65.83; H,
7.37; N, 17.06. Found: C, 65.98; H, 7.78; N, 17.27.
En zym a tic Hyd r olysis of DL-P h en yla la n in e Nitr ile (1j).
L-(+)-2-Methyphenylglycine 3e as white powder: 12 h (42%);
[R]25 +172 (c 0.5, 1 N HCl); ee >99% (HPLC); mp 211 °C; IR
D-(-)-Phenylalanine amide (2j) as a white powder: 0.7 h (48%);
D
[R]25 -9 (c 1.0, MeOH); ee 77% (HPLC); mp 135-137 °C; IR
(KBr) 2620-3148, 1675, 1632, 1618 cm-1; 1H NMR (D2O) δ 7.23-
7.30 (m, 4H, ArH), 4.93 (s, 1H, CH), 2.37 (s, 3H, CH3); MS (EI)
m/z 120 ([M - CO2H]+, 100%). Anal. Calcd for C9H11NO2: C,
65.44; H, 6.71; N, 8.48. Found: C, 65.29; H, 6.67; N, 8.32.
En zym ztic Hyd r olysis of DL-4-Meth oxyp h en ylglycin e
Nitr ile (1f). D-(-)-4-Methoxyphenylglycine amide (2f) as white
powder: 1.5 (45%); [R]25D -80 (c 0.25, MeOH); ee >99% (HPLC);
mp 159 °C; IR (KBr) 3404, 3368, 3359, 3205, 1634 cm-1; 1H NMR
(DMSO-d6) δ 7.39 (s, 1H, CONHH), 7.27 (d, J ) 8.5 Hz, 2H,
ArH), 6.94 (s, 1H, CONHH), 6.84 (d, J ) 8.5 Hz, 2H, ArH), 4.19
(s, 1H, CH), 3.70 (s, 3H, CH3O); MS (EI) m/z 136 ([M - CONH2]+,
100%); MS (FAB) 166 ([M + 1]+). Anal. Calcd for C9H12N2O2:
C, 59.99; H, 6.71; N, 15.55. Found: C, 59.64; H, 6.86; N, 15.34.
L-(+)-4-Methoxyphenylglycine (3f) as white powder: 1.5 h
(51%); [R]25 +153 (c 0.9, 1 N HCl) [lit.21 [R]23 +156 (c 1.0, 3N
D
(KBr) 3338, 3182, 1667 cm-1; H NMR (DMSO-d6) δ 7.16-7.31
1
(m, 6H, ArH and CONHH), 6.96 (s, 1H, CONHH), 3.29-3.34
(m, 1H, CH), 2.90 (dd, J ) 13.3, 5.1 Hz, 1H), 2.58 (dd, J ) 13.3,
8.4 Hz, 1H); MS (FAB) m/z 164 (M+), 163 ([M - 1]+). Anal. Calcd
for C9H12N2O: C, 65.83; H, 7.37; N, 17.06. Found: C, 65.43; H,
7.53; N, 16.98.
L-Phenylalanine (3i) as a white solid: 0.7 h (42%); [R]25 -2
D
20
(c 1.0, 1 N HCl) (lit.2424 [R]D +7.07 (c 1.0, 1 N HCl), D-(+)-
phenylalanine); ee 51% (HPLC); mp 258 °C dec (lit.24 mp 271
°C dec, dL-phenylalanine); IR (KBr) 2554-3339, 1589 cm-1; 1H
NMR (D2O) δ 6.98-7.13 (m, 5H, ArH), 4.04 (dd, J ) 7.5, 5.9 Hz,
1H, CH), 3.04 (dd, J ) 14.9, 5.6 Hz, 1H), 2.90 (dd, J ) 14.9, 7.7
Hz, 1H).
En zym a tic Hyd r olysis of DL-Va lin e Nitr ile (1k ). D-Valine
amide (2k ) as a white solid: 1.2 h (48%); ee 22% (HPLC analysis
of the corresponding N-benzoylated compound); mp 80-83 °C;
IR (KBr) 3356, 1664, 1607 cm-1; 1H NMR (DMSO-d6) δ 7.22 (s,
1H, CONHH), 6.87 (s, 1H, CONHH), 2.82 (d, J ) 5.0 Hz, 1H,
CH), 1.72-1.88 (m, 1H, CH), 0.82 (d, J ) 6.8 Hz, 3H, CH3), 0.73
(d, J ) 6.8 Hz, 3H, CH3); MS (FAB) m/z 116 (M+), 115 ([M -
1]+). Anal. Calcd for C5H12N2O: C, 51.70; H, 10.41; N, 24.12.
Found: C, 51.73; H, 10.52; N, 24.02.
L-Valine 3k as a white solid: 1.2 h (47%); ee 34% (HPLC
analysis of the corresponding N-benzoylated compound); mp 210
°C dec [lit.24 mp 298 °C dec, DL-valine]; IR (KBr) 2585-3164,
1595 cm-1; 1H NMR (D2O) δ 3.48 (d, J ) 4.4 Hz, 1H, CH), 2.13-
2.16 (m, 1H), 0.91 (d, J ) 7.0 Hz, 3H, CH3), 0.85 (d, J ) 7.0 Hz,
3H, CH3).
D
D
HCl)]; ee 96% (HPLC); mp 248-249 °C [lit.15 mp 246 °C]; IR
(KBr) 2508-3043, 1615 cm-1; 1H NMR (D2O) δ 7.13 (d, J ) 8.7
Hz, 2H, ArH), 6.78 (d, J ) 8.7 Hz, 2H, ArH), 4.87 (s, 1H, CH),
3.56 (s, 3H, CH3O).
En zym a tic Hyd r olysis of DL-3-Meth oxyp h en ylglycin e
Nitr ile (1g). D-(-)-3-Methoxyphenylglycine amide (2g) as white
powder: 1.5 h (47%); [R]25 -64 (c 0.5, MeOH); ee 77% (HPLC);
D
mp 85 °C; IR (KBr) 3382, 3348, 3326, 3284, 1674 cm-1; 1H NMR
(DMSO-d6) δ 7.47 (s, 1H, CONHH), 7.21 (t, J ) 7.7 Hz, 1H, ArH),
7.02 (s, 1H, CONHH), 6.97 (s, 1H, ArH), 6.95 (d, J ) 7.7 Hz,
1H, ArH), 6.79 (d, J ) 7.7 Hz, 1H, ArH), 4.23 (s, 1H, CH), 3.73
(s, 3H, CH3O); MS (EI) m/z 136 ([M - CONH2]+, 100%). Anal.
Calcd for C9H12N2O2: C, 59.99; H, 6.71; N, 15.55. Found: C,
59.50; H, 6.78; N, 15.45.
Ch em ica l H yd r olysis of D-(-)-P h en ylglycin e Am id e
(2a ). D-(-)-Phenylglycine amide 2a (1 mmol, ee>99%) was
refluxed in hydrochloric acid solution (6 N) for 2.5 h to give, after
the workup as for L-(+)-3a , pure D-(-)-phenylglycine 4 in 99%
yield. [R]25 -121 (c 0.8, 1 N HCl) (lit.19 [R]25 -155 (c 1.0,
L-(+)-3-Methoxyphenylglycine (3g) as white powder: 1.5 h
(46%); [R]25 +176 (c 0.5, 1 N HCl) (lit.22 [R]23 -115 (c 1.0, 1 N
D
D
NH4OH), D-(-)-3-methoxyphenylglycine); ee >99% (HPLC); mp
202 °C; IR (KBr) 2607-3157, 1610 cm-1; 1H NMR (D2O) δ 7.33
(t, J ) 7.9 Hz, 1H, ArH), 6.95-6.97 (m, 3H, ArH), 4.61 (s, 1H,
CH), 3.76 (s, 3H, CH3O); MS (EI) m/z 120 ([M - CO2 - NH3]+,
100%). Anal. Calcd for C9H11N2O2: C, 59.66; H, 6.12; N, 7.73.
Found: C, 59.32; H, 6.21; N, 7.79.
D
D
1 N HCl), D-(-)-phenylglycine); ee >99% (HPLC); mp 239 °C
(lit.20 mp 246 °C).
Ack n ow led gm en t. This work was supported by the
Major Basic Research Development Program (No.
2000077506), the National Nature Science Foundation
of China, and the Chinese Academy of Sciences. M.X.W.
thanks O. Meth-Cohn and J . Colby for discussions.
En zym a tic Hyd r olysis of DL-3,4-Meth ylen ed ioxyp h en yl-
glycin e Nit r ile (1h ). D-(-)-3,4-Methylenedioxyphenylglycine
amide (2h ) as a yellowy powder: 2.2 h (39%); [R]25 -45 (c 0.4,
D
MeOH); ee >99% (HPLC); mp 128-129 °C; IR (KBr) 3381, 3309,
3293 (sh), 3013, 1665, 1653 cm-1;1H NMR (DMSO-d6) δ 7.38 (s,
1H, CONHH), 6.96 (s, 1H, CONHH), 6,93 (s, 1H, ArH), 6.82 (d,
J ) 8.0 Hz, 1H, ArH), 6.79 (d, J ) 8.0 Hz, 1H, ArH), 5.94 (s,
2H, OCH2O), 4.17 (s, 1H, CH); MS (EI) m/z 150 ([M - CONH2]+,
100%). Anal. Calcd for C9H10N2O3: C, 55.67; H, 5.19; N, 14.43.
Found: C, 55.62; H, 5.36; N, 14.28.
Su p p or tin g In for m a tion Ava ila ble: Preparation of ra-
cemic R-amino nitrile hydrochlorides 1, N-benzoylation of
valine, cyclohexylglycine, and their amides, HPLC analysis of
amino acids 3 and amides 2, and results of biotransformation
of racemic R-amino acid amides 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
L-(-)-3,4-Methylenedioxyphenylglycine (3h ) as a yellow pow-
der: 2.2 h (48%); [R]25 +120 (c 0.4, 1 N HCl); ee 99% (HPLC);
D
J O0256282
mp 229 °C; IR (KBr) 2598-3155, 1610 cm-1; H NMR (D2O) δ
1
6.73-6.78 (m, 3H, ArH), 5.78 (s, 2H, OCH2O), 4.88 (s, 1H, CH);
MS (EI) m/z 195 (M+, 5%), 150 ([M - CO2H]+, 100). Anal. Calcd
for C9H9NO4: C, 55.39; H, 4.65; N, 7.18. Found: C, 55.29; H,
4.68; N, 7.11.
(23) Beller, M.; Eckert, M.; Geissler, H.; Napierski, B.; Rebenstock,
H.-P.; Holla, E. W. Chem.-Eur. J . 1998, 4, 935.
(24) Oversly L. B.; Ingersole, A. M. J . Am. Chem. Soc. 1951, 73,
3363.
J . Org. Chem, Vol. 67, No. 18, 2002 6545