Highly efficient and enantioselective synthesis of L-arylglycines and D-arylglycine amides from biotransformations of nitriles

Article abstract of DOI:10.1016/S0040-4039(01)01439-3

Under very mild conditions, the Rhodococcus sp. AJ270-catalysed biotransformation of arylglycine nitriles 1, prepared easily from the reaction of substituted benzaldehydes, ammonium chloride and potassium cyanide, proceeded efficiently to produce optically active D-arylglycine amides 2 and L-arylglycines 3 in excellent yields with enantiomeric excesses higher than 99%.

Full text of DOI:10.1016/S0040-4039(01)01439-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6925–6927
Highly efficient and enantioselective synthesis of
L
-arylglycines
and
D
-arylglycine amides from biotransformations of nitriles
Mei-Xiang Wang* and Shuang-Jun Lin
Centre for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China
Received 29 May 2001; accepted 3 August 2001
Abstract—Under very mild conditions, the Rhodococcus sp. AJ270-catalysed biotransformation of arylglycine nitriles 1, prepared
easily from the reaction of substituted benzaldehydes, ammonium chloride and potassium cyanide, proceeded efficiently to
produce optically active
© 2001 Elsevier Science Ltd. All rights reserved.
D-arylglycine amides 2 and L-arylglycines 3 in excellent yields with enantiomeric excesses higher than 99%.
Arylglycines, a type of nonproteinogenic amino acid,
and their derivatives have attracted much attention
because such compounds exhibit intriguing biological
activity. A number of a-arylglycines, for example, have
been used as antagonists of metabotropic glutamate
sis of optically active arylglycines and their amides
utilising readily available starting materials should be of
great interest.
Nitriles are important organic compounds and they are
easily prepared and chemically transformed.9 For
example, the formation of an a-amino nitrile from an
aldehyde or ketone, ammonium chloride and sodium
cyanide, followed by hydrolysis (Strecker synthesis), is a
simple method for preparing racemic a-amino acids.
Unfortunately, chemical hydrolysis of nitriles including
amino nitriles needs harsh conditions such as using
strong acid or base, which always results in a low
chemical yield and by-products. Biotransformations of
nitriles using both microbial cells and enzymes have
been demonstrated as being unique and environmen-
tally benign methods for the synthesis of chiral car-
boxylic acids and their derivatives due to the excellent
selectivity and very mild reaction conditions.10 Our
earlier work11 has demonstrated that Rhodococcus sp.
AJ270, a robust nitrile hydratase/amidase-containing
microorganism, was able to hydrolyse a wide range of
structurally diverse mono- and di-nitriles with excellent
chemo- and regioselectivities. Very recently, we have
found that Rhodococcus sp. AJ270 could efficiently and
enantioselectively catalyse the hydrolysis of a number
of racemic a-substituted phenylacetonitriles12 and 2-
arylcyclopropanecarbonitriles13 to produce the corre-
sponding enantiopure carboxylic acids and amides in
high yields. The nitrile hydratase and/or amidase
involved in Rhodococcus sp. AJ270 could also recognise
the enantiotopic cyano group during the course of
desymmetrisation of 3-arylglutaronitriles.14 Our interest
in understanding the reaction scope and limitations of
both nitrile hydratase and amidase involved in Rhodo-
receptors,1 while
D-phenylglycine amide and its 4-
hydroxyphenylglycine amide analogue have been used
successfully to produce penicillin and cephalosporin
antibiotics.2 Arylglycines and their amides are also
powerful and versatile building blocks in organic syn-
thesis, and they have been employed widely as chiral
auxiliaries and ligands in asymmetric reactions.3 Syn-
theses of optically active a-amino acids including aryl-
glycines and their derivatives are well documented in
the literature.4 Although asymmetric chemical syntheses
of amino acid derivatives have advanced tremendously
in recent years, enzymatic reactions still play an impor-
tant part in the preparation and production of amino
acids. Lipase- and esterase-catalyzed kinetic resolution
of racemic amino acid esters and of N-acylated amino
acids, for instance, has been extensively studied.2,5,6
or -Hydantoinases catalyse an efficient ring opening
reaction of hydantoin to afford - or -amino acids,
respectively, a process that has been developed into
commercial production of
-amino acids.7 Amidase has
D-
L
D
L
D
also been reported to effect amino acid synthesis
through kinetic resolution of a racemic amino amide.8
All methods reported, however, are limited to certain
types of substrate and often require the preparation of
the precursors or derivatives of the amino acids, which
are sometimes laborious. A general asymmetric synthe-
* Corresponding author. Tel.: +8610-62554628; fax: +8610-62569564;
e-mail: mxwang@infoc3.icas.ac.cn
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01439-3

6926
M.-X. Wang, S.-J. Lin / Tetrahedron Letters 42 (2001) 6925–6927
out in a phosphate buffer at pH 7.13. Although the
transformation proceeded in 8 h at pH 7.13, which is
slower than that at pH 7.62, no detrimental effect such
as decomposition of the substrate 1a was observed. The
amidase involved in Rhodococcus sp. AJ270 appeared
coccus sp. AJ270, and in preparing chiral arylglycines
and their amides has led us to undertake the current
study.
Few examples of enantioselective biotransformations of
a-amino nitriles have been reported. The nitrilase of
Rhodococcus rhodochrous PA-3415 and a nitrilase-con-
taining Acinetobacter sp. culture16 have the ability to
highly
and
L
-selective, as the complete conversion of both
L-
D
-amide was only effected after more than 1 day
and with low substrate concentration. To test its useful-
ness in practical organic synthesis, we tried a gram-scale
biotransformation of 1a (6.75 mmol, 1.14 g), which
produced almost identical chemical yields of 2a and 3a
with e.e.s >99%.
convert several aliphatic DL-a-amino nitriles into
amino acids in moderate to excellent enantiomeric
excesses, while the nitrilase-catalysed hydrolysis of DL
phenylglycine nitrile using Aspergillus furmigatus has
been reported to form
-phenylglycine in 80% e.e.17
Using a mutant of Brevibacterium sp. R312, Arnaud et
al.18 claimed to obtain a
-specific amidase which trans-
formed aliphatic DL-amino amides into -amino acids
and -amino amides.
L-
-
L
In order to investigate the effect of the substituent on
the phenyl ring on the reactivity and enantioselectivity
of the biotransformation, we extended our range of
substrates to other arylglycine nitriles that were pre-
pared readily following the Strecker reaction from sub-
stituted benzaldehydes.19 In the aqueous buffer with pH
7.13, however, the hydration of the substituted phenyl-
glycine nitriles 1b–g was slower than their parent ana-
logue 1a, and consequently they underwent
decomposition to release aldehyde and cyanide that in
turn caused inhibition of nitrile hydratase. The bio-
transformation of 1b–g was then accelerated when the
pH of the buffer was adjusted to 7.62 (Scheme 1). As
illustrated in Table 1, the reaction rate and enantiose-
lectivity were dependent on both the electronic and
steric effects of the substituent. For example, the pres-
ence of a 2-methyl group on the phenyl ring retarded
the reaction (entries 8–9), while 3-chlorophenylglycine
nitrile 1c led to amino acid 3c in an enantiomeric excess
of 87%. It is also worth noting that excellent enantiose-
lectivities and high yields were obtained for most of the
L
L
D
To begin our study, we first examined the biotransfor-
mation of DL-phenylglycine 1a, a commercially avail-
able chemical, under various conditions (entries 1–4 in
Table 1). Considering the optimal working pH for
nitrile hydratase of Rhodococcus sp. AJ270 and to
prevent the decomposition of phenylglycine nitrile in
aqueous buffer, the biotransformation was performed
in a phosphate buffer with pH 7.62. The hydration of
nitrile was found to be very rapid with all the amino
nitrile being consumed in 25 min. Quenching the reac-
tion after 2.5 h gave
D
-phenylglycine amide 2a and
L
-phenylglycine 3a in ca. 50% yield each. The enan-
tiomeric excesses obtained for amide 2a and acid 3a
were not satisfactory, being 74 and 93%, respectively.
The enantioselectivity of the biotransformation was
then improved to >99% when the reaction was carried
Table 1. Enantioselective biotransformation of arylglycine nitriles20
Entry
Sub. 1
Ar
Conditionsa
Arylglycine amide 2
Arylglycine 3
Yieldb (%) e.e.c (%)
Yieldb (%)
e.e.c (%)
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1b
1c
1d
1e
1e
1f
C6H5
C6H5
C6H5
C6H5
4-ClC6H4
3-ClC6H4
4-MeC6H5
2-MeC6H5
2-MeC6H5
4-MeOC6H5
3-MeOC6H5
3,4-OCH2OC6H3
2 mmol, pH 7.62, 2.5 h
2 mmol, pH 7.13, 8 h
2 mmol, pH 7.13, 9.5 h
1 mmol, pH 7.13, 25 h
2 mmol, pH 7.62, 4 h
2 mmol, pH 7.62, 3 h
2 mmol, pH 7.62, 3 h
2 mmol, pH 7.62, 5 h
2 mmol, pH 7.62, 12 h
2 mmol, pH 7.62, 3.5 h
2 mmol, pH 7.62, 1.5 h
2 mmol, pH 7.62, 2 h
49
43
23
–
42
26
42
62
45
11
47
35
74
\99
\99
–
\99
93
86
17
38
\99
71
47
52
58
93
52
40
50
30
42
47
46
41
93
\99
81
5
97
87
\99
\99
\99
\99
\99
\99
9
10
11
12
1g
1h
62
a Rhodococcus sp. AJ270 cells (2 g wet weight) in phosphate buffer (0.1 M, 50 ml) were used. The reaction conditions were not optimised.
b Isolated yield.
c Determined by chiral HPLC analysis.21
Scheme 1.

M.-X. Wang, S.-J. Lin / Tetrahedron Letters 42 (2001) 6925–6927
6927
Scheme 2.
arylglycines 3d–h, while only with para-substituted
phenylglycine nitriles such as 1b and 1f was good to
excellent enantiocontrol observed for the corresponding
amino amides.
2. Wegman, M. A.; Hacking, M. A. P. J.; Rops, J.; Pereira,
P.; van Rantwijk, F.; Sheldon, R. A. Tetrahedrom: Asym-
metry 1999, 10, 1739 and references cited therein.
3. For a review, see: Coppola, G. M.; Schuster, H. F.
Asymmetric Synthesis, Construction of Chiral Molecules
Using Amino Acids. Wiley: New York, 1987.
4. For reviews, see: (a) Duthaler, R. O. Tetrahedron 1994,
50, 1539; (b) Williams, R. M. Synthesis of Optically Active
h-Amino Acids; Pergamon: Oxford, 1989; (c) Williams, R.
M.; Hendrix, J. A. Chem. Rev. 1992, 92, 889.
5. For reviews: see: (a) Enzyme Catalysis in Organic Synthe-
sis; Drauz, K.; Waldmann, H., Eds.; VCH: Weinheim,
1995; (b) Wong, C.-H.; Whitesides, G. M. Enzymes in
Synthetic Organic Chemistry; Pergamon, 1994; (c) Prepar-
ative Biotransformations: Whole Cell and Isolated Enzymes
in Organic Chemistry; Roberts, S. M., Ed.; Wiley: New
York, 1993.
6. Baker, S. R.; Goldsworthy, J.; Harden, R. C.; Salhoff, C.
R.; Schoepp, D. D. Bioorg. Med. Chem. Lett. 1995, 5, 223.
7. Garcia, M. J.; Azerad, R. Tetrahedron: Asymmetry 1997,
8, 85 and references cited therein.
8. Hermes, H. F. M.; Tandler, R. F.; Sonke, T.; Dijkhuizen,
L.; Meijer, E. M. Appl. Environ. Microbiol. 1994, 60, 153.
9. The Chemistry of the Cyano Group; Rappoport, Z., Ed.;
Wiley Interscience: New York, 1970.
To shed further light on the process, the racemic
phenylglycine amide 2a, obtained from the chemical
hydration of phenylglycine nitrile 1a using concentrated
H2SO4 (98%), was fed to Rhodococcus sp. AJ270 under
identical reaction conditions. The reaction was termi-
nated at 50% conversion to give
D
-phenylglycine amide
2a in 84% e.e. and
L
-phenylglycine 3a in 85% e.e.
(Scheme 2).
The outcome of the biotransformations of arylglycine
nitriles 1 and of phenylglycine amide 2a indicated that
the amidase involved in Rhodococcus sp. AJ270 is
highly
L
-
or S-enantioselective, while the nitrile
- or S-enantioselec-
hydratase probably also exhibits
L
tivity but to a low degree.22 Additionally, the enantiose-
lectivity of nitrile hydratase is probably affected greatly
by the structure of the amino nitriles. Therefore double
S-enantioselections of nitrile hydratase and amidase
resulted in the enantiopure arylglycines 3 and optically
active arylglycine amides 2 with varied enantiomeric
excesses depending on the substrates.
10. For recently reviews, see: (a) Sugai, T.; Yamazaki, T.;
Yokoyama, M.; Ohta, H. Biosci. Biotech. Biochem. 1997,
61, 1419; (b) Crosby, J.; Moiliet, J.; Parratt, J. S.; Turner,
N. J. J. Chem. Soc., Perkin Trans. 1 1994, 1679.
11. Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin
Trans. 1 1997, 1099; J. Chem. Soc., Perkin Trans. 1 1997,
3197.
12. Wang, M.-X.; Lu, G.; Ji, G.-J.; Huang, Z.-T.; Meth-
Cohn, O.; Colby, J. Tetrahedron: Asymmetry 2000, 11,
1123.
In conclusion, we have shown a very efficient, conve-
nient and scale-upable method for the preparation of
optically active L-arylglycines and D-arylglycine amides
from the biotransformation of racemic arylglycine
nitriles using Rhodococcus sp. AJ270 cells under very
mild conditions. The overall enantioselectivity of the
reaction was derived from the combined effects of a
high L-enantioselective amidase and a low L-enantiose-
lective nitrile hydratase involved in the cell.
13. Wang, M.-X.; Feng, G.-Q. Tetrahedron Lett. 2000, 41,
6501.
14. Wang, M.-X.; Liu, C.-S.; Li, J.-S.; Meth-Cohn, O. Tetra-
hedron Lett. 2000, 41, 8549.
15. Bhalla, T. C.; Miura, A.; Wakamoto, A.; Ohba, Y.;
Furuhashi, K. Appl. Microbiol. Biotechnol. 1992, 37, 184.
16. Macdam, A. M.; Knowles, C. J. Biotechnol. Lett. 1985, 7,
865.
17. Choi, S. Y.; Goo, Y. M. Arch. Pharm. Res. 1986, 9, 45.
18. Arnaud, A.; Galzy, P.; Jallageas, J. Bull. Soc. Chim. Fr.
1980, 87.
Acknowledgements
This work was supported by the Major Basic Research
Development Program (No. 2000077506), the National
Natural Science Foundation of China and the Chinese
Academy of Sciences. M.-X.W. thanks O. Meth-Cohn
and J. Colby for discussion.
19. Preparation of amino nitriles, see: Hanafusa, T.; Ichihara,
J.; Ashida, T. Chem. Lett. 1987, 687.
20. For general procedure of biotransformation, see: Ref. 12.
21. Chiral HPLC analysis was performed using a CROWN-
PAK CR(+) column (150 mm×4 mm) with H2O–HClO4
(pH 1.5–1.9) solution as an eluent.
22. Similar low S-enantioselectivity of nitrile hydratase was
observed in the biotransformation of 2-aryl-3-methylbuty-
ronitriles. Wang, M.-X.; Li, J.-J.; Ji, G.-J.; Li, J.-S. J.
Mol. Catal. B-Enzym. 2001, 14, 81.
References
1. (a) Watkins, J.; Collingridge, G. TiPS 1994, 15, 333; (b)
Sekiyama, N.; Hayashi, Y.; Nakanishi, S.; Jane, D. E.;
Tse, H.-W.; Birse, E. F.; Watkins, J. C. Br. J. Pharmacol.
1995, 117, 1593.