An unusual β-vinyl effect leading to high efficiency and enantioselectivity of the amidase, nitrile biotransformations for the preparation of enantiopure 3-arylpent-4-enoic acids and amides and their applications in synthesis

Article abstract of DOI:10.1021/jo061664f

(Chemical Equation Presented) Biotransformations of 3-arylpent-4- enenitriles catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase/amidase-containing microbial whole-cell catalyst were studied, and an unusual β-vinyl effect of the substrate on

Full text of DOI:10.1021/jo061664f

acrylamide from biocatalytic hydration of acrylonitrile.2 Recent
studies have demonstrated that biotransformations of nitriles
complement the existing asymmetric chemical and enzymatic
methods for the synthesis of chiral carboxylic acids and their
derivatives.3,4 The distinct features of enzymatic transformations
of nitriles are the straightforward generation of enantiopure
amides, valuable organonitrogen compounds in synthetic chem-
istry, in addition to the formation of enantiopure carboxylic
acids. For example, we3c have shown that Rhodococcus eryth-
ropolis AJ270,5 a nitrile hydratase/amidase-containing whole
cell catalyst, is able to efficiently and enantioselectively
transform nitriles including R-aminonitriles,6 R-alkyl-7 and
R-allyl-substituted arylacetonitriles,8 cyclopropanecarbonitriles,9
and oxiranecarbonitriles10 into the corresponding useful poly-
functionalized chiral carboxylic acids and amides. In contrast
to the successful enantioselective nitrile biotransformations for
the preparation of chiral carboxylic acids and amide derivatives
bearing an R-stereocenter,3-10 biotransformations of substrates
having a chiral center remote from the cyano or the amido
functional group have been reported to proceed with, in most
cases, disappointingly low enantioselectivity and chemical
yield11-15 except for some biocatalytic desymmtrization reac-
tions of 3-substituted glutoranitrile derivatives.16 For instance,
biotransformations of the Baylis-Hillman nitriles11 and its one-
carbon homologated nitriles12 gave moderate enantioselectivity
whereas â-phenylbutyronitrile,13 â-,14 γ-, or δ-hydroxylated
alkanenitriles15 gave no or extremely low enantiocontrol. It is
generally believed that the movement of a stereocenter from
the reactive site (R-position to functional group) to a remote
place gives rise to the decrease of enantioselectivity in asym-
metric reactions. However, this notion may not be true for
An Unusual â-Vinyl Effect Leading to High
Efficiency and Enantioselectivity of the Amidase,
Nitrile Biotransformations for the Preparation of
Enantiopure 3-Arylpent-4-enoic Acids and
Amides and Their Applications in Synthesis
Ming Gao, De-Xian Wang, Qi-Yu Zheng, and
Mei-Xiang Wang*
Beijing National Laboratory for Molecular Sciences, Laboratory
of Chemical Biology, Institute of Chemistry, Chinese Academy
of Sciences, Beijing 100080, China
mxwang@iccas.ac.cn
ReceiVed August 10, 2006
Biotransformations of 3-arylpent-4-enenitriles catalyzed by
Rhodococcus erythropolis AJ270, a nitrile hydratase/amidase-
containing microbial whole-cell catalyst were studied, and
an unusual â-vinyl effect of the substrate on the biocatalytic
efficiency and enantioselectivity of the amidase was ob-
served. While 3-arylpent-4-enenitriles and 3-phenylpen-
tanenitrile were efficiently hydrated by the action of the less
R-enantioselective nitrile hydratase, the amidase showed
greater activity and higher enantioselectivity against 3-aryl-
pent-4-enoic acid amides than 3-arylpentanoic acid amides.
Under very mild conditions, nitrile biotransformations pro-
vided an efficient synthesis of highly enantiopure (R)-3-
arylpent-4-enoic acids and (S)-3-arylpent-4-enoic acid amides,
and their applications were demonstrated by the synthesis
of chiral γ-amino acid, 2-pyrrolidinone, and 2-azepinone
derivatives.
(3) For reviews, see: (a) Sugai, T.; Yamazaki, T.; Yokoyama, M.; Ohta,
H. Biosci. Biotech. Biochem. 1997, 61, 1419 and references therein. (b)
Martinkova, L.; Kren, V. Biocatal. Biotrans. 2002, 20, 73. (c) Wang, M.-
X. Top. Catal. 2005, 35, 117.
(4) For recent examples, see: (a) DeSantis, G.; Zhu, Z.; Greenberg, W.
A.; Wong, K.; Chaplin, J.; Hanson, S. R.; Farwell, B.; Nicholson, L. W.;
Rand, C. L.; Weiner, D. P.; Robertson, D. E.; Burk, M. J. J. Am. Chem.
Soc. 2002, 124, 9024. (b) Effenberger, F.; Oâwald, S. Tetrahedron:
Asymmetry 2001, 12, 279. (c) Hann, E. C.; Sigmund, A. E.; Fager, S. K.;
Cooling, F. B.; Gavagan, J. E.; Ben-Bassat, A.; Chauhan, S.; Payne, M. S.;
Hennessey, S. M.; DiCosimo, R. AdV. Synth. Catal. 2003, 345, 775. (d)
Preiml, M.; Hillmayer, K.; Klempier, N. Tetrahedron Lett. 2003, 44, 5057.
(e) Yokoyama, M.; Kashiwagi, M.; Iwasaki, M.; Fushuku, K.; Ohta, H.;
Sugai, T. Tetrahedron: Asymmetry 2004, 15, 2817.
(5) (a) Blakey, A. J.; Colby, J.; Williams, E.; O’Reilly, C. FEMS
Microbiol. Lett. 1995, 129, 57. (b) Colby, J.; Snell, D.; Black, G. W.
Monatsh. Chem. 2000, 131, 655. (c) O’Mahony, R.; Doran, J.; Coffey, L.;
Cahill, O. J.; Black, G. W.; O’Reilly, C. Antonie Van Leeuwenhoek 2005,
87, 221.
(6) (a) Wang, M.-X.; Lin, S.-J. J. Org. Chem. 2002, 67, 6542. (b) Wang,
M.-X.; Lin, S.-J.; Liu, J.; Zheng, Q.-Y. AdV. Synth. Catal. 2004, 346, 439.
(7) (a) Wang, M.-X.; Lu, G.; Ji, G.-J.; Huang, Z.-T.; Meth-Cohn, O.;
Colby, J. Tetrahedron: Asymmetry 2000, 11, 1123. (b) Wang, M.-X.; Li,
J.-J., Ji, G.-J.; Li, J.-S. J. Mol. Cat. B: Enzym. 2001, 14, 77.
Biotransformations of nitriles, either through a direct conver-
sion from a nitrile to a carboxylic acid catalyzed by a nitrilase
or through the nitrile hydratase-catalyzed hydration of a nitrile
followed by the amide hydrolysis catalyzed by the amidase, have
become effective and environmentally benign methods for the
production of carboxylic acids and their amide derivatives.1 One
of the well-known examples is the industrial production of
(8) (a) Wang, M.-X.; Zhao, S.-M. Tetrahedron Lett. 2002, 43, 6617. (b)
Wang, M.-X.; Zhao, S.-M. Tetrahedron: Asymmetry 2002, 13, 1695.
(9) (a) Wang, M.-X.; Feng, G.-Q. Tetrahedron Lett. 2000, 41, 6501. (b)
Wang, M.-X.; Feng, G.-Q. New J. Chem. 2002, 1575. (c) Wang, M.-X.;
Feng, G.-Q. J. Org. Chem. 2003, 68, 621-624. (d) Wang, M.-X.; Feng, G.
Q. J. Mol. Catal. B: Enzym. 2002, 18, 267. (e) Wang, M.-X.; Feng, G.-Q.;
Zheng, Q.-Y. AdV. Synth. Catal. 2003, 345, 695; (f) Wang, M.-X.; Feng,
G.-Q.; Zheng, Q.-Y. Tetrahedron: Asymmetry 2004, 15, 347.
(10) (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.; Deng, G.; Wang, D.-X.
Zheng, Q.-Y. J. Org. Chem. 2005, 70, 2439.
* To whom correspondence should be addressed. Tel: +86-10-62565610.
Fax: +86-10-62564723.
(1) (a) Faber, K. Biotransformations in Organic Chemistry, 3rd ed.;
Spring-Verlag: New York, 1997; pp 136-145. (b) Kobayashi, M.; Shimizu,
S. FEMS Microbiol. Lett. 1994, 120, 217 and references therein. (c) Meth-
Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin Trans. 1 1997, 1099. (d)
Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin Trans. 1 1997, 3197
and references therein.
(2) Nagasawa, T.; Schimizu, H.; Yamada, H. Appl. Microbiol. Biotechnol.
1993, 40, 189.
10.1021/jo061664f CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/11/2006
9532
J. Org. Chem. 2006, 71, 9532-9535

TABLE 1. Enantioselective Biotransformations of Racemic
enzymatic reactions since the chiral recognition site of an
enzyme might be located in some distance to the catalytic center.
In other words, the chiral recognition between the enzyme and
a substrate might occur at a remote pocket from the reaction
site. If this hypothesis works, it could lead to highly enanti-
oselective biocatalytic reactions of the substrates that bear a
remotely positioned chiral center. Very recently, we17 have
shown that a simple O-benzyl protection group on â-hydroxy
nitriles acted probably as a docking group to enhance the chiral
recognition by the enzymes and therefore resulted in the
dramatic increase of enantioselectivity of biocatalysis. To further
test this hypothetic remote chiral recognition mechanism, and
also to explore the application of nitrile biotransformations in
the synthesis of enantiopure valuable and polyfunctionalized
intermediates, we undertook the investigation of biotransfor-
mation of 3-arylpent-4-enenitriles. Different from the previously
used protection/docking strategy,17,18 we introduced a vinyl
substituent into the â-position of 3-arylpropionitriles, hoping
that such an intrinsic â-functional moiety might increase the
substrates’ affinity and chiral interactions with the enzymes and
therefore lead to highly efficient and enantioselective biotrans-
formations. Gratifyingly, the presence of a â-vinyl group within
the nitrile substrates plays indeed a significant role in the
improvement of biocatalytic efficiency and enantioselectivity.
Herein, we report the highly enantioselective biotransformations
of 3-arylpent-4-enenitriles and an unusual â-vinyl substituent
effect on the activity and selectivity of the amidase involved in
the R. erythropolis AJ270 strain. The applications of the
biotransformation products in the synthesis of chiral γ-amino
acid and chiral N-heterocyclic compounds will also be discussed.
Initially, we tested the biocatalytic hydrolysis of racemic
3-phenylpent-4-enenitrile 1a in the presence of R. erythropolis
AJ270 whole cells. Under the very mild conditions, 1a was
found to undergo efficient reaction to yield optically active
carboxylic acid 2a and amide 3a. The nitrile hydratase catalyzed
hydration was very fast, and the complete conversion of nitrile
1a (40 mM) into the amide 2a was effected within several hours,
while the hydrolysis of amide 2a into acid 3a, with the aid of
the amidase, took a slightly longer period (entries 1 and 2, Table
1). Excellent chemical yields and enantiomeric excess values
were obtained for the (S)-3-phenylpent-4-enoic acid amide 2a
and (R)-3-phenylpent-4-enoic acid 3a after 15.5 h incubation
3-Arylpent-4-enenitriles 1a-h
2
3
reaction conditionsa yieldb
eec
(%)
yieldb
(%)
eec
entry
1
R
(mmol, h)
(%)
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1a
1a
1a
1a
1a
H
H
H
H
H
2, 0.5d
2, 9
2, 15.5
1, 6.5
13, 24.3e
1, 9
1, 10.5
1, 4.75
1, 5.5
1, 8.5
1, 10f
0.5, 3.5
0.5, 4
1, 9
74.5
72
48
50
45
49
49
50.5
49
47
0
44.1
95.2
90.5
>99.5
>99.5
94.9
94.5
97.4
93.2
>99.5
78.9
>99.5
59.0
3
26
47
46
49
50
50
48
50
49
55
44
55
48
98.8
98.4
95.4
94.6
92.5
90.0
95.0
90.0
91.0
94.2
57.1
92.9
77.3
58.1
1b 4-OMe
1c 4-Me
1d 4-F
1e 4-Cl
1f 3-Cl
1g 2-Cl
1g 2-Cl
1g 2-Cl
1h 4-Br
25
51
42
49
a Biotransformation was carried out in a suspension of Rhodococcus
erythropolis AJ270 cells (2 g wet weight) in phosphate buffer (50 mL, pH
7.0) at 30 °C. b Isolated yield. c Determined by HPLC analysis using a
chiral column (see the Supporting Information). d Nitrile 1a (yield 18%, ee
15.8%) was recovered. e In this multigram-scale biotransformations, 14 g
of wet weight cells was used. f Nitrile 1g (yield 11%, ee 39.4%) was
recovered.
with the cell catalyst (entry 3, Table 1). Under identical
conditions, a more efficient and highly enantioselective biotrans-
formation was observed when the concentration of 1a was
halved (entry 4, Table 1).
To examine the scope of the reaction and the influence of
the substituent attached on the benzene ring on the efficiency
and enantioselectivity of biotransformations, a number of
racemic 3-arylpent-4-enenitriles 1b-h were subjected to incu-
bation with R. erythropolis AJ270 (Table 1). As illustrated by
the results compiled in Table 1, all substrates, irrespective of
the nature of the substituent on the benzene ring, underwent
efficient biotransformations within about 10 h to afford almost
quantitative yields of the corresponding amide and acid products,
except 3-(2-chlorophenyl)pent-4-enenitrile 1g. which required
a lower concentration to fulfill effective hydrolysis (entries 11-
13). It indicates that only when the bulkiness of the benzene
ring at the ortho position increased did hydrolysis proceed
slowly. The steric effect, on the other hand, did not influence
the enantioselectivity of the biotransformations, as almost all
substrates gave excellent chemical yields of the products with
high enantiomeric purity. The only exception is the hydrolysis
of 3-(4-bromophenyl)pent-4-enenitrile 1h which showed moder-
ate enantiocontrol (entry 14, Table 1).
(11) Wang, M.-X.; Wu, Y. Org. Biomol. Chem. 2003, 1, 535.
(12) Zhao, S.-M.; Wang, M.-X. Chin. J. Chem. 2002, 20, 1291.
(13) Gradley, M. L.; Knowles, C. J. Biotechnol. Lett. 1994, 16, 41.
(14) (a) Klempier, N.; de Raat, A.; Faber, K.; Griengl, H. Tetrahedron
Lett. 1991, 32, 341. (b) In the case of biotransformation of â-benzoyloxy-
pentanenitrile, good enantioselectivity was obtained. Yokoyama, M.; Imai,
N.; Sugai, T. Ohta, H. J. Mol. Catal. B: Enzym. 1996, 1, 135.
(15) Taylor, S. K.; Chmiel, N. H.; Simons, L. J.; Vyvyan, J. R. J. Org.
Chem. 1996, 61, 9084.
(16) (a) Kakeya, H.; Sakai, N.; Sano, A.; Yokoyama, M.; Sugai, T.; Ohta,
H. Chem. Lett. 1991, 1823. (b) Crosby, J. A.; Parratt, J. S.; Turner, N. J.
Tetrahedron: Asymmetry 1992, 3, 1547. (c) Maddrell, S. J.; Turner, N. J.;
Kerridge, A.; Willetts, A. J.; Crosby, J. Tetrahedron Lett. 1996, 37, 6001.
(d) Vink, M. K.; Schortinghuis, C. A.; Luten, J.; van Maarseveen, J. H.;
Schoemaker, H. E.; Hiemstra, H.; Rutjes, F. P. J. T. J. Org. Chem. 2002,
67, 7869. (e) DeSantis, G.; Wong, K.; Farwell, B.; Chatman, K.; Zhu, Z.;
Tomlinson, G.; Huang, H.; Tan, X.; Bibbs, L.; Chen, P.; Kretz, K.; Burk,
M. J. J. Am. Chem. Soc. 2003, 125, 11476. (f) Robertson, D. E.; Chaplin,
J. A.; DeSantis, G.; Podar, M.; Madden, M.; Chi, E.; Richardson, T.; Milan,
A.; Miller, M.; Weiner, D. P.; Wong, K.; McQuaid, J.; Farwell, B.; Preston,
L. A.; Tan, X.; Snead, M. A.; Keller, M.; Mathur, E.; Kretz, P. L.; Burk,
M. J.; Short, J. M. Appl. EnViron. Microbiol. 2004, 70, 2429. (g) Wang,
M.-X.; Liu, C.-S.; Li, J.-S. Tetrahedron: Asymmetry 2001, 12, 3367. (h)
Wang, M.-X.; Liu, C.-S.; Li, J.-S.; Meth-Cohn, O. Tetrahedron Lett. 2000,
41, 8549.
Encouraged by the highly efficient and enantioselective
biotransformations of 3-arylpent-4-enenitriles, we then extended
study to racemic 3-phenylpentanenitrile 4, a substrate devoid
only of a double bond compared to 3-phenyl-pent-4-enenitrile
1a. To shed light on the stereochemical outcomes, and to
examine the effect of the â-vinyl group on the biocatalytic
(17) Ma, D.-Y.; Zheng, Q.-Y.; Wang, D.-X.; Wang, M.-X. Org. Lett.
2006, 8, 3231.
(18) (a) Braunegg, G.; De Raadt, A.; Feichtenhofer, S.; Griengl, H.;
Kopper, I.; Lehmann, A.; Weber, H.-J. Angew. Chem., Int. Ed. 1999, 38,
2763. (b) De Raadt, A.; Griengl, H.; Weber, H. Chem. Eur. J. 2001, 7, 27.
J. Org. Chem, Vol. 71, No. 25, 2006 9533

TABLE 2. Enantioselective Biotransformations of Racemic Nitriles 1a and 4 and Amides 2a and 5
1 or 4
2 or 5
3 or 6
yield,
ee,
yield,
ee,
yield,
ee,
b
c
b
c
b
c
ent
subs
R
EWG
CN
CN
CONH2
CN
timea
%
%
%
%
%
%
1
2
3
4
5
6
1a
1a
2a
4
4
5
vinyl
vinyl
vinyl
Et
Et
Et
30 min
15.5 h
15.5 h
20 min
26 h
18
18
15.8
13.4
74.5
47.5
48
79
48
<1
3
47
49
trace
47
48
98.8
95.4
95.1
N.D.
82.2
87.6
95.2
97.1
<1
CN
CONH2
85.8
81.6
23 h
48
a Substrate (2 mmol) was incubated with a suspension of R. erythropolis AJ270 cells (2 g wet weight) in phosphate buffer (50 mL, pH 7.0) at 30 °C.
b Isolated yield. c Determined by HPLC or GC analysis using the chiral columns (see the Supporting Information).
efficiency and enantioselectivity of both the nitrile hydratase
and the amidase within R. erythoropolis AJ270, cell-catalyzed
kinetic resolution of racemic amide 2a and 5 was also
investigated (Table 2). Surprisingly, under conditions identical
to those for the reaction of 1a, biotransformation of 4 proceeded
slowly with only modest enantioselectivity. For example, only
after 26 h incubation were equal amounts of optically active
(R)-amide 5 and (S)-acid 6 obtained with ee values of 85.8%
and 82.2%, respectively (entry 5, Table 2). Careful examination
of the nitrile hydration reaction of racemic 1a and 4 revealed
that the nitrile hydratase in the microbial cells exhibited high
enzymatic activity but low enantioselectivity toward both
3-phenylpent-4-enenitrile 1a and 3-phenylpentanenitrile 4, as
the hydration of 1a and 4 took place very rapidly and the ee
values for both recovered nitriles 1a and 4 were around 15%
(entries 1 and 4, Table 2). In other words, the nitrile hydratase
showed loose discrimination power between â-vinyl and â-ethyl
substituents within the substrates 1a and 4, respectively. On
the other hand, as indicated in Table 2, the kinetic resolution
of racemic amide 2a finished in 15.5 h, furnishing excellent
enantioselectivity with an enantiomeric ratio (E)19 up to 164
(entry 3, Table 2), whereas the kinetic resolution of racemic
amide 5 took almost 1 day to afford modest enantioselectivity
with E around 39 (entry 6, Table 2). It is obvious that the overall
efficiency and enantioselectivity of the nitrile biotransformations
(entries 2 and 5) were determined by a combination of the effects
of the nitrile hydratase and the amidase, with the latter playing
a dominant role. The different efficiencies and enantioselec-
tivities shown by the amidase against between 3-phenylpent-
4-enoic acid amide 2 and 3-phenylpentanoic acid amide 5
strongly suggested that the amidase involved in R. erythropolis
AJ270 is highly sensitive toward the structures of the substrates.
A slight structure variation of the substrate, such as the change
from a â-ethyl in amide 5 to a â-vinyl in amide 2, led to a
significant enhancement of the biocatalytic efficiency and
enantioselectivity. It is worth noting that, to the best of our
knowledge, it is rare that a carbon-carbon double bond at the
â-position to the amido functional group can facilitate the
amidase-catalyzed hydrolysis and can drastically increase the
enantioselectivity.
variety of nitrile substrates containing either an R-3 or a â-chiral
center.11-15 These properties of the nitrile hydratase, such as
having a broad substrate spectrum and possessing low or none
enantioselectivity, are determined by its enzyme structure in
which there is a spacious pocket near the active site.20 In other
words, marginal difference in structures between nitriles 1a and
4 was not recognized by the nitrile hydratase, and almost
identical biocatalytic hydration reactions were therefore ef-
fected.21
The high sensitivity of the amidase toward the structure of
the amide substrates is intriguing. To interpret the biocatalysis
of various racemic amides bearing an R-stereocenter, amidase
of a deeply buried and highly steric demanding active site has
been proposed.9 Taking the similarity between 3-phenylpent-
4-enoic acid amide 2a and 3-phenylpentanoic acid amide 5 into
consideration, the rationalization for the different catalytic
efficiencies and enantioselectivities of the amidase displayed
for two substrates remains a challenge at this stage because of
the lack of molecular structure of the amidase.22 However, it is
most probably that the amidase may have another binding
domain slightly away from the amido reactive site. This binding
domain seems able to discriminate the substituents of slight
electronic and/or steric difference, such as between vinyl and
ethyl. As a consequence, in addition to the interaction between
the active site of the amidase and the amido and/or aryl moieties
of the substrate, the selective binding of the â-vinyl with the
specific vinyl binding domain of the enzyme may contribute
greatly to the acceleration of the reaction and to the drastic
improvement of the enantioselectivity. Much more effort is
certainly needed to elucidate the amidase’s action on the
molecular level. Nevertheless, the outcomes of our study have
shown that, in contrast to the general notion, the biotransfor-
mations of nitriles and amides are in fact useful in the
preparation of highly enantiopure carboxylic acids and amides
with a â-chiral center, provided the substrates are carefully
designed.
As multifunctionalized molecules, chiral 3-aryl-pent-4-enoic
acids and their amide derivatives are useful in organic synthesis.
(20) (a) Huang, W. J.; Jia, J.; Cummings, J.; Nelson, M.; Schneider, G.;
Lindqvist, Y. Structure 1997, 5, 691. (b) Nagashima, S.; Nakasako, M.;
Dohmae, N.; Tsujimura, M.; Takio, K.; Odaka, M.; Yohda, M.; Kamiya,
N.; Endo, I. Nat. Struct. Biol. 1998, 5, 347.
(21) Only in the case of biotransformations of sterically well-defined
cyclopropanecarbonitrile substrates was good enantioselectivity obtained.
See ref 10b,c.
The outcomes of the current study are in agreement with
almost all previous observations that nitrile hydratases are a
type of highly active and less selective enzyme against a wide
(19) (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294. (b) Program “Selectivity” by Faber, K.; Hoenig, H.
http://www.cis.TUGraz.at/orgc/.
(22) No molecular structure of amidase involved in nitrile-hydrolyzing
microorganisms has been reported. Our study on the isolation and
crystallization of the amidase in R. erythropolis AJ270 is making progress.
9534 J. Org. Chem., Vol. 71, No. 25, 2006

SCHEME 1. Synthetic Applications of Nitrile
Biotransformation Products
enoic acids (R)-3 and (S)-3-arylpent-4-enoic acid amides (S)-2.
While the nitrile hydratase exhibits low R-enantioselectivity
against 3-arylpent-4-enenitriles, the amidase is highly R-
enantioselective toward 3-arylpent-4-enoic acid amide substrates.
Moreover, when the vinyl group in 3-phenylpent-4-enenitrile
was replaced by an ethyl group, biotransformations of racemic
3-phenylpentanenitrile 4 proceeded slowly and gave a dimin-
ished enantioselectivity, owing to the greater enzyme activity
and higher enantioselectivity of the amidase toward 3-phenyl-
pent-4-enoic acid amide 2a than toward 3-phenylpentanoic acid
amide 5. The results have expanded the application of nitrile
and amide biotransformations in the synthesis of carboxylic
acids and amides that have a â or even more remote chiral
center, provided the substrates are carefully engineered. The
resulting enantiopure 3-arylpent-4-enoic acids (R)-3 and amide
derivatives (S)-2, which are not easily available from asymmetric
chemical reactions, can serve as the versatile chiral intermediates
in organic synthesis. Their synthetic applications have been
demonstrated by the convenient preparations of chiral γ-amino
acid, five-membered 2-pyrrolidinone and seven-membered
2-azepinone derivatives.
It was noteworthy that optically active acid 3a, the only known
compound in the literature in the series of 2 and 3, was obtained
from lengthy asymmetric syntheses using chiral auxiliaries.23
Our nitrile biotransformation approach, however, provided a
straightforward and scale-able route to highly enantiopure (S)-
3-phenylpent-4-enoic acid amide (S)-2a and (R)-3-phenylpent-
4-enoic acid (R)-3a in the laboratory (entry 5, Table 1).
Chemical hydrolysis of (S)-3-phenylpent-4-enoic acid amide in
refluxing hydrochloride (6 N) resulted in a quantitative yield
of (S)-phenylpent-4-enoic acid (S)-3a, the antipode of the
biotransformation product (R)-3a (Scheme 1). A large number
of structurally diversified chiral organic compounds are feasible
on the basis of the functional group transformations of both
enantiopure intermediates 2 and 3.23,24 To demonstrate their
synthetic values, we synthesized chiral γ-amino-R-phenylbutyric
acid 9, a γ2-amino acid potentially useful for peptide mimetics,25
simply by reducing the carboxamido group of (S)-2a followed
by oxidation of the vinyl group. Upon treatment of diaz-
omethane, γ-amino acid 9 was easily converted into a five-
membered chiral lactam 10.26 A seven-membered enantiopure
ꢀ-lactam, (4S)-1-benzyl-4-phenyl-1,3,4,7-tetrahydro-azepin-
2(2H)-one 13,27 was conveniently prepared in an excellent
overall yield from the coupling reaction of (S)-3a with N-
allylbenzylamine 11 followed by ring-closure metathesis using
the first-generation Grubbs’ catalyst.28 Its 4R-enantiomer, 15
was also obtained similarly when (R)-3-phenylpent-4-enoic acid
(R)-3a was employed as the starting material (see the Supporting
Information). It is worth noting that in all chemical transforma-
tions depicted in Scheme 1, no racemization was observed.
In summary, we have shown that biotransformations of
racemic 3-arylpent-4-enenitriles, catalyzed by the R. erythropolis
AJ270 whole cell catalyst under mild conditions, provide an
efficient preparation of highly enantiopure (R)-3-arylpent-4-
Experimental Section
General Procedure for the Biotransformations of Nitriles or
Amides. To an Erlenmeyer flask (150 mL) with a screw cap were
added Rhodococcus sp. AJ270 cells5 (2 g wet weight) and potassium
phosphate buffer (0.1 M, pH 7.0, 50 mL), and the resting cells
were activated at 30 °C for 0.5 h with orbital shaking. Racemic
nitriles or amide were added in one portion to the flask, and the
mixture was incubated at 30 °C using an orbital shaker (200 rpm).
The reaction, monitored by TLC and HPLC, 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 adjusted to pH 12 with aqueous NaOH solution (2 N)
and extracted with ethyl ether (50 mL × 3). After drying (MgSO4)
and removing solvent under vacuum, the residue was subjected to
a silica gel column using a mixture of petroleum ether and ethyl
acetate (from 2:1 to 1:2) as the mobile phase to give pure amide
product and, in some cases, the recovered nitrile. The aqueous phase
was then adjusted to pH 2 with hydrochloric acid (2 N) and
extracted with ethyl ether (50 mL × 3). The organic phase was
combined. After drying (MgSO4) and removing solvent under
vacuum, the residue was chromatographied on a silica gel column
using a mixture of petroleum ether and ethyl acetate (from 20:1 to
4:1) as an eluent to give pure acid product. The structures of all
products were fully characterized by spectroscopic data and
microanalyses. The absolute configurations of acids (R)-3a, (S)-
3a, and (R)-3e were determined by comparing the directions of
their optical rotations with that of the authentic samples,23 while
the absolute configurations of other acids (R)-3 were assigned
assuming that the introduction of a substituent on the benzene ring
did not change the direction of optical rotation. The absolute
configuration of amide products was obtained from the optical
rotation of their corresponding acids.
(23) (a) Brown, E.; Deroye, C.; Touet, J. Tetrahedron: Asymmetry 1998,
9, 1605. (b) Mitsui, K.; Sato, T.; Urabe, H.; Sato, F. Angew. Chem., Int.
Ed. 2004, 43, 490.
(24) (a) Karla, R.; Ebert, B.; Thorkildsen, C.; Herdeis, C.; Johansen, T.
N.; Nielsen, B.; Krogsgaard-Larsen, P. J. Med. Chem. 1999, 42, 2053. (b)
Belda, O.; Lundgren, S.; Moberg, C. Org. Lett. 2003, 5, 2275.
(25) For a comprehensive review of peptide mimetics, see: Seebach,
D.; Beck, A. K.; Bierbaum, D. J. Chem. BiodiV. 2004, 1, 1111.
(26) Kim, B. J.; Park, Y. S.; Beak, P. J. Org. Chem. 1999, 64, 1705.
(27) For a review of medium ring N-heterocycles, see: Evans, P. A.;
Holmes, A. B. Tetrahedron 1991, 47, 9131.
Acknowledgment. We thank the MOST (2003CB716005),
the NNSC, and the CAS for financial support.
Supporting Information Available: Spectroscopic data of 1-3
1
and 7-13. H and 13C NMR spectra of 2a, 3a, 10, and 13. HPLC
analysis of all chiral products. This material is available free of
charge via the Internet at http://pubs.acs.org.
(28) (a) Grubbs, R. H.; Miller, S. J.; Fu, G. C. Acc. Chem. Res. 1995,
28, 446. (b) Fu, G. C.; Nguyen, S. T.; Grubbs, R. H. J. Am. Chem. Soc.
1993, 115, 9856.
JO061664F
J. Org. Chem, Vol. 71, No. 25, 2006 9535