Nitrile biotransformations for the synthesis of highly enantioenriched β-hydroxy and β-amino acid and amide derivatives: A general and simple but powerful and efficient benzyl protection strategy to increase enantioselectivity of the amidase

Article abstract of DOI:10.1021/jo800074k

(Chemical Equation Presented) Biotransformations of a number of racemic β-hydroxy and β-amino nitrile derivatives were studied using Rhodococcus erythropolis AJ270, the nitrile hydratase and amidase-containing microbial whole cell catalyst, under very mild conditions. The overall enantioselectivity of nitrile biotransformations was governed predominantly by the amidase whose enantioselectivity was switched on remarkably by an O- and a N-benzyl protection group of the substrates. While biotransformations of β-hydroxy and β-amino alkanenitriles gave low yields of amide and acid products of very low enantiomeric purity, introduction of a simple benzyl protection group on the β-hydroxy and β-amino of nitrile substrates led to the formation of highly enantioenriched β-benzyloxy and β-benzylamino amides and acids in almost quantitative yield. The easy protection and deprotection operations, high chemical yield, and excellent enantioselectivity render the nitrile biotransformation a useful protocol in the synthesis of enantiopure β-hydroxy and β-amino acids.

Full text of DOI:10.1021/jo800074k

Nitrile Biotransformations for the Synthesis of Highly
Enantioenriched ꢀ-Hydroxy and ꢀ-Amino Acid and Amide
Derivatives: A General and Simple but Powerful and Efficient
Benzyl Protection Strategy To Increase Enantioselectivity of the
Amidase
Da-You Ma, De-Xian Wang, Jie Pan, Zhi-Tang Huang, 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 January 12, 2008
Biotransformations of a number of racemic ꢀ-hydroxy and ꢀ-amino nitrile derivatives were studied using
Rhodococcus erythropolis AJ270, the nitrile hydratase and amidase-containing microbial whole cell catalyst,
under very mild conditions. The overall enantioselectivity of nitrile biotransformations was governed
predominantly by the amidase whose enantioselectivity was switched on remarkably by an O- and a
N-benzyl protection group of the substrates. While biotransformations of ꢀ-hydroxy and ꢀ-amino
alkanenitriles gave low yields of amide and acid products of very low enantiomeric purity, introduction
of a simple benzyl protection group on the ꢀ-hydroxy and ꢀ-amino of nitrile substrates led to the formation
of highly enantioenriched ꢀ-benzyloxy and ꢀ-benzylamino amides and acids in almost quantitative yield.
The easy protection and deprotection operations, high chemical yield, and excellent enantioselectivity
render the nitrile biotransformation a useful protocol in the synthesis of enantiopure ꢀ-hydroxy and ꢀ-amino
acids.
Introduction
building blocks for the construction of catalysts in asymmetric
synthesis.1,2 As a consequence, enormous efforts have been
spent on the synthesis of optically active ꢀ-hydroxy and ꢀ-amino
acids and their derivatives, and a large number of synthetic
methods have been reported.1–3 However, general approaches
to the efficient and catalytic synthesis of enantiopure ꢀ-hydroxy
and ꢀ-amino acids and their derivatives are still rare.
Enantiomerically pure ꢀ-hydroxy and ꢀ-amino acids and their
derivatives are important chemical entities because they are
prevalent in natural products and synthetic pharmaceuticals.1
They are also used as catalysts and the chiral ligands or essential
Biotransformations of nitriles,4 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
* To whom correspondence should be addressed. Tel: +86-10-62565610. Fax:
+86-10-62564723.
(1) (a) Noyori, R. Asymmetric Catalysis in Organic Synthesis; John Wiley
& Sons: 1994; pp 56-82. (b) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am.
Chem. Soc. 1996, 118, 7237. (c) Labeeuw, O.; Blanc, D.; Phansavath, P.;
Ratovelomanana-Vidal, V.; Genet, J.-P. Eur. J. Org. Chem. 2004, 2352. (d) Herb,
C.; Bayer, A.; Maier, M. E. Chem. Eur. J. 2004, 10, 5649.
(2) EnantioselectiVe Synthesis of ꢀ-Amino Acids; Juaristi, E., Soloshonok,
V. A., Eds.; John Wiley & Sons: New York, 2005.
10.1021/jo800074k CCC: $40.75
Published on Web 05/07/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 4087–4091 4087

Ma et al.
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. One
of the well-known examples is the industrial production of
acrylamide from biocatalytic hydration of acrylonitrile.5 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.6 One of the distinct features of enzymatic trans-
formations of nitriles is the straightforward generation of
enantiopure amides, valuable organonitrogen compounds in
synthetic chemistry, in addition to the formation of enantiopure
carboxylic acids. For example, we6c have shown that Rhodo-
coccus erythropolis AJ270,7 a nitrile hydratase/amidase-contain-
ing whole cell catalyst, is able to efficiently and enantioselec-
tively transform a variety of racemic nitriles into highly
enantiopure carboxylic acids and amides. Using the nitrile
biotransformation approach, many structurally diverse R-amino
acids and amides of high enantiomeric purity have been
synthesized.8
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
asymmetric reactions. However, this notion may not be true
for 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. To test this hypothetic remote
chiral recognition mechanism, and also to explore the application
of nitrile biotransformations in the synthesis of enantiopure
valuable ꢀ-hydroxy and ꢀ-amino acids and their amide deriva-
tives, we undertook the current study.15 We have revealed that
the biotransformations of nitriles that contain a ꢀ-stereocenter
can take place enantioselectively to afford the corresponding
amide and acid products with excellent enantiomeric purity,
provided the substrates are carefully engineered. Herein, we
report a very simple, general, but powerful substrate engineering
strategy to increase the enantioselectivity. Introduction of an
O- and N-benzyl group into the ꢀ-hydroxy and ꢀ-amino nitrile
and amide substrates, respectively, has been shown to enhance
dramatically the enantioselectivity of the amidase and therefore
to lead efficient biotransformations of nitriles to afford highly
enantioenriched ꢀ-hydroxy and ꢀ-amino acid and amide
derivatives.
In contrast to the successful enantioselective nitrile biotrans-
formations for the preparation of chiral carboxylic acids and
amide derivatives that bear an R-stereocenter,6c,9,10 biotrans-
formations 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 enantioselec-
tivity and chemical yield11–13 except for some biocatalytic
desymmtrization reactions of 3-substituted glutaronitrile deriva-
tives.14 Biotransformations of the Baylis-Hillman nitriles and
their one-carbon homologated nitriles, for example, gave only
moderate enantioselectivity,12 whereas ꢀ-phenylbutyronitrile11a
or ꢀ-, γ-, or δ-hydroxylated alkanenitriles yielded no or
extremely low enantiocontrol.11d
Results and Discussion
Biotransformations of Racemic ꢀ-Hydroxy- and ꢀ-Ben-
zyloxyalkanenitriles. We initially investigated the biotransfor-
mations of racemic 3-hydroxy alkanenitriles 1, expecting the
(10) (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. Catal. B: Enzym. 2001, 14, 77. (c) Wang, M.-X.; Zhao, S.-M.
Tetrahedron Lett. 2002, 43, 6617. (d) Wang, M.-X.; Zhao, S.-M. Tetrahedron:
Asymmetry 2002, 13, 1695. (e) Wang, M.-X.; Feng, G.-Q. Tetrahedron Lett.
2000, 41, 6501. (f) Wang, M.-X.; Feng, G.-Q. New J. Chem. 2002, 1575. (g)
Wang, M.-X.; Feng, G.-Q. J. Org. Chem. 2003, 68, 621–624. (h) Wang, M.-X.;
Feng, G.-Q. J. Mol. Catal. B: Enzym. 2002, 18, 267. (i) Wang, M.-X.; Feng,
G.-Q.; Zheng, Q.-Y. AdV. Synth. Catal. 2003, 345, 695. (j) Wang, M.-X.; Feng,
G.-Q.; Zheng, Q.-Y. Tetrahedron: Asymmetry 2004, 15, 347. (k) Wang, M-X.;
Lin, S.-J.; Liu, C.-S.; Zheng, Q.-Y.; Li, J.-S. J. Org. Chem. 2003, 68, 4570. (l)
Wang, M.-X.; Deng, G.; Wang, D.-X.; Zheng, Q.-Y. J. Org. Chem. 2005, 70,
2439. (m) Wang, J.-Y.; Wang, D.-X.; Zheng, Q.-Y.; Huang, Z.-T.; Wang, M.-
X. J. Org. Chem. 2007, 72, 2040. (n) Wang, J.-Y.; Wang, D.-X.; Pan, J.; Huang,
Z.-T.; Wang, M.-X. J. Org. Chem. 2007, 72, 9391. (o) Gao, M.; Wang, D.-X.;
Zheng, Q.-Y.; Huang, Z.-T.; Wang, M.-X. J. Org. Chem. 2007, 72, 6060.
(11) (a) Gradley, M. L.; Knowles, C. J. Biotechnol. Lett. 1994, 16, 41. (b)
Klempier, N.; de Raat, A.; Faber, K.; Griengl, H. Tetrahedron Lett. 1991, 32,
341. (c) Yokoyama, M.; Imai, N.; Sugai, T,.; Ohta, H. J. Mol. Catal. B: Enzym.
1996, 1, 135. (d) Taylor, S. K.; Chmiel, N. H.; Simons, L. J.; Vyvyan, J. R. J.
Org. Chem. 1996, 61, 9084. (e) Wu, Z.-L.; Li, Z.-Y. J. Mol. Catal. B: Enzym.
2003, 22, 105.
(3) For leading references, see: (a) Genet, J.-P. Acc. Chem. Res. 2003, 36,
908. (b) Hagemann, B.; Junge, K.; Enthaler, S.; Michalik, M.; Riermeier, T.;
Monsees, A.; Beller, M. AdV. Synth. Catal. 2005, 347, 1978. (c) Touati, R.;
Gmiza, T.; Jeulin, S.; Deport, C.; Ratovelomanana-Vidal, V.; Hassine, B. B.;
Genet, J.-P. Synlett 2005, 2478. (d) Wang, Z.; Zhao, C.; Pierce, M. E.; Fortunak,
J. M. Tetrahedron: Asymmetry 1999, 10, 225. (e) Nelson, S. G. Tetrahedron:
Asymmetry 1998, 9, 357. (f) Denmark, S. E.; Beutner, G. L.; Wynn, T.; Eastgate,
M. D. J. Am. Chem. Soc. 2005, 127, 3774. (g) Denmark, S. E.; Fan, Y.; Eastgate,
M. D. J. Org. Chem. 2005, 70, 5235. (h) Yamashita, Y.; Ishitani, H.; Shimizu,
H.; Kobayashi, S. J. Am. Chem. Soc. 2002, 124, 3292. (i) Ocampo, R.; Dolbier,
W. R. Tetrahedron 2004, 60, 9325. (j) Ribeiro, C. M. R.; Farias, F. M. C. Mini-
ReV. Org. Chem. 2006, 3, 1.
(4) For overviews, see: (a) Kobayashi, M.; Shimizu, S. FEMS Microbiol.
Lett. 1994, 120, 217. (b) Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin
Trans. 1 1997, 1099. (c) Meth-Cohn, O.; Wang, M.-X. J. Chem. Soc., Perkin
Trans. 1 1997, 3197. and references therein.
(5) Nagasawa, T.; Schimizu, H.; Yamada, H. Appl. Microbiol. Biotechnol.
1993, 40, 189.
(6) For reviews, see: (a) Sugai, T.; Yamazaki, T.; Yokoyama, M.; Ohta, H.
Biosci. Biotechnol. Biochem. 1997, 61, 1419. (b) Martinkova, L.; Kren, V. Biocat.
Biotrans. 2002, 20, 73. (c) Wang, M.-X. Top. Catal. 2005, 35, 117.
(7) (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.
(12) (a) Wang, M.-X.; Wu, Y. Org. Biomol. Chem. 2003, 1, 535. (b) Zhao,
S.-M.; Wang, M.-X. Chin. J. Chem. 2002, 20, 1291.
(13) The presence of a ꢀ-vinyl group of the substrate was found to increase
the enantioselectivity of biotransformations: Gao, M.; Wang, D.-X.; Zheng, Q.-
Y.; Wang, M.-X. J. Org. Chem. 2006, 71, 9532.
(8) (a) Wang, M.-X.; Lin, S.-J. Tetrahedron Lett. 2001, 42, 6925. (b) Wang,
M.-X.; Lin, S.-J. J. Org. Chem. 2002, 67, 6542. (c) Wang, M.-X.; Lin, S.-J.;
Liu, J.; Zheng, Q.-Y. AdV. Synth. Catal. 2004, 346, 439. (d) Wang, M.-X.; Liu,
J.; Wang, D.-X.; Zheng, Q.-Y. Tetrahedron: Asymmetry 2005, 16, 2409.
(9) (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.; Oꢀwald, 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.
(14) (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.
(15) Part of the work of the biotransformations of ꢀ-hydoxy nitriles has
appeared as a preliminary communication: Ma, D.-Y.; Zheng, Q.-Y.; Wang, D.-
X.; Wang, M.-X. Org. Lett. 2006, 8, 3231.
(16) (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/.
4088 J. Org. Chem. Vol. 73, No. 11, 2008

Synthesis of ꢀ-Hydroxy and ꢀ-Amino Acid and Amide DeriVatiVes
SCHEME 1. Biotransformation of Racemic ꢀ-Hydroxy and ꢀ-Benzyloxy Nitrilesa
a Key: (i) R. erythropolis AJ270, phosphate buffer, pH 7.0, 30 °C.
SCHEME 2. Biotransformation of Functionalized
formation of enantiomerically pure 3-hydroxy acids and amide
derivatives. In the presence of Rhodococcus erythropolis AJ270
whole cell catalyst under very mild conditions, the biotrans-
formation proceeded rapidly. Unfortunately, however, only
disappointingly low enantiomeric excesses (ee) were obtained
for amide 2 and acid 3 products along with the low chemical
yields (Scheme 1). The low enantioselectivity of the biotrans-
formations indicated that both the nitrile hydratase and the
amidase involved in R. erythropolis AJ270 display low enantio-
differentiating power toward the 3-hydroxyalkanenitriles and
3-hydroxyalkaneamides, respectively, while the loss of mass
balance was most probably attributable to the further metabo-
lization of the resulting small ꢀ-hydroxy acid and amide
derivatives, which was catalyzed by other enzymes within
microbial whole cells, and to the difficulty of their extraction
from aqueous reaction media because of their high polarity and
hydrophilicity.
ꢀ-Benzyloxy Nitriles 6a-ca
a Key: (i) R. erythropolis AJ270, phosphate buffer, pH 7.0, 30 °C.
catalyst. Finally, for the biotransformations of UV-inactive
3-hydroxy alkanenitrile substrates, a UV-active protection group
should be benificial for facile analysis and monitering of the
reaction. To meet these criteria, a benzyl protection group
appeared ideal.
The observation of low enantioselectivity of the biotransfor-
mation of ꢀ-hydroxy nitriles was not totally unexpected. Except
for highly enantioselective biotransformations of some prochiral
dinitriles14 and few racemic nitriles that bear a ꢀ-stereogenic
center,13 most of the hydrolyses of ꢀ-chiral center-containing
racemic nitriles reported to date gave appallingly poor
enantioselectivity.11,12 The low enantiocontrol of biotransfor-
mation is mainly originated from less efficient chiral recognition
of the substrate by the enzyme. It is most probably that neither
the nitrile hydratase nor the amidase are able to discriminate a
pair of alkanenitrile and alkaneamide enantiomers, respectively,
that have a free hydroxyl group ꢀ-positioned to cyano and amido
functionality.
Inspired by the docking strategy in drug discovery study and
in biocatalysis,17–19 we envisioned that the introduction of a
removable protection group on the free hydroxyl moiety as a
docking moiety in nitrile substrate might result in its enhanced
chiral recognition by the enzyme. It might thus lead to the
improved enantioselectivity in biotransformations. To choose
a protection or a docking group, several criteria need to be
considered. First, the O-protection group should be readily
introduced and removed, and it should not cause contamination
to the products. Second, no racemerization is allowed when the
protection group is cleaved. This requires very mide conditions
for deprotection reaction. Additionally, the protection group
should be stable or resistant to other enzymes such as esterases,
lipases, and oxidoreductases that are present in the whole
To our delight, introduction of a benzyl group onto the
hydroxy of nitrile substrates, which was readily achieved by
the reaction with benzyl bromide in the presence of Ag2O (see
the Supporting Information), led to a dramatic increase of
enenatioselectivity of biotransformations albeit over a longer
reaction period. In comparison to the biotransformations of
hydroxylated nitriles 1 (R′ ) H), for example, R. erythropolis
AJ270-catalyzed hydrolysis of their corresponding O-benzylated
3-hydroxyalkanenitriles 1 (R′ ) Bn) gave excellent enantiose-
lectivity, affording highly enantioenriched amide 4 and acid 5
products (Scheme 1). In addition to the drastic enhancement of
enantioselectivity, protection of the free hydroxy by a benzyl
group also resulted in a remarkable increase of isolated yield
of amide and acid products due to most probably the increase
of the hydrophobicity of products (Scheme 1). The high
enantioselectivity of the overall biotransformation of O-benzy-
lated nitriles 1 is the result of combined actions of a highly
enantioselective amidase and a lowly enantioselective nitrile
hydratase, which was evidenced by the low enantioselectivity
of the nitrile hydratase-catalyzed kinetic resolution of racemic
nitriles.15 Both amide and acid products underwent efficient
debenzylation reaction to afford highly enantiopure ꢀ-hydroxy
amides and acids. This also allowed us to assign the absolute
configurations of amides 4 and acids 5 on the basis of the
comparison of the optical rotation of ꢀ-hydroxy amides and
acids with that of authentic samples.15
(17) (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.
(18) (a) De Raadt, A.; Fetz, B.; Griengl, H.; Klingler, M. F.; Kopper, I.;
Krenn, B.; Mu¨unzer, D. F.; Ott, R. G.; Plachota, P.; Weber, H. J.; Braunegg,
G.; Mosler, W.; Saf, R. Eur. J. Org. Chem. 2000, 3835. (b) De Raadt, A.; Fetz,
B.; Griengl, H.; Klingler, M. F.; Krenn, B.; Mereiter, K.; Mu¨unzer, D. F.;
Plachota, P.; Weber, H.; Saf, R. Tetrahedron 2001, 57, 8151.
To further demonstrate the application of nitrile biotransfor-
mations in synthesis, functionalized ꢀ-benzyloxylated nitriles
6a-c were subjected to R. erythropolis AJ270-catalyzed hy-
drolysis. As illustrated in Scheme 2, biotransformations of
racemic 4-chloro-3-benzyloxypropanenitrile 6a gave optically
(19) (a) De Raadt, A.; Feichtenhofer, S.; Griengl, H.; Klingler, M. F.; Weber,
H. J. Mol. Catal. B: Enzym. 2001, 11, 361. (b) De Raadt, A.; Feichtenhofer, S.;
Griengl, H.; Klingler, M. F.; Weber, H. J. Mol. Catal. B: Enzym. 2001, 11, 361.
J. Org. Chem. Vol. 73, No. 11, 2008 4089

Ma et al.
TABLE 1. Biotransformations of Racemic ꢀ-Benzylamino Nitriles 12a
13
14
entry
12
R
R′
time
yieldb (%)
eec (%)
yieldb (%)
eec (%)
Ed
1e
2
3
12a
12b
12b
12c
12d
12d
12e
Me
Me
Me
Et
H
100 min
80 min
85 min
2 h
2 h
25 h
30
55
46
47
44
45
46
56.2
74.2
93.2
>99.5
12.6
43
45
53
50
20.8f
86.6
61.4
Bn
Bn
Bn
Bn
Bn
Bn
31
13
>200
4
>99.5
5g
6
iPr
iPr
c-Pr
>99.5
92.4
48
45
90.2
96.4
99
161
7
14 h
a Biotransformation was carried out by incubating substrate (2 mmol) 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 chiral HPLC analysis. d Calculated following a literature method.16 e Both
amide and acid products were isolated as N-Cbz-protected derivatives. f Determined after esterification using CH2N2. g Nitrile 12d (47% yield, ee 14.2%)
was recovered.
SCHEME 3. Biotransformation of Racemic Nitrile 9
SCHEME 4. Determination of Absolute Configurations of
ꢀ-Amino Acids
active amide S-7a and γ-lactone R-8a in excellent yields with
ee of 58.4% and 63.2%, respectively. The formation of γ-lactone
R-8a is most likely due to the spontaneous cyclization of acid
formed from hydrolysis of nitrile 6a. While the reaction of
racemic 3-benzyloxypent-4-enenitrile 6b afforded corresponding
amide S-7b and acid R-8b with modest enantioselectivity,
racemic 3-benzyloxyhex-5-enenitrile 6c underwent effective
biotransformations to furnish amide R-7c and acid S-8c in
excellent chemical yields and high optical yields. Highly
enantioenriched amide R-7c and acid S-8c were also prepared
easily from a multigram scale biotransformation of racemic 6c
(see the Supporting Information).
It was interesting to note that, in sharp contrast to highly
enantioselective biotransformations of nitrile 6c (Scheme 2), the
same biocatalytic reaction of racemic nitrile 9, an isomer of 6c,
gave very low enantiopurity of amide 10 and acid 11 (Scheme
3). The results showed the sensitivity of the enzyme toward
the structure of substrates in term of enantioselection. The
interexchange of benzyl and allyl groups within nitrile structure
led to a significant drop of enantioselectivity. It might imply a
preferential binding domain of the enzyme for a benzyloxy
moiety rather than for a benzyl group in chiral recognition.
Biotransformations of Racemic ꢀ-Amino- and ꢀ-Benzy-
laminoalkanenitriles. To examine the generality of the protec-
tion/docking strategy to improve the enantioselectivity of
biocatalysis and also to prepare highly enantiopure ꢀ-amino acid
and amide derivatives, R. erythropolis AJ270-catalyzed reaction
of racemic ꢀ-amino- and ꢀ-benzylaminoalkanenitriles 12 was
studied (Table 1). Analogous to the racemic ꢀ-hydroxy nitriles
(Scheme 1), racemic 3-aminobutyronitrile 12a underwent bio-
catalytic hydrolysis to give R-3-aminobutyramide 13a and S-3-
aminobutyric acid 14a with ee of 56.2% and 20.8%, respectively
(entry 1, Table 1). Introduction of a benzyl group into the free
amino group of 3-aminobyturonitrile gave rise to a similar
remarkable enhancement of enantioselectivity of the reaction
of 12b, affording a quantitative yield of amide R-13b and acid
S-14b with high enantiomeric excess values (entry 2, Table 1).
Under the identical biocatalytic conditions, all other racemic
ꢀ-benzylaminoalkanenitriles examined were transformed ef-
ficiently into highly enantiopure ꢀ-benzylamino amides 13c-e
and ꢀ-benzylamino acids 14c-e in excellent yield (entries 4,
6, and 7, Table 1). Similar to the biotransformations of
ꢀ-benzyloxyalkanenitriles, the nitrile hydratase within R. eryth-
ropolis AJ270 did not show good enantioselectivity against
ꢀ-benzylaminoalkanenitrile substrates. This has been exemplified
by the observation of very poor enantioselectivity of the kinetic
resolution of racemic 3-benzylamino-4-methylpentanenitrile 12d
(entry 5, Table 1). The excellent enantioselectivity of the overall
nitrile biotransformations is therefore originated from the
dominant effect of a highly enantioselective amidase within R.
erythropolis AJ270.
To determine the absolute configurations of ꢀ-benzylamino
amide and acid products, the resulting enantioenriched acids
14b and 14d were converted into compounds S-1720 and R-1821
through esterification using CH2N2 followed by N-benzylation
and N-debenzylation, respectively (Scheme 4). Comparison of
their optical rotation directions with that of authentic samples20,21
indicated the biotransformation products 14b and 14d have S-
(20) Etxebarria, J.; Vicario, J. L.; Badia, D.; Carrillo, L. J. Org. Chem. 2004,
69, 2588.
(21) Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G. Tetrahedron 1995,
51, 12337.
4090 J. Org. Chem. Vol. 73, No. 11, 2008

Synthesis of ꢀ-Hydroxy and ꢀ-Amino Acid and Amide DeriVatiVes
SCHEME 5. Enantiomeric Selection of the Amidase against
ꢀ-Benzyloxy and ꢀ-Benzylamino Amides
the residue was subjected to a silica gel column using a mixture of
petroleum ether and acetone as the mobile phase to give pure amide
product and, in some cases, the recovered nitrile. The aqueous phase
was then adjusted to pH 4 with hydrochloric acid (2 N) and
extracted with ethyl ether (3 × 50 mL). The organic phase was
combined. After drying (MgSO4) and removal of the solvent under
vacuum, the residue was chromatographed on a silica gel column
using a mixture of petroleum ether and acetone as an eluant to
give pure acid product. For biotransformations of ꢀ-benzylamino
nitriles, the resulting aqueous solution was distilled under vacuum
to remove water. Hydrochloric acid (1 N, 5 mL) was then added
to the resulting residue. After being allowed to stand at room
temperature for 30 min, the acidic solution was filtrated. The filtrate
was loaded on a cation-exchange resin (10 mL) and eluted with
water (200 mL) followed by aqueous ammonia solution (1%, 200
mL). The ammonia eluant was concentrated and then chromato-
graphed on a C-18 silica gel column using a gradient eluant [from
pure water to a mixture of methanol and water (7:3)] to give pure
acid and amide products. The structures of all products were fully
characterized by spectroscopic data and microanalyses. The ee
values were determined by HPLC analyses using chiral stationary
phase (see the Supporting Information).
and R-configurations, respectively. The stereochemistry of nitrile
biotransformations observed in current study suggests that the
dominant amidase within R. erythropolis AJ270 whole cell
catalyst exhibits the same sense of enantiomeric selection against
both ꢀ-benzyloxy and ꢀ-benzylamino amides. In addition, the
amidase shows excellent enantioselectivity toward both ꢀ-ben-
zyloxy and ꢀ-benzylamino amides, irrespective of the nature
of an ether (X ) O) or imino (X ) NH) linkage in substrates
(Scheme 5). Apparently, the steric effect, viz. the fitting of the
substrate into the chiral pocket of the enzyme, may play a crucial
role in determining the enantioselectivity.
For biotransformation of racemic 12c, enantiopure R-13c and
S-14c were obtained in 47% and 50% yield, respectively. (R)-3-
(Benzylamino)pentanoic acid amide (13c): oil; [R] -21.4 (c
4.300, MeOH); ee >99.5% (chiral HPLC analysis of its N-methyl
derivative using a Chiralcel OJH column with a mixture of hexane
and 2-propanol (9:1) at a flow rate 0.5 mL/min as the mobile phase,
Conclusion
1
In summary, we have shown a remarkable method of
dramatically enhancing the enantioselectivity of biotransforma-
tions of nitriles using a general and simple but powerful
protection/docking strategy. A simple and convenient protection
of free hydroxyl and amino by a benzyl group gave rise to a
significant increase of enantioselectivity of biocatalytic hydroly-
sis of ꢀ-hydroxy- and ꢀ-aminoalkanenitriles. The excellent
enantioselectivity of overall nitrile biotransformations stemmed
from the synergistic effect of a low enantioselective nitrile
hydratase and a high enantioselective amidase within R.
erythropolis AJ270. The current study provided an efficient
approach to highly enantiopure ꢀ-hydroxy and ꢀ-amino acids
and their amide derivatives that are valuable chiral intermediates
in organic synthesis.
oven temperature 15 °C, t1 ) 17.7 min, t2 ) 20.9 min); H NMR
(300 MHz, DMSO-d6) 7.40 (H, br), 7.34-7.20 (5H, m), 6.74 (1H,
br), 3.69 (2H, s), 2.77 (1H, quin, J ) 6.0 Hz), 2.19 (1H, dd, J )
14.3, 6.5 Hz), 2.13 (1H, dd, J ) 14.3, 6.1 Hz), 2.12 (1H, br),
1.46-1.37 (2H, m), 0.85 (3H, t, J ) 7.3 Hz); 13C NMR (75 MHz,
CDCl3) δ 173.6, 141.2, 128.0, 127.8, 126.4, 55.1, 49.9, 39.3, 26.0,
9.6 ppm; IR (KBr) ν 3313, 3186 (NH, CONH2), 1666 (CdO) cm-1
;
MS (ESI+) m/z 207.1 [M + 1]+. Anal. Calcd for C12H18N2O: C,
69.87; H, 8.80; N, 13.58. Found: C, 69.70; H, 8.87; N, 13.59. (S)-
3-(Benzylamino)pentanoic acid (14c): mp152-153 °C; [R] +32.4
(c 2.655, H2O); ee >99.5% (chiral HPLC analysis of its methyl
ester using a Chiralcel OD column with a mixture of hexane and
2-propanol (9:1) at a flow rate 0.8 mL/min as the mobile phase,
1
oven temperature 20 °C, t1 ) 7.0 min, t2 ) 8.8 min); H NMR
(300 MHz, D2O) 7.43 (5H, s), 4.21 (2H, s), 3.39-3.30 (1H, m),
2.60 (1H, dd, J ) 16.9, 4.9 Hz), 2.43 (1H, dd, J ) 16.9, 7.3 Hz),
1.86-1.77 (1H, m), 1.67-1.57 (1H, m), 0.91 (3H, t, J ) 7.5 Hz);
13C NMR (75 MHz, D2O) δ178.2, 131.2, 129.5, 129.3, 56.8, 47.9,
35.4, 23.4, 8.9 ppm; IR (KBr) ν 3281-2375 (NH, COOH), 1697,
1594 cm-1; MS (ESI+) m/z 208.2 [M + 1]+; HR-FAB m/z calcd
for C12H17NO2 207.1259, found 208.1334.
Experimental Section
General Procedure for the Biotransformations of Nitriles. To
an Erlenmeyer flask (150 mL) with a screw cap was added
Rhodococcus sp. AJ270 cells7,10a (2 g wet weight), which were
prepared in bulk from a 5 L fermentor using acetamide as both the
carbon and nitrogen source and stored at -20 °C in a freezer, 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 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 or HPLC, was quenched after a
specified period of time (see Table 1 and the Supporting Informa-
tion) by removing the biomass through a Celite pad filtration. For
biotransformations of ꢀ-hydroxy and ꢀ-benzyloxy nitriles, the
resulting aqueous solution was adjusted to pH 12 with aqueous
NaOH solution (2 N) and extracted with ethyl ether (3 × 50 mL).
After drying (MgSO4) and removal of the solvent under vacuum,
Acknowledgment. We thank the Ministry of Science and
Technology (2003CB716005, 2006CB806106), the National
Natural Science Foundation of China, and the Chinese Academy
of Sciences for financial support.
Supporting Information Available: Preparation of starting
nitriles and their spectroscopic data; full characterization of
1
products; H and 13C NMR spectra of 2-5, 7, 8, 10, 11, 13,
and 14; HPLC analysis of all chiral products. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO800074K
J. Org. Chem. Vol. 73, No. 11, 2008 4091