Unexpected stereorecognition in nitrilase-catalyzed hydrolysis of β-hydroxy nitriles

Article abstract of DOI:10.1021/ol061542+

Biocatalytic enantioselective hydrolysis of β-hydroxy nitriles to corresponding (S)-enriched β-hydroxy carboxylic acids has been achieved for the first time by an isolated nitrilase bII6402 from Bradyrhizobium japonicum USDA110. This offers a new "green" approach to optically pure β-hydroxy nitriles and β-hydroxy carboxylic acids. The observed remote stereorecognition is surprising because this nitrilase shows no enantioselectivity for the hydrolysis of α-hydroxy nitriles such as mandelonitrile.

Full text of DOI:10.1021/ol061542+

ORGANIC
LETTERS
2006
Vol. 8, No. 20
4429-4431
Unexpected Stereorecognition in
Nitrilase-Catalyzed Hydrolysis of
â
-Hydroxy Nitriles
Sukanta Kamila, Dunming Zhu, Edward R. Biehl, and Ling Hua*
Department of Chemistry, Southern Methodist UniVersity, Dallas, Texas 75275
lhua@smu.edu
Received June 22, 2006
ABSTRACT
Biocatalytic enantioselective hydrolysis of
â
-hydroxy nitriles to corresponding (S)-enriched
â
-hydroxy carboxylic acids has been achieved for
the first time by an isolated nitrilase bll6402 from Bradyrhizobium japonicum USDA110. This offers a new “green” approach to optically pure
-hydroxy nitriles and -hydroxy carboxylic acids. The observed remote stereorecognition is surprising because this nitrilase shows no
enantioselectivity for the hydrolysis of -hydroxy nitriles such as mandelonitrile.
â
â
r
Optically pure â-hydroxy carboxylic acids and their deriva-
tives are precursors of â-blockers and 1,3-amino alcohols,
which are important intermediates for the synthesis of natural
products, antibiotics, and chiral auxiliaries.1-3 In addition,
chiral â-hydroxy carboxylic acids are widely used to prepare
copolyesters for film, fiber, molding, and coating applica-
tions.4 The utility of chiral â-hydroxy carboxylate compounds
as useful synthons has stimulated the development of new
methodologies for their construction, and a variety of
methods have been reported.5-10 A straightforward approach
to optically pure â-hydroxy carboxylic acids is enantiose-
lective hydrolysis of â-hydroxy nitriles, which are readily
accessible by the cyanization of R-haloketones with sodium
cyanide followed by NaBH4 reduction11 or ring opening of
epoxides by sodium cyanide.12 However, chemical hydrolysis
of nitriles usually requires strong basic or acidic conditions
and an elevated reaction temperature that often result in the
undesirable elimination of OH for nitriles with a â-hydroxy
group, yielding unsaturated byproducts.13 To solve these
problems, biocatalytic hydrolysis of nitriles becomes the
choice because the reaction can be performed at a neutral
pH condition and room temperature, offering the possibility
of hydrolyzing nitriles bearing functionalities that cannot
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62, 1079-1094.
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(4) Goodman, I. In Concise Encyclopedia of Polymer Science and
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pp 793-799.
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Menschik, J.; Nicolau, G.; Sloboda, A. E.; Watts, D. E. J. Med. Chem.
1979, 22, 1385-1389.
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(13) 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, 775-782.
(5) Yakura, T.; Tanaka, T.; Ikeda, M.; Uenishi, J. I. Chem. Pharm. Bull.
2003, 51, 471-473.
(6) Padhi, S. K.; Pandian, N. G.; Chadha, A. J. Mol. Catal. B: Enzymatic
2004, 29, 25-29.
(7) Huerta, F. F.; Baeckvall, J.-E. Org. Lett. 2001, 3, 1209-1212.
(8) Wang, Y.-C.; Yan, T.-H. J. Org. Chem. 2000, 65, 6752-6755.
10.1021/ol061542+ CCC: $33.50
© 2006 American Chemical Society
Published on Web 09/08/2006

tolerate harsh conditions. An additional advantage of bio-
catalytic hydrolysis of nitriles is its high chemo-, regio-, and
stereoselectivity.14-17 In this context, there have been a few
reports on the biocatalytic hydrolysis of â-hydroxy nitriles
to corresponding â-hydroxy carboxylic acids and amides
using microorganisms possessing nitrile hydratase and ami-
dase activity.13,18-21 In these studies, the microorganisms with
nitrilase activity were also tested but showed no or extremely
low activity toward the hydrolysis of â-hydroxy nitriles.13,18-20
To the best of our knowledge, there are only two reports
dealing with the hydrolysis of â-hydroxy nitriles with isolated
nitrilases. One is the report from DeSantis et al., in which
the desymmetrization of 3-hydroxyglutaronitrile has been
achieved with genetically engineered nitrilases.22 The other
one is our recent studies on nitrilase ZmNIT2 from maize
(Zea mays), which catalyzed the hydrolysis of â-hydroxy
nitriles to a mixture of â-hydroxy carboxylic amides and
acids, with the amides being the major products (63-88%
yields).23 Herein, we present the results on the enantiose-
lective hydrolysis of â-hydroxy nitriles to (S)-enriched
â-hydroxy carboxylic acids catalyzed by an isolated nitrilase
from Bradyrhizobium japonicum USDA110.
Figure 1. â- and R-hydroxy nitriles.
from unreacted â-hydroxy nitriles by preparative thin-layer
chromatography.
The ee values of both the product acids and the recovered
nitriles were measured by chiral HPLC analysis, and their
absolute configurations were determined by comparing the
sign of optical rotation with the literature data. The enan-
tiomeric ratios (E) were calculated using the equations
proposed by Sih et al.26 The results are presented in Table 1
Recently, we have cloned and purified a nitrilase (bll6402)
from Bradyrhizobium japonicum USDA110 and demon-
strated that it was an efficient catalyst for the hydrolysis of
mandelonitrile and its derivatives.24 To further explore its
synthetic application, this nitrilase was examined for the
hydrolysis of â-hydroxy nitriles (Figure 1). â-Hydroxy
nitriles were prepared by the cyanization of R-bromoketones
with sodium cyanide followed by NaBH4 reduction (see
Supporting Information).11 The obtained â-hydroxy nitriles
were treated with purified nitrilase bll6402 in potassium
phosphate buffer (100 mM, pH 7.0), and the reaction mixture
was incubated overnight at 30 °C. The mixture was then
saturated with NaCl and extracted with ethyl acetate.25 The
extract was dried over sodium sulfate, and evaporation of
the solvent under reduced pressure afforded the crude
products. The â-hydroxy carboxylic acids were separated
Table 1. Enantioselective Hydrolysis of â-Hydroxy Nitriles
Catalyzed by Nitrilase bll6402
recovered nitrile (R)-1 product acid (S)-2
â-hydroxy
entry
nitrile
yield (%)a
ee (%) yield (%)a ee (%) Eb
1
2
3
4
5
6
7
8
9
1a
1b
1c
1d
1e
1f
1g
1h
1i
41
37
40
35
57
46
46
40
42
0
53
74
53
76
66
75
67
75
37
-
36
38
32
40
27
36
35
32
39
98
48
60
65
42
90
43
91
84
59
0
5
9
8
5
43
5
52
27
13
-
(14) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic
Synthesis: Regio- and StereoselectiVe Biotransformations, 2nd ed.; Wiley-
VCH: Weinheim, 2006.
(15) Wang, M.-X. Top. Catal. 2005, 35, 117-130.
(16) Martinkova, L.; Kren, V. Biocatal. Biotransform. 2002, 20, 73-
93.
c
c
10
1j
(17) Sugai, T.; Yamazaki, T.; Yokoyama, M.; Ohta, H. Biosci., Biotech-
nol., Biochem. 1997, 61, 1419-1427.
a Isolated yield. b The enantiomeric ratio is calculated by the equation
E ) ln[(1 - c)(1 - ee(S))]/ln[(1 - c)(1 + ee(S))], where c is calculated
by the equation c ) [ee(S) + eeo]/[ee(S) + ee(P)], ee(S) is the ee of the
substrate, eeo is the initial ee of the substrate, and ee(P) is the ee of the
product.26 c Not applicable.
(18) Wu, Z.-L.; Li, Z.-Y. J. Mol. Catal. B: Enzymatic 2003, 22, 105-
112.
(19) Brady, D.; Beeton, A.; Zeevaart, J.; Kgaje, C.; van Rantwijk, F.;
Sheldon, R. A. Appl. Microbiol. Biotechnol. 2004, 64, 76-85.
(20) Wang, M.-X.; Wu, Y. Org. Biomol. Chem. 2003, 1, 535-540.
(21) Ma, D.-Y.; Zheng, Q.-Y.; Wang, D.-X.; Wang, M.-X. Org. Lett.
2006, 8, 3231-3234.
(22) 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, 11476-11477.
(23) Mukherjee, C.; Zhu, D.; Biehl, E. R.; Parmar, R. R.; Hua, L.
Tetrahedron 2006, 62, 6150-6154.
together with the data for the hydrolysis of mandelonitrile
under the same conditions.27
Nitrilase bll6402 catalyzed the enantioselective hydrolysis
of aromatic â-hydroxy nitriles to give (S)-enriched â-hydroxy
(24) Zhu, D.; Mukherjee, C.; Biehl, E. R.; Hua, L. Appl. Microbiol.
Biotechnol. 2006, submitted.
(25) Addition of NaCl into the reaction mixture facilitated the extraction
of acid products. Because workup procedures were not optimized, causing
the loss of acid products, the total yields of acids and unreacted nitriles
were not high in some cases.
(26) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294-7299.
(27) Substrate 1a was tested under the same conditions except without
nitrilase bll6402, and no hydrolysis was detected.
4430
Org. Lett., Vol. 8, No. 20, 2006

carboxylic acids with recovery of (R)-enriched â-hydroxy
nitriles. The substituent on the benzene ring of â-hydroxy
nitriles did not significantly affect the enzyme activity but
exerted some effect on the enantioselectivity. Among the
para-substituted aryl â-hydroxy nitriles, the hydrolysis of
the substrate with the para-methoxy group (1e) showed the
highest enantioselectivity with E being 43. The position of
the substituent on the benzene ring also affected the
enantioselectivity. For example, the enantiomeric ratios E
for the substrates with a para- and meta-methoxy substituent
were 43 (1e) and 52 (1g), respectively, whereas that of their
counterpart with an ortho-methoxy group (1f) was only 5.
In contrast, this nitrilase showed higher enantioselectivity
for â-hydroxy nitrile with an ortho-chloro group (1h) than
the one with a para-chloro substituent (1c). Thus, the
enantioselectivity of this nitrilase was influenced by both
the steric and electronic factors of the substituents on the
benzene ring.
As we previously reported, nitrilase bll6402 showed no
enantioselectivity for the hydrolysis of R-hydroxy nitriles
such as mandelonitrile.24 Therefore, the observed stereodis-
crimination in the hydrolysis of â-hydroxy nitriles was
surprising because it was in contrast to the usual observation
that a chiral carbon atom at the â-position to the reaction
center would be recognized with much more difficulty than
the one at the R-position.18,23
To test the role that the â-hydroxy group plays in the
enantioselective hydrolysis of â-hydroxy nitriles, 3-phenyl-
butyronitrile (in which the hydroxy group was replaced with
a methyl group) was treated with nitrilase bll6402. However,
3-phenylbutyronitrile was not hydrolyzed by nitrilase bll6402
under the same conditions. This indicates that the hydroxy
group of â-hydroxy nitriles not only promotes the stereo-
discrimination at the â-position but also plays a critical role
in determining the enzyme activity.
Nitrilase possesses a cysteine residue in its catalytic site,
and the proposed reaction mechanism for the hydrolysis of
nitriles involves the formation of an enzyme-substrate
complex where the thiol residue of the enzyme forms a
covalent bond with nitrile carbon. Addition of a H2O
molecule to the complex generates a tetrahedral intermediate.
Loss of ammonia from this tetrahedral intermediate gives
an acyl-enzyme intermediate, which is further hydrolyzed
to produce carboxylic acid and release the enzyme for the
next catalytic cycle.28,29 Hydrogen bond formation, which is
a very common feature in enzyme catalysis, occurs when a
polar amine/hydroxy group either from the substrate or from
the enzyme residue is in close proximity to another amine/
hydroxy group from a similar environment.30,31 The observed
stereorecognition in the hydrolysis of â-hydroxy nitriles
might be due to hydrogen bonding between the â-hydroxy
group with a NH2 or a OH group of enzymes. The substrate
is thus anchored on the surface of the enzyme by hydrogen
bonding so that the CN group of the (S)-enantiomer is in
closer proximity to the cysteine residue of the catalytic site
than that of the (R)-enantiomer. This allows the formation
of an enzyme-substrate complex favorable for the (S)-
enantiomer over (R)-configured â-hydroxy nitriles, thus
leading to (S)-configured â-hydroxy carboxylic acids as the
major enantiomer. Because the crystal structure of nitrilase
bll6402 is not known, further studies are needed to determine
the effect of substrate-enzyme hydrogen bonding on activity
and enantioselectivity.
In conclusion, kinetic resolution of aromatic â-hydroxy
nitriles to the corresponding enantiomerically enriched â-hy-
droxy carboxylic acids and â-hydroxy nitriles has been
achieved by an isolated nitrilase from Bradyrhizobium
japonicum USDA110. In addition, nitrilase bll6402 shows
surprisingly unusual stereorecognition in the hydrolysis of
â-hydroxy nitriles, whereas the hydrolysis of R-hydroxy
nitriles is not enantioselective. Nitrilase-catalyzed enantio-
selective hydrolysis is superior to the lipase-catalyzed kinetic
resolution of â-hydroxy nitriles because the latter is usually
followed by chemical hydrolysis that is difficult to be
performed without side reactions.10 Therefore, nitrilase-
catalyzed enantioselective hydrolysis offers a new “green”
approach to optically pure â-hydroxy nitriles and â-hydroxy
acids, although a further improvement in enantioselectivity
is needed.
Acknowledgment. We thank Southern Methodist Uni-
versity for start-up support and Robert Welch Foundation
for financial support.
Supporting Information Available: General procedures
for the preparation of â-hydroxy nitriles and their hydrolysis,
characterization data, and 13C NMR spectra for enantiomeri-
cally enriched â-hydroxy nitriles and acids. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL061542+
(29) Pace, H. C.; Brenner, C. GenomeBiology 2001, 2, (http://
genomebiology.com/2001/2/1/reviews/0001).
(30) Zheng, Y.-J.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
4285-4288.
(31) Shan, S.-O.; Herschlag, D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
14474-14479.
(28) O’Reilly, C.; Turner, P. D. J. Appl. Microbiol. 2003, 95, 1161-
1174.
Org. Lett., Vol. 8, No. 20, 2006
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