Biocatalytic strategy toward asymmetric β-hydroxy nitriles and γ-amino alcohols

Article abstract of DOI:10.1016/j.tetlet.2011.03.009

A library of 20 bakers' yeast reductases, that are overexpressed in Escherichia coli, were screened against a variety of β-keto nitriles. Enzymes from the aldose reductase and the short chain dehydrogenase family displayed activity toward these substrates. All of the seven substrates were reduced with high enantioselectivities and in some cases both antipodes could be synthesized in high ees. These whole-cell reactions afforded gram quantities of asymmetric compounds that could ultimately lead to scaleable and simple synthesis to new drug analogs of serotonin reuptake inhibitors and β-adrenergic blocking agents.

Full text of DOI:10.1016/j.tetlet.2011.03.009

Tetrahedron Letters 52 (2011) 2440–2442
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Biocatalytic strategy toward asymmetric b-hydroxy nitriles and
c-amino alcohols
Randall W. Nowill a, Trisha J. Patel a, David L. Beasley a, Jose A. Alvarez a, Elizah Jackson III a,
Todd J. Hizer a, Ion Ghiviriga b, Scott C. Mateer a, Brent D. Feske a,
⇑
a Department of Chemistry and Physics, Armstrong Atlantic State University, 11935 Abercorn St. Savannah, GA 31419, USA
b Department of Chemistry, University of Florida, PO box 117200, Gainesville, FL 32611, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of 20 bakers’ yeast reductases, that are overexpressed in Escherichia coli, were screened against a
variety of b-keto nitriles. Enzymes from the aldose reductase and the short chain dehydrogenase family
displayed activity toward these substrates. All of the seven substrates were reduced with high enantiose-
lectivities and in some cases both antipodes could be synthesized in high ees. These whole-cell reactions
afforded gram quantities of asymmetric compounds that could ultimately lead to scaleable and simple
synthesis to new drug analogs of serotonin reuptake inhibitors and b-adrenergic blocking agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 3 December 2010
Revised 14 February 2011
Accepted 1 March 2011
Available online 6 March 2011
Keywords:
Reductase
Bakers’ yeast
b-Keto nitrile
Biocatalysis
1. Introduction
OH
*
O
1) Ac2O
Asymmetric b-hydroxy nitriles are excellent chiral precursors
due to their reactive diversity (Scheme 1). These asymmetric prod-
ucts have been used to make a variety of chiral natural products
and pharmaceuticals.1–3 The most popular example is the utiliza-
2) RMgBr
R
R
OH
*
OH
H2 / Pd/C
N
tion of b-hydroxy nitriles 1 or c-amino alcohols 2 in the synthesis
NH2
R
R *
of the serotonin reuptake inhibitors4–6 and b-adrenergic blocking
agents.7–9
1
2
Chiral organic10–12 and inorganic13–20 catalysts have both been
investigated in the synthesis of these molecules. The main disad-
vantage to these strategies is that the catalysts often involve a dif-
ficult synthesis and have toxicity issues especially when
considering the use of heavy metals for the synthesis of pharma-
ceuticals. In addition, high enantioselectivity is often difficult to
achieve.
Biocatalytic strategies utilizing isolated enzymes have also been
a strategy toward asymmetric b-hydroxy nitriles 1.21–23 The most
popular examples use a lipase or nitrilase to catalyze the kinetic
resolution of the racemic b-hydroxy nitrile.4,24–27 However, these
enzymes are inherently limited to a 50% yield when resolving the
racemate. The reduction of b-keto nitriles using isolated keto
reductases has recently been investigated; however, the cofactor
must be supplied or a cofactor regeneration system used when
working with pure enzymes.28,29
H3O+
OH
*
O
R
OH
Scheme 1. Examples of b-hydroxy nitriles synthetic utility.
Whole-cell biocatalytic strategies have also been investigated
for the reduction of b-keto nitriles. This is advantageous because
the cell will supply the cofactors needed for the reduction resulting
in affordable and simple reaction conditions. However, studies
using Curvularia lunata and bakers’ yeast (Saccharomyces cerevisiae)
to reduce these substrates have been plagued by a dominant alkyl-
ating mechanism (Scheme 2).30–35
Bakers’ yeast (S. cerevisiae) has been a popular biocatalytic tool
that has been investigated to achieve a variety of reductions.36 Its
ability to reduce an assortment of ketone substrates is ultimately
due to the many keto reductases this organism contains.37 Unfor-
tunately, this large number of reductases often leads to a mixture
of products formed by competing enzymes. To circumvent this
⇑
Corresponding author. Tel.: +1 912 344 3210; fax: +1 912 344 3433.
E-mail address: brent.feske@armstrong.edu (B.D. Feske).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.009

R. W. Nowill et al. / Tetrahedron Letters 52 (2011) 2440–2442
2441
area represents the short chain dehydrogenase family. Interest-
ingly, the other two families did not show activity toward this class
of substrates. Comparison shows that the short chain dehydroge-
nase family tends to accept more of these substrates when com-
pared to the aldose reductase family. In previous investigations
YNL331c has been considered part of the aldose reductase fam-
ily;43 however, based on our observed broad range of substrate
selectivity YNL331c acts more like the short-chain dehydrogenase
family members because it reduces all of the ketone substrates.
This is in contrast to the aldose family members which exhibited
a narrow range of substrate acceptance. Interestingly, comparison
of YNL331c amino acid sequence to the members of the short-
chain dehydrogenase family or the aldose reductase family does
not exhibit a high degree of similarity to either reductase family.
However, when the amino acid relationship is viewed in terms of
a phylogenetic tree it suggests that YNL331c is more closely related
to the short-chain dehydrogenase family than the aldose reductase
family (see Supplementary data).
O
O
Bakers' Yeast
OH
CN
R
CN
CN
or
R
R
Curvularia lunata
major product
Scheme 2. Ethylated product is formed during wild-type whole-cell reactions using
Curvularia lunata and bakers’ yeast.
problem, GST-reductase chimeras were engineered and placed into
Escherichia coli creating a bakers’ yeast reductase library.37–39 We
have used this system to screen the stereospecificity of a single
reductase for a given substrate by use of the pure fusion protein
or in whole-cells.39–41
2. Results and discussion
This manuscript reports the screening of this heterologous li-
brary for its ability to asymmetrically reduce a variety of b-keto ni-
triles. The simplicity of this system lies in the fact that it uses
whole-cells to supply the cofactors, minimizes competing reac-
tions by overexpressing a single yeast reductase, and is scaleable
thus it is well suited for the synthetic chemist who desires gram
quantities.
The asymmetric reduction of a variety of alkyl and aryl substi-
tuted b-keto nitriles were investigated (Scheme 3). Many of these
were commercially available or they were synthesized using acid
chlorides and cyanoacetic acid following the procedure by Kirsten
and Schrader.42 Each substrate was screened using whole-cell reac-
tion conditions with the engineered E. coli and each enzyme’s ste-
reoselectivity was recorded (Table 1).
Most importantly, this screening shows that using these en-
zymes to reduce b-keto nitriles is synthetically useful. Every sub-
strate can be reduced affording a product that is at least 99% ee.
Moreover, every substrate apart from 4a, we find that the enzyme
library can select for both the
trast to similar substrates that have been reduced by these en-
zymes in which they predominantly select for the isomer
only.43 In fact, the aldose reductase family only yields the
enan-
L and the D isomers, which is in con-
L
D
tiomer when reducing this class of substrates. Even more interest-
ing, is that for substrates 5a, 7a, 8a, and 9a an enzyme can be
chosen to afford either enantiomer in high optical purity. These re-
sults suggest that b-keto nitriles may be more synthetically useful
with these reductases than other ketone substrates investigated
because of the diverse stereoselectivity that is observed.
This enzyme library can be broken down into four families ( -
D
After each screening, the enzyme that affords the best enanti-
oselectivity was then chosen for scale up using whole-cell non-
growing conditions (Table 2), which has been previously shown
to result in gram quantities and clean reactions.40,41
hydroxy dehydrogenase, medium chain dehydrogenase, short
chain dehydrogenase, and aldose reductase). The shaded area on
Table 1 represents the aldose reductase family and the non-shaded
These whole-cell reactions were conducted in a non-growing
media (100 mM PO4À, pH = 7, 5 g/L glucose, OD600 = 18). The final
concentration of these reactions varied between 0.5 and 1.0 g/L.
Since a 1 L reaction yielded enough product for characterization
we did not work on condition optimization by manipulating the
protein expression, non-growing reaction conditions, or found it
necessary to utilize cell lysates with a cofactor regeneration sys-
tem. As mentioned previously, whole-cell reactions of b-keto
B.Y. Reductase
OH
*
O
library
CN
CN
R
R
3a - 9a
3b - 9b
Scheme 3. The bakers’ yeast reductase library was screened against a variety of
b-keto nitrile substrates.
Table 1
Enzyme library screening results reported in eea
O
Cl
O
F
O
White row––Short chain dehydrogenase family. Light gray row––undetermined. Dark gray row––Aldose reductase family. Absolute configuration was determined by NMR
and each product was fully characterized (see Supplementary data) and is represented as
L and D in the table to better identify enzyme–substrate binding patterns.
a
Percent calculated by chiral GC analysis.

2442
R. W. Nowill et al. / Tetrahedron Letters 52 (2011) 2440–2442
Table 2
Results from the whole-cell reaction scale up
Substrate
Enzyme
Yield (%)
Final concentration
Ethylated producta (%)
Bakers yeast (%) ethylateda
3a
4a
5a
6a
7a
8a
9a
YOL151w
YOL151w
YOL151w
YOL151w
YOL151w
YOL151w
YGL039w
98
86
96
89
91
89
91
0.66 g/L
0.69
1.01
0.41
0.55
<1
<1
4
2
2
38
28
90
78
63
71
41
0.57
0.49
<1
<1
The percent of ethylated product formed is compared to the percent ethylated by bakers’ yeast.
a
Percent calculated by GC analysis.
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nitriles afford an alkylated major product when using C. lunata and
bakers’ yeast. We found that this heterologous whole-cell system
can limit the alkylated product to less than 5% and in some cases
no ethylated product is found. We observed the bakers yeast con-
version to be very slow and after 24 h there was usually less than
15% conversion (3 mM); whereas, using the overexpression system
usually achieved 100% conversion in 12 h. This is most likely due to
the difference in expression levels of the reductases in the bakers’
yeast versus the engineered cells.
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Asymmetric b-hydroxy nitriles are chiral precursors which have
been applied to the synthesis of serotonin reuptake inhibitors4–6
and b-adrenergic blocking agents.7–9 We have shown a simple
and scaleable approach to make a variety of asymmetric b-hydroxy
nitriles while avoiding the alkylated product often seen with
whole-cell biocatalysis. In most cases these products are reduced
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Acknowledgments
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This work was supported by NSF-RUI grant CHE-0848708 from
the Organic and Macromolecular Chemistry Program and NSF-MRI
CHE-0923153. Additional thanks to Jon Stewart at the University of
Florida for donating the enzyme library in these studies.
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.03.009.
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