Stereocomplementary bioreduction of β-ketonitrile without ethylated byproduct

Article abstract of DOI:10.1021/ol402733y

α-Ethylation is competing with the biocatalytic reduction of aromatic β-ketonitriles in a whole-cell system. Use of two newly mined robust and stereocomplementary carbonyl reductases in a biphasic system has completely eliminated the competing byproduct.

Full text of DOI:10.1021/ol402733y

ORGANIC
LETTERS
2013
Vol. 15, No. 21
5408–5411
Stereocomplementary Bioreduction of
β‑Ketonitrile without Ethylated Byproduct
Guo-Chao Xu, Hui-Lei Yu,* Zhi-Jun Zhang, and Jian-He Xu*
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory
of Bioreactor Engineering, East China University of Science and Technology,
130 Meilong Road, Shanghai 200237, People’s Republic of China
huileiyu@ecust.edu.cn; jianhexu@ecust.edu.cn
Received August 10, 2013
ABSTRACT
R-Ethylation is competing with the biocatalytic reduction of aromaticβ-ketonitriles in a whole-cell system. Use of two newly mined robust and stereo-
complementary carbonyl reductases in a biphasic system has completely eliminated the competing byproduct. For the first time, both enantiomers of
fluoroxetine precursors were obtained at 0.5 M with >99% ee and excellent chemoselectivity, without addition of any external cofactors.
Optically pure β-hydroxynitriles are important building
blocks for the synthesis of a variety of natural compounds
and pharmaceutically relavant products because of their
facileconversionintochiral1,3-amino alcohols, β-hydroxy
acids, etc.1 The most important application has been found
to be the precursors of the popular serotonin/norepinephrine
reuptake inhibitors (SNRIs), commercially known as Prozac
(fluoxetine), Straterra (atomoxetine), and nisoxetine. These
medicines are employed for relieving people from inception
and other disorders, including anxiety, chronic pain, alco-
holism, migraine headaches, urinary incontinence, sleep and
memory disorders, obesity, and bulimia.2
reactions with acetonitrile and benzaldehyde, β-boration
of R,β-unsaturated nitriles followed by oxidation, borane
reductions, and hydrogen transferofβ-ketonitriles.3 Silica-
immobilized Ru-TsDPEN catalysts were developed by
Tu et al. for asymmetric transfer hydrogenation of aryl
ketones with relatively high activity and 97% ee.3f Iridium
diamine catalysts were designed by Carreira et al. for the
reduction of aryl ketonitrile with 47ꢀ95% ee.3h However,
the main disadvantage of these strategies is the time- and
cost-intensive synthesis of catalysts employed and the
toxicity issues especially when considering the use of heavy
metals for the synthesis of pharmaceuticals. It is often dif-
ficult to achieve a very high enantioselectivity for chemical
routes.
Several chemicalrouteshave been developed foryielding
chiral β-hydroxy nitriles, including asymmetric aldol-type
Asymmetric synthesis of optically active compounds
employing biocatalysts represents an effeicent alternative
to chemical strategies. A classic example is the kinetic resolu-
tion of racemic β-hydroxy nitrile catalyzed by a lipase or
nitrilase.1c,4 However, these enzymes are inherently limited
to a theoretical yield of 50% when resolving the racemate.
The oxidoreductase-catalyzed asymmetric reduction of
β-ketonitriles provides another route to preparing β-hydroxy
nitrile with almost 100% yield. However, a common side
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r
10.1021/ol402733y
Published on Web 10/21/2013
2013 American Chemical Society

employment of recombinant whole cells, especially those
coexpressing reductase with glucose dehydrogenase, could
achieve high reduction efficiency and internal cofactor
regeneration.8 We thus hope that the use of these newly
designed whole cells in the biphasic system can make the
bioreduction process independent of metabolic activity and
avoid the formation of a byproduct.
Scheme 1. Asymmetric Reduction of Benzoylacetonitrile with
Recombinant E. coli BL21(DE3) Cells Harboring Carbonyl
Reductase and Glucose Dehydrogenase
Exploring nature’s diversity is a promising approach to
discovering novel and efficient biocatalysts with desired
properties.9 A carbonyl reductase toolbox, consisting of
30 oxidoreductases, has been designed and developed
using a genome mining strategy as previously described.10
These reductases show moderate identities (40ꢀ70%)
with known oxidoreductases. Of these oxidoreductases,
17 belong to the short-chain dehydrogenase/reductase
(SDR) family, 7 belong to the aldo/keto reductase family
(AKR), and 6 belong to the zinc-containing alcohol
dehydrogenase family (ADH_SF_Zn-type). Six oxidore-
ductases from this toolbox were selected because of their
higher specific activity shown in the asymmetric reduction
of aryl ketones (Table S2). Among them, two reductases,
DhCR from Debaryomyceshansenii (GenBank accession
No. CAG87931) and CgCR from Candida glabrata (No.
CAG60239), displayed complementary enantioselectivity
toward benzoylacetonitrile. In addition, both reductases
exhibited excellent chemical selectivity in reducing the
carbonyl group, without forming any ethylated byproduct
asreported in the literature.5g Therefore, DhCRand CgCR
were selected for further characterization in asymmetric
reduction of benzoylacetonitrile, whose products (either
(R)- or (S)-β-hydroxynitrile) are very important chiral
synthons for the production of many pharmaceuticals
including fluoroxetine.
To establish an efficient route for asymmetric bio-
reduction of benzoylacetonitrile, a glucose dehydrogenase
from Bacillus megaterium (BmGDH)11 was introduced
for the internal regeneration of the cofactor (NADPþ/
NADPH) and coexpressed in E. coli with DhCR and
CgCR, respectively. The asymmetric reductions of five
different benzoylacetonitriles with high ee values by re-
combinant DhCRꢀBmGDH and CgCRꢀBmGDH whole
cells are shown in Table 1. A higher conversion rate was
found with CgCR (data not shown) than DhCR. However,
in the case of substrates 3, 4, and 5 catalyzed by DhCR,
reaction of R-ethylation of β-ketonitriles employing “whole-
cell system” biocatalysts was diadvantageous for the appli-
cation in the scale-up synthesis of chiral β-hydroxy nitrile.
This competing R-ethylation was even found in the recom-
binant E. coli BL21(DE3) system overexpressing carbonyl
reductases from baker’s yeast.2a,5 This ethylated product was
supposed to come from aldol type condensation between
acetaldehyde produced by the metabolism of cells and the
R-cynaoketon of substrates, followed by reduction of the
resultant alkene.5a Although the ethylated byproduct can
be avoided by lowering the reaction temperature to 4 °C5f or
employing purified carbonyl reductase,5g low catalytic effi-
ciency, the requirement of expensive nicotiamide cofactors,
the tedious purification procedure, and the low stability of
isolated enzyme posed new issues.
To eliminate the concomitant R-ethylated byproduct
and improve the productivity of β-hydroxy nitriles, we
then made an effort to search for new carbonyl reductases
with higher catalytic efficiency and employed a biphasic
system of aqueous/organic solvent for the bioreduction
of β-ketonitrile with recombinant “designer cells” coex-
pressing carbonyl reductase (CR) and glucose dehydro-
genase (GDH).6 Biphasic systems are usually employed to
improve the substrate solubility and to avoid the sponta-
neous hydrolysis and substrate inhibition.7 Additionally,
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Ghivirga, I.; Mateer, S. C.; Feske, B. D. Tetrahedron Lett. 2011, 52,
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Org. Lett., Vol. 15, No. 21, 2013
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Table 1. Relative Activity of DhCR and CgCR towards
Various Aryl β-Ketonitriles and the Percentage of R-Ethylated
Byproduct Detected in Aqueous System
Figure 1. Asymmetric reduction of benzoylacetonitrile with
recombinant BmGDHꢀDhCR whole cells in aqueous/toluene
biphasic system. (b) 30.0 g/L, (9) 50.0 g/L, (2) 72.5 g/L.
Reactions were carried out with 0.30, 0.50, and 0.725 g of
benzoylacetonitrile dissolved in 5.0 mL of toluene, 0.50 g of
DhCR-BmGDH whole cells, and 1.50 equiv of glucose in 5.0 mL
of PBS buffer (pH6.5) at 25 °C, 180 rpm with titration of 2.0 M
NaOH to control pH at 6.5 until completion.
a The reductase activity was spectrophotometrically estimated with
1 mM aryl β-ketonitriles, 0.1 mM NADPH, and a proper amount of
purified enzymes at 30 °C. The activity of DhCR toward benzoylaceto-
nitrile, 60 nmol/min/mg, was considered as 100%. b Reactions were
carried out with 10 mM benzoylacetonitrile derivatives (dissolved in
DMSO), 10 g/L recombinant whole cells of BmGDHꢀDhCR or BmGDHꢀ
CgCR with 1.5 equiv glucose in 0.5 mL PBS (pH 6.5, 100 mM) at 30 °C,
900 rpm for 12 h (Thermomixer compact, Eppendorf). Samples were
analyzed by GC equipped with Chiralsil-CP column.
diffused into the aqueous phase increasingly and then was
asymmetrically reduced by the carbonyl reductase.
Tendifferent organicsolvents with different Log Pvalues
were examined for their performance as the organic phase.
As shown in Table S3, with ethyl butyrate, ethyl hexanoate,
butyl acetate, or dibutyl phathlate, the β-ketonitrile was
efficiently extracted into the organic layer. And DhCR,
CgCR, and BmGDH also showed high residual activities in
all of the above-mentioned organic solvents except dibutyl
phathlate. Taking the optimized partition coefficient, low
toxicity, and moderate boiling point into account, toluene
was chosen as the organic phase. To our delight, as shown
in Table 2, none of the ethylated byproduct was detected
with this biphasic system employing recombinant whole
cells harboring pET28-BmGDH-DhCR. Within 24 h, all
the tested substrates were asymmetrically and chemoselec-
tively reduced into optically pure form with >82% yield.
To work as an efficient tool in organic synthesis, it is essen-
tial to establish a bioreduction process with high substrate
loadings and most importantly, without the requirement of
external cofactor addition. We then sought to investigate
the application of DhCR in the synthesis of (R)-3-hydroxy-
3-phenylpropanenitrile, a key synthon for fluoroxetine, at
high substrate loading. As shown in Figure 1, within 30 h,
as much as 0.5 M benzoylacetonitrile (145 g/L in toluene
phase) was asymmetrically reduced into optically pure (R)-
3-hydroxy-3-phenylpropanenitrile without external cofac-
tor addition. Compared with other reported bioreduction
systems in Table 3, these two enantiocomplemetary reduc-
tases in the toluene/aqueous biphasic system provide several
additional advantages, including high substrate loading,
no ethylated byproduct, no addition of external cofactor,
and easy operation, which make them more competitive and
Table 2. Asymmetric Reduction of Aryl β-Ketonitriles in
Toluene/Aqueous System
BmGDHꢀDhCRa)
BmGDHꢀCgCRa)
time conversion eeb) yield time conversion eeb) yield
substrate [h]
[%]
[%] [%] [h]
[%]
[%] [%]
1
2
3
4
5
6
>99
>99
>99
>99
>99
>99/R 85
>99/R 82
>99/R 84
>99/R 84
>99/R 82
4
>99
>99
>99
>99
>99
>99/S 89
>99/S 87
>99/S 85
>99/S 85
>99/S 87
15
24
6
12
24
6
12
12
a) Reaction was carried out at 10 g/L substrate concentration
dissolved in 5 mL of toluene, 1.5 equiv of glucose, and a proper amount
of dried BmGDHꢀDhCR (50 g/L) or BmGDHꢀCgCR (20 g/L) whole
cells in 5 mL of PBS buffer (pH 6.5, 100 mM) at 25 °C until completion.
b) ee was measured using chiral GC, the absolute configuration was
determined according to optical rotation data, and the standard product
samples were prepared by reduction with NaBH4.
some ethylated byproducts (4ꢀ9%) were detected. No
ethylated product was detected in the reactions mediated
by CgCR. The relatively lower specific activity of DhCR
(20ꢀ30% of CgCR) to β-ketonitrile derivatives may ac-
count for the appearance of the ethylated byproduct. The
unreduced substrates in aqueous phase were liable to be
ethylated by the cell metabolism of E. coli. Inspired by
the purpose of avoiding the competing R-ethylation of
the unreduced substrate in the aqueous phase, an organic
phase was introduced. As shown in Scheme 1, in the
biphasic system, the majority of the substrate was located
in the organic phase. The substrate in the organic phase
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Org. Lett., Vol. 15, No. 21, 2013

Table 3. Comparative Preparation of Optically Active 3-Hydroxy-3-phenylpropanenitrile Using Biocatalytic Asymmetric Reduction
a I. E.: isolated enzyme. b W. C.: Whole cells. c Conversion rate. d Percentage of R-ethylated byproduct. e Space-time yield.
promising tools in the preparation of both enantiomers of
chiral alcohols. This study also provides an effective proof
for the discovery of naturally evolved biocatalysts.
system. Protein engineering of these two carbonyl reduc-
tases to further improve their catalytic activity are now
underway in our laboratory, in order to make these two
newly discovered reductases very competitive and promis-
ing tools for the preparation of key building blocks of
fluoroxetine.
In summary, to screen for enzymes with high specific
activity, enantioselectivity, and substrate/product tolerance
for the asymmetric reduction of aromatic β-ketonitriles,
two stereocomplementary carbonyl reductases DhCR and
CgCR were identifid with desired properties from a newly
established oxidoreductase toolbox. Effective asymmetric
reduction of aromatic β-ketonitrile was achieved with
DhCR and CgCR. Ethylated byproducts were successfully
eliminated in a toluene/aqueous biphasic system employing
the recombinant E. coli whole cells coexpressing reductase
and BmGDH. As much as 145 g/L benzoylacetonitrile in
the toluene phase was reduced into optically pure (R)-3-
hydroxy-3-phenylpropanenitrile without external cofactor
addition and with no ethylated byproduct. Either the
high catalytic activity of reductases or the use of a bi-
phasic system was considered as the key to eliminate the
competing R-ethylated byproduct when using a whole cell
Acknowledgment. This work was financially supported
by the National Natural Science Foundation of China
(No. 21276082), Ministry of Science and Technology, P.R.
China (Nos. 2011CB710800 and 2011AA02A210), China
National Special Fund for State Key Laboratory of Bior-
eactor Engineering (No. 2060204), and Huo Yingdong
Education Foundation.
Supporting Information Available. General experimen-
tal procedures and spectra data. This material is avaiable
free of charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 21, 2013
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