Asymmetric bioreduction of activated carbon-carbon double bonds using Shewanella yellow enzyme (SYE-4) as novel enoate reductase

Article abstract of DOI:10.1016/j.tet.2012.05.092

Shewanella yellow enzyme (SYE-4), a novel recombinant enoate reductase, was screened against a variety of different substrates bearing an activated double bond, such as unsaturated cyclic ketones, diesters, and substituted imides. Dimethyl- and ethyl esters of 2-methylmaleic acid were selectively reduced to (R)-configured succinic acid derivatives and various N-substituted maleimides furnished the desired (R)-products in up to >99% enantiomeric excess. Naturally occurring (+)-carvone was selectively reduced to (-)-cis- dihydrocarvone and (-)-carvone was converted to the diastereomeric product, respectively. Overall SYE-4 proved to be a useful biocatalyst for the selective reduction of activated CC double bonds and complements the pool of synthetic valuable enoate reductases.

Full text of DOI:10.1016/j.tet.2012.05.092

Tetrahedron 68 (2012) 7619e7623
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Asymmetric bioreduction of activated carbonecarbon double bonds using
Shewanella yellow enzyme (SYE-4) as novel enoate reductase
a
a
b
a,
ꢀ b
*
Naseem Iqbal , Florian Rudroff , Ann Brige , Jozef Van Beeumen , Marko D. Mihovilovic
a Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, 163-OC, A-1060 Vienna, Austria
b Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Shewanella yellow enzyme (SYE-4), a novel recombinant enoate reductase, was screened against a variety
of different substrates bearing an activated double bond, such as unsaturated cyclic ketones, diesters, and
substituted imides. Dimethyl- and ethyl esters of 2-methylmaleic acid were selectively reduced to
(R)-configured succinic acid derivatives and various N-substituted maleimides furnished the desired
(R)-products in up to >99% enantiomeric excess. Naturally occurring (þ)-carvone was selectively reduced
to (ꢀ)-cis-dihydrocarvone and (ꢀ)-carvone was converted to the diastereomeric product, respectively.
Overall SYE-4 proved to be a useful biocatalyst for the selective reduction of activated C]C double bonds
and complements the pool of synthetic valuable enoate reductases.
Received 7 March 2012
Received in revised form 3 May 2012
Accepted 22 May 2012
Available online 28 May 2012
Dedicated to Professor Manfred Reetz on
the occasion of receiving the Tetrahedron
Prize
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Enoate reductase
Shewanella yellow enzyme
Bioreduction
Biocatalysis
1. Introduction
common substrates for the majority of the old yellow enzyme (OYE)
family, producing optically active saturated ketones.14,15
Biocatalytic conversion of alkenes to chiral alkanes has received
significant attention in recent years as a complementary strategy to
metal-assisted hydrogenation reactions. This biotransformation is
merely a nucleophilic attack by hydride originating from NAD(P)H
across an activated C]C double bond via a Michael-type reac-
tion.1e4 Enzymes involved are referred to as enoate reductases
(EREDs).5 These biocatalysts are able to reduce a wide variety of
Much less is known about the asymmetric bioreduction of car-
boxylic acid derivatives. Whereas simple alkyl esters seem to be
accepted to a certain degree, ester hydrolysis represents a major
side reaction in whole-cell bioreductions.16,17 Another interesting
substrate class is N-protected a-methylmaleimides that are ster-
eoselectively reduced by plant cell cultures (tobacco, liverwort) or
whole cells of the cyanobacterium Synechococcus sp. to yield (R)-2-
methylsuccinimide.18
substrates, such as conjugated enals, enones, a,b-unsaturated car-
boxylic acids, imides, nitroalkenes, and ynones (Fig. 1).1,6e8
Recently, old yellow flavoprotein (OYE) from Saccharomyces
carlsbergensis was re-discovered for the chemo- and stereoselective
conversion of enones and nitro-olefins into saturated
compounds.9,10 Ligand binding properties of related enzymes and
mechanistic aspects of the bioreduction were investigated
extensively.11,12 The catalytic cycle was found to proceed via
a pingepong biebi mechanism. The catalytic potential (reducing
‘activated’ alkenes in the presence of NADPH) and possible
synthetic applications of EREDs were partly discovered in the last
decade.13
a,b-Unsaturated cyclic ketones represent the most
* Corresponding author. Tel.: þ43 1 58801 15420; fax: þ43 1 58801 15499; e-mail
Fig. 1. Schematic overview of enoate reductase mediated reduction of activated C]C
double bonds.
address: mmihovil@pop.tuwien.ac.at (M.D. Mihovilovic).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.05.092

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N. Iqbal et al. / Tetrahedron 68 (2012) 7619e7623
Although the remarkable synthetic potential of enoate re-
recently published data on a similar set of substrates by the group of
Stewart et al. the SYE-4 enzyme shows a better performance with
respect to conversion and selectivity.32
ductases has been recognized long ago,13,19 preparative scale ap-
plications were severely impeded by two major problems:
application of simple to use whole-cell systems (most prominent
baker’s yeast, but also fungi and yeasts, such as Geotrichum candi-
dum, Rhodotorula rubra, Beauveria bassiana, Aspergillus niger, etc.) is
largely compromised by undesired side reactions, such as carbonyl
reduction (catalyzed by competing alcohol dehydrogenases/car-
bonyl reductases),20e23 or ester hydrolysis (mediated by carboxyl
ester hydrolases).24 On the other hand, the first generation of iso-
lated (cloned) enoate reductases was obtained from (strict or fac-
ultative) anaerobe microbes, which were inapplicable to
preparative-scale transformations owing to their sensitivity to-
ward traces of molecular oxygen. Recently, this bottleneck was
resolved by providing oxygen-stable OYEs from yeasts.7,9,25,26
Homologs of old yellow enzyme from Shewanella oneidensis
(SYEs) were shown to be catalytically active when recombinantly
expressed as GST fusionproteins. S. oneidensis is an important model
organism in bioremediation studies because it is characterized by
unique respiratory capabilities, such as the possibility to reduce
heavy metals.27 Recently, the biochemical properties of different
SYE originating enzymes were compared to those of other OYE
family members28 and also the structure of SYE-4 was described.29
We investigated the substrate scope of the novel enoate re-
ductase from Shewanella sp. in details by using a diverse range of
activating groups, such as ketones, imides, carboxylic ester moie-
ties, as well as by varying cyclic and acyclic structural scaffolds.
Furthermore, we studied the substrate conversion of SYE-4
enzyme on maleic acid derivatives and homologs bearing diester
functionalities. Maleic acid analogs 1j and 1k were fully converted
and gave optically pure succinic acid derivatives 2j and 2k (entries
10 & 11).20 Dimethyl 2-methylmaleate (1j) was reduced to (2R)-
dimethyl 2-methylsuccinate (2j) in very good yield (85%) and op-
tical purity (98%ee) (½a D22
þ7.4 (c 2.9, CHCl3)). Accordingly, (2R)-
ꢁ
diethyl 2-methylsuccinate (2k) was isolated from the bioreduction
of diethyl 2-methylmaleate (1k) in 83% yield and with 99% enan-
tiomeric excess (½a D22
þ3.1 (c 0.9, CHCl3)). Absolute configuration
ꢁ
was determined by comparing the sign of optical rotation of liter-
ature known reference compounds to the isolated bio-products.
The absolute configuration of dimethyl 2-methylsuccinate (2j)
and diethyl 2-methylsuccinate (2k) was assigned to (R) based on
a comparison of specific rotation.
In contrast to maleic acid derivatives, exo-methylene analogs 1l
and 1m were found to be rather poor substrates for SYE-4. In the
case of 1l, low conversion (19%) and of 1m, no conversion at all was
observed (entries 12 & 13). Although a,b-unsaturated esters were
suspected to be good substrates for enoate reductases,6 a long re-
action time and overall slow enzyme kinetics were observed.
Whole-cell mediated bioreduction was dominated by ester hydro-
lysis, leading to the more reactive maleic acid derivatives, which
undergo subsequent reduction to the corresponding succinates.
This drawback was successfully circumvented by using crude cell
lysate, entirely eliminating undesired ester hydrolysis.
2. Results and discussion
Finally, compounds with an imide functionality (1neu, Table 1,
entries 14e21) were investigated to extend the substrate profile of
SYE-4 protein. According to the literature, imides are good substrates
for several enoate reductases, such as OPR1, OPR3, YqjM and other
OYEs,26,33 but the investigated substrate scope was limited to de-
rivatives substituted at C-3 (Table 1, entries 14e21; compounds
bearing an R1 group). A more comprehensive examination of the in-
fluence of different nitrogen protecting substituents (R2) on substrate
The results from all biotransformations of various substrates with
SYE-4 protein are summarized inTable 1. All biotransformations were
performed with the crude cell extract of a SYE-4 over expressing
Escherichia coli strain and in the presence of a NADPþ/G6P/G6PDH
cofactor recycling system. The bioreduction of 2-methylcyclopent-2-
enone (1a) gave 92% of the desired product with a moderate enan-
tiomeric purity of 52% after 6 h reaction time. The time screening for
substrate 1a showed a gradual decrease in enantiomeric excess
acceptance and stereospecificity was performed. These
a-methyl-
caused by either the acidity of
ductase type enzyme of the host (data not shown). Interestingly,
neither 3-methylcyclopenten- (1b) nor cyclohexenone (1c) yielded
the desired bioreduction products, even not after
transformation time, which is in contrast to the well-known EREDs
from Saccharomyces cerevisiae (OYE1-3) and Zymomonas mobilis
(NCR).30 Since substrate acceptance for 3-substituted cyclic ketones
wasrathernarrowand 2-substitutedketones gave reasonableresults,
we expanded the substrate scope toward more sterically demanding
2-substituted cyclopenten- and hexenones (entries 4 & 5).31
a
-protons or a competing enoate re-
maleimides turned out to be excellent substrates for SYE-4 and gave
almost 100% conversions with a high stereopreference in a reasonable
timeframe (Table 1, entries 14e19). Sterically more demanding sub-
strates were accepted, as well as less demanding structures, and no
significant substitution effect on the stereopreference was observed.
All products showed the (R)-configuration; this assignment is based
on a preparative scale experiment of 1s by SYE-4 to 2s (76% isolated
6 h bio-
yield and an optical rotation of ½a D22
þ12.7 (c 2.4, CHCl3)). The absolute
ꢁ
configuration of imides was determined by comparing the signs of
optical rotation of isolated products with reference compounds from
literature.34,35 All other imides were assigned accordingly, due to
similar properties and chromatographic behavior.
Surprisingly, compound 1d was hardly converted at all, whereas
the six-membered analog 1e gave almost full conversion (92%) after
6 h reaction time with a moderate enantiomeric excess (51% ee).
Compounds 1a, 1d and 1e possess structural similarities, so they
behave in a similar fashion upon enzyme mediated bioreduction;
these findings are also in accordance with previous observations by
Faber et al. testing the same substrates on different enoate re-
ductases.6,30 Furthermore, we investigated cyclic ketones 1f and 1g,
which were reduced to saturated ketones 2f and 2g by the SYE-4
protein in reasonable to good yields (Table 1, entries 6 & 7).
We continued our investigations by examining enantiopure car-
vone analogues, which were selectively reduced to dihydrocarvones.
(R)-Carvone (1h) was converted to (þ)-(2R,5R)-trans-dihy-
drocarvone (2h) with excellent stereoselectivity (59% isolated yield;
Additional substrates 1r and 1t bearing two unsaturated C]C
moieties were tested to investigate functional group tolerance. As
expected, activated double bonds were attacked in a ‘Michael-type’
addition whereas non-activated C]C bonds remained unaffected.
3. Conclusions
Within this study we have demonstrated the synthetic potential
of SYE-4, an enoate reductase from Shewanella sp. We investigated
the anti bioreduction of different activated alkenes, such as
a,b-
unsaturated ketones, -unsaturated esters and -methyl-
a,
b
a
maleimides. In general, the catalytic performance was very good
and most of the desired products were obtained in good yields and
optical purities. Remarkable results were obtained for the optically
pure terpenes (þ) and (ꢀ) carvone, which were selectively reduced
to the cis and trans diastereoisomers. Furthermore, we could
97% ee; ½a 2D2
þ14.2 (c 0.8, CHCl3) and (S)-carvone (1i) gave
ꢁ
(ꢀ)-(2R,5S)-cis-dihydrocarvone (2i) quantitatively without com-
promising the optical purity of the precursor (53% isolated yield;
½
a 2D2
ꢁ
ꢀ16.9 (c 1.2, CHCl3) Table 1, (entries 8 & 9). According to

N. Iqbal et al. / Tetrahedron 68 (2012) 7619e7623
7621
Table 1
Bioreduction of different substrates with SYE-4 proteins
Entry
Substrate
Time
Conv. (%)a
ee (%)
Abs. config.
Product
Ketones
1
2
3
1a (R¼2-Me)
1b (R¼3-Me)
1c
6 h
6 h
6 h
92
nc
nc
52
na
na
nd
na
na
2a
2b
2c
4
5
6
7
8
9
1d (n¼1)
6 h
6 h
6 h
6 h
1 h
1 h
4
rac
51
na
2d
2e
2f
1e (n¼2)
92
30
71
100
100
nd
1f (n¼1)
na
na
1g (n¼2)
na
na
2g
2h
2i
1h (þ)-Carvone
1i (ꢀ)-Carvone
97b
95c
2R,5R
2R,5S
Diesters
10
1j (R¼Me)
1k (R¼Et)
1l (R¼Me)
1m (R¼Et)
6 h
6 h
6 h
6 h
99
99
19
nc
98
99
90
na
R
2j
11
12
13
R
2k
2j
R
na
2k
Imides
14
1n (R1¼Me; R2¼Me)
1 h
99
99
R
2n
15
16
17
18
19
20
21
1o (R1¼Me; R2¼Et)
1p (R1¼Me; R2¼Propyl)
1q (R1¼Me; R2¼Butyl)
1r (R1¼Me; R2¼Allyl)
1s (R1¼Me; R2¼Bn)
1t (R1¼H; R2¼Allyl)
1u (R1¼H; R2¼Bn)
3 h
3 h
3 h
3 h
3 h
1 h
1 h
99
>99
99
99
99
99
98
99
99
99
99
99
na
na
R
R
R
R
R
na
na
2o
2p
2q
2r
2s
2t
2u
nd¼not determined; na¼not applicable.
a
Conversion based on GC.
Diastereomeric excess (trans).
Diastereomeric excess (cis).
b
c
demonstrate that the active site of SYE-4 does accept sterically
more demanding residues. Investigations of N-substituted mal-
eimides clearly showed that neither the stereoselectivity nor the
substrate acceptance was affected by the N-residue. Overall SYE-4 is
a versatile and synthetically useful biocatalyst for the reduction of
various activated C]C double bonds and will complement the
biocatalytic toolbox of already known enoate reductases.
solvents were distilled prior to use. Screening experiments
were performed in sterile multi-well plates. Flash column
chromatography was performed on silica gel 60 from Merck
(40e63
mm). NMR spectra were recorded on a Bruker AC 200
(200 MHz) spectrometer; chemical shifts are reported in parts per
million using TMS as internal standard. Enantiomeric purity was
determined by chiral phase GC using
a BGB 175 column
(30 mꢂ0.25 mm ID, 0.25
m
m film) on a Thermo Finnigan Trace or
4. Experimental
4.1. General
Focus chromatograph and compared to reference material
obtained by chemical conversion, if applicable. GCeMS analyses
were carried out on a GCeMS Thermo Scientific DSQ II using
a standard capillary column BGB5 (30 mꢂ0.25 mm ID, 0.25
mm
Chemicals and microbial growth media were purchased from
film). Specific rotation was determined using a Perkin Elmer
commercial suppliers and used without further purification. All
Polarimeter 241.

7622
N. Iqbal et al. / Tetrahedron 68 (2012) 7619e7623
4.2. Typical screening procedure for bioreductions
(3H, s). 13C NMR (50 MHz; CDCl3; Me4Si):
(d), 51.5 (q), 51.7 (q), 172.0 (s), 175.5 (s).
d
¼16.8 (q), 35.6 (t), 37.3
Initially, SYE-4 was overexpressed in a BL21(DE3) E. coli strain
carrying the pGEX-4 T-2 plasmid. The protein was obtained from
recombinant cell cultures according to previous literature
protocols.28
4.3.4. (2R)-Diethyl
2-methylsuccinate
(2k).38 Diethyl
2-methylmaleate (1k) (20 mg in ethanol/water (2:1)) was reduced to
(2R)-diethyl 2-methylsuccinate (2k) (16 mg, 83%) (½a D22
þ3.1 (c 0.9,
ꢁ
An Erlenmeyer flask containing sterile TB medium (200 mL)
CHCl3)). 1H NMR (200 MHz; CDCl3; Me4Si):
d
¼1.22 (3H, d, J¼7 Hz),
supplemented with chloroamphenicol (34
m
g/ml) (200
m
L) was
1.26 (6H, dt, J¼7.2, 1.2 Hz), 2.33e2.44 (1H, m), 2.66e2.92 (2H,
incubated with a 2% vol of an overnight culture grown on LB
m), 4.08e4.20 (4H, dq, J¼7.2, 2.4 Hz), 13C NMR (50 MHz;
medium (chloroamphenicol) up to an OD600¼1 within 3 h at
CDCl3; Me4Si):
171.6 (s), 175.1 (s).
d
¼14.0 (q), 16.8 (q), 35.7 (d), 37.6 (t), 60.3 (t), 60.4 (t),
28 ꢃC. Enzyme synthesis was induced using 160
m
L
IPTG
(0.5 mM). The flask was shaken (120 rpm) at 28 ꢃC for 24 h. The
cell pellet was collected by centrifugation (6000 rpm for 15 min),
washed with 10 mM PBS buffer (30 mL), centrifuged (6000 rpm
for 4 min) again, and finally for further use resuspended in PBS
(5 mL) for further use. Protease inhibitor (phenyl methane sul-
fonyl fluoride) was added to a final conc. of 0.2 mM (0.2 M stock
in EtOH). Cells were ruptured by sonication (6 cycles, pulse for
10 s, 60 s cooling). The cell debris was centrifuged (10,000 rpm
for 15 min) to obtain the crude cell lysate, which was stored at
ꢀ20 ꢃC until further use. A Bradford protein assay was conducted
to calculate the protein concentration in the cell lysate. Bio-
transformations were performed with SYE-4 protein (25 mg/mL)
4.3.5. (3R)-1-Benzyl-3-methylpyrrolidine-2,5-dione
Benzyl-3-methyl-1H-pyrrole-2,5-dione (1s) (30 mg in ethanol/
(2s).39 1-
water
(2:1))
was
bioreduced
to
(3R)-1-benzyl-3-
methylpyrrolidine-2,5-dione (2s) (24 mg, 76%) (½a D22
þ12.7 (c 2.4,
ꢁ
CHCl3)). 1H NMR (200 MHz; CDCl3; Me4Si),
d
¼1.28e1.32 (3H, d,
J¼7.2 Hz), 2.14e2.37 (1H, m), 2.80e2.90 (2H, m), 4.62 (2H, s),
7.25e7.34 (5H, m). 13C NMR (50 MHz; CDCl3; Me4Si):
d
¼16.7 (q),
34.7 (t), 36.4 (d), 42.3 (t), 127.9 (d), 128.6 (d), 128.7 (d), 135.8 (s),
176.1 (s), 180.2 (s). GC: MS, m/z¼203 (Mþ, 100), 174 (23), 160 (70),
104 (61), 91 (58).
in sterile multi-well plates (500
TriseHCl (50 mM, pH 8), NADPþ (200
(4 mM), glucose-6-phosphate dehydrogenease (1 unit), and
substrate 2 mM, 0.8 L (stock solution in EtOH/H2O (2:1)) at
m
L each well) in the presence of
Acknowledgements
m
M), glucose-6-phosphate
This project was in part funded by the Austrian Science Fund
FWF (P-I723). N.I. acknowledges financial support by the Higher
Education Commission, Pakistan, for receiving an international PhD
fellowship.
m
28 ꢃC for 6 h. Samples were collected after 1 h, 3 h and 6 h. The
reaction mixture was extracted with ethyl acetate containing the
internal standard methyl benzoate and samples were analyzed
by chiral GC and GCeMS (Table 1).
References and notes
4.3. Typical procedure for preparative biotransformations
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4.3.1. (þ)-(2R,5R)-2-Methyl-5-(prop-1-en-2-yl)cyclohexanone
(2h).36 (ꢀ)-Carvone (10 mg in ethanol/water (2:1)) was reduced,
applying the general bioreduction protocol to (þ)-trans-dihy-
drocarvone (2h) (6 mg, 59%) (½a D22
ꢁ
þ14.2 (c 0.8, CHCl3). 1H NMR
(200 MHz; CDCl3; Me4Si),
d
¼1.02 (3H, d, J¼6.8 Hz), 1.46e1.84 (4H,
m), 1.66 (3H, s), 2.28e2.56 (4H, m), 4.62e4.76 (2H, m). 13C NMR
(50 MHz; CDCl3; Me4Si):
d
¼15.6 (q), 21.5 (q), 26.3 (t), 30.6 (t), 43.9
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(d), 44.1 (d), 44.6 (t), 111.5 (t), 146.8 (s), 214.0 (s). GC: MS, m/z¼152
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(2i). (þ)-Carvone (10 mg in ethanol/water (2:1)) was converted to
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(ꢀ)-cis-dihydrocarvone (2i) (5.4 mg, 53%) (½a D22
ꢁ
ꢀ16.9 (c 1.2,
¼0.98 (3H, d,
CHCl3)). 1H NMR (200 MHz; CDCl3; Me4Si),
d
J¼6.5 Hz), 1.28e1.90 (4H, m), 1.46 (3H, s), 2.01e2.18 (4H, m),
4.45e4.48 (2H, m). 13C NMR (50 MHz; CDCl3; Me4Si):
20.4 (q), 30.7 (t), 34.8 (t), 44.6 (d), 46.8 (d), 46.9 (t), 109.5 (t), 147.5
(s), 212.4 (s). GC: MS, m/z¼152 (Mþ, 15), 137 (11), 95 (66), 81 (43),
67 (100).
d
¼14.3 (q),
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4.3.3. (2R)-Dimethyl
2-methylsuccinate
(2j).37 Dimethyl
2-
methylmaleate (1j) (20 mg in ethanol/water (2:1)) was reduced
to (2R)-dimethyl 2-methylsuccinate (2j) (18 mg, 85%) (½a D22
þ7.4 (c
ꢁ
2.9, CHCl3)). 1H NMR (200 MHz; CDCl3; Me4Si):
d
¼1.23 (3H, d,
J¼7.2 Hz), 2.35e2.47 (1H, m), 2.68e2.94 (2H, m), 3.68 (3H, s), 3.70

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