NAD(P)h-independent asymmetric C=C bond reduction catalyzed by ene reductases by using artificial co-substrates as the hydrogen donor

Article abstract of DOI:10.1002/chem.201303897

To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems.

Full text of DOI:10.1002/chem.201303897

DOI: 10.1002/chem.201303897
Full Paper
&
Biocatalysis
NAD(P)H-Independent Asymmetric C=C Bond Reduction Catalyzed
by Ene Reductases by Using Artificial Co-substrates as the
Hydrogen Donor
Christoph K. Winkler, Dorina Clay, Marcello Entner, Markus Plank, and Kurt Faber*[a]
Abstract: To develop a nicotinamide-independent single fla-
voenzyme system for the asymmetric bioreduction of C=C
bonds, four types of hydrogen donor, encompassing more
than 50 candidates, were investigated. Six highly potent,
cheap, and commercially available co-substrates were identi-
fied that (under the optimized conditions) resulted in con-
versions and enantioselectivities comparable with, or even
superior to, those obtained with traditional two-enzyme nic-
otinamide adenine dinucleotide phosphate (NAD(P)H)-recy-
cling systems.
Introduction
the oxidized flavin cofactor is recycled by NAD(P)H. The latter
has to be regenerated through a second redox cycle, requiring
an additional dehydrogenase (such as formate, glucose,
glucose-6-phosphate, alcohol, or phosphite dehydrogenase),
and the corresponding natural co-substrate, which serves as
the ultimate hydride source.[5–7]
Flavin-dependent ene reductases from the “old yellow
enzyme” (OYE) family have become frequently used for catalyz-
ing the asymmetric reduction of activated C=C bonds.[1,2] In
recent years, these enzymes have been widely applied to the
asymmetric synthesis of pharmaceutically relevant targets and
industrial intermediates.[3] Despite the excellent stereoselectivi-
ties often achieved and the possibility to control the stereo-
chemical outcome of the bioreduction,[4] the overall hydrogen
transfer of the commonly employed coupled-enzyme system[5]
is rather complex (Scheme 1). After reduction of the substrate,
To find more economically advantageous systems, a variety
of alternative flavin mononucleotide (FMN)-regeneration sys-
tems, such as direct, light-mediated recycling,[8,9] have recently
been developed, which still have to prove their viability in
preparative-scale applications.[10,11] In contrast to the coupled-
enzyme method, the coupled-substrate system[5] is appealingly
simple because it requires only a single protein together with
a suitable, cheap co-substrate, serving as the hydrogen donor
for the direct recycling of the flavin cofactor. In this context,
we have recently proposed
a nicotinamide-independent
system, which was developed from the flavoprotein-catalyzed
disproportionation of conjugated enones[12]—historically
also termed “dismutase activity” or “aromatase activity”
(Scheme 2).[13–15] The desaturation of the co-substrate is ther-
modynamically unfavorable because it requires a strong exter-
nal driving force for the breakage of CÀH s bonds, which are
not energetically compensated for by the newly formed C=C p
bond. However, during the dehydrogenation of cyclohex-2-
enones, the newly formed dienone quickly tautomerizes to
form the corresponding phenol, which provides a large energy
Scheme 1. Hydrogen-transfer pathways in the bioreduction of C=C bonds
activated by an electron-withdrawing group (EWG): indirect hydrogen trans-
fer from a natural hydrogen donor through nicotinamide catalyzed by a de-
hydrogenase (coupled-enzyme system); nicotinamide-independent direct
hydrogen transfer from an artificial hydrogen donor catalyzed by a single
ene reductase (coupled-substrate system).
gain of approximately À30 kcalmolÀ1 [13–15]
.
Alternatively, elevat-
ed temperatures,[16] artificial flavin cofactors with strongly ele-
vated redox potentials,[17] and synthetic nicotinamide ana-
logues have been employed as the hydride source.[18]
[a] Dr. C. K. Winkler, D. Clay, M. Entner, M. Plank, Prof. Dr. K. Faber
Department of Chemistry, Organic and Bioorganic Chemistry
University of Graz, Heinrichstrasse 28, 8010 Graz (Austria)
Fax: (+43)316-380-9840
In addition to the typical ene reductase activities, OYEs also
show NAD(P)H oxidase activity, in the course of which H2O2 is
generated through oxidation of reduced FMNH2 by molecular
oxygen. Depending upon the type of substrate, hydrogen per-
oxide thus formed may cause spontaneous Weitz–Scheffer ep-
oxidation of the activated C=C bond,[19,20] which can be pre-
vented by working under an inert atmosphere.[21]
E-mail: kurt.faber@uni-graz.at
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303897.
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons At-
tribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
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donors in the test reaction; several even proved to
be superior to those previously described (1c,
34c).[12] Furthermore, numerous trends could be de-
lineated from the co-substrate screening (Scheme 4):
1) For type I donors, the molecular shape appears
to be critical. The small co-substrate 1c was a poor
Scheme 2. Flavoprotein-catalyzed disproportionation of conjugated enones.
hydrogen donor, whereas the larger analogue 13c
gave conversions of up to 65%; surprisingly, closely
Although the nicotinamide-independent, coupled-substrate,
hydrogen-transfer system could be successfully demonstrated,
it suffered from incomplete conversion (ꢀ65%) due to the
enzyme inhibition exerted by the co-product, phenol, which
forms a strong charge-transfer complex with FMN.[15,22–27] Al-
though this drawback could be overcome by in situ co-prod-
uct removal using solid-phase phenol scavengers, the macro-
scopic polymeric resins caused undesired racemization of chir-
ally sensitive products, such as a-substituted ketones (e.g.,
1a).[21] To develop a more robust and widely applicable cou-
pled-substrate system, we initiated a search for “artificial” hy-
drogen donors that would form (quasi)aromatic, but non-inhib-
iting co-products.
related structures 14c–17c were not accepted at all, nor were
the 4-substituted derivatives 9c and 10c. Even more puzzling,
compound 3c is a weak hydrogen donor (up to 14% conver-
sion by using XenA at pH 7.5), but 2c, lacking a distant para-
methyl group, shows no activity. Large bicyclic structures 21c,
22c, and 24c–28c (but not 23c) acted favorably and proved
to be active hydrogen donors. The tricyclic analogue 29c was
apparently too bulky for this reaction.
2) In addition to steric constraints, electronic activation of
the a-carbon atom seems to play a major role, as demonstrat-
ed by both co-substrates bearing an additional electron-with-
drawing acetyl group in the a-position (30c and 31c) being
accepted in the reaction. Likewise, compounds 18c and 19c
were found to be weak hydrogen donors. Although 20c con-
tains two electron-withdrawing substituents, steric restrictions
seem to override the electronic activation. In contrast, enol
ethers in the a-position (4c, 7c, 8c), a b-enamine (5c), or a b-
halo derivative (11 c) were unsuitable for the reaction, although
the a-enol ether analogue (6c) was shown to be a weak
donor. Type II derivatives lack a conjugated C=C bond, but
Results and Discussion
For our screening of co-substrates, we chose 4-ketoisophorone
(1a) as the substrate, which yields, upon bioreduction, chirally
sensitive (R)-levodione (1b). The latter is an important inter-
mediate for the synthesis of carotenoids (Scheme 3).[28] To ac-
count for the broad diversity of
ene reductases, OYE1 from Sac-
charomyces pastorianus[29,30] and
XenA
from
Pseudomonas
putida[31] were selected as repre-
sentative candidates due to their
distant sequence relationship
(27% identity, 55% similarity).
Both reductases displayed decid-
edly different activities in pre-
liminary studies.[21] Because activ-
ities have been shown to be
strongly dependent on the pH
of the reaction mixture, hydro-
gen donors were tested at
pH 7.5 and 9. The hydrogen
donors can be classified into
four groups: type I: derivatives
of cyclohex-2-enone, yielding
phenols; type II: 1,2-, 1,3-, and
1,4-cyclohexanediones, furnish-
ing hydroquinones; type III:
N-, O-, and S-ketoheterocycles,
forming heteroaromatics; and
type IV: 1,3- and 1,4-cyclohexa-
diene derivatives, leading to
nonphenolic co-products.
Surprisingly, co-substrates of
Scheme 3. Screening of four different types of hydrogen donor (1c–52c) in the NAD(P)H-independent bioreduc-
all types served as hydrogen tion of 4-ketoisophorone (1a).
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in organocatalytic C=C reduction
reactions.[32,33] In contrast, the
majority of the five-membered
heterocycles showed moderate
to high activities; in particular,
N-Boc-pyrrolidinone (46c) and 2-
methyltetrahydrofuranone (48c)
gave 78 and 82% yields of (R)-
levodione (1b), respectively.
However, low conversion was
observed with the thiophenone
bearing an additional activating
nitrile moiety (49c; 8% by using
XenA at pH 7.5). The surprising
performance of five-membered
ketoheterocycles as hydrogen
donors can be attributed to two
things: First, type IV hydrogen
donors bear an electron-donat-
ing nitrogen or oxygen heteroa-
tom in the g-position, which fa-
cilitates the hydride departure
from the b-carbon atom. Sec-
ondly, enzyme inhibition occurs
due to formation of a charge-
transfer complex between FMN
and a phenolate anion,[34] which
was shown in crystal structures
of OYE1 in a complex with para-
hydroxybenzaldehyde (Protein
Data Bank (PDB), entry 1OYB)[35]
and of the OYE1 mutant W116A
in a complex with 2-methyl-5-
(prop-1-en-2-yl)phenol (PDB, en-
tries 4GBU and 4GXM). Clearly,
the five-membered hydroxyhe-
teroaromatics formed after hy-
drogen abstraction from 46c
and 48c result in less favorable
p
interactions
with
FMN
than phenols or hydroqui-
nones.[24,34,36–38]
Scheme 4. Co-substrates used as hydrogen donors in the NAD(P)H-independent bioreduction of 4-ketoisophorone
(1a) to form (R)-levodione (1b) by using OYE1 and XenA enzymes at pH 7.5 and pH 9 (Boc=tert-butoxycarbonyl).
4) Not surprisingly, all co-sub-
strates of type IV, lacking an
possess an enolizable carbonyl group, and hence are generally
less suitable for this reaction and none of the tested co-sub-
strates showed conversions of more than 20%. Interestingly,
only 1,2-cyclohexanediones (32c, 33c) and 1,4-cyclohexane-
diones (34c, 35c) were accepted by the enzymes, whereas all
of the 1,3-cyclohexanediones were inactive, regardless of their
substitution pattern or the presence of electron-withdrawing
groups (37c–42c).
3) Of the heterocyclic type III co-substrates, none of the six-
membered-ring-containing substrates were accepted, including
the dihydrouracil derivatives (44c, 45c) and the well-known
“Hantzsch ester” 43c, which has a structural resemblance to re-
duced nicotinamide and is widely applied as a hydride donor
electron-withdrawing group attached to the alkene moiety
(51c, 52c), were inactive. Only compound 50c, bearing an ac-
tivating group in the exo-position, gave a moderate conver-
sion.
In summary, co-substrates from all four classes were active
as hydrogen donors and their reverse (reduction) reaction was
observed as a minor side reaction, if a side reaction occurred
(<3% conversion). Steric hindrance plays an important role in
the reaction with monocyclic cyclohexenones as the co-sub-
strates, while bicyclic hexenones were more favorable in the
reaction. Electronic activation through the presence of an addi-
tional electron-withdrawing group (such as an acetyl group)
on Ca facilitates proton abstraction, whereas electron-donating
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groups at Cb support hydride departure to flavin. In contrast to
six-membered heterocycles, five-membered rings were suc-
cessful co-substrates. The presence of an activating carbonyl
group is necessary for the acceptance of a co-substrate.
ketone (R)-levodione (1b) is prone to racemization, which
occurs even faster at elevated pH and renders substrate 1a
a challenging candidate.[39] At pH 7.5, however, ee values of
more than 60% were generally obtained.
In the next step, the hydrogen donors that performed best
in the co-substrate screening reactions were selected for fur-
ther optimization studies by using a set of eight ene reductas-
es, which have previously shown the highest acceptance of un-
natural co-substrates (other than nicotinamide;[21] Table 1).
Generally, all of the selected enzymes were able to accept the
six co-substrates (13c, 24c, 25c, 30c, 46c, 48c) and showed
up to 88% conversion (NCR with 46c) in the bioreduction of
compound 1a. Among the enzymes, XenA exhibited the
broadest co-substrate scope, with conversions of 59–78% with
all hydrogen donors except 48c. Other favorable enzyme–
co-substrate combinations were OYE1 and OYE2 with 25c (57
and 59% conversion at pH 9, respectively) and EBP1 with 48c
(68% conversion). Ene reductases from thermophilic microor-
ganisms showed good activities, yielding conversions of up to
64 (CrS with 25c at pH 9) and 56% (GkOYE with 30c), al-
though the corresponding stereoselectivities for (R)-1b ranged
from low to moderate. As previously observed,[21] the a-chiral
Co-substrates 13c, 30c, and 48c are chiral and were used in
racemic form. With the exception of the chirally unstable b-di-
ketone rac-30a, enzymatic dehydrogenation of hydrogen
donors rac-13c and rac-48c should proceed with kinetic reso-
lution, yielding the corresponding achiral aromatic oxidation
co-products and the remaining (slower reacting) co-substrate
enantiomer. Indeed, ee values of up to greater than 99% were
observed for (S)-13c and (R)-48c, indicating excellent enantio-
selectivities with enantiomeric ratios (E values) up to >200.
Owing to the high enantioselectivities for co-substrates rac-
13c and rac-48c, only 50% of the hydrogen source is available
for the reaction. Consequently, higher conversions should be
reached in the presence of two or more equivalents of the co-
substrate (Table 2). The apparent imbalance between the ee
values of (S)-13c and (R)-48c and the conversion is due to
their limited stability after extended reaction times.
Inspired by these results, the enzyme/co-substrate combina-
tions giving the highest conversions with substrate 1a were
Table 1. Selection of the best hydrogen donors and ene reductases in the NAD(P)H-independent reduction of 4-ketoisophorone (1a) to form (R)-levodione
(1b).[a]
Co-substrate
pH
7.5
OYE1
OYE2
YqjM
XenA
NCR
32
47
19
EBP1
GkOYE
CrS
46
61
80
c. [%]
<1
n.d.
n.d.
3
n.c.
n.d.
n.d.
3
14
53
25
34
73
40
1
n.d.
n.d.
1
15
91
18
ee (R)-1b [%]
ee (S)-13c [%]
c. [%]
15
65
40
47
35
ee (R)-1b [%]
ee (S)-13c [%]
c. [%]
ee (R)-1b [%]
c. [%]
9
n.d.
n.d.
3
n.d.
9
n.d.
n.d.
3
n.d.
12
<10
22
44
76
14
<10
85
47
73
64
<10
70
6
86
20
28
n.d.
n.d.
2
n.d.
3
6
96
56
83
56
<10
rac
99
45
77
36
7.5
9
ee (R)-1b [%]
19
19
<10
<10
n.d.
<10
c. [%]
ee (R)-1b [%]
c. [%]
ee (R)-1b [%]
c. [%]
ee (R)-1b [%]
c. [%]
ee (R)-1b [%]
c. [%]
ee (R)-1b [%]
c. [%]
2
n.d.
41
rac
10
57
57
rac
13
75
2
n.d.
32
rac
2
n.d.
59
rac
16
2
n.d.
23
Rac
4
n.d.
33
Rac
30
13
55
69
rac
22
54
59
rac
45
60
78
7
42
9
rac
13
63
8
rac
72
70
88
12
7
66
5
n.d.
16
57
5
n.d.
4
n.d.
14
<10
1
n.d.
2
n.d.
2
n.d.
1
n.d.
17
9
54
54
rac
19
70
64
rac
31
65
37
11
7.5
9
7.5
9
7.5
9
68
38
<10
61
35
<10
70
49
<10
30
<10
ee (R)-1b [%]
<10
c. [%]
21
82
22
35
16
99
8
75
n.d.
51
19
>99
6
74
n.d.
4
n.d.
n.d.
5
n.d.
n.d.
12
<10
<10
7
73
n.d.
25
18
38
24
78
51
68
11
92
4
n.d.
n.d.
10
16
n.d.
6
79
12
43
19
90
ee (R)-1b [%]
ee (R)-48c [%]
c. [%]
ee (R)-1b [%]
ee (R)-48c [%]
7.5
9
[a] Conversions of optimal enzyme–co-substrate combinations are highlighted in bold. Standard conditions: substrate 1a (10 mm), enzyme (100 mgmLÀ1),
co-substrate 13c, 24c, 25c, 30c, 46c, 48c (10 mm), OYE1 (Saccharomyces pastorianus), OYE2 (Saccharomyces cerevisiae), YqjM (Bacillus subtilis), NCR (nicoti-
namide-dependent cyclohexenone reductase; Zymomonas mobilis), Xenobiotic reductase XenA (Pseudomonas putida), EBP1 (estrogen binding protein, Can-
dida albicans), GkOYE (Geobacillus kaustophilus DSM 7263), CrS (chromate reductase, Thermus scotoductus SA-01); c.=conversion; ee=enantiomeric excess;
n.d.=not determined; n.c.=no conversion.
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Table 2. Nicotinamide-independent asymmetric bioreduction of activated alkenes 1a–6a by using selected artificial hydrogen donors, 24c, 25c, 30c, 46c,
48c (additional data are given in the Supporting Information).
Substrate
Co-substrate [mM]
Enzyme [mgmLÀ1
]
pH
Conversion [%]
eeP [%]
24c
24c
25c
25c
25c
30c
30c
46c
46c
46c
48c
10
50
10
50
10
10
50
50
50
50
50
XenA
CrS
XenA
CrS
300
100
300
100
300
300
100
100
100
100
100
9
9
9
9
9
7.5
9
7.5
9
9
94
>99
>99
98
94
93
94
98
93
>99
>99
rac
rac
<10 (R)
rac
rac
77 (R)
10 (R)
88 (R)
27 (R)
29 (R)
21 (R)
CrS
GkOYE
GkOYE
NCR
XenA
NCR
EBP1
9
46c
50
NCR
100
9
>99
>99 (S)
24c
25c
10
50
XenA
CrS
300
100
9
9
>99
>99
>99 (R)
>99 (R)
46c
48c
50
50
NCR
EBP1
100
100
9
9
44
21
>99 (S)
>99 (S)
24c
46c
48c
50
50
50
CrS
XenA
EBP1
100
100
100
9
9
9
>99
>99
>99
>99 (R)
>99 (R)
>99 (R)
25c
30c
50
50
CrS
GkOYE
100
100
9
9
>99
>99
96 (R)
96 (R)
further optimized with respect to enzyme loading and co-sub-
strate concentration, which finally allowed conversions to
reach completion (>99%) and also improved the enantioselec-
tivities for (R)-1b (Table 2). To demonstrate the practical applic-
ability of the optimized system, several types of substrate—
enal 2a, enone 6a, a,b-unsaturated esters 4a and 5a, and the
cyclic imide 3a—were tested (Table 2).
available reference compounds 13d, 24d, 25d, and 30d and
with independently synthesized reference materials 46d and
48d.
Conclusion
(S)-Citronellal (2b) was obtained from citral (2a) by using
NCR with 46c as the hydrogen donor with quantitative conver-
sion and excellent stereoselectivity (>99% ee). Likewise, com-
pound 3a was reduced quantitatively by using XenA and CrS
at elevated enzyme loading or in the presence of a five-fold
excess of 24c as the hydrogen donor. With (E)-b-cyanoacrylic
ester 4a, only enzymes NCR and EBP1 were active, and both
gave similar conversions and stereoselectivities to the classic
NAD(P)H system.[40] Diester 5a and a-methylcyclohex-2-enone
(6a) were quantitatively reduced with excellent stereoselectivi-
ties with various enzyme–co-substrate combinations. The ee
value of 96% for 6b was caused by imperfect stereoselectivity
and not due to racemization, as in case of 1b. The absolute
configurations of products 1b–6b were determined as previ-
ously reported,[29,40,42,43] and those of 13c and 48c were deter-
mined through co-injection on a GC with an independently
synthesized reference material (see the Experimental Section
for details). Aromatic co-products from the biotransformations
were identified by co-injection on a GC with commercially
Four types of H-donor—encompassing more than 50 candi-
dates consisting of cyclohex-2-enones, cyclohexanediones, 5-
and 6-membered N-, O- and S-ketoheterocycles and dienes—
were screened in the coupled-substrate, nicotinamide-inde-
pendent bioreduction of C=C bonds by using flavin-dependent
ene reductases. Six co-substrates were identified that (under
optimized conditions) resulted in conversions and enantiose-
lectivities comparable with, or even superior to, those obtained
in the presence of an excess of nicotinamide cofactor or in
combination with traditional NAD(P)H recycling.[29,39,41–44] These
results prove the practical applicability of the NAD(P)H-inde-
pendent, single-enzyme, hydrogen-transfer system by using
cheap (commercially available), artificial hydrogen donors. Al-
though the in situ recycling of hydrogen donors is presently
not feasible, the co-substrate costs for this reaction are
modest.[45]
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was allowed to warm to 08C and stirred for 20 min at this temper-
ature. The solvent was evaporated and a solution of methyl acry-
late (1 mL, 11.0 mmol) in DMSO (4 mL) was added to the residue.
The reaction was stirred for 20 h at ambient temperature. The mix-
ture was poured into cold, aqueous sulfuric acid (5%) and extract-
ed three times with diethyl ether (40 mL). The organic layers were
combined, washed with saturated aqueous NaHCO3 (20 mL) and
brine (20 mL), dried over MgCO3 and concentrated by evaporation
of the solvent. The residue was purified by column chromatogra-
phy (hexane/ethyl acetate 20:1), which yielded methyl 5-methyl-4-
oxotetrahydrofuran-3-carboxylate (990 mg, 6.26 mmol, 64%). TLC
results were viewed by using a KMnO4 staining solution or UV254
(Rf =0.34, hexane/ethyl acetate 2:1).
Experimental Section
General
TLC plates were run on silica gel Merck 60 (F254). Silica gel 60 from
Merck was also used for flash column chromatography. GC-MS
analyses were performed on an HP 6890 Series GC system
equipped with a 5973 mass selective detector and a 7683 Series in-
jector using a (5% phenyl) methylpolysiloxane capillary column
(HP-5MS, 30 mꢁ0.25 mm, 0.25 mm film). GC-FID analyses were car-
ried out on a Varian 3800 and on an Agilent 7890A by using H2 as
the carrier gas (14.5 psi). NMR measurements were performed on
a Bruker Avance III 300 MHz NMR spectrometer. Chemical shifts are
reported relative to trimethylsilane (TMS, d=0.00 ppm) and cou-
pling constants (J) are given in Hz.
Synthesis of (R)-2-methyldihydrofuran-3(2H)-one [(R)-48c][48]
General procedure for the nicotinamide-independent
anaerobic enzymatic C=C reduction reaction
Methyl
5-methyl-4-oxotetrahydrofuran-3-carboxylate
(200 mg,
1.3 mmol) was added to sulfuric acid (10%, 5 mL) and the mixture
was stirred for 3.5 h at 708C. The reaction mixture was then cooled
to ambient temperature, poured into saturated aqueous NaHCO3
(50 mL), and extracted three times with ethyl acetate (30 mL). The
organic layers were combined, washed with saturated aqueous
NaHCO3 (20 mL) and brine (20 mL), dried with MgSO4, concentrat-
ed by evaporation of the solvent and purified by column chroma-
tography (hexane/ethyl acetate, 5:1) to yield (R)-2-methyldihydro-
furan-3(2H)-one [(R)-48c, 20 mg, 0.2 mmol, 94% ee]. TLC results
were viewed by using a KMnO4 staining solution (Rf =0.36, hexane/
ethyl acetate 2:1). Spectroscopic data were in agreement with
those of the commercially available reference compound rac-48c.
An aliquot of the isolated enzyme (OYE1, OYE2, CrS, EBP1, NCR,
XenA, YqjM, GkOYE; protein purity >90%, protein content in reac-
tion 100 mgmLÀ1) was added to a screw-top glass vial (2 mL) con-
taining a degassed buffer solution (0.8 mL, 50 mm, tris(hydroxyme-
thyl)aminomethane·HCl (TrisHCl) buffer; pH 7.5 or pH 9), the sub-
strate (1a–6a, 10 mm), and the hydrogen donor (1c–52c; 10 mm).
The vial was flushed with argon, and sealed with a teflon-coated
septum and a lid. The mixture was shaken for 24 h at 308C and
120 rpm by using an Infors Unitron shaker and the products were
extracted with ethyl acetate (2ꢁ0.7 mL). The combined organic
phase was dried over Na2SO4 and analyzed on a GC to determine
the conversion and stereoselectivity. On a preparative scale, prod-
ucts could be easily separated from excess hydrogen donor and
phenolic byproducts by simple silica gel filtration due to the large
difference in Rf values.
Preparation of tert-butyl 3-oxo-2,3-dihydro-1H-pyrrole-1-car-
boxylate (46d)
An aliquot of isolated NCR (protein purity >90%, protein content
in reaction 200 mgmLÀ1) was added to 30 screw-top glass vials
(2 mL) containing a degassed buffer solution (0.8 mL, 50 mm,
TrisHCl buffer; pH 7.5 or pH 9), 4-ketoisophorone(1a, 30 mm),
acting as the hydrogen acceptor, and tert-butyl 3-oxopyrrolidine-1-
carboxylate (46c; 10 mm). The vials were flushed with argon,
sealed with a teflon-coated septum and a lid. The mixtures were
shaken for 24 h at 308C and 120 rpm by using an Infors Unitron
shaker. After the transformation, all phases were collected and the
products were extracted with ethyl acetate (2ꢁ30 mL). The com-
bined organic phase was dried over Na2SO4, concentrated, and the
product was purified by column chromatography (hexane/ethyl
acetate, 5:1) to yield tert-butyl 3-oxo-2,3-dihydro-1H-pyrrole-1-car-
boxylate (46d; 10.5 mg). TLC results were viewed by using
a KMnO4 staining solution (Rf =0.65, hexane/ethyl acetate 2:1).
1H NMR (300 MHz, CDCl3): d=8.33 (d, J=4.1 Hz, 2H), 5.65 (d, J=
4.2 Hz, 2H), 4.01 (s, 2H), 1.54 ppm (s, 9H).
Synthesis of a-(+)-3,4-epoxycarene[46]
A solution of meta-chloroperbenzoic acid (1.037 g, 6.0 mmol in
CHCl3 (12 mL)) was added dropwise to a stirred solution of
(+)-carene (0.508 g, 3.7 mmol) in chloroform (6 mL) over a period
of 75 min. The reaction was stirred for a further 40 min and then
quenched with aqueous sodium bisulfite (40%, 2 mL). The organic
layer was separated, washed with saturated aqueous NaHCO3
(15 mL) and brine (15 mL), dried with Na2SO4, and concentrated by
evaporation of the solvent to give a-(+)-3,4-epoxycarene as a light
yellow oil (0.561 g, 3.68 mmol).
Synthesis of (S)-3-isopropyl-6-methylcyclohex-2-enone
[(S)-13c][47]
Crude a-(+)-3,4-epoxycarene (355 mg, 2.6 mmol) was dissolved in
dichloromethane (10 mL) and cooled to À788C (N2(l)/EtOH). Trime-
thylsilyl triflate (TMSOTf; 44 mL) was added and the reaction was
stirred for 3 h. Saturated aqueous NaHCO3 (5 mL) and diethyl ether
(10 mL) were then added. The organic layer was separated,
washed twice with brine (10 mL), dried with Na2SO4 and concen-
trated by evaporation of the solvent to give (S)-3-isopropyl-6-meth-
ylcyclohex-2-enone [(S)-13c; 45 mg, 0.3 mmol, 12%, 25% ee]. Spec-
troscopic data were in agreement with those of the commercially
available reference compound rac-13c.
Synthesis of 2-methylfuran-3(2H)-one (48d)
2-Methyldihydrofuran-3(2H)-one (48c, 2 g, 20 mmol, 1.93 mL) was
dissolved in dry THF (100 mL) and cooled to À808C (liquid N2/
EtOH) under an argon atmosphere. N,N-Diisopropylethylamine
(7.78 g, 60 mmol, 10.4 mL) was then added over 10 min and the
mixture was stirred for 10 min, followed by slow addition of trime-
thylsilyl trifluoromethanesulfonate (8.89 g, 40 mmol, 7.23 mL) over
a further 10 min. The mixture was stirred and kept at between
À608C and À808C for 90 min and then allowed to warm to room
temperature over 90 min. The solution was then cooled to À608C,
and N-bromosuccinimide (4 g in 50 mL of dry THF) was added,
turning the yellow solution red. The mixture was stirred for 60 min
Synthesis of methyl 5-methyl-4-oxotetrahydrofuran-3-car-
boxylate[48]
Methyl l-lactate (1.0 g, 9.8 mmol) was dissolved in diethyl ether
(4 mL) and added to a cooled (À388C, N2(l)/EtOH) suspension of
NaH (267 mg, 50%, 5.6 mmol) in diethyl ether (6 mL). The mixture
Chem. Eur. J. 2014, 20, 1403 – 1409
www.chemeurj.org
1408ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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at this temperature and then the reaction was quenched by addi-
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Keywords: alkene reduction
reductases · enzyme catalysis · hydrogen donors
· artificial biocatalysis · ene
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Received: October 4, 2013
Published online on December 30, 2013
Chem. Eur. J. 2014, 20, 1403 – 1409
www.chemeurj.org
1409ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim