A substrate-driven approach to determine reactivities of α,β-unsaturated carboxylic esters towards asymmetric bioreduction

Article abstract of DOI:10.1002/chem.201200990

The degree of C=C bond activation in the asymmetric bioreduction of α,β-unsaturated carboxylic esters by ene-reductases was studied, and general recommendations to render these "borderline-substrates" more reactive towards enzymatic reduction are proposed. The concept of "supported substrate activation" was developed. In general, an additional α-halogenated substituent proved to be beneficial for enzymatic activity, whereas β-alkyl or β-aryl substituents were detrimental for the reactivity of nonhalogenated substrates, and α-cyano groups showed little effect. The alcohol moiety of the ester functionality was found to have a strong influence on the reaction rate. Overall, activities were determined by both steric and electronic effects. Biotransformation: The asymmetric bioreduction of α,β-unsaturated carboxylic esters by ene-reductases could be tuned by varying the degree of C=C bond activation (see scheme). An additional α-halogenated substituent proved to be beneficial for enzymatic activity, whereas β-alkyl or β-aryl substituents were detrimental for the reactivity of nonhalogenated substrates. Copyright

Full text of DOI:10.1002/chem.201200990

FULL PAPER
DOI: 10.1002/chem.201200990
A Substrate-Driven Approach to Determine Reactivities of a,b-Unsaturated
Carboxylic Esters Towards Asymmetric Bioreduction
Gꢀbor Tasnꢀdi,[a] Christoph K. Winkler,[b] Dorina Clay,[b] Nargis Sultana,[b]
Walter M. F. Fabian,[b] Mꢁlanie Hall,[b] Klaus Ditrich,[c] and Kurt Faber*[b]
Abstract: The degree of C=C bond ac-
tivation in the asymmetric bioreduction
of a,b-unsaturated carboxylic esters by
ene-reductases was studied, and gener-
al recommendations to render these
“borderline-substrates” more reactive
towards enzymatic reduction are pro-
posed. The concept of “supported sub-
strate activation” was developed. In
general, an additional a-halogenated
substituent proved to be beneficial for
enzymatic activity, whereas b-alkyl or
b-aryl substituents were detrimental for
the reactivity of nonhalogenated sub-
strates, and a-cyano groups showed
little effect. The alcohol moiety of the
ester functionality was found to have
a strong influence on the reaction rate.
Overall, activities were determined by
both steric and electronic effects.
Keywords: biocatalysis · ene-reduc-
tase · enzymes · substituent effects ·
substrate activation
Introduction
creased attention due to its exquisite chemo-, regio-, and
stereoselectivity, further strengthened by mild operational
conditions and independence from heavy metals.[12] The en-
zymes catalyzing this reaction are ene-reductases
[EC 1.3.1.X], members of the “Old Yellow Enzyme” (OYE)
family.[13] Over the past few years, these flavoproteins have
been proven to be powerful catalysts for the synthesis of
valuable chiral building blocks, amino acid derivatives, ter-
penoids, and fragrances.[14] Ene-reductases catalyze the re-
duction of C=C bonds that are electronically activated by
a conjugated electron-withdrawing group (EWG), which
also serves as anchor for substrate binding in the enzyme
active site; nonactivated (isolated) alkenes are unreactive
(Scheme 1). Well-accepted substrates include enals, enones,
Modern synthetic strategies rely on stereoselective transfor-
mations to increase molecular complexity through genera-
tion of optical activity. In this context, asymmetric reduction
involves the transformation of prochiral sp2-centers into
chiral sp3-moieties, which creates complexity and added
value in the formed product. Hence, reduction is widely ap-
plied in the chemical and pharmaceutical industry, in which
single enantiomers are highly desired.[1–3] The asymmetric
catalytic reduction of C=C bonds is particularly powerful,
because it (theoretically) leads to the generation of up to
two stereogenic centers.[1] On industrial scale, transition-
metal-catalyzed processes are most sophisticated,[4–6] where-
as organocatalytic asymmetric transfer hydrogenations are
still at the stage of development.[7–11] The biocatalytic var-
iant, that is, the asymmetric bioreduction, is gaining in-
[a] Dr. G. Tasnꢀdi
ACIB GmbH c/o
Biocatalytic Synthesis, University of Graz
Heinrichstrasse 28, 8010-Graz (Austria)
Scheme 1. Asymmetric bioreduction of a,b-unsaturated esters by using
ene-reductases.
[b] C. K. Winkler, D. Clay, N. Sultana, Prof. Dr. W. M. F. Fabian,
Dr. M. Hall, Dr. K. Faber
Department of Chemistry, Organic & Bioorganic Chemistry
University of Graz, Heinrichstrasse 28, 8010-Graz (Austria)
Fax : (+43)316-380-9840
a,b-unsaturated carboxylic acids and esters, nitriles, and ni-
troalkenes. Although enzyme- and substrate-based stereo-
control techniques are now well understood, no rules re-
garding substrate activation have been defined, which would
allow the prediction of reactivities and thus assist to use
these enzymes more systematically.[15] Based on their high
degree of polarization, enals, enones, and nitroalkenes con-
stitute excellent Michael acceptors and are thus well accept-
ed by OYEs. On the other hand, carboxylic acids and deriv-
atives thereof, such as esters and nitriles, are less activated
and often behave as “borderline substrates”. The data avail-
E-mail: Kurt.Faber@Uni-Graz.at
[c] Dr. K. Ditrich
Fine Chemicals & Biocatalysis Research
BASF SE
Carl-Bosch-Strasse 38, 67056 Ludwigshafen (Germany)
Supporting information for this article contains description of the
source of enzymes, chemicals and materials, chiral and nonchiral ana-
lytics, synthesis of substrates, and reference compounds, determina-
tion of absolute configurations, additional data on organic cosolvents,
and DFT calculations and is available on the WWW under http://
dx.doi.org/10.1002/chem.201200990.
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able to date indicate that alkenes with a single carboxylic
acid or ester group are generally poorly converted by
OYEs.[16–18] The insufficient degree of substrate activation in
a,b-unsaturated carboxylic acids may be overcome by using
so-called “enoate-reductases”, which possess an [Fe4S4]
cubane cluster in addition to the flavin cofactor. Unfortu-
nately, this motif renders these enzymes extremely sensitive
towards traces of O2, which makes them inapplicable to
preparative-scale bioreductions.[19,20]
variations in the alcohol part of the ester functionality were
investigated, and the degree of C=C bond activation was re-
lated to enzymatic activity. Additionally, both reaction rate
and stereoselectivity could be tuned by a switch of substrate
E/Z-configuration and type of organic co-solvent to produce
enantiopure a-halo esters.
Results
Although a single carboxylic acid/ester/nitrile moiety is
barely sufficient for alkene activation, addition of a second
electron-withdrawing moiety may help to enhance the
degree of polarization of the C=C bond to facilitate the bio-
reduction of these “borderline-substrates”. Recently, OYEs
1–3 were shown to catalyze the stereoselective bioreduction
of methyl a-halo-2-alkenoates and 3-arylacrylates.[21–23] Com-
parable methods for the preparation of chiral a-halo esters
include organocatalytic[24–26] and metal-catalyzed[27] a-halo-
genation, stereoselective substitution[28–31] as well as resolu-
tion of racemic esters by enzymatic hydrolysis.[32,33] Asym-
metric hydrogenation of alkenes is restricted to fluoro deriv-
atives[34,35] due to reductive dehalogenation in the presence
of reducing metal agents. Additionally, stereoselective bro-
mination leads to enantiopure a,b-dihalogenated com-
pounds.[36,37] Chiral a-halo esters have found application in
the synthesis of biologically active compounds[38,39] and her-
bicides[40] as well as in natural-product synthesis,[41] whereas
stereocontrolled nucleophilic substitution on enantiopure a-
halo esters gives access to optically active N-, O-, and S-con-
taining compounds.[42–47]
Seven isolated ene-reductases (OYEs 1–3, YqjM, NCR,
OPR1, and OPR3) known to display broad substrate accept-
ance in the reduction of a,b-unsaturated compounds were
investigated by using a series of acrylic ester derivatives 1a–
8a (Scheme 1 and Table 1). The overall poor reactivity of
acrylonitrile (1a; only NCR, OYE1, and OYE2 showed
modest activities, Table 1, entry 1)[48] indicated that the ni-
trile moiety was a poor activating group and prompted us to
test various alkyl acrylates (2a–7a). The change from nitrile
to carboxylic ester as activating group had a huge influence
on the reaction rates, because alkyl esters 2a–4a were
almost quantitatively converted by the ene-reductases
tested, except with YqjM and OPR1 (Table 1, entries 2–4).
Surprisingly, the chain length of the alcohol moiety had
a strong positive effect on YqjM and OPR1 activities, in
which conversions improved from 27 to 91% and <1 to
55% going from methyl to n-butyl. An analogous effect was
observed with Rocheꢂ ester precursors and a-alkoxy-func-
tionalized enones,[49,50] which may be caused by alternative
substrate-binding modes induced by steric effects. The
Herein, we propose the concept of “supported substrate
activation” as a tool to probe the reactivity of a,b-unsaturat-
ed carboxylic esters with ene-reductases and to test the in-
fluence of additional substituents on the C=C bond activa-
tion. The electronic properties and the size of the a- and b-
substituents of the unsaturated acid moiety together with
stericACTHNGUTERNaUNG lly cumbersome tert-butyl ester (5a) was only accept-
ed by NCR (87% conversion), whereas the other enzymes
were poorly active (cmax =7%; Table 1, entry 5). The pres-
ence of an a-methyl group in methyl methacrylate (6a) di-
minished the reactivity of OYE2, OYE3, NCR, and OPR3
(2a: 89–99% conversion; 6a: 14–91% conversion; Table 1,
Table 1. Bioreduction of acrylic-ester derivatives with ene-reductases.
Entry
Substrate
Product
Cofactor
OYE1
Conv. ee
[%]
OYE2
Conv. ee
[%]
OYE3
Conv. ee
[%]
YqjM
Conv. ee
[%]
NCR
Conv. ee
[%]
OPR1
Conv. ee
[%]
OPR3
Conv. ee
[%]
1
2
3
4
5
NADH
NADH
NADH
NADH
NADH
31
>99
>99
>99
7
30
>99
>99
98
<1
97
80
98
n.c.
<1
27
42
91
1
9
n.c.
<1
5
55
<1
<1
89
98
>99
7
>99
>99
>99
87
6
6
NADH
>99
91
58
46
89
26
14
98
>99
83
>99
>99
>99
33
7
NADH
40 (S)
45 (S)
83 (S)
86 (R)
>99 (R)
93 (R)
>99 (R)
93
>99 (S)
83
99 (S)
97
39 (R)
14
75 (R)
90
>99 (S)
97
98 (S)
92
43 (R)
79
14 (R)
>99
>99 (S)
88
>99 (S)
>99
57 (S)
9
9
34
57 (R)
74
72 (R)
>99
97 (R)
77
21
53 (R)
19
54 (R)
87
90 (R)
19
8
NADH
n.c.
>99 (S)
37
98 (S)
33
93 (R)
54
69 (R)
4
9
NAD+/GDH
NADH
45 (S)
35
69 (R)
56
10
11
NAD+/GDH
59 (S)
94 (R)
84 (R)
94 (R)
Reaction conditions: substrate (10 mm), NADH (15 mm) in Tris-HCl buffer (50 mm, pH 7.5), 308C, 24 h; n.c.=no conversion; NAD+/GDH=glucose/glu-
cose dehydrogenase was used for cofactor recycling.
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Reactivities of a,b-Unsaturated Carboxylic Esters
FULL PAPER
entries 2 and 6). Changing the electron-donating a-methyl
moiety to an electron-withdrawing a-chloro substituent
translated into greatly improved reactivity: all enzymes
showed enhanced conversion with methyl 2-chloroacrylate
(7a) compared with 6a, in particular OYE3, YqjM, OPR3,
and OPR1, which displayed a 1.4 to 3.8-fold improvement in
conversions (Table 1, entry 7). It is tempting to relate this
effect of electronic activation to the mechanism of OYEs,
because the reaction proceeds through a Michael-type addi-
tion through nucleophilic attack of a hydride onto the b-
carbon followed by proton addition onto the a-position.[51]
Thus, the additional electron-withdrawing a-chloro group
(in contrast to the methyl moiety) enhances the degree of
C=C polarization, thereby favoring the overall addition of
[2H]. However, comparing 7a with unsubstituted methyl ac-
rylate 2a, it appears that also steric factors play a role. Al-
though (electronically expected) higher reaction rates were
achieved with YqjM and OPR1, OYE3 and OPR3 showed
reduced conversion with nonactivated 7a. Moderate-to-high
that the a-cyano group would enhance the C=C bond polari-
zation (Figure 1B). Unfortunately, substrates 15a–17a un-
derwent decomposition and/or polymerization under screen-
ing conditions (data not shown) and were not further inves-
tigated.
As an alternative, we tested b-substituted a-haloacrylic
ester derivatives. To our delight, methyl 2-chloropentenoate
[(Z)-8a] was nicely accepted and yielded (S)-8b with excel-
lent stereoselectivity (conversion up to>99%, >99% ee) in
the presence of OYEs 1–3 and YqjM (Table 1, entry 8). This
supports the hypothesis that C=C bond activation can be
tuned by the presence of a-halo substituents, because the
corresponding a-unsubstituted derivative [(E)-12a] was not
converted at all. By using the glucose/glucose dehydrogen-
ase (NAD+/GDH) cofactor recycling system, 97% conver-
sion and 98% ee were obtained by using OYE2 (Table 1,
entry 9). Interestingly, NCR and OPR1 showed moderately
expressed opposite stereopreference [ee up to 72% for (R)-
8b]. The E-isomer was similarly well accepted, and the
change of isomer geometry had a strong influence on the
stereochemical outcome for some of the enzymes: OYE1,
OYE2, YqjM, and OPR3 showed a strong switch, that is,
(Z)-8a gave (S)-8b and (E)-8a led to (R)-8b. In contrast,
OYE3 always formed (S)-8b, and NCR and OPR1 were
always R-selective, regardless of the E/Z-configuration of
substrate 8a (Table 1, entry 10). This behavior has been pre-
viously observed with OYEs.[53] The cofactor recycling
system yielded noticeable variations in conversion and ee
values (Table 1, entry 11). Such an influence of the nicotin-
S-stereoACHTUNGTRENNUNGselectivity was obtained with OYEs 1–3 (40–83%
enantiomeric excess (ee)), whereas YqjM, NCR, and OPRs
showed excellent opposite stereopreference (86 up to
>99% ee for (R)-7b).
Next, we investigated the effects of b-substituents. Inter-
estingly, the addition of an alkyl substituent, regardless of its
size, totally deactivated the system (compounds 9a–12a,
<1% conversion, Figure 1A). Herein, the result can also be
rationalized by considering the reaction mechanism. Indeed,
the electron-donating group diminished the d+ at the b-
carbon; in addition, the b-position may be particularly sensi-
tive towards steric hindrance, because the hydride has to be
delivered at this point from N5 of the flavin cofactor. In
need of additional activation of unreactive b-alkyl- or b-
aryl-substituted acrylic esters, we looked for potential a-acti-
vating groups. Because methyl (Z)-2-ethoxy-3-(4-methoxy-
phenyl)propenoate with a moderately electron donating a-
alkoxy group was not converted by OYE3,[52] a-cyano-sub-
stituted acrylates, such as methyl 2-cyanoacrylate (15a),
methyl 2-cyano-3,3-dimethylacrylate (16a), and methyl 2-
cyano-3-phenylpropenoate [(E)-17a], were tested, expecting
AHCTUNGTREGaNNNU mide regeneration system on the bioreduction reaction has
been observed in numerous cases.[54–57] Although not fully
understood, it may result from differences in the (bi-bi Ping-
Pong) kinetics between ene-reductase and recycling enzyme,
leading to variations in the ratio oxidized/reduced cofactor.
Prompted by these results, a series of a-halogenated cin-
namic esters (18a–20a) were tested to overcome the insuffi-
cient degree of activation in (unreactive) methyl cinnamate
((E)-13a, <1% conversion, Figure 1A). Again, the intro-
duction of an a-halo substituent proved to be successful, be-
cause all substrates were converted with OYEs 1–3 (Table 2,
entries 1–8). Among them, OYE3 showed highest activities
(conversion 36–97%) and S-stereoselectivity (79% up to
>99% ee) with methyl a-chloro-, bromo-, and iodocinna-
mates. The ability of OYE3 to accept larger substrates is
likely due to an enlarged binding pocket caused by an ex-
change of phenylalanine F296 (in OYE1 and OYE2) by
serine S297 in OYE3.[58,59] The type of halogen had a strong
effect on the activity of OYE3: although a-fluoro-substitu-
tion led to an unreactive substrate ((Z)-14a, <1% conver-
sion, Figure 1A), the Cl-, Br-, and I-analogues (Z)-18a, (Z)-
19a, and (Z)-20a were successfully converted at different
rates, and the a-bromo derivative (Z)-19a was the most re-
active of the series (Table 2, entry 3). The S-stereoprefer-
ence of OYEs 1–3 was conserved throughout the series 18a–
20a, reduced stereoselectivity was observed with the steri-
cally most demanding iodo analogue (Z)-20a (ee 79–80%).
The E/Z-configuration of the brominated substrate had no
Figure 1. A) Nonsubstrates for ene-reductases (<1% conversion, data
not shown). B) Substrates undergoing polymerization or decomposition.
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K. Faber et al.
Table 2. Bioreduction of cinnamic-ester derivatives with ene-reductases.
Entry
Substrates
Product
Cofactor
OYE1
Conv. ee
[%]
OYE2
Conv. ee
[%]
OYE3
Conv. ee
[%]
YqjM
Conv. ee
[%]
NCR
Conv. ee
[%]
OPR1
Conv. ee
[%]
OPR3
Conv. ee
[%]
9
7
36
98 (S)
14
>99 (S)
79
99 (S)
18
99 (S)
97
98 (S)
39
95 (S)
43
79 (S)
12
80 (S)
58
5
5
1
2
3
4
5
6
7
8
9
NADH
n.c.
n.c.
n.c.
n.c.
92 (S)
>99 (S)
1
n.d.
11
95 (S)
18
97 (S)
9
92 (S)
33
90 (S)
7
85 (S)
8
84 (S)
3
>99 (S)
<1
n.d.
94 (R)
<1
n.d.
15
90 (R)
8
95 (R)
45
66 (S)
29
74 (S)
2
n.d.
8
n.d.
<1
NAD+/GDH
NADH
n.c.
n.c.
<1
n.d.
<1
n.d.
<1
n.d.
8
6
<1
n.d.
48 (S)
10
94 (S)
13
>99 (R)
42
>99 (R)
<1
4
NAD+/GDH
NADH
n.c.
98 (S)
15
90 (S)
15
89 (S)
24
85 (S)
12
85 (S)
14
96 (S)
16
4
99 (R)
28
>99 (R)
NAD+/GDH
NADH
>99 (R)
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.d.
<1
n.d.
NAD+/GDH
NADH
n.c.
n.c.
93 (S)
21
98 (S)
98 (S)
9
98 (S)
n.d.
10
NAD+/GDH
n.c.
n.c.
n.c.
96 (S)
<1
n.d.
1
<1
n.d.
2
<1
n.d.
3
11
12
NADH
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
n.c.
NAD+/GDH
n.d.
n.d.
n.d.
Reaction conditions: substrate (10 mm), NADH (15 mm) or cofactor-recycling system in Tris-HCl buffer (50 mm, pH 7.5), 308C, 24 h; n.c.=no conver-
sion; n.d.=not determined; NAD+/GDH=glucose/glucose dehydrogenase was used for cofactor recycling.
effect on the stereorecognition by OYEs 1–3 (Table 2, en-
tries 3–6), which is in line with similar observations on
methyl a-bromo-butenoate, hexenoate, and heptenoate.[23]
In contrast, NCR and OPR1 converted the geometric E/Z-
isomers of 19a in a stereocomplementary fashion (Table 2,
entries 3–6). YqjM and OPR3 were not active on (Z)-18a–
20a, whereas (E)-19a was reduced slowly (8 and 28% con-
version) with excellent ee to (R)-19b. The use of a cofactor
recycling system improved the performance of OYE2,
YqjM, NCR, and OPR3 in the bioreduction of (E)-19a
giving access to (R)-19b in up to 42% conversion and
>99% ee.
alcohol functionality in cinnamic ester derivatives was detri-
mental; only methyl esters reacted, ethyl esters of 19a, 21a,
and 22a were not accepted by the enzymes (data not
shown).
To summarize the observed trends, the properties of the
a-halo substituent in methyl cinnamates, covalent radius and
electronegativity, were plotted against reactivity (i.e., con-
version) and stereoselectivity (ee; Figure 2). Although the
electronegativity of the a-halogen substituent correlates
with the reactivity from bromo to iodo, it fails to predict the
reactivity of the fluoro and chloro derivatives. Likewise, the
size of the substituent[60] explains the drop in reactivity
Additionally, two a-halo-cinnamic ester derivatives [(Z)-
21a and (Z)-22a] with electron-withdrawing substituents on
the aromatic ring were tested (Table 2, entries 9–12). Inter-
estingly, the presence of two substituents on the phenyl
moiety enhanced the reactivity of (Z)-21a compared with
(Z)-18a, but did not influence the enzyme stereoselectivity.
OYE1 and OYE3 showed exactly the same trend with
NADH as cofactor: 55% enhanced conversion through ring
activation. This effect was more pronounced with OYE2 in
presence of a recycling system (Table 2, entries 9–10). All
enzymes tested were inactive on (Z)-22a. It is an interesting
aspect that the o-nitro group supports an (aci-nitro-like)
mesomeric form leading to the delocalization of positive
charge on the 1’-phenyl carbon and a-C thereby reducing
the electrophilicity on b-C (which is impossible with the m-
nitro group, cf. substrate 21a). Although beneficial on small-
er unsubstituted alkyl acrylates (2a–4a), the variation of the
Figure 2. Effect of a-halogen substituent on the bioreduction of methyl
(Z)-a-halo cinnamates by OYE3 (14a: X=F; 18a: X=Cl; 19a: X=Br,
20a: X=I).
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Reactivities of a,b-Unsaturated Carboxylic Esters
FULL PAPER
(from bromo to iodo) going in hand with a drop in stereo-
other substrates were poorly converted in the presence of
this cosolvent. An even more pronounced increase was ach-
ieved with (Z)-22a, unreactivity of which in neat aqueous
buffer was completely overcome by addition of TBME or
DIPE (ꢀ96% conversion, >99% ee). Interestingly, THF,
EtOAc, acetone, TBME, DIPE, toluene, and dichlorome-
thane provided usually higher conversions for substrates
with substituents on the aromatic ring [(Z)-21a and (Z)-
22a], in contrast to unsubstituted derivatives [(Z)-18a–
20a].[22] Overall, DMF was a poor cosolvent.
ACHTUNGTRENNUNGselectivity, pointing at binding issues in the active site and
imperfect substrate recognition.[49,59] Thus, electronic effects
influencing C=C bond polarization alone cannot account for
observed trends in substrate reactivity—steric effects obvi-
ously play an important role.
We envisaged to rationalize the “supported-substrate acti-
vation” concept by using conceptual DFT[61] to estimate the
reactivity of the investigated compounds towards nucleo-
philic addition at b-C in a,b-unsaturated carboxylic esters.
The global electrophilicity index (w) was used to provide
a reactivity scale for a series of different molecules. Overall,
no strong relationship with enzyme activity was found,
though clusters of enzyme–substrate combinations could be
identified, in which electrophilicity reflected substrate reac-
tivity (Figures S1 and S2 in the Supporting Information).
Because all substrates tested possess low solubility in
aqueous media, we tested various organic cosolvents (10%
v/v) to enhance their solubility thereby overcoming mass-
transfer limitations. In addition, organic cosolvents are
known to modulate the catalytic properties of enzymes.[62]
We selected OYE3 for the cosolvent study with substrates
showing particularly low-to-moderate conversions [(Z)-18a–
22a] (Figure 3). Although stereoselectivity of OYE3 was un-
changed, significant increase in reactivity was observed, es-
pecially in biphasic systems (Figure 3 and Table S3 in the
Supporting Information), which supports the beneficial
effect of water-immiscible solvents on the performance of
OYEs as well as enzyme robustness.[63] In general, tert-butyl
methyl ether (TBME), di-isopropyl ether (DIPE), and n-
hexane afforded superior conversion: notable improvements
were observed in the case of (Z)-18a and (Z)-20a with n-
hexane (from 36 to 84%, from 43 to 67%, respectively),
only a slight enhancement was obtained with (Z)-19a (from
79 to 84%). Compound (Z)-21a also showed enhanced re-
action rate with most cosolvents including EtOAc, although
Conclusion
Investigation of the de/activating effects of a- and b-sub-
stituents on the asymmetric bioreduction of acrylic and cin-
namic esters by using ene-reductases was investigated and
condensed in the following trends: 1) the tuning of the reac-
tivity is possible to some extent by influencing the polariza-
tion of the C=C bond through electronic substituent effects;
2) the presence of an a-halogen atom strongly improved re-
action rates, whereas 3) a-cyano groups were largely ineffec-
tive; 4) an electron-donating group on b-C tended to deacti-
vate nonhalogenated derivatives. However, electronic effects
could not be disentangled from steric effects. Though struc-
turally highly related, ene-reductases yield specific binding
modes with each substrate, thus preventing a general state-
ment on specific interactions involved with a given com-
pound. Overall, a-halo-substituted acrylic and cinnamic
esters were reduced with high-to-excellent conversion and
stereoselectivity, occasionally even in a stereocomplementary
fashion through enzyme- or substrate-based stereocontrol.
Additionally, organic cosolvents had a strong enhancing
effect on enzyme activity.
Experimental Section
Source of enzymes, chemicals and ma-
terials, chiral and nonchiral analytics,
synthesis of substrates, and reference
compounds, determination of absolute
configurations, additional data on or-
ganic co-solvents, and DFT calcula-
tions are available in the Supporting
Information.
General procedure for the bioreduc-
tion: An aliquot of enzyme (OYE 1–3,
YqjM, NCR, OPR1, OPR3; 6.6–31 mL
of the stock solution, final protein con-
centration 75–125 mgmLÀ1), was added
to a Tris-HCl buffer solution (0.8 mL,
50 mm, pH 7.5) containing the sub-
strate (10 mm) and the cofactor
NADH (15 mm). The mixture was
shaken at 308C and 120 rpm. After
24 h, the products were extracted with
EtOAc (2ꢃ0.5 mL). The combined or-
ganic phases were dried over Na2SO4
and analyzed on achiral GC to deter-
Figure 3. Effect of cosolvents (10% v/v) on the bioreduction of a,b-unsaturated carboxylic esters with OYE3;
water-miscible solvents (blue); water-immiscible solvents (red).
Chem. Eur. J. 2012, 00, 0 – 0
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K. Faber et al.
mine the conversion and on chiral GC or HPLC, respectively, to deter-
mine the ee. For cofactor recycling, the oxidized form of the cofactor
(NAD+, 100 mm), the cosubstrate (glucose 20 mm), and the recycling
enzyme (glucose dehydrogenase, 10 U) were used.
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Published online: && &&, 0000
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Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Reactivities of a,b-Unsaturated Carboxylic Esters
FULL PAPER
Biocatalysis
G. Tasnꢀdi, C. K. Winkler, D. Clay,
N. Sultana, W. M. F. Fabian, M. Hall,
K. Ditrich, K. Faber* ........ &&&& — &&&&
Biotransformation: The asymmetric
bioreduction of a,b-unsaturated car-
boxylic esters by ene-reductases could
be tuned by varying the degree of C=C
bond activation (see scheme). An addi-
tional a-halogenated substituent
proved to be beneficial for enzymatic
activity, whereas b-alkyl or b-aryl sub-
stituents were detrimental for the reac-
tivity of nonhalogenated substrates.
A Substrate-Driven Approach to
Determine Reactivities of a,b-Unsatu-
rated Carboxylic Esters Towards
Asymmetric Bioreduction
Chem. Eur. J. 2012, 00, 0 – 0
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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&
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These are not the final page numbers!