Journal of Biotechnology 162 (2012) 381–389

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Asymmetric bioreduction of activated alkenes to industrially relevant optically

active compounds
Christoph K. Winkler b , Gábor Tasnádi a,b , Dorina Clay b , Mélanie Hall b , Kurt Faber b,∗
a
ACIB Co. c/o, Heinrichstrasse 28, 8010 Graz, Austria
b
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Ene-reductases from the ‘Old Yellow Enzyme’ family of flavoproteins catalyze the asymmetric reduction
Received 31 October 2011 of various ␣,␤-unsaturated compounds at the expense of a nicotinamide cofactor. They have been applied
Received in revised form 23 March 2012 to the synthesis of valuable enantiopure products, including chiral building blocks with broad indus-
Accepted 28 March 2012
trial applications, terpenoids, amino acid derivatives and fragrances. The combination of these highly
Available online 4 April 2012
stereoselective biocatalysts with a cofactor recycling system has allowed the development of cost-
effective methods for the generation of optically active molecules, which is strengthened by the
Keywords:
availability of stereo-complementary enzyme homologues.
Asymmetric reduction
Biocatalysis © 2012 Elsevier B.V. All rights reserved.
Ene-reductase
Old Yellow Enzyme
Chiral building blocks

1. Introduction stereoselective reduction of a broad variety of ␣,␤-unsaturated

compounds, affording excellent yields and enantiomeric excess,
The increasing demand for small enantiopure molecules as while working under mild conditions of pH and temperature. A
chiral building blocks for the synthesis of biologically active com- whole set of homologous enzymes has been developed and several
pounds (most notably active pharmaceutical ingredients – API) industrially relevant molecules could be obtained in nonracemic
has contributed to the development of highly specific synthetic form. This review focuses on this new enzyme platform, present-
strategies. The reduction of alkenes, for instance, is a powerful ing pertinent examples while stressing on general rules that should
tool in modern asymmetric synthesis and various approaches are help chemists incorporate ene-reductases in the design of asym-
now available on industrial scale. Transition-metal based homoge- metric synthetic routes.
neous catalysis has reached high standards (Knowles, 2002; Noyori,
2002), and related fields of catalysis are now becoming competitive 2. System
in this area. Metal-free organocatalysis uses general acid-type cata-
lysts to perform stereoselective transfer hydrogenation, but suffers 2.1. Reaction mechanism
from low atom economy due to the requirement for molar amounts
of the ‘Hantzsch ester’ used as reductant (List and Yang, 2006; The mechanism of the ene-reductase-catalyzed reduction of
Yang et al., 2005). Nature, on the other hand, provides an attractive ␣,␤-unsaturated compounds has been studied in great detail (Kohli
sustainable and cost-effective alternative. The biocatalytic analog and Massey, 1998). The reaction was shown to proceed via the
relies on ene-reductases to perform the reduction of activated C C stereoselective transfer of a hydride (derived from the reduced
bonds. These enzymes belong to the ‘Old Yellow Enzyme’ family flavin-cofactor) onto C␤, while a Tyr-residue adds a proton (ulti-
of nicotinamide-dependent flavoproteins and have been intensely mately derived from the solvent) onto C␣ from the opposite side
investigated over the past five years in view of their applica- (Fig. 1). The overall addition of [2H] onto a C C bond resem-
bility in preparative-scale biotransformations (Hall et al., 2010; bles a Michael-type addition of a complex hydride and results
Stuermer et al., 2007; Toogood et al., 2010). They catalyze the highly with exclusive relative trans-stereospecificity.1 Reduction of the

∗ Corresponding author. Tel.: +43 316 380 5332; fax: +43 316 380 9840. 1
Rare cases for cis-addition were observed with plant cell cultures and flavin-
E-mail address: [email protected] (K. Faber). independent reductases: Shimoda, K., Ito, D.I., Izumi, S., Hirata, T., 1996. Novel

0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.03.023
382 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389

[H -]
R2 R1 H
R2 R1
R3 EWG R33 H EWG
[H +] ene-
FlavinH2 Flavin
reductase
[H-]:hydride from flavin
[H+]: proton from Tyr-residue
or solvent

NAD(P)+ NAD(P)H

HCO 2H FDH CO2

Glucose GDH Gluconic acid
Glucose-6-phosphate G6PDH 6-Phosphogluconate
2-Propanol ADH Acetone
Phosphite PTDH
Phosphate
cofactor recycling system

EWG = electron withdrawing group

aldehyde, ketone, nitrile,carboxylic acid
ester,lactone, cyclic imide,nitro
Fig. 1. Asymmetric bioreduction of activated alkenes using ene-reductases.

oxidized flavin cofactor at the expense of NAD(P)H closes the cat- nitroalkanes thus formed are stable, ␣-analogs are somewhat
alytic cycle (Fig. 1). Ene-reductases often show relaxed specificities labile due to the acidity of the ␣-H.
for NADH or NADPH as cofactor, which allows to choose the recy- (iv) Depending on their degree of activation, ␣,␤-unsaturated
cling system on a case-to-case basis. The enzymes have been shown carboxylic acids or esters behave as ‘borderline’-substrates:
to tolerate organic co-solvents very well, especially water immis- whereas simple ␣,␤-unsaturated mono-carboxylic acids
cible ones, in up to 50%, v:v (Stueckler et al., 2010a; Yanto et al., or -esters are not easily reduced by ene-reductases, they
2011). are good substrates for ‘enoate-reductases’ from anaerobic
organisms, which possess an additional (oxygen-sensitive)
2.2. Substrates ferredoxin Fe4 S4 -cofactor (Ferraboschi et al., 1987; Tischer
et al., 1979). However, mono-acids or -esters can be activated
Only C C-bonds that are electronically activated by a by an additional electron-withdrawing group, such as a second
conjugated electron-withdrawing group (EWG) are reduced, acid- or ester-group, a halogen or a nitrile (Brenna et al., 2011c;
non-activated (isolated) alkenes are unreactive. The following func- Kitazume and Ishikawa, 1984). Consequently, di-carboxylic
tional groups may serve as ‘activators’: acids and -esters are well accepted. Cyclic imides, bearing two
activating carbonyl groups next to the C C bond are good
(i) ␣,␤-Unsaturated carboxaldehydes (enals) are good substrates substrates in general.
and yield the expected saturated aldehydes as products when (v) ␣,␤-Unsaturated nitriles are only slightly activated and also
pure ene-reductases are used (Stueckler et al., 2010a). How- count as ‘borderline’-substrates, although complex nitrile-
ever, in whole-cell biotransformations (using e.g. baker’s containing molecules have been successfully reduced (Kosjek
yeast), carbonyl reduction is a dominant side reaction form- et al., 2008).
ing saturated prim-alcohols via over-reduction of the product
or allylic alcohols by depleting the substrate (Hall et al., 2006;
2.3. Enzymes
Mueller et al., 2006).
(ii) ␣,␤-Enones are usually well accepted, competing carbonyl
Enzymes from the ‘Old Yellow Enzyme’ family are widely dis-
reduction is less dominant as with enals.
tributed in microorganisms and in plants. Some of them occur in
(iii) Conjugated nitroalkenes are highly activated and are thus
well-defined pathways, e.g. in the biosynthesis of jasmonic acid
readily reduced. Whereas chiral centers in the ␤-position of the
or the metabolism of morphine (Barna et al., 2002; Schaller et al.,
2000), others are involved in the detoxification of xenobiotics
(Williams et al., 2004), such as trinitrotoluene (TNT, Barna et al.,
reductase participation in the syn-addition of hydrogen to the C C bond of enones 2001). Over recent years, a great variety of new homologues has
in the cultured cells of Nicotiana tabacum. J. Chem. Soc., Perkin Trans. 1. 355–358; been identified and their potential as biocatalysts was investigated.
Bougioukou, D.J., Stewart, J.D., 2008. Opposite stereochemical courses for enzyme-
mediated alkene reductions of an enantiomeric substrate pair. J. Am. Chem. Soc. 130,
Table 1 gives a summary of ene-reductases used in isolated form in
7655–7658. asymmetric bioreduction reactions.
 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389 383

Table 1
Ene-reductases from the ‘Old Yellow Enzyme’ family.

Enzyme Organism

Fungi
Old yellow enzyme 1 (OYE1) Saccharomyces pastorianus (formerly S. carlsbergensis) (Saito et al., 1991)
Old yellow enzyme 2 and 3 (OYE2 and 3) Saccharomyces cerevisiae (Karplus et al., 1995)
Old yellow enzyme (OYE) Candida macedoniensis AKU4588 (Kataoka et al., 2002, 2004)
Estrogen binding protein (EBP1) Candida albicans (Buckman and Miller, 1998)
Kluyveromyces lactis yellow enzyme 1 (KYE1) Kluyveromyces lactis (Chaparro-Riggers et al., 2007)
Old yellow enzyme 2.6 (OYE 2.6) Pichia stipitis CBS 6054 (Padhi et al., 2009)
Bacteria
YqjM Bacillus subtilis (Fitzpatrick et al., 2003)
NAD(P)H-dependent 2-cyclohexen-1-one reductase (NCR) Zymomonas mobilis (Mueller et al., 2007)
Xenobiotic reductase A (XenA) Pseudomonas putida II-B (Blehert et al., 1999)
Xenobiotic reductase B (XenB) Pseudomonas fluorescens I-C (Blehert et al., 1999)
Pentaerythritol tetranitrate reductase (PETNr) Enterobacter cloacae PB2 (French et al., 1996)
TOYE Thermoanaerobacter pseudoethanolicus E 39 (Adalbjornsson et al., 2010)
SYE1-4 Shewanella oneidensis (Brige et al., 2006)
GkOYE Geobacillus kaustophilus DSM 7263 (Schittmayer et al., 2010)
Chromate reductase (CrS) Thermus scotoductus SA-01 (Opperman et al., 2008, 2010)
Morphinone reductase (MR) Pseudomonas putida M10 (French and Bruce, 1994)
YersER Yersinia bercovieri (Chaparro-Riggers et al., 2007)
Gluconobacter oxidans ene-reductase Gluconobacter oxidans DSM 2343 (Richter et al., 2011)
N-ethylmaleimide reductase (NemR) Escherichia coli (Miura et al., 1997)
Glycerol trinitrate reductase (NerA) Agrobacterium radiobacter (Snape et al., 1997)
Plants
12-Oxophytodienoate reductase 1–3 (OPR1-3) Arabidopsis thaliana (Biesgen and Weiler, 1999; Costa et al., 2000; Schaller and Weiler, 1997)
12-Oxophytodienoate reductase 1–3 (LeOPR1-3) Solanum lycopersicum (formerly Lycopersicon esculentum) (Strassner et al., 1999, 2002)
Commercially available
ERED101-114 Source not available (Kosjek et al., 2008)

2.4. Cofactor regeneration 6-phosphogluconate respectively, both unstable compounds that

hydrolyse spontaneously. This renders all four systems practically
A major limitation to the broad application of nicotinamide- irreversible and thereby shifts the equilibrium towards reduction.
dependent enzymes for synthetic purpose lies in the prohibitive Phosphite dehydrogenase (PTDH) (Vrtis et al., 2002; Woodyer et al.,
cost of these natural cofactors, especially the reduced form 2003) has also been applied to the reduction of ␣,␤-unsaturated
(∼500 D /g NADH and 1400 D /g NADPH, from chemical suppli- nitriles, which provides in situ pH-control as phosphate is being
ers). Fortunately, advances in cofactor regeneration techniques produced throughout the reaction (Kosjek et al., 2008).
now allow the use of catalytic amounts of NAD(P)H and various While all these systems represent coupled-enzyme approaches
systems have been exploited with ene-reductases for in situ recy- requiring two proteins, the first example of a coupled-substrate
cling (Fig. 1) (Faber, 2011; Hall and Bommarius, 2011), while often single-enzyme approach applied to ene-reductases was recently
serving as driving force to overcome thermodynamic equilibrium published, where advantage was taken of the disproportionation
limitations (Park et al., 2011). of enones catalyzed by a single ene-reductase. With the enone
A common strategy for the regeneration of NADH is the formate substrate being reduced to the corresponding saturated ketone, a
dehydrogenase (FDH)-catalyzed oxidation of formate to CO2 . FDH sacrifial enone co-substrate served as artificial H-donor and was
has been successfully employed with ene-reductases, although oxidized, thereby rendering the reduced flavin for a subsequent
substrate and product depletions were observed with an enal (cit- catalytic cycle. 2-Enones and 1,4-diones were particularly good co-
ronellal), resulting from carbonyl reduction caused by prim-ADH substrates as their oxidized forms spontaneously tautomerized to
impurities in the commercial FDH preparation; likewise, racemi- phenol and hydroquinone derivatives, respectively, thus driving
sation of ␣-substituted cycloalkanones was observed (Hall et al., the reaction to the desired product side (Stueckler et al., 2010b).
2008a). Recently, alcohol dehydrogenase ADH-‘A’ was successfully Nonconventional regeneration methods are also being devel-
combined with several ene-reductases, using only 2 equivalents of oped (Hollmann et al., 2010). A light-driven system was designed
2-propanol as H-donor, thereby producing acetone. As above, alde- with YqjM, where irradiation with white light in the presence
hydes are not suitable substrates due to over-reduction of the CH O of external free flavin and EDTA allowed the full conversion of
moiety (Tauber et al., 2011). Glucose-6-phosphate dehydrogenase 4-ketoisophorone to levodione. The system, however, was plagued
(G6PDH) and glucose dehydrogenase (GDH) are commonly used by the non-stereoselective background reaction catalyzed by free
as cofactor recycling systems for ene-reductases (Hall et al., 2007, flavin, leading to reduced product enantiopurity (Taglieber et al.,
2008b). While G6PDH only accepts NADP+ , GDH can be employed 2008).
with both nicotinamide cofactors.
Occasionally, the nature of the substrate had a dramatic influ- 3. Applications
ence on the efficiency of the recycling system. For instance, FDH,
GDH and G6PDH were inactive in presence of a cis-configurated 3.1. Synthesis of amino acid derivatives
␣,␤-unsaturated dicarboxylic acid (citraconic acid), which acts as
strong chelator for divalent metal ions. The addition of metals (e.g. Natural and non-natural amino acids are valuable building
Ca2+ , Mg2+ or Zn2+ ) to the reaction medium proved necessary to blocks and key intermediates for a number of pharmaceuticals,
overcome deactivation of the recycling enzymes and to make this heterocycles or modified peptides (Goodman et al., 2007; Hughes
substrate amenable to bioreduction (Stueckler et al., 2007). and Moody, 2007; Trabocchi et al., 2005). Ene-reductases have
While FDH produces CO2 and ADH forms highly volatile ace- been successfully used for the synthesis of various ␣- and ␤-amino
tone, GDH and G6PDH furnish gluconolactone/gluconic acid and acid analogs. In a process developed by Swiderska and Stewart
384 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389

R H H outcome by OYE3 was due to a flipped “bottom/top” orientation

1. OYE1, NADPH
1 Me R R of the substrate, resulting in an exchange of the activating ester as
2 O2N 2. H2, Raney-Ni HCl .H2N
Et R docking group in the active site. This switch of the activating group
3 n-Pr COOEt 3. HCl, Δ COOH opens new perspectives for the asymmetric synthesis of ␤-amino
4 i-Pr 1a - 4a 1b - 4b acids.

Fig. 2. Route to ␤2 -amino acids.

3.2. Terpenoids

R1 R1 Terpenoids are one of the largest classes of natural products

offering a great variety of biologically active compounds and chi-
OYE1-3, YqjM
* ral intermediates. Enantiomers of dihydrocarvone (12b) are minor
MeO 2C NH NADH MeO 2C NH components of essential oils produced by plants and have been used
as chiral starting compounds in the synthesis of natural products
O R2 O R2 (e.g. striatenic acid, pechueloic acid) (Aubin et al., 2006; Blay et al.,
5a - 11a (R)- or (S)-5b - 11b 2007; Harrowven et al., 2005), antimalarial drugs (Dong et al., 2010)
R1 R2 and valuable chiral synthons (de Rouville et al., 2009; Krawczyk
et al., 2007). In the course of exploring the substrate specificity of
5 H Me
PETN reductase (Fryszkowska et al., 2009), (5R)- and (5S)-carvone
6 CO2Me H (12a) were quantitatively reduced into the diastereomeric products
7 CO2Me Me (12b) with the same absolute (R)-configuration on the newly gener-
8 CO2Me Et ated centre at C2 in 95% and 88% diastereomeric excess, respectively
9 CO2Me Pr (Fig. 4).
Both enantiomers of citronellal (13b), a key intermediate in
10 CO2Me Ph
menthol synthesis, have been prepared with excellent ee values
11 CO2Me Bn (> 95%) starting from (E/Z)-citral (13b) using various OYEs (Fig. 5)
(Bougioukou et al., 2010; Fryszkowska et al., 2009; Hall et al., 2007,
Fig. 3. Reduction of ␣,␤-dehydroamino acid derivatives by ene-reductases.
2008a,b; Mueller et al., 2010). While (S)-citronellal [(S)-13b] could
be produced quantitatively, (R)-citronellal [(R)-13b] was obtained
(2006), ␤-nitroacrylates 1a–4a were stereoselectively reduced by with 69% conversion. It was observed that the (E/Z)-configuration
OYE1 to the corresponding ␤-nitro carboxylic acid esters as the of citral played a crucial role in the stereoselectivity of OYEs 1–3
key step in the asymmetric synthesis of optically active ␤2 -amino (Mueller et al., 2007). Whereas whole cells generally led to over-
acids (Fig. 2). Since the bioreduction of the C C bond proceeded reduction of the product to the corresponding saturated alcohol
chemoselectively, the nitro group was subsequently reduced with (Hall et al., 2006; Mueller et al., 2006), isolated OYE-enzymes fur-
Raney-Ni. Ethyl ␣-alkyl-␤-nitroacrylates were reduced with high nished the aldehyde 13b as single product.
stereoselectivity (ee ≥ 87%) and ␤2 -amino acids were isolated as
their hydrochloride salts (1b–4b) in good overall yield (57–73%). 3.3. Fragrance compounds
␤-Alkyl-␤-nitroacrylates (␤3 -amino acid precursors) on the other
hand were reduced with low stereoselectivities, most likely due to ␣-Methyl dihydrocinnamaldehyde derivatives (14b and 15b)
the ␣-protonation occurring after product release from the active are of commercial importance (Brenna et al., 2003), with 14b
site. being the olfactory principle of the lily-of-the-valley odor (Enders
Recently, ␣,␤-dehydroamino acid derivatives have been iden- and Dyker, 1990), marketed under the trade name LilialTM or
tified as novel substrates for members of the OYE family LysmeralTM , while 15b, marketed as HelionalTM or TropionalTM ,
(Fig. 3) (Stueckler et al., 2011). While an ␣-amino acid is the active ingredient of various perfumes (Enders and Backes,
precursor having an additional methyl group at C␤ (methyl 3- 2004). A convenient enzymatic strategy for the synthesis of 14b
methyl-2-acetamidoacrylate) and an ␣-alanine precursor (methyl and 15b was developed (Stueckler et al., 2010a) via bioreduc-
3-acetamidoacrylate) were unreactive, N-acyl derivative of alanine tion of ␣-methyl cinnamaldehydes (14a and 15a) with OYEs. The
(5a) and aspartic acid ester (8a) were reduced by YqjM to the (S)-antipodes were produced with OYE1-3 in an aqueous-organic
corresponding (S)-enantiomers (5b, 41% conv., 97% ee; 8b, up to biphasic system (containing 20% t-BuOMe) in >95% ee and quanti-
quantitative yield and 99% ee). A switch of stereopreference in the tative yield (Fig. 6).
reduction of aspartic acid derivatives 6a–11a could be induced with
OYE3 via substrate engineering by variation of the size of the N- 3.4. Chiral building blocks
acyl protective group. While 6a, 7a and 10a were reduced to the
(S)-amino acid derivatives (23% to >99% ee), the (R)-enantiomers (6R)-Levodione (16b), obtained by asymmetric bioreduction of
were obtained from 8a, 9a and 11a (61% up to 92% ee). 2 H-labelling 4-ketoisophorone (16a), represents an important industrial inter-
experiments in D2 O revealed that the opposite stereochemical mediate for carotenoide synthesis (e.g. zeaxanthin, cryptoxanthin,

H
H
O O
PETNR O
COOH
NADPH

HOOC
(R)- or (S)-12a (2R,5R)- or (2R,5S)-12b (+)-striatenic acid pechueloic acid

Fig. 4. Total asymmetric synthesis of striatenic and pechueloic acid via ene-reductase catalyzed reduction of carvone 12a producing the key intermediate 12b.
 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389 385

CHO ene-reductase CHO

NAD(P)H *
(E/Z)-13a (R)- or (S)-13b OH
citral citronellal

enzyme config. e.e. (-)-menthol

OYE 2.6 (R) 98
OPR1 (S) >95
OPR3 (S) >95
NCR (S) >95
YqjM (S) >95
NemA (S) 66

Fig. 5. Bioreduction of citral (13a) to citronellal (13b).

R2 CHO OYE1-3 R2 S CHO epoxides, aminoalcohols, hydroxylamines, and haloketones (Fig. 8).
In addition to the classical asymmetric synthesis involving N-
NADH, 20% t-BuOMe
R1 R1 sulfonyloxaziridines (Davis and Chen, 1992; Davis et al., 1986;
Hughes et al., 2005), and several biocatalytic systems (Adam et al.,
14a, 15a 14b: LilialTM, 15b: HelionalTM
1999; Demir et al., 2007; Patel, 2006), an additional biocatalytic
R1 R2
alternative was recently provided through the asymmetric reduc-
14 t-Bu H
tion of ␣,␤-unsaturated alkoxy ketones (Winkler et al., 2010).
15 O-CH 2 -O
Stereocomplementary routes to O-protected acyloins were devel-
Fig. 6. Fragrance production with ene-reductases.
oped via substrate engineering through variation of the size of the
O-protecting group. Both enantiomers of ␣-alkoxy enones could be
obtained in up to >99% ee, while ␤-analogs were not converted. The
xanthoxin) (Demole and Enggist, 1974). So far, all OYE family O-protected acyloins thus obtained can be used in further synthetic
members have yielded strictly the (R)-enantiomer (up to >99% ee) steps; particularly allyl- or benzyl-moieties can be easily removed
(Toogood et al., 2010). A one-pot two-step enzymatic cascade was under mild conditions.
developed leading to (4R,6R)-actinol (16c). The first ene-reduction Enantiopure lactones are valuable synthetic precursors. For
was catalyzed by OYE2, expressed in E. coli and used as cell extract, instance, ␥-butyrolactone (22c) has been utilized as building block
to furnish (6R)-levodione (16b) as intermediate. The latter was in the synthesis of natural products such as milbemycin ␤3 , jas-
subsequently reduced at the carbonyl group to actinol (16c) with plakinolide and amphidinolides (Fig. 9) (Korpak and Pietruszka,
levodione reductase from Corynebacterium aquaticum M-13, also 2011). Two of its four possible stereoisomers were recently
expressed in E. coli. Glucose dehydrogenase was used for the regen- obtained via an enzymatic two-step one-pot cascade. In the first
eration of NADH, which allowed the quantitative formation of step, OYE1 was employed for the generation of the first stereocen-
(4R,6R)-actinol (16c) in 94% ee (Fig. 7) (Wada et al., 2003). ter, where reduction of the two (E/Z)-isomers of starting material
Due to its broad acceptance as a substrate by a large number of 22a was stereoconvergent and yielded the (R)-enantiomer 22b. In
OYE homologues, 4-ketoisophorone (16a) emerged as a standard the second step, various alcohol dehydrogenases (ADH) were used
test-substance for the characterization of ene-reductases [(OPR1 for carbonyl reduction leading to the ␥-hydroxy ester, followed by
and OPR3 (Hall et al., 2007, 2008a), YqjM (Hall et al., 2008a), OYE1- spontaneous lactonization to 22c (Korpak and Pietruszka, 2011).
3 and NCR (Hall et al., 2008b), PETNr (Fryszkowska et al., 2009; The carbonyl reduction proceeded with enzyme-based stereocon-
Mueller et al., 2010), NemR, MR and EBP1 (Mueller et al., 2010), Glu- trol, where proper choice of the catalyst allowed both (2R,4S)-22c
conobacter oxidans ER (Richter et al., 2011), XenA (Chaparro-Riggers (with Prelog-type ADH-T from Thermoanaerobacter species) and
et al., 2007; Yanto et al., 2010), TOYE (Adalbjornsson et al., 2010), (2R,4R)-22c (with anti-Prelog-type ADH-LK from Lactobacillus kefir)
CrS (Opperman et al., 2010), YersER and KYE1 (Chaparro-Riggers in good yields (up to 80%) and perfect stereoselectivity (>99% ee).
et al., 2007), OYE from Candida macedoniensis (Kataoka et al., 2004)], (R)-3-Hydroxy-2-methylpropanoate (23b), commonly denoted
the screening for novel ene-reductase activity in organisms (Goretti as ‘Roche-Ester’, is a popular chiral building block for the syn-
et al., 2011; Raimondi et al., 2010) and the development of novel thesis of vitamins (vitamin E), fragrance compounds (muscone),
cofactor regeneration systems (Taglieber et al., 2008, 2010; Tauber antibiotics (rapamycin) and natural products (Stueckler et al.,
et al., 2011). 2010c). Prominent routes for its preparation include enzymatic
Chiral acyloins (17b–21b) are important building blocks in oxidation of prochiral diols (Molinari et al., 2003) or the tran-
asymmetric synthesis (Adam et al., 1999; Demir et al., 2007; sition metal-catalyzed asymmetric hydrogenation of acrylate
Patel, 2006). They can be converted into nonracemic diols, esters using Rh- (Holz et al., 2008; Qiu et al., 2009; Wassenaar

O O O
levodione Carotenoids:
ene-reductase reductase
Zeaxanthin
GDH/glucose/NAD+ GDH/glucose/NAD+ Cryptoxanthin
Xanthoxin
O O OH
16a (R)-16b (4R,6R)-16c
4-ketoisophorone levodione actinol

Fig. 7. Ene-reductase catalyzed reduction of 4-ketoisophorone (16a) to (R)-levodione (16b) and (4R,6R)-actinol (16c).
386 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389

O O nonracemic
n( ) ene-reductase n( ) diols, epoxides,
NADH aminoalcohols,
OR OR haloketones

17a-21a (S)-17b-20b or (R)-21b

n R
17 1 Me
18 2 Allyl
19 2 n-propyl
20 2 Benzyl
21 2 Me

Fig. 8. Production of chiral acyloins via ene-reductases using OYE1-3, YqjM, NerA, OPR1, OPR3, XenA, XenB, EBP1 and NCR.

Fig. 9. A two-step one-pot cascade leading to ␥-butyrolactones (22c).

et al., 2008) or Ru-catalysts (Pautigny et al., 2008). A biocat- up to 20%), while electron-withdrawing groups increased con-
alytic equivalent was shown using ene-reductases. The reaction version levels (28a–30a, conversion 58–91%) in comparison with
proceeded via strict (R)-stereoselective reduction of methyl 2- the non-substituted derivatives (27a and 32a, conversion 37–38%).
hydroxymethylacrylate derivatives (>99% ee in almost all cases; The latter can be explained by the varying degree of polarization
Fig. 10), with ene-reductases showing overall broad acceptance of the C C bond. Both chloro- and bromo-substituents at the ␣-
for this type of compounds (Stueckler et al., 2010c). Substrate position were accepted by the enzyme. (S)-␣-Chlorocinnamates
engineering via hydroxyl-group protection (allyl-, benzyl- or 28b and 30b were recovered from baker’s yeast fermentation
TBDMS-ethers) enhanced the reaction rate significantly (up to and subsequently transformed into non-natural d-phenylalanine
>99% conversion) and hence allowed direct access to protected derivatives (28c and 30c), thus offering a new route to enan-
(R)-‘Roche-Ester’ (23b), a convenient intermediate for further syn- tiomerically pure non-natural ␣-amino acid derivatives. A library of
thesis. ␣,␤-unsaturated ␣-halo esters bearing various alkyl chains was also
Chiral ␣-halogenated carboxylic acids and esters are useful tested (34–37a, Fig. 11) (Brenna et al., 2011c). Most interestingly,
synthons since they can be transformed into a broad range of in contrast to the opposite stereopreference observed in baker’s
derivatives by stereospecific nucleophilic substitution reactions yeast-mediated reduction of (E/Z)-isomers of ␣,␤-unsaturated ␣-
with nitrogen (Righi et al., 2006), oxygen (Hesek et al., 2009; chloroesters [(Utaka et al., 1989), also confirmed with isolated
Yang et al., 2001) and sulfur (Narendra et al., 2010; Seki et al., OYE1-3 acting on methyl 2-chloro-4-methylpent-2-enoate (Brenna
2000) nucleophiles. Enantiopure ␣-haloesters in particular are et al., 2011a)], both (E/Z)-isomers of the ␣-bromo-analogs were
valuable chiral synthons for the synthesis of several therapeu- converted to the (S)-product (ee up to 97%).
tic agents used for the treatment of non-insulin dependent type Enantiopure nitriles are versatile chiral building blocks due
2 diabetes mellitus (T2DM) (Brenna et al., 2011c). Brenna and to their chemical reactivity, allowing further transformation into
co-workers investigated the bioreduction of various methyl ␣- numerous functional groups (e.g. carboxylic acids, amines or
halo-␤-substituted acrylates using isolated OYE1-3 and baker’s aldehydes). For instance, nitrile 42b contains a spiropiperidine
yeast (Fig. 11) (Brenna et al., 2011b). OYE3 furnished the cor- backbone and is relevant for pharmaceutical research (Fig. 12) (Jia
responding (S)-products in good to excellent stereoselectivity et al., 2007; Limanto et al., 2008; Lu et al., 2007). In a study with
(ee ≥ 88%). The conversion strongly depended on the substitution commercially available ene-reductases, the C C bond of a series
pattern of the aromatic ring. In general, electron-donating groups of ␣,␤-unsaturated nitriles were reduced in high yields and stere-
on the ring lowered the reaction rate (31a and 33a, conversion oselectivities (up to 99% ee, Fig. 12) (Kosjek et al., 2008). While all

R O O
ene-reductase Vitamin E
23 H Rapamycin
24 allyl RO OMe NADH RO OMe
Muscone
25 benzyl
26 TBDMS 23a-26a (R)-23b-26b

Fig. 10. Roche ester production via ene-reductases using OYE1-3, YqjM, NCR, NerA, OPR1, OPR3 and XenA.
 C.K. Winkler et al. / Journal of Biotechnology 162 (2012) 381–389 387

Fig. 11. Ene-reductase-catalyzed reduction of ␣-halo-esters and further transformation into chiral products.

CN CN
R R
ERED101-114 ERED101-114
CN CN
NAD(P)H NAD(P)H Cl
Cl
R
38a-41a (R)-38b-41b 42a N 42b N
38 H H H
39 CH3
40 OCH3
41 Cl

Fig. 12. Ene-reductase-catalyzed production of enantiopure nitriles.

enzymes showed (R)-selectivity for substrates 38a–41a, the abso- Acknowledgements

lute configuration of 42b was not assigned.
This study was performed in cooperation within the Austrian
Centre of Industrial Biotechnology (ACIB, funded through the FFG-
4. Concluding remarks
COMET-Program) and project P22722 of the FWF and support by
the FWF, BMWFJ, BMVIT, SFG, Standortagentur Tirol, ZIT and BASF
Ene-reductases from the ‘Old Yellow Enzyme’ family of flavo-
SE (Ludwigshafen) is gratefully acknowledged.
proteins have attracted increasing interest from synthetic chemists
over the last years due to their exquisite chemo-, regio-, and
stereoselectivities. Their use in the reduction of various ␣,␤-
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