The substrate spectra of pentaerythritol tetranitrate reductase, morphinone reductase, N-ethylmaleimide reductase and estrogen-binding protein in the asymmetric bioreduction of activated alkenes

Article abstract of DOI:10.1002/adsc.200900832

Four flavoproteins from the old yellow enzyme (OYE) family, pentaerythritol tetranitrate (PETNR) reductase, N-ethylmaleimide reductase (NEMR), morphinone reductase (MorR) and estrogen-binding protein (EBP1), exhibited a broad substrate tolerance by accepting conjugated enals, enones, imides, dicarboxylic acids and esters, as well as a nitroalkene and therefore can be employed for the asymmetric bioreduction of carbon-carbon double (C=C) bonds. In particular, morphinone reductase and estrogen-binding protein often showed a complementary stereochemical preference in comparison to that of previously investigated OYES.

Full text of DOI:10.1002/adsc.200900832

FULL PAPERS
DOI: 10.1002/adsc.200900832
The Substrate Spectra of Pentaerythritol Tetranitrate Reductase,
Morphinone Reductase, N-Ethylmaleimide Reductase and
Estrogen-Binding Protein in the Asymmetric Bioreduction of
Activated Alkenes
Nicole J. Mueller,a Clemens Stueckler,a Bernhard Hauer,b Nina Baudendistel,b
Hazel Housden,c Neil C. Bruce,c and Kurt Fabera,*
a
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Fax : (+43)-316-380-9840; e-mail: kurt.faber@uni-graz.at
BASF AG, GVF/E-B9,d-67056 Ludwigshafen, Germany
Department of Biology, University of York, PO Box 373, York, U.K.
b
c
Received: November 30, 2009; Revised: January 13, 2010; Published online: February 9, 2010
Abstract: Four flavoproteins from the old yellow double (C=C) bonds. In particular, morphinone re-
enzyme (OYE) family, pentaerythritol tetranitrate ductase and estrogen-binding protein often showed a
(PETNR) reductase, N-ethylmaleimide reductase complementary stereochemical preference in com-
(NEMR), morphinone reductase (MorR) and estro- parison to that of previously investigated OYEs.
gen-binding protein (EBP1), exhibited a broad sub-
strate tolerance by accepting conjugated enals,
enones, imides, dicarboxylic acids and esters, as well Keywords: alkene bioreduction; biotransformations;
as a nitroalkene and therefore can be employed for enoate reductase; estrogen-binding protein; morphi-
the asymmetric bioreduction of carbon-carbon none reductase; NEM reductase; PETN reductase
Introduction
and 3 from Lycopersicon esculentum (tomato),[12]
YqjM from Bacillus subtilis[12] and an OYE homo-
During the past few years, the asymmetric bioreduc- logue from Candida macedoniensis.[9] The substrate-
tion of alkenes bearing an electron-withdrawing (acti- selectivity pattern of further candidates, such as
vating) group catalysed by flavoproteins from the old XenA from Pseudomonas putida, KYE1 from Klyver-
yellow enzyme (OYE) family has emerged as a valua- omyces lactis and Yers-ER from Yersinia bercovieri is
ble tool for the biocatalytic synthesis of non-racemic unknown.[10] Although several enoate reductases are
a- or b-substituted aldehydes, ketones, carboxylic available from commercial sources, the inavailability
acids/esters, nitriles and nitroalkanes.[1] In OYE-type of biochemical and structural data prevents the pre-
enzymes, the relative stereochemistry of the addition diction and rationalisation of their substrate tolerance
of [2H] is dictated by the enzyme mechanism and in- and stereoselectivities, which severely limits their ap-
variably proceeds in a trans-specific fashion.[2,3] In the plicability.[11] The data available so far indicate that a-
past, the majority of asymmetric C=C-bioreductions substituted 5- and 6-membered cyclic enones are
were performed using whole fermenting cells, usually freely accepted with stereoselectivities varying from
bakerꢀs yeast.[4] Although the resulting stereoselectivi- low to excellent, whereas b-substituted analogues are
ties were often excellent, undesired carbonyl reduc- converted much more slowly. A critical question re-
tion caused by competing carbonyl reductases often garding the applicability of these enzymes in asym-
led to substrate and/or product depeletion causing metric synthesis is the availability to access both ste-
low overall yields.[5] It was only recently that cloned reoisomers. The latter could be achieved for selected
and overexpressed oxygen-stable enoate reductases[6] types of substrates either by choice of the appropriate
became available in sufficient amounts. The most (iso)enzyme[12] or by the (E/Z)-stereochemistry of the
promising enzymes described so far are OYEs isoen- substrate.[8,13] These strategies have been denoted as
zymes 1–3 from Saccharomyces pastorianus (formerly enzyme- and substrate-based stereocontrol, respec-
S. carlsbergensis) and S. cerevisiae, respectively,[7] tively, and were recently complemented by the design
NCR from Zymomonas mobilis,[8] OPR isoenzymes 1 of stereodivergent enzyme mutants.[14] However, for
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Nicole J. Mueller et al.
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many substrates, stereocontrol including the tech- straight-chain and cyclic alkenes bearing an aldehyde,
nique of substrate-engineering failed, which severely ketone, carboxylic acid or ester, cyclic imide and nitro
limits the practical value of these enzymes, which moiety as electron-withdrawing (activating) group
allow one to access to a- and b-chiral carbonyl com- (Scheme 1, Table 1). Initial tests were performed
pounds, which are important intermediates or prod- using equimolar amounts of cofactor [NAD(P)H] and
ucts for flavour and fragrance applications. In order promising enzyme-substrate combinations were re-
to circumvent the limitations mentioned above and to peated with NADH-recycling using the glucose dehy-
extend the number of enoate reductases for prepara- drogenase/glucose system.
tive biotransformations, we extended our search to-
wards biochemically and structurally well described good substrates for all enzymes, providing the sub-
OYE homologues. stituent was located in the a-position (substrates 1a
Cyclic 5- and 6-membered ring enones proved to be
Many bacterial enzymes with amino acid sequence and 3a). The smaller and more flexible 2-methylcyclo-
homology to OYE have now been reported. Pentaery- pent-2-enone (1a) gave lower stereoselectivities [(S)-
thritol tetranitrate reductase (PETNR) from Entero- 1b eemax 64% using PETNR] as compared to the steri-
bacer cloacae strain PB2 was originally isolated from cally more demanding 2-methylcyclohex-2-ene ana-
soil contaminated with explosives,[15] whilst morphi- logue 3a [(R)-3b eemax 88% using NEMR]. A (weakly
none reductase (MorR) was obtained from an opi- exhibited) switch of stereopreference for (S)-3b was
ates-degrading strain of Pseudomonas putida.[16] N- observed with MorR depending on the choice of co-
Ethylmaleimide reductase (NEMR) is a close relative factor NADH or NADPH. a-Alkylcycloalkanones 1b
of PETNR from Escherichia coli.[17] Since estrogen- and 3b are stereochemically somewhat labile and
binding protein from Candida albicans[2b,18] was re- became racemised by proteinogenic impurities in
ported to catalyse the conjugate reduction of cyclo- crude enzyme preparations, such as crude EBP1. This
hex-2-enone and its disproportionation yielding cyclo- undesired side activity, however, could be eliminated
hexanone and phenol – denoted as ꢂaromataseꢀ activi- by using pure EBP1. The same effect was also ob-
ty in the formation of the phenolic A ring of ste- served when impure (commercial) enzyme prepara-
roids[19] – we speculated that it might also exhibit tions were used for cofactor recycling.[5a,12a] However,
enoate reductase activity on non-natural substrates.
it was recently observed that (depending on the reac-
The crystal structures of PETNR and MorR have tion conditions) product racemisation for a-chiral car-
been determined in complex with various ligands and bonyl compounds may also be non-enzymatic.[29]
the active site structures show high structural similari-
In contrast, b-substituted cycloalkenones 2a and 4a
ty to old yellow enzyme from yeast.[20,21,22] These fla- were transformed at reduced reaction rates, which is
voenzymes contain the prosthetic group FMN bound presumably due to steric hindrance exerted by the b-
at the top of an 8-fold b/a barrel. The active site acid substituent upon hydride attack from the bulky flavin
identified in OYE1 (Tyr196) is replaced by Cys191 in cofactor onto C-b, combined with enhanced stereose-
MorR, which has been shown not to have an involve- lectivities for the corresponding (S)-configurated
ment in the mechanism of reduction of the olefinic products 2b and 4b (up to >99% ee using NEMR and
bond found in 2-cyclohexen-1-one and morphinone. crude EBP1). For these substrates, no racemisation
The source of proton reduction of a/b-unsaturated was detected.
carbonyl compounds by MorR is, therefore, currently
In terms of reaction rate and stereoselectivity, the
unclear.[21,23] In PETNR Tyr-186 or water act as the cyclic imide 5a was an excellent substrate for PETNR
proton donor.[24] The roles of His181 and His184 in and NEMR yielding (R)-5b in 97 to >99% ee at full
substrate binding in PETNR have been investigated conversion. An attempt to force the stereochemical
and these residues position substrates such that they outcome of the bioreduction by addition of a large N-
are aligned with the flavin N-5 atom in an orientation phenyl protective group (6a),[25] which would force
that is favourable for hydride transfer.[20] The histidine the substrate to adopt a flipped orientation within the
residue at position 181 is strongly conserved across active site[26] was successful for the first time for
the OYE family, whilst residue 184 varies between enoate reductases: whereas the unmodified substrate
histidine and asparagine. In PETNR the residue is 5a gave (R)-5b in 49% ee, the bulky N-phenyl deriva-
histidine, whilst in OYE and MorR, the residue is as- tive 6a furnished the mirror-image product (S)-6b
paragine.[21,22]
with MorR using NADH, albeit in low ee (10%). It is
noteworthy that this strategy failed completely for
OPR1, OPR3, YqjM,[4b] OYE 1–3 and NCR-reduc-
Results and Discussion
tase,[7a] which invariably gave (R)-configurated prod-
ACHTUNGTRENNUNG
ucts in excellent ee. The variation of the stereoselec-
In order to obtain a reasonably broad picture of the tivity during the reduction of 6a using MorR by
reactivities and stereoselectivities of our candidate en- switching from NADH to NADPH [(S)- versus (R)-
zymes, we selected a series of structurally diverse 6b with an ee of 10% and 92%, respectively] is strik-
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The Substrate Spectra of Pentaerythritol Tetranitrate Reductase, Morphinone Reductase
Scheme 1. Asymmetric bioreduction of alkenes 1a–16a using pentaerythritol tetranitrate reductase (PETNR), N-ethylmale-
ACHTUNGTRENNUNGimide reductase (NEMR), morphinone reductase (MorR) and estrogen-binding protein (EBP1).
ing. Such a behaviour has been noted before for duced, yielding (R)-9b in 99% ee, while diacid 11a
OYEs and has been attributed to allosteric effects of proved to be unreactive.
the cofac
G
The antimicrobial terpene citral (13a) is a relatively
Due to their low carbonyl activity, which limits inexpensive commodity material, which is widely used
their electron-withdrawing effect, dicarboxylic acids in the flavour and fragrance industry. In contrast, its
have to be considered as borderline-substrates for chiral reduction product citronellal (13b) is an impor-
typical OYEs.[27] (E)-Dicarboxylate 7a, which was pre- tant ingredient in the perfumery industry and is used
viously shown to be unreactive with all OYEs tested as the starting material for the synthesis of non-race-
previously,[10a,13a] was successfully reduced to (S)-7b in mic menthol,[28] consequently, its value is approxi-
50% ee by EBP1 for the first time. In contrast, the mately 20 times higher.[8b] Whereas (S)-13b could be
(Z)-analogue 8a was a much better substrate, yielding accessed in excellent ee via bioreduction of 13a using
the mirror-image counterpart (R)-7b in up to >99% OPR1, OPR3, YqjM and NCR-reductase,[4,7a,8] (R)-
ee using PETNR. As previously observed, the addi- 13b was formed in varying ees by using OYEs 1–3
tion of an equimolar amount of a divalent metal ion, (eemax 87%). While PETNR and NEMR exhibited the
such as Ca2+ or Mg2+, was required to avoid enzyme more commonly observed preference for (S)-13b
deactivation of the cofactor recycling enzyme (eemax 66%), EBP1 gave the desired (more valuable)
GDH.[13a,10a] Due to their enhanced carbonyl activities, (R)-13b in 53% ee. The importance of using pure en-
the corresponding dimethyl esters proved to be better zymes is highlighted by over-reduction of (R)-13b at
substrates: diesters (E)-9a and (Z)-10a were readily the carbonyl moiety yielding (R)-citronellol when
reduced to (R)-9b in up to >99% ee by all enzymes, crude EBP1 was used.
except for MorR, which displayed no activity. Most
Whereas the majority of enzymes reduced nitroal-
remarkably, no stereochemical switch depending on kene (E)-14a to (R)-14b in up to 97% ee, again,
the (E/Z)-configuration of substrate, as observed for MorR acted in a stereocomplementary fashion by
YqjM [(E)-9a!(S)-9b] and OPR1 [(Z)-9a!(R)-9b] forming (S)-14b (eemax 46%). Using PETNR, the ste-
was observed.[13a] In line with the above-mentioned reoselectivity of this transformation is remarkably
observations, dimethyl ester 12a was successfully re- sensitive to the reaction conditions, and by switching
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The Substrate Spectra of Pentaerythritol Tetranitrate Reductase, Morphinone Reductase
and 300 mM NaCl, pH 8). Cells were lysed by ultrasonica-
between aerobic to anaerobic conditions or exchang-
ing the cofactor from NADH to NADPH, the stereo-
chemical outcome may even switch to (S).[29] Finally,
sterically demanding substrates, such as ketoisophor-
one (15a) and (R)-carvone (16a) were reduced with
good to excellent (dia)stereoselectivities to yield (R)-
15b (eemax 82%) and (R,R)-16b (de up to 95%). Over-
all comparison of the results from a study which was
performed independently at about the same time on
PETN reductase[29b] reveals that the stereoselectivities
were either identical (substrates 2a, 4a, 5a, 6a, 13a,
16a), or somewhat lower (1a, 3a, 14a) or higher (15a).
All deviations, however, can be attributed to stereo-
chemically unstable products which are prone to a
certain degree of spontaneous (or protein-catalysed)
racemisation or to cases where substrates are subject
to in-situ E/Z-isomerisation (13a, 14a), which high-
lights the importance of the reaction conditions for
this type of biotransformation.
tion (Soniprep, MSE) at an amplitude of 12 mm for 6 cycles
of 15 seconds on followed by 30 seconds off. The cell sus-
pension was cooled during sonication in a cracked-ice bath,
and insoluble material removed by centrifugation followed
by filtration (0.22 mm). The His-tagged proteins were loaded
onto a nickel-affinity column (His-Select, from Sigma Al-
drich), washed with buffer A, then eluted with buffer B (as
A, but with 250 mM imidazole). Purified protein was then
dialysed into 50 mM sodium phosphate buffer pH 7 and
stored frozen (ꢀ208C). Protein purities were checked by
SDS-PAGE.
Preparation of Crude and Purified EBP1
Estrogen binding protein from Candida albicans[2b] with C-
terminal His-tag was cloned in pDHE and transformed into
Escherichia coli TG10+ as reported.[32] Preculturing was per-
formed by inoculation of 100 mL LB medium [Trypton
(10 g/L, Oxoid L0042), yeast extract (5 g/L, Oxoid L21) and
NaCl (5 g/L, Roth 9265.1)] with ampicillin (100 mgmLꢀ1),
spectomycin (50 mgmLꢀ1) and chloramphenicol (10 mgmLꢀ1)
and incubated at 378C and 120 rpm to OD600 =1. Preculture
(10 mL) was added to 1-L flasks containing 1000 mL LB
medium, ampicillin (100 mgmLꢀ1), spectomycin (50 mgmLꢀ1)
and chloramphenicol (10 mgmLꢀ1), rhamnose (0.34 g/L) and
IPTG (20 mmol/L) and grown overnight (16 h) at 378C and
120 rpm. The next morning the cells were harvested by cen-
trifugation (8000 rpm, 20 min, 48C), washed with Tris-HCL
buffer solution (5 mL, 50 mM, pH7.5) and recentrifugated
under the same conditions to afford a cell pellet. For a
crude enzyme preparation, the cells were resuspended in
Tris-HCL buffer solution (5 mL, 50 mM, pH7.5), shock-
frozen in liquid nitrogen and lyophilised. The preparation
could be stored at 48C for several months without signifi-
cant loss of activity. Purification of His-tagged EBP1 was
done using an Ni-affinity column (HisTrap FF, GE Health-
care) according to the manufacturerꢀs protocol, the enzyme
solution had a protein content of 4.1 mgmLꢀ1. Activities of
the same cell line containing the empty plasmid were low
(ꢁ5%).
Conclusions
Investigation of the substrate spectrum of four flavo-
proteins from the OYE family, pentaerythritol tetrani-
trate reductase (PETNR), N-ethylmaleimide reduc-
tase (NEMR), morphinone reductase (MorR) and es-
trogen-binding protein (EBP1), revealed that these
enzymes readily accept conjugated enals, enones,
imides, dicarboxylic acids and esters, as well as a ni-
troalkene. Depending on the enzyme-substrate combi-
nation, the stereochemical preference often comple-
mented that of previously investigated OYEs both in
terms of their stereoselectivity and stereopreference,
which (in some cases) proved to be opposite. In addi-
tion, the strategy of substrate engineering by variation
of a protective group regarding size and polarity in
order to control the stereochemical outcome of the
bioreduction was successful with OYEs for the first
time. This behaviour was most pronounced for MorR
and EBP1, which makes them promising candidates
for the generation of active-site variants showing al-
tered stereoselectivities.
General Procedure for the Bioreduction of 1a–16a in
the presence of a Molar Equivalent of NADH or
NADPH, and by using GDH-Recycling
General procedure for the enzymatic bioreduction of 1a–
16a: An aliquot of enzyme [PETN-reductase, NEM-reduc-
tase, Morphinone reductase, or EBP1 corresponding to a
final protein concentration of 125 mgmLꢀ1; in the case of
crude EBP1, lyophilised whole cells of E. coli TG10+ con-
taining EBP1 (20 mg) were used] was added to a Tris-HCl
buffer solution (0.8 mL, 50 mM, pH7.5) containing the sub-
strate (10 mM) and the cofactor NADH or NADPH
(15 mM). N-Phenyl-2-methylmaleimide (6a) was added as a
0.5M DMF solution (1% final DMF concentration) to over-
come its poor solubility in water. The mixture was shaken at
308C and 120 rpm. After 24 h the products 1a–6a, 10a and
12a–16a were extracted with EtOAc (2ꢃ0.5 mL). The com-
bined organic phases were dried over Na2SO4 and analysed
on achiral and chiral GC. Dicarboxylic acids 7a, 8a and 11a
were freeze-dried, and analysed as dimethyl esters after de-
Experimental Section
Preparation of PETNR, NEMR and MorR
The genes for PETNR, NEMR and MorR were sub-cloned
into the vector pET16B (NOVAGEN) as previously report-
ed.[31] All three proteins were produced in the following
way: E.coli DH5 alpha cells transformed with the required
plasmid were grown to OD600 at 378C in LB broth also con-
taining 0.1 g/L carbenicillin. They were then cooled to 208C
and induced by the addition of 1 mM IPTG (final) and incu-
bated overnight at 208C. Cells were harvested by centrifuga-
tion, resuspended in buffer A (50 mM sodium phosphate
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rivatisation.[5a] Products were identified by comparison with
independently synthesised authentic reference materials via
co-injection on GC-MS and achiral GC.
propyl-phenyl phase capillary column (J&W Scientific DB-
1701, 30 m, 0.25 mm, 0.25 mm), detector temperature 2508C,
split ratio 30:1. Temperature programme for 15a: 1108C
hold 5 min, 308C minꢀ1 to 2008C, hold 2 min. Retention
times were as follows: epoxide 6.82 min, 15a 7.14 min and
15b 7.73 min. Temperature programme for 16a: 1108C,
158C minꢀ1 to 2008C, hold 10 min. Retention times were as
follows: 16b 4.61 min and 4.70 min (diastereomers), carveol
4.81 and 16a 5.03 min.
General procedure for cofactor recycling for the reduction
of 1a–16a: The procedure described above was modified by
replacing the molar equivalents of NAD(P)H by catalytic
amounts of NAD+ (100 mM), the cosubstrate glucose
(20 mM) and the recycling enzyme (glucose dehydrogenase,
10 U). In the case of dicarboxylic acids 7a, 8a and 11a a di-
valent metal ion (CaCl2 or MgCl2) was added to the system
in equimolar amounts to the substrate (10 mM). The reac-
tion was performed and worked up as described above.
Determination of enantiomeric excess and absolute con-
figuration: The enantiomeric excess of 2-methylcyclopentan-
1-one 1b[4b] 3-methylcyclopentan-1-one 2b, 2-methylcyclo-
hexan-1-one 3b, 3-methylcyclohexan-1-one 4b and 2-methyl-
succinimide 5b[4b] was determined using a modified b-cyclo-
dextrin capillary column (Chiraldex B-TA, 40 m, 0.25 mm).
Detector temperature 2008C, injector temperature 1808C,
split ratio 25:1. Temperature program for 2b: 708C hold
10 min, 18C minꢀ1 to 808C, 158C minꢀ1 to 1608C hold 5 min.
Retention times: (S)-2b and (R)-2b 16.92 and 17.33 min, re-
spectively. Temperature programme for 3b and 4b: 708C
hold 10 min, 158C minꢀ1 to 1808C, hold 1 min. Retention
times: (R)-3b and (S)-3b 14.03 and 14.24 min, (R)-4b and
(S)-4b 14.86 and 15.03 min, respectively. The enantiomeric
excess of 6b was determined via HPLC analyses using a Shi-
madzu system equipped with a Chiracel OD-H column
(25 cmꢃ0.46 cm), using n-heptane/EtOH, 95:5 (isocratic,
flow rate of 1 mL minꢀ1, 188C). Retention times: (R)-6b and
(S)-6b 23.72 min and 25.43 min, respectively. The enantio-
meric excess of 1-nitro-2-phenylpropane 14b[4] and levo-
dione 15b[10a] was determined using a b-cyclodextrin capilla-
ry column (CP-Chirasil-DEX CB, 25 m, 0.32 mm, 0.25 mm
film). The enantiomeric excess of methylsuccinic acid 7b (as
dimethyl ester 9b), methylsuccinic dimethyl ester 9b and cit-
ronellol was determined using a modified b-cyclodextrin ca-
pillary column (Hydrodex-b-6TBDM, 25 m, 0.25 mm). De-
tector temperature 2008C, injector temperature 1808C, split
ratio 20:1. Temperature programme for 9b: 908C hold
4 min, 0.58C minꢀ1 to 958C, 158C minꢀ1 to 1308C, hold
3 min. Retention times: (R)-9b and (S)-9b 6.57 and
6.98 min, respectively. Temperature programme for citronel-
lol: 908C hold 80 min, 0.58C minꢀ1 to 958C, 158C minꢀ1 to
1608C, hold 5 min. Retention times: (R)-citronellol and (S)-
citronellol 83.31 and 84.85 min, respectively. The enantio-
meric excess of citronellal 13b[10a] was determined using a
modified b-cyclodextrin capillary column (Hydrodex-b-
TBDAc, 25 m, 0.25 mm). Detector temperature 2008C, in-
jector temperature 1808C, split ratio 20:1. The diastereo-
meric excess of (+)-dihydrocarvone 16b was determined by
GC-FID using a 14% cyanopropyl-phenyl phase capillary
column (J&W Scientific DB-1701, 30 m, 0.25 mm, 0.25 mm),
detector temperature 2508C, split ratio 30:1. Temperature
programme for 16b: 1108C, 15 8C minꢀ1 to 2008C, hold
10 min. Retention times were as follows: (5R,2R)-16b and
(5R,2S)-16b 4.61 min and 4.70 min, respectively.
Synthesis of Substrates and Non-Racemic Reference
Compounds
2-Methyl-2-cyclopenten-1-one 1a, (R)-3-methylcyclopentan-
1-one (R)-2b, 3-methyl-2-cyclohexen-1-one 4a, (R)-3-methyl-
cyclohexan-1-one (R)-4b, N-phenyl-2-methylmaleimide 6a,
(S)-methylsuccinic acid (S)-7b, mesaconic acid 7a, (R)-citro-
nellal (R)-13b, (S)-citronellal (S)-13b, (S)-citronellol, (+)-di-
hydrocarvone 16b (mixture of diastereomers) were pur-
chased from Sigma Aldrich, 3-methyl-2-cyclopenten-1-one
2a and itaconic acid 11a were purchased from Fluka. 2-
Methyl-2-cyclohexen-1-one 3a, (E)-citral (E)-13a and (R)-
carvone (R)-16a were kindly provided by BASF (Ludwig-
shafen). Citraconic acid 8a was purchased from Alfa Aesar,
ketoisophorone 15a was purchased from ABCR. NADH,
NAD+, glucose dehydrogenase (475 UmLꢀ1) were from Co-
dexis and NADPH was from Biocatalytics. 2-Methylmalei-
mide 5a, dimethyl esters 9a, 10a and 11a were synthesised as
previously reported.[5a,10a] (Z)-1-Nitro-2-phenylprop-1-ene
(Z)-14a was synthesised as previously reported.[4] rac-2,3-
Epoxy-3,5,5-trimethyl-1,4-cyclohexanedione was synthesised
as previously reported.[31]
Analytical Methods
Determination of conversion: Conversions of 2-methyl-2-cy-
clopenten-1-one 1a,[4b] 3-methyl-2-cyclopenten-1-one 2a,[4b]
2-methyl-2-cyclohexen-1-one 3a, 3-methyl-2-cyclohexen-1-
one 4a,[10a] 2-methylmaleimide 5a, N-phenyl-2-methylmale-
ACHTUNGTRENNUNG
imide 6a,[4b] mesaconic acid 7a (analysed as dimethyl ester
9b, see below),[5a] citraconic acid 8a (analysed as dimethyl
ester 9b, see below),[5a] mesaconic dimethyl ester 9a,[5a] citra-
conic dimethyl ester 10a,[5a] itaconic acid 11a (analysed as di-
methyl ester 9b, see below),[5a] itaconic dimethyl ester 12a,[5a]
and 1-nitro-2-phenylprop-1-ene 14a,[4] were analysed by GC-
FID using a 6% cyanopropyl-phenyl phase capillary column
(Varian CP-1301, 30 m, 0.25 mm, 0.25 mm), detector temper-
ature 2508C, split ratio 30:1. Temperature programme for
3a: 808C hold 10 min, 308C minꢀ1 to 2008C, hold 2 min. Re-
tention times were as follows: 3b 5.98 min and 3a 7.83 min.
Temperature programme for 5a: 808C hold 2 min,
208C minꢀ1 to 1608C, 308C minꢀ1 to 2208C, hold 2 min. Re-
tention times were as follows: 5a 5.78 min and 5b 6.59 min.
Products 7b, 8b and 11b were analysed as the corresponding
dimethyl ester 9b after derivatization using BF3-methanol
solution.[5a] Conversion of citral 13a[10a] was determined by
GC-FID using a PEG-phase capillary column (Varian CP-
Wax 52 CB, 30 m, 0.25 mm, 0.25 mm), detector temperature
2508C, split ratio 20:1. Conversion of ketoisophorone 15a
and (R)-carvone 16a was determined using a 14% cyano-
The absolute configurations of 2b, 4b, 9b, citronellol, 13b
and 16b were determined by co-injection with reference ma-
terials of known absolute configuration. The absolute con-
figuration of 3b or, respectively, 1b was determined by co-in-
jection with enantioenriched reference material obtained by
reduction of 3a or, respectively, 1a with YqjM.[4a] The abso-
lute configuration of 5b was determined by co-injection with
enantioenriched reference material obtained by reduction of
392
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Adv. Synth. Catal. 2010, 352, 387 – 394

The Substrate Spectra of Pentaerythritol Tetranitrate Reductase, Morphinone Reductase
5a with OPR1.[4b] The absolute configuration of 6b was de-
termined by co-injection with enantioenriched reference ma-
terial by reduction of 6a with OPR1.[7a] The absolute con-
Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K.
Gruber, B. Hauer, R. Stuermer, W. Kroutil, P. Macher-
oux, K. Faber, Adv. Synth. Catal. 2008, 350, 411–418.
[13] a) C. Stueckler, M. Hall, H. Ehammer, E. Pointner, W.
Kroutil, P. Macheroux, K. Faber, Org. Lett. 2007, 9,
5409–5411; b) M. Utaka, S. Konishi, A. Mizuoka, T.
Ohkubo, T. Sakai, S. Tsuboi, A. Takeda, J. Org. Chem.
1989, 54, 4989–4992.
ACHTUNGTRENNUNGfiguration of 14b or, respectively, 15b was determined by co-
injection with enantioenriched reference material ob-
tained by reduction of 14a or, respectively, 15a with bakerꢀs
yeast.[8c,10a]
[14] S. K. Kumar, D. J. Bougioukou, J. D. Stewart, J. Am.
Chem. Soc. 2009, 131, 3271–3280.
[15] P. R. Binks, C. E. French, S. Nicklin, N. C. Bruce, Appl.
Environ. Microbiol. 1996, 62, 1214–1219.
References
[1] R. Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin.
Chem. Biol. 2007, 11, 203–213.
[16] A. M. Hailes, N. C. Bruce, Appl. Environ. Microbiol.
1993, 59, 2166–2170.
[2] Delivery of a hydride from the flavin cofactor onto Cb
goes in hand with donation of a proton onto Ca from
the opposite side via a Tyr or Cys residue; see: a) R. E.
Williams, N. C. Bruce, 2002, 148, 1607–1614; b) P. A.
Karplus, K. M. Fox, V. Massey, FASEB J. 1995, 9,
1518–1526.
[3] cis-Specific bioreduction of alkenes has been observed
with flavin-independent enzymes, see: a) K. Shimoda,
D. I. Ito, S. Izumi, T. Hirata, J. Chem. Soc. Perkin
Trans. 1 1996, 355–358; b) D. J. Bougioukou, J. D.
Stewart, J. Am. Chem. Soc. 2008, 130, 7655–7658.
[4] For reviews see: a) I. L. Gatfield, Adv. Biochem. Eng./
Biotechnol. 1997, 55, 221–238; b) S. Servi, Synthesis
1990, 1–25; c) C. Fuganti, Pure Appl. Chem. 1990, 62,
1449–1452.
[5] a) A. Mꢄller, B. Hauer, B. Rosche, J. Mol. Catal. B:
Enzym. 2006, 38, 126–130; b) M. Hall, B. Hauer, R.
Stuermer, W. Kroutil, K. Faber, Tetrahedron: Asymme-
try 2006, 17, 3058–3062; c) H. G. W. Leuenberger, W.
Boguth, E. Widmer, R. Zell, Helv. Chim. Acta 1976, 59,
1832–1849.
[6] Enoate reductases from anaerobic organisms have
been shown to be sensitive towards O2 and are thus of
limited practical applicability; see: a) H. Simon, H.
Gꢄnther, J. Bader, W. Tischer, Angew. Chem. 1981, 93,
897–898; b) R. Eck, H. Simon, Tetrahedron 1994, 50,
13631–13640; c) H. Gꢄnther, H. Simon, Biocatal. Bio-
transform. 1995, 12, 1–26.
[7] a) M. Hall, C. Stueckler, B. Hauer, R. Stuermer, T.
Friedrich, M. Breuer, W. Kroutil, K. Faber, Eur. J. Org.
Chem. 2008, 1511–1516; b) M. A. Swiderska, J. D.
Stewart, Org. Lett. 2006, 8, 6131–6133; c) M. A. Swi-
derska, J. D. Stewart, J. Mol. Catal. B: Enzym. 2006, 42,
52–54.
[8] A. Mꢄller, B. Hauer, B. Rosche, Biotechnol. Bioeng.
2007, 98, 22–29.
[9] a) M. Kataoka, A. Kotaka, R. Thiwthong, M. Wada, S.
Nakamori, S. Shimizu, J. Biotechnol. 2004, 114, 1–9;
b) M. Kataoka, A. Kotaka, A. Hasegawa, M. Wada, A.
Yoshizumi, S. Nakamori, S. Shimizu, Biosci. Biotechnol.
Biochem. 2002, 66, 2651–2657.
[17] K. Miura, Y. Tomioka, H. Suzuki, M. Yonezawa, T.
Hishinuma, M. Mizugaki, Biol. Pharm. Bull. 1997, 20,
110–112.
[18] a) J. Buckman, S. M. Miller, Biochemistry 1998, 37,
14326–14336; b) A. D. N. Vaz, S. Chakraborty, V.
Massey, Biochemistry 1995, 34, 4246–4256.
[19] For instance, the formation of 17b-estradiol from the
corresponding enone-precursor 19-nortestosterone.
[20] B. M. Barna, H. Khan, N. C. Bruce, I. Barsukov, N. S.
Scrutton, P. C. E. Moody, J. Mol. Biol. 2001, 310, 433–
447.
[21] T. Barna, L. F. Messiha, C. Petosa, N. C. Bruce, N. S.
Scrutton, P. C. E. Moody, J. Biol. Chem. 2002, 277,
30976–30983.
[22] K. M. Fox, P. A. Karplus, Structure 1994, 2, 1089–1105.
[23] H. F. Messiha, A. W. Munro, N. C. Bruce, I. Barsukov,
N. S. Scrutton, J. Biol. Chem. 2005, 280, 10695–10709.
[24] H. Khan, T. Barna, N. C. Bruce, A. W. Munro, D. Leys,
N. S. Scrutton, FEBS J. 2005, 272, 4660–4671.
[25] The strategy to control the stereoselectivity of an
enzyme by altering the substrate structure through var-
iation of protective groups or by modification of a
docking (’anchor’) group has been successfully demon-
strated for various enzymes classes, for example see:
a) A. de Raadt, H. Griengl, Curr. Opin. Biotechnol.
2002, 13, 537–542; b) B. V. Nguyen, O. Nordin, C.
Vçrde, E. Hedenstrçm, H. E. Hçgberg, Tetrahedron:
Asymmetry 1997, 8, 983–986; c) K. Adachi, S. Kobaya-
shi, M. Ohno, Chimia 1986, 40, 311–314; d) K. Faber,
G. Ottolina, S. Riva, Biocatalysis 1993, 8, 91–132.
[26] For a review on stereocomplementary enzymes see: P.
Mugford, U. Wagner, Y. Jiang, K. Faber, R. Kazlauskas,
Angew. Chem. 2008, 120, 8912–8923; Angew. Chem.
Int. Ed. 2008, 47, 8782–8793.
[27] a,b-Unsaturated mono-carboxylic acids (which are gen-
erally not accepted by OYEs) can be efficiently re-
duced by 2-enoate reductases, which are conjugated Fe/
S-flavoproteins, from (strict or facultative) anaerobic
organisms, such as Clostridia and Proteus spp., see
ref.[9]
[28] a) A. F. Trasarti, A. J. Marchi, C. R. Apesteguia, J.
Catal. 2004, 224, 484–488; b) P. Mꢅki-Arvela, N.
Kumar, D. Kubicka, A. Nasir, T. Heikkilꢅ, V. P. Lehto,
R. Sjçholm, T. Salmi, D. Y. Murzin, J. Mol. Catal. A:
Chem. 2005, 240, 72–81.
[29] a) H. S. Toogood, A. Fryszkowska, V. Hare, K. Fisher,
A. Roujeinikova, D. Leys, J. M. Gardiner, G. M. Ste-
phens, N. S. Scrutton, Adv. Synth. Catal. 2008, 350,
[10] J. F. Chaparro-Riggers, T. A. Rogers, E. Vaquez-Figuer-
oa, K. M. Polizzi, A. S. Bommarius, Adv. Synth. Catal.
2007, 349, 1521–1531.
[11] B. Kosjek, F. J. Fleitz, P. G. Dormer, J. T. Kuethe, P. N.
Devine, Tetrahedron: Asymmetry 2008, 19, 1403–1406.
[12] a) M. Hall, C. Stueckler, W. Kroutil, P. Macheroux, K.
Faber, Angew. Chem. 2007, 119, 4008–4011; Angew.
Chem. Int. Ed. 2007, 46, 3934–3937; b) M. Hall, C.
Adv. Synth. Catal. 2010, 352, 387 – 394
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
393

Nicole J. Mueller et al.
FULL PAPERS
2789–2803; b) A. Fryszkowska, H. Toogood, M.
Sakuma, J. M. Gardiner, G. M. Stephens, N. S. Scrutton,
Adv. Synth. Catal. 2009, 351, 2976–2990.
Mueller, C. Stueckler, M. Hall, P. Macheroux, K. Faber,
Org. Biomol. Chem. 2009, 7, 1115–1119.
[31] R. A. Williams, D. A. Rathbone, N. S. Scrutton, N. C.
Bruce, Appl. Environ. Microbiol. 2004, 70, 3566–3574.
[32] C. Stueckler, T. C. Reiter, N. Baudendistel, K. Faber,
Tetrahedron 2010, 66, 663–667.
[30] Non-enzymatic epoxidation of C=C bonds may occur
through H2O2, which is generated by flavoproteins via
reduction of O2 at the expense of NAD(P)H; see: N. J.
394
asc.wiley-vch.de
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2010, 352, 387 – 394