Epoxidation of conjugated C=C-bonds and sulfur-oxidation of thioethers mediated by NADH:FMN-dependent oxidoreductases

Article abstract of DOI:10.1039/b819057g

Three FMN-dependent oxidoreductases, YcnD and YhdA from Bacillus subtilis and Lot6p from Saccharomyces cerevisiae, oxidised α,β-unsaturated carbonyl compounds and a thioether, respectively, to furnish the corresponding racemic epoxides or sulfoxide, respectively. The mechanism of this enzyme-mediated (rather than enzyme-catalysed) oxidation was shown to proceed via the NADH-dependent reduction of O₂, forming H₂O ₂, which acted as oxidant in a spontaneous (non-enzymatic) fashion. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b819057g

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=
Epoxidation of conjugated C C-bonds and sulfur-oxidation of thioethers
mediated by NADH:FMN-dependent oxidoreductases
Nicole Jasmin Mueller,a Clemens Stueckler,a Melanie Hall,a Peter Macherouxb and Kurt Faber*a
Received 27th October 2008, Accepted 28th November 2008
First published as an Advance Article on the web 26th January 2009
DOI: 10.1039/b819057g
Three FMN-dependent oxidoreductases, YcnD and YhdA from Bacillus subtilis and Lot6p from
Saccharomyces cerevisiae, oxidised a,b-unsaturated carbonyl compounds and a thioether, respectively,
to furnish the corresponding racemic epoxides or sulfoxide, respectively. The mechanism of this
enzyme-mediated (rather than enzyme-catalysed) oxidation was shown to proceed via the
NADH-dependent reduction of O2, forming H2O2, which acted as oxidant in a spontaneous
(non-enzymatic) fashion.
chiral 1a is an inexpensive commodity material, its a,b-reduction
product ‘citronellal’ is highly valuable since it serves as important
ingredient of perfumes. The latter can be obtained via asymmetric
bioreduction using enoate reductases.10,2c When we tested YcnD,
Lot6p and YhdA for this transformation in the presence of NADH
Introduction
During the course of our studies on the asymmetric biore-
duction of activated alkenes,1 we sought to extend the set of
readily available enzymes beyond the typical enoate reductases
from the old-yellow-enzyme (OYE) family, such as OYEs 1–3
from Saccharomyces and Zymomonas mobilis NCR-reductase2 or
OPR-isoenzymes from tomato and YqjM from Bacillus subtilis.3
Our candidates of interest were oxidoreductases which utilize
NAD(P)H as hydride source to reduce the flavin-cofactor FMN
in their active site. The flavoprotein YcnD from Bacillus subtilis
was assumed to be responsible for the delivery of reduced FMN
to enzymes that require a reduced cofactor for activity, such as
luciferase.4 From the same organism, thermostable YhdA was
=
as hydride source, we were surprised to find that the a,b-C C bond
of 1a was not reduced, but epoxidised in up to 36% yield (Scheme 1,
Table 1).
This unexpected outcome of the reaction was verified with
cyclohexenone (2a), which gave 2,3-epoxycyclohexanone 2b in
20–60%. In a similar fashion, ketoisophorone (3a) was not
reduced to levodione (2,2,6-trimethylcyclohexane-1,4-dione) as
expected,2a,3b,c but furnished epoxydione 3b (28–75%). Finally,
menadione (4a, vitamin K311) gave epoxymenadione 4b in up to
69% yield. In nature, the latter is formed as oxidation product
from vitamin K3 during the post-translational g-carboxylation of
glutamyl residues of vitamin K-dependent proteins, which play a
key role in signal transduction, growth control, and haemostasis.12
Overall, YcnD and YhdA showed superior activities than Lot6p.
The short-chain aldehyde 5a could not be epoxidised due to
decomposition of its presumed epoxy-product 5b. Epoxidation
of 5a using chemical methods failed for the same reason.
The requirement for an electron-withdrawing (activating)
group, such as an aldehyde or ketone moiety, for epoxidation
seemed to be rather strict, since epoxidation of 1a occurred only
=
recently shown to reduce nitroaromatics, chromate and the –N N–
double bond of various azo-dyes.5 The structural homolog Lot6p
from Saccharomyces cerevisiae catalyses a two-electron reduction
of various quinones.6 These three enzymes react via a ping-pong
bi-bi mechanism, where the nicotinamide cofactor reduces first the
FMN, followed by the subsequent reduction of the substrate. This
mechanistic similarity with enoate reductases7 together with the
undefined physiological role of these enzymes led us to investigate
=
their substrate-spectrum in the bioreduction of activated C C
bonds.8
=
Results and discussion
at the a,b-C C bond, whereas the terminal (non-activated) olefin
remained intact. In line with this observation, styrene 8a and a-
methylstyrene 9a proved to be unreactive (Scheme 2).
Citral (1a) is a non-chiral terpene which occurs as E/Z-mixture of
isomers, denoted as geranial and neral, respectively.9 While non-
Since alkene-epoxidation and thioether-sulfoxidation are often
catalysed by the same class of (flavin-dependent) enzyme,13 we
tested YcnD, YhdA and Lot6p for the oxidation of thioanisol 6a.
In particular, YcnD furnished sulfoxide 6b in 80% yield, without
formation of sulfone resulting from over-oxidation. However,
thioether 7a, which serves as synthetic precursor for the anti-ulcer
aDepartment of Chemistry, Organic & Bioorganic Chemistry, University
of Graz, Heinrichstrasse 28, Austria. E-mail: Kurt.Faber@Uni-Graz.at;
Fax: +43-316-380-9840; Tel: +43-316-380-5332
bInstitute of Biochemistry, Graz University of Technology, Petersgasse 12,
A-8010, Graz, Austria
Scheme 1 Enzyme-mediated alkene epoxidation and sulfoxidation of thioether.
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Table 1 Enzyme-mediated epoxidation of alkenes and thioether oxidation
Substrate Product
Enzyme
Conditions
Conversion [%]
YcnD
Lot6p
YhdA
—
Lot6p
YhdA
NADH
NADH
NADH
H2O2
H2O2
H2O2
30
22
36
42
43
41
YcnD
Lot6p
YhdA
—
Lot6p
YhdA
NADH
NADH
NADH
H2O2
H2O2
H2O2
60
20
35
24
26
25
YcnD
Lot6p
YhdA
—
NADH
NADH
NADH
H2O2
75
28
42
33
YcnD
Lot6p
YhdA
—
NADH
NADH
NADH
H2O2
22
24
69
77
YcnD
Lot6p
YhdA
NADH
NADH
NADH
decomposition
YcnD
Lot6p
YhdA
—
NADH
NADH
NADH
H2O2
80
25
25
36
Scheme 2 Non-substrates for enzyme-mediated epoxidation and sulfoxidation.
agent omeprazol (7b) was unreactive (<1%), which is presumably
due to the electron-withdrawing effect of the imidazole moiety.
The fact that all of the oxidation products were formed in
racemic (or near racemic) form14 led us to the assumption, that
the oxidation reaction was proceeding without direct enzyme
catalysis, similar to enzymatic halogenation catalysed by heme-
or vanadium-depending haloperoxidases.15 The most plausible
oxidant would be H2O2, which could arise through enzyme-
catalysed reduction of molecular oxygen at the expense of NADH,
an activity which is common for flavin-dependent NAD(P)H-
oxidases16 (Scheme 3). In order to verify this hypothesis, oxidation
of 1a and 2a was performed using YcnD in presence of (H2O2-
degrading) catalase, and secondly, by exclusion of O2 under an
atmosphere of Ar. No epoxides were detected in either case. In
a complementary fashion, spontaneous epoxidation occurred at
comparable rates in presence of H2O2 in the absence of enzymes.
Finally, blank-experiments in the absence of NADH (with
Scheme 3 Mechanism of enzyme-mediated epoxidation of alkenes and
sulfoxidation of thioethers.
external H2O2 added) showed no significant rate acceleration in the
epoxidation of substrates 1a and 2a using Lot6p and YhdA. These
data exclude the possibility that both flavoproteins could catalyse
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the direct alkene-epoxidation using H2O2 as oxidant. These data
prove that this process is ‘enzyme-mediated’ rather than ‘enzyme-
catalysed’.
24.2, 25.6, 33.4, 38.3, 63.5, 64.1, 64.6, 122.4, 122.6, 132.7, 133.3,
198.8, 199.5.
rac-2,3-Epoxy-1-cyclohexanone (2b). To a stirred mixture of
cyclohexenone (2a) (240 mg, 2.5 mmol), sec-butylamine (8 ml,
3%mol) and methanol (5 ml) an aqueous 30% H2O2 (1.25 g,
11 mmol) solution was slowly added. The mixture was stirred
at room temperature for 12 h. Diethyl ether (10 ml), H2O
(1.5 ml) and brine (3 ml) were added. The organic layer was
separated, washed with brine, dried (Na2SO4) and concentrated
by evaporation at atmospheric pressure and room temperature.
Silica gel chromatography followed (eluent pentane/diethyl ether,
10:1) and afforded 2b in 57% yield (160 mg, 1.43 mmol).19
1H-NMR (CDCl3): d 1.26–2.58 (m, 6H) 3.59–3.60 (m, 1H), 3.23
(d, 1H, J = 3.9 Hz). 13C-NMR (CDCl3): d 17.0, 22.9, 36.4, 55.1,
55.9, 205.9.
In this context it should be mentioned that the enzymes used
in this study (YcnD, YhdA and Lot6p) play an essential role in
oxidative stress response by two-electron-reduction of quinones to
furnish the corresponding hydroquinones.4,5a,6 In other words, in
vivo the electrons are passed onto a quinone substrate (rather than
oxygen) and hence alkene epoxidation and sulfoxidation observed
here are adventitious and most probably do not occur in a cellular
environment.
Overall, this epoxidation- and sulfoxidation-reaction bears
some resemblance to the lipase-catalysed formation of peroxy-
carboxylic acids (from H2O2 and fatty acids), which in turn act as
oxidants in the epoxidation of alkenes, sulfoxidation of thioethers
and the Baeyer–Villiger reaction in a non-enzymatic and thus non-
stereoselective fashion.17
rac-2,3-Epoxy-3,5,5-trimethyl-1,4-cyclohexanedione (3b). To a
stirred mixture of ketoisopherone (3a) (380 mg, 2.5 mmol), sec-
butylamine (8 ml, 3%mol) and methanol (5 ml) an aqueous 30%
H2O2 (1.25 g, 11 mmol) was slowly added. The mixture was
stirred at room temperature for 12 h. Diethyl ether (10 ml), H2O
(1.5 ml) and brine (3 ml) were added. The organic layer was
separated, washed with brine, dried (Na2SO4) and concentrated
by evaporation at atmospheric pressure and room temperature.
Silica gel chromatography followed (eluent pentane/diethyl ether,
25:1) and afforded 3b in 45% yield (190 mg, 1.13 mmol).
1H-NMR (CDCl3): d 1.05 (s, 3H), 1.26 (s, 3H), 1.53 (s, 3H), 2.13
(d, 1H, J = 13.4 Hz), 3.13 (d, 1H, J = 13.4 Hz), 3.47 (s, 1H).
13C-NMR (CDCl3): d 16.0, 26.1, 27.0, 45.5, 47.1, 62.9, 64.8, 204.2,
205.6.
Experimental section
General
Citral (1a), menadione (4a), prenal (5a), omeprazole sulfide (7a)
and omeprazole (7b) were provided by BASF (Ludwigshafen),
cyclohexenone (2a) was from Fluka, ketoisophorone (3a) was
purchased from ABCR, thioanisole (6a) and styrene (8a) were
from Aldrich, a-methylstyrene (9a) was purchased from Lactan,
NADH and NADPH were purchased from Biocatalytics/Codexis,
sodium metaperiodate was from Lancaster and catalase from
bovine liver was from Sigma.
Bacillus subtilis YcnD and YhdA and Saccharomyces cerevisiae
Lot6p were expressed and purified as recently reported.4–6
GC-MS analyses were performed on a HP 6890 Series GC
system equipped with a 5973 mass selective detector and a 7683
Series injector using a (5%-phenyl)-methylpolysiloxane capillary
column (HP-5Msi, 30 m, 0.25 mm ID, 0.25 mm film). GC-FID
analyses were carried out on a Varian 3800 using H2 as carrier
gas (14.5 psi). HPLC analyses were performed using a Shimadzu
system equipped with a Chiralcel AD column (25 cm, 0.46 cm).
NMR spectra were measured on a Bruker AMX spectrometer at
360 MHz.
rac-2,3-Epoxy-2-methyl-1,4-naphthoquinone (4b). A solution
of menadione (4a) (200 mg, 1.16 mmol) and sodium percarbonate
(365 mg, 1.16 mmol) in ethanol (4 ml) and H2O (1.2 ml) was stirred
at room temperature for 10 min. H2O (20 ml) was added, the
suspension filtered, washed and dried in vacuo. White crystalline
product in 76% yield (166 mg, 0.88 mmol) was obtained.20
1H-NMR (DMSO): d 1.61 (s, 3H), 4.11 (s, 1H), 7.84–7.96 (m,
4H). 13C-NMR (DMSO): d 14.8, 61.5, 62.0, 126.7, 127.2, 132.2,
132.2, 134.9, 135.1, 192.1.
rac-Methyl phenyl sulfoxide (6b). A mixture of thioanisole 6a
(248 mg, 2 mmol) and aqueous 30% H2O2 (227 mg, 2 mmol)
was stirred at 35 ◦C for 18 h. The solution was saturated with
NaCl and extracted with ethyl acetate (3 ¥ 5 ml). The combined
organic phases were washed with saturated aqueous Na2S2O3
(5 ml), dried (Na2SO4) and evaporated to yield 6b in 85% yield
(238 mg, 1.7 mmol).21
Synthesis of reference material
diastereomeric-rac-2,3-Epoxy-3,7-dimethyl-6-octenal (1b). To
a stirred mixture of citral 1a (380 mg, 2.5 mmol), sec-butylamine
(8 ml, 3 %mol) and methanol (5 ml) an aqueous 30% H2O2
(1.25 g, 11 mmol) solution was slowly added. The mixture was
stirred at room temperature for 12 h. Diethyl ether (10 ml), H2O
(1.5 ml) and brine (3 ml) were added. The organic layer was
separated, washed with brine, dried (Na2SO4) and concentrated
by evaporation at atmospheric pressure and room temperature.
Silica gel chromatography followed (eluent petroleum ether/ethyl
acetate, 10:1) and afforded 1b in 36% yield (151 mg, 0.9 mmol).18
1H-NMR (CDCl3): d 1.41 (s, 3H), 1.43 (s, 3H), 1.50–1.76 (m,
16H), 2.06–3.18 (m, 4H), 3.13 (d, 1H, J = 5.1 Hz), 3.17 (d, 1H,
J = 5.0 Hz), 5.03–5.06 (m, 2H), 9.41 (d, 1H, J = 5.1 Hz), 9.44 (d,
1H, J = 5.0 Hz). 13C-NMR (CDCl3): d 17.2, 17.6, 17.6, 22.1, 23.4,
1H-NMR (CDCl3): d 2.67 (s, 3H), 7.42–7.50 (m, 3H), 7.59–7.61
(m, 2H). 13C-NMR (CDCl3): d 43.9, 123.5, 129.3, 131.0, 145.6.
General procedure for the enzymatic oxidation
An aliquot of the isolated enzyme YcnD, YhdA or Lot6p (protein
purity >90%, protein content 90–110 mg/mL) was added to a
Tris-HCl buffer solution (0.8 mL, 50 mM, pH 7.5) containing
the substrate (5 mM) and the cofactor NADH (10 mM). The
mixture was shaken at 30 ◦C and 120 rpm for 48 h and the
products were extracted with EtOAc (2 ¥ 0.5 mL). The combined
organic phases were dried (Na2SO4) and the resulting samples were
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analyzed on achiral GC. Products were identified by comparison
with authentic reference materials which were either commercially
available or were independently synthesized as described above,
via co-injection on GC-MS and achiral GC.
column (Varian Chiraldex G-PN, 25 m, 0.32 mm). Detector
temperature 200 ◦C, injector temperature 180 ◦C, split ratio
20:1. Temperature program: 80 ◦C, hold for 2 min, 5 ◦C/min
to 120 ◦C, hold for 2.50 min, 10 ◦C/min to 160 ◦C, hold for 2 min.
Retention times were as follows: diastereomeric-rac-2,3-epoxy-3,7-
dimethyl-6-octenal (1b) 7.78 min and 8.08 min (diastereomers) and
(E/Z)-citral (1a) 8.43 min and 9.14 min.
General procedure for reaction with the YcnD/catalase system.
To the reaction mixture was added catalase from bovine liver
(20–25 U) and the reaction was carried out as described above.
The enantiomeric excess of rac-2,3-epoxy-1-cyclohexanone (2b)
was analysed by GC-FID using a modified b-cyclodextrin cap-
illary column (Hydrodex-b-TBDAc, 25 m, 0.25 mm). Detector
temperature 200 ◦C, injector temperature 180 ◦C, split ratio
General procedure for reaction under Ar with YcnD. The
medium was flushed with argon for 10 min and after addition
of YcnD, the reaction mixture was stirred under protective
atmosphere for 24 h and worked up as described above.
◦
◦
20:1. Temperature program: 65 C, hold for 10 min, 30 C/min
to 180 ◦C, hold for 2 min. Retention times were as follows:
cyclohexenone (2a) 13.63 min and rac-2,3-epoxy-1-cyclohexanone
(2b) 13.95 min and 14.33 min.
General procedure for the blank-reaction in presence of H2O2.
Blank-reaction in presence of H2O2 was performed in a Tris-HCl
buffer solution (0.8 mL, 50 mM, pH 7.5) containing the substrate
(5 mM) and H2O2 (10 mM). The reaction and workup was carried
out as described in the general procedure.
The enantiomeric excesses of rac-2,3-epoxy-3,5,5-trimethyl-
1,4-cyclohexanedione (3b) and rac-2,3-epoxy-2-methyl-1,4-
naphthoquinone (4b) were analysed by GC-FID using
a
General procedure for the blank-reaction in presence of H2O2
and enzyme. An aliquot of the isolated enzyme YcnD, YhdA
or Lot6p (8–10 ml, protein purity >90%, protein content 90–
110 mg/mL) was added to a Tris-HCl buffer solution (0.8 mL,
50 mM, pH 7.5) containing the substrate (5 mM) and H2O2
(10 mM). The reaction and workup was carried out as described
in the general procedure.
b-cyclodextrin capillary column (CP-Chirasil-DEX-CB, 25 m,
0.32 mm, 0.25 mm). Detector temperature 200 ◦C, injector
temperature 180 ◦C, split ratio 20:1. Temperature program for
rac-2,3-epoxy-3,5,◦5-trimethyl-1,4-◦cyclohexanedione (3b)◦: 90 ◦C,
◦
hold for 2 min, 4 C/min to 115 C, 20 C/min to 180 C, hold
for 2 min. Temperature program for rac-2,3-epoxy-2-methyl-
1,4-naphthoquinone (4b): 80 ◦C, hold for 2 min, 5 ◦C/min
to 140 ◦C, hold for 2 min, 20 ◦C/min to 160 ◦C, hold for
2 min. Retention times were as follows: rac-2,3-epoxy-3,5,5-
trimethyl-1,4-cyclohexanedione (3b) 7.29 min and 8.98 min,
ketoisopherone (3a) 8.65 min, menadione (4a) 16.80 min and
rac-2,3-epoxy-2-methyl-1,4-naphthoquinone (4b) 17.11 min and
17.44 min.
The enantiomeric excess of rac-methyl phenyl sulfoxide (6b) was
analysed by HPLC using a Chiralcel AD column (25 cm, 0.46 cm).
Eluent n-heptane/ethanol 95:5, flow 1 ml/min, temperature 24 ◦C
(40 min isocratic). Retention times were as follows: thioanisole
(6a) 3.74 min and rac-methyl phenyl sulfoxide (6b) 23.92 min and
25.58 min.
The enantiomeric excess of omeprazole (7b) was analysed by
HPLC using a Chiralcel AD column (25 cm, 0.46 cm). Eluent
n-heptane/isopropanol 65:35, flow 0.3 ml/min, temperature 18 ◦C
(60 min isocratic). Retention times were as follows: omeprazole
sulfide (7a) 20.13 min and omeprazole (7b) 26.50 min and
33.43 min.
Analytical procedures
Determination of conversion. Conversions for citral (1a) and
cyclohexenone (2a) were 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 250 ◦C, split
ratio 30:1. Temperature program for citral (1a) and cyclohexenone
(2a): 110 ◦C, hold for 5 min, 10 ◦C/min to 200 ◦C, hold for 2 min.
Retention times were as follows: diastereomeric-rac-2,3-epoxy-3,7-
dimethyl-6-octenal (1b) 9.10 min and 9.31 min, citral (E/Z) (1a)
9.54 min and 10.10 min, cyclohexenone (2a) 4.13 min and rac-2,3-
epoxy-1-cyclohexanone (2b) 5.97 min.
Conversions for ketoisopherone (3a), menadione (4a) and
thioanisole (6a) were analysed by GC-FID using a 6% cyano-
propyl-phenyl phase capillary column (Varian CP-1301, 30 m,
0.25 mm, 0.25 mm), detector temperature 250 ◦C, split ratio 30:1.
Temperature program for ketoisopherone (3a) and thioanisole
(6a): 110 ◦C, hold for 5 min, 30 ◦C/min to 200 ◦C, hold
for 2 min. Temperature program for menadione (4a): 160 ◦C,
5 ◦C/min to 180 ◦C, 20 ◦C/min to 250 ◦C. Retention times were
as follows: rac-2,3-epoxy-3,5,5-trimethyl-1,4-cyclohexanedione
(3b) 6.00 min, ketoisopherone (3a) 6.19 min, menadione
(4a) 5.55 min, rac-2,3-epoxy-2-methyl-1,4-naphthoquinone (4b)
5.78 min, thioanisole (6a) 4.98 min and rac-methyl phenyl
sulfoxide (6b) 8.51 min. The conversion of omeprazole sulfide
(7a) was analysed by HPLC using a Chiralcel AD column (25 cm,
0.46 cm). Eluent n-heptane/isopropanol 65:35, flow 0.3 ml/min,
temperature 18 ◦C (60 min isocratic). Retention times were as
follows: omeprazole sulfide (7a) 20.13 min and (R,S)-omeprazole
(7b) 26.50 min and 33.43 min.
Acknowledgements
Financial support by BASF AG (Ludwigshafen) is gratefully
acknowledged. Bernhard Hauer, Rainer Stu¨rmer, Steffen Maurer
(Ludwigshafen), Heidemarie Ehammer, Sonja Sollner and Peter
Remler (Graz) are cordially thanked for the provision of substrates
and enzymes, respectively, and thanks go to Christoph Goebl for
his excellent technical assistance.
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