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was allowed to warm to 08C and stirred for 20 min at this temper-
ature. The solvent was evaporated and a solution of methyl acry-
late (1 mL, 11.0 mmol) in DMSO (4 mL) was added to the residue.
The reaction was stirred for 20 h at ambient temperature. The mix-
ture was poured into cold, aqueous sulfuric acid (5%) and extract-
ed three times with diethyl ether (40 mL). The organic layers were
combined, washed with saturated aqueous NaHCO3 (20 mL) and
brine (20 mL), dried over MgCO3 and concentrated by evaporation
of the solvent. The residue was purified by column chromatogra-
phy (hexane/ethyl acetate 20:1), which yielded methyl 5-methyl-4-
oxotetrahydrofuran-3-carboxylate (990 mg, 6.26 mmol, 64%). TLC
results were viewed by using a KMnO4 staining solution or UV254
(Rf =0.34, hexane/ethyl acetate 2:1).
Experimental Section
General
TLC plates were run on silica gel Merck 60 (F254). Silica gel 60 from
Merck was also used for flash column chromatography. GC-MS
analyses were performed on an HP 6890 Series GC system
equipped with a 5973 mass selective detector and a 7683 Series in-
jector using a (5% phenyl) methylpolysiloxane capillary column
(HP-5MS, 30 mꢁ0.25 mm, 0.25 mm film). GC-FID analyses were car-
ried out on a Varian 3800 and on an Agilent 7890A by using H2 as
the carrier gas (14.5 psi). NMR measurements were performed on
a Bruker Avance III 300 MHz NMR spectrometer. Chemical shifts are
reported relative to trimethylsilane (TMS, d=0.00 ppm) and cou-
pling constants (J) are given in Hz.
Synthesis of (R)-2-methyldihydrofuran-3(2H)-one [(R)-48c][48]
General procedure for the nicotinamide-independent
anaerobic enzymatic C=C reduction reaction
Methyl
5-methyl-4-oxotetrahydrofuran-3-carboxylate
(200 mg,
1.3 mmol) was added to sulfuric acid (10%, 5 mL) and the mixture
was stirred for 3.5 h at 708C. The reaction mixture was then cooled
to ambient temperature, poured into saturated aqueous NaHCO3
(50 mL), and extracted three times with ethyl acetate (30 mL). The
organic layers were combined, washed with saturated aqueous
NaHCO3 (20 mL) and brine (20 mL), dried with MgSO4, concentrat-
ed by evaporation of the solvent and purified by column chroma-
tography (hexane/ethyl acetate, 5:1) to yield (R)-2-methyldihydro-
furan-3(2H)-one [(R)-48c, 20 mg, 0.2 mmol, 94% ee]. TLC results
were viewed by using a KMnO4 staining solution (Rf =0.36, hexane/
ethyl acetate 2:1). Spectroscopic data were in agreement with
those of the commercially available reference compound rac-48c.
An aliquot of the isolated enzyme (OYE1, OYE2, CrS, EBP1, NCR,
XenA, YqjM, GkOYE; protein purity >90%, protein content in reac-
tion 100 mgmLÀ1) was added to a screw-top glass vial (2 mL) con-
taining a degassed buffer solution (0.8 mL, 50 mm, tris(hydroxyme-
thyl)aminomethane·HCl (TrisHCl) buffer; pH 7.5 or pH 9), the sub-
strate (1a–6a, 10 mm), and the hydrogen donor (1c–52c; 10 mm).
The vial was flushed with argon, and sealed with a teflon-coated
septum and a lid. The mixture was shaken for 24 h at 308C and
120 rpm by using an Infors Unitron shaker and the products were
extracted with ethyl acetate (2ꢁ0.7 mL). The combined organic
phase was dried over Na2SO4 and analyzed on a GC to determine
the conversion and stereoselectivity. On a preparative scale, prod-
ucts could be easily separated from excess hydrogen donor and
phenolic byproducts by simple silica gel filtration due to the large
difference in Rf values.
Preparation of tert-butyl 3-oxo-2,3-dihydro-1H-pyrrole-1-car-
boxylate (46d)
An aliquot of isolated NCR (protein purity >90%, protein content
in reaction 200 mgmLÀ1) was added to 30 screw-top glass vials
(2 mL) containing a degassed buffer solution (0.8 mL, 50 mm,
TrisHCl buffer; pH 7.5 or pH 9), 4-ketoisophorone(1a, 30 mm),
acting as the hydrogen acceptor, and tert-butyl 3-oxopyrrolidine-1-
carboxylate (46c; 10 mm). The vials were flushed with argon,
sealed with a teflon-coated septum and a lid. The mixtures were
shaken for 24 h at 308C and 120 rpm by using an Infors Unitron
shaker. After the transformation, all phases were collected and the
products were extracted with ethyl acetate (2ꢁ30 mL). The com-
bined organic phase was dried over Na2SO4, concentrated, and the
product was purified by column chromatography (hexane/ethyl
acetate, 5:1) to yield tert-butyl 3-oxo-2,3-dihydro-1H-pyrrole-1-car-
boxylate (46d; 10.5 mg). TLC results were viewed by using
a KMnO4 staining solution (Rf =0.65, hexane/ethyl acetate 2:1).
1H NMR (300 MHz, CDCl3): d=8.33 (d, J=4.1 Hz, 2H), 5.65 (d, J=
4.2 Hz, 2H), 4.01 (s, 2H), 1.54 ppm (s, 9H).
Synthesis of a-(+)-3,4-epoxycarene[46]
A solution of meta-chloroperbenzoic acid (1.037 g, 6.0 mmol in
CHCl3 (12 mL)) was added dropwise to a stirred solution of
(+)-carene (0.508 g, 3.7 mmol) in chloroform (6 mL) over a period
of 75 min. The reaction was stirred for a further 40 min and then
quenched with aqueous sodium bisulfite (40%, 2 mL). The organic
layer was separated, washed with saturated aqueous NaHCO3
(15 mL) and brine (15 mL), dried with Na2SO4, and concentrated by
evaporation of the solvent to give a-(+)-3,4-epoxycarene as a light
yellow oil (0.561 g, 3.68 mmol).
Synthesis of (S)-3-isopropyl-6-methylcyclohex-2-enone
[(S)-13c][47]
Crude a-(+)-3,4-epoxycarene (355 mg, 2.6 mmol) was dissolved in
dichloromethane (10 mL) and cooled to À788C (N2(l)/EtOH). Trime-
thylsilyl triflate (TMSOTf; 44 mL) was added and the reaction was
stirred for 3 h. Saturated aqueous NaHCO3 (5 mL) and diethyl ether
(10 mL) were then added. The organic layer was separated,
washed twice with brine (10 mL), dried with Na2SO4 and concen-
trated by evaporation of the solvent to give (S)-3-isopropyl-6-meth-
ylcyclohex-2-enone [(S)-13c; 45 mg, 0.3 mmol, 12%, 25% ee]. Spec-
troscopic data were in agreement with those of the commercially
available reference compound rac-13c.
Synthesis of 2-methylfuran-3(2H)-one (48d)
2-Methyldihydrofuran-3(2H)-one (48c, 2 g, 20 mmol, 1.93 mL) was
dissolved in dry THF (100 mL) and cooled to À808C (liquid N2/
EtOH) under an argon atmosphere. N,N-Diisopropylethylamine
(7.78 g, 60 mmol, 10.4 mL) was then added over 10 min and the
mixture was stirred for 10 min, followed by slow addition of trime-
thylsilyl trifluoromethanesulfonate (8.89 g, 40 mmol, 7.23 mL) over
a further 10 min. The mixture was stirred and kept at between
À608C and À808C for 90 min and then allowed to warm to room
temperature over 90 min. The solution was then cooled to À608C,
and N-bromosuccinimide (4 g in 50 mL of dry THF) was added,
turning the yellow solution red. The mixture was stirred for 60 min
Synthesis of methyl 5-methyl-4-oxotetrahydrofuran-3-car-
boxylate[48]
Methyl l-lactate (1.0 g, 9.8 mmol) was dissolved in diethyl ether
(4 mL) and added to a cooled (À388C, N2(l)/EtOH) suspension of
NaH (267 mg, 50%, 5.6 mmol) in diethyl ether (6 mL). The mixture
Chem. Eur. J. 2014, 20, 1403 – 1409
1408ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim