First chemo-enzymatic synthesis of the (R)-Taniguchi lactone and substrate profiles of CAMO and OTEMO, two new Baeyer–Villiger monooxygenases

Article abstract of DOI:10.1007/s00706-016-1873-9

Abstract: This study investigates the substrate profile of cycloalkanone monooxygenase and 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase, two recently discovered enzymes of the Baeyer–Villiger monooxygenase family, used as whole-cell biocatalysts. Biooxidations of a diverse set of ketones were performed on analytical scale: desymmetrization of substituted prochiral cyclobutanones and cyclohexanones, regiodivergent oxidation of terpenones and bicyclic ketones, as well as kinetic resolution of racemic cycloketones. We demonstrated the applicability of the title enzymes in the enantioselective synthesis of (R)-(?)-Taniguchi lactone, a building block for the preparation of various natural product analogs such as ent-quinine. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1873-9

Monatsh Chem
DOI 10.1007/s00706-016-1873-9
ORIGINAL PAPER
First chemo-enzymatic synthesis of the (R)-Taniguchi lactone
and substrate profiles of CAMO and OTEMO, two new Baeyer–
Villiger monooxygenases
Florian Rudroff1
Michael J. Fink1,2 Ramana Pydi1 Uwe T. Bornscheuer3
Marko D. Mihovilovic1
•
•
•
•
Received: 4 October 2016 / Accepted: 6 November 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract
Abstract This study investigates the substrate profile of
cycloalkanone monooxygenase and 2-oxo-D3-4,5,5-
Graphical abstract
trimethylcyclopentenylacetyl-coenzyme
A monooxyge-
nase, two recently discovered enzymes of the Baeyer–
Villiger monooxygenase family, used as whole-cell bio-
catalysts. Biooxidations of a diverse set of ketones were
performed on analytical scale: desymmetrization of sub-
stituted prochiral cyclobutanones and cyclohexanones,
regiodivergent oxidation of terpenones and bicyclic
ketones, as well as kinetic resolution of racemic cycloke-
tones. We demonstrated the applicability of the title
enzymes in the enantioselective synthesis of (R)-(-)-
Taniguchi lactone, a building block for the preparation of
various natural product analogs such as ent-quinine.
Keywords Biotransformation Á
Baeyer–Villiger oxidation Á Taniguchi lactone
Introduction
Baeyer–Villiger
monooxygenases
are well-known
enzymes, promoting the oxidation of cyclic and linear
ketones into the corresponding lactones or esters by acti-
vating molecular oxygen in water at ambient temperature.
This enzyme family catalyzes the biological equivalent to
the chemical Baeyer–Villiger oxidation, discovered by
Adolf Bayer and Victor Villiger in 1899 [1], which requires
peracids and rather harsh reaction conditions. Major
drawbacks of the classic stoichiometric synthetic route are
the poor tolerance of other functional groups (e.g., towards
double bonds and heteroatoms), the need of potentially
explosive reagents (e.g., meta-chloroperbenzoic acid), and
the reduced possibility of chiral induction [2–10].
& Florian Rudroff
florian.rudroff@tuwien.ac.at
1
Institute of Applied Synthetic Chemistry, TU Wien,
Getreidemarkt 9/163-OC, 1060 Vienna, Austria
2
Present Address: Department of Chemistry and Chemical
Biology, Harvard University, 12 Oxford St, Cambridge, MA
02138, USA
The resulting esters and, especially if obtained optically
enriched, chiral lactones are promising building blocks for
the synthesis of valuable intermediates of drugs and natural
products [11]. During the last decades, applicability of
BVMOs has been demonstrated in multiple accounts:
3
Department of Biotechnology and Enzyme Catalysis,
Institute of Biochemistry, University of Greifswald, 17487
Greifswald, Germany
123

F. Rudroff et al.
desymmetrization of prochiral substrates, kinetic resolu-
tions of racemic ketones, and regioselective
and OTEMO in the phylogenetic tree (Fig. 1) of BVMOs
motivated our efforts of in-depth substrate profiling of
these new biocatalysts.
transformations [12–19]. We had previously profiled the
substrate scope of a range of BVMOs [20–24], allowing us
to correlate their stereopreference with protein sequence
[25]. This led to a significant clustering of BVMOs into
subgroups, and eventually to the development of a decision
guidance tool [24], enabling a pre-selection of best-per-
forming biocatalysts for a particular substrate class.
Furthermore, we wished to demonstrate the applica-
bility of both title enzymes by developing a new
biocatalytic approach for the synthesis of optically
pure (R)-(-)-Taniguchi lactone. The enantiomeric
(S)-(?)lactone [29] has been used as a building block for
the synthesis of various natural products, e.g., salicifo-
liol, threosyl-50-deoxyphosphonic acid, and purine
analogs [30], including the first stereoselective synthesis
of quinine [31]. Several attempts succeeded to obtain the
Taniguchi lactone in optically pure form via chiral res-
olution by chiral agents [32–34]. Synthetic accessibility
of the (R)-(-)-Taniguchi lactone will provide a novel
approach for the synthesis of, e.g., ent-quinine and ent-
salicifoliol, respectively.
Camphor monooxygenase (CAMO) from the ascomy-
cete Cylindrocarpon radicicola ATCC 11011 was recently
reported as the first recombinant BVMO originating from a
eukaryotic organism [26]; it possesses a general activity for
both cyclic and linear ketones. In contrast, recombinant
2-oxo-D3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A
monooxygenase (OTEMO) from Pseudomonas putida
NCIMB 10007 showed activity for cyclic ketones, prefer-
entially [27, 28]. Whenever new BVMOs are discovered, it
is of particular interest to know their activity, catalytic
performance, and selectivity in relation to previously
characterized enzymes. The distinct positions of CAMO
Here, we present a comprehensive substrate profile of
two novel BVMOs (CAMO, OTEMO) and the first chemo-
enzymatic synthesis of the (R)-(-)-Taniguchi lactone in
enantiopure form via a desymmetrization step.
Fig. 1 Phylogenetic tree of
BVMOs. The sequences of the
investigated enzymes CAMO
and OTEMO are highlighted.
CAMO belongs to the
CHMOAcineto subgroup,
whereas OTEMO clustered
separately from known groups
of correlated sequence and
stereopreference
123

First chemo-enzymatic synthesis of the (R)-Taniguchi lactone and substrate profiles of CAMO and…
including phenyl 3a and benzyl 4a residues (Scheme 1). The
enantiomeric ratio E was estimated using Sih’s equation.
E = [ln(1 - eeS) - ln(1 ? eeS/eeP)]/
Results and discussion
We investigated the sequence homology of CAMO and
OTEMO. Their divergent positioning in the phylogenetic
analysis (CAMO clustered with CHMO-type enzymes,
whereas OTEMO branched off at an early point; Fig. 1) sug-
gested a distinctly different substrate profile between the
biocatalysts. To determine the catalyst performance a diverse
array of substrates (e.g., 2- and 4-substituted cyclohexanones,
3-substituted cyclobutanones, and terpenones) was investi-
gated. The experiments were conducted on analytical scale and
the results obtained were compared and referenced to published
values. The substrates were grouped into kinetic resolutions,
regiodivergent oxidations, and desymmetrization reactions.
[ln(1 ? eeS) - ln(1 ? eeS/eeP)],
from ee of substrate (eeS) and ee of product (eeP)
values, and compared to already published reference
biotransformations (Table 1). Since CAMO is closely
related in sequence to CHMO-type enzymes, we expec-
ted its stereopreference to align with the cluster; the
hypothesis was found to be true with all tested com-
pounds of the class. Also, OTEMO had the same sense of
chiral induction. Nevertheless, except for the transfor-
mation of 2-phenylcyclohexanone with CAMO
(E [ 200), the title enzymes were only poorly selective
(E = 3–69), when compared to the best reference results
using CDMO or CHMOs.
Kinetic resolutions
We tested a set of a-substituted cyclohexanones, starting from
small methyl 1a, to allyl 2a, and more bulky substituents,
Regiodivergent biotransformations
The second set of substrates was composed of fused
bicyclic cyclobutanones and monocyclic terpenones to test
the regioselectivity of CAMO and OTEMO. For these
particular substrate classes the formation of two regioiso-
mers is often observed: the ‘‘normal’’ lactone (n), based on
the nucleophilicity-driven rearrangement, and the ‘‘abnor-
mal’’ lactone (abn) governed by stereoelectronic effects
(Scheme 2) [36].
Scheme 1
Table 1 Kinetic resolutions of 2-substituted cyclohexanones
Substrate
R
Comp. no
CAMO
OTEMO
Conv (%)a
% ee S/% ee P
Eb
Conv (%)a
% ee S/% ee P
Eb
Me
Allyl
Ph
1
2
3
4
56
46
48
54
86(-)/60(?)
53(-)/88(-)
96(-)/99(?)
62(-)/91(-)
11
28
33
60
32
58
23(-)/40 (?)
43(-)/50(-)
43(-)/95(?)
73(-)/94(-)
3
5
O
[200
41
64
69
R
Bn
Substrate
R
Comp. no
Reference biotransformation
Conv (%)a
% ee S/% ee P
Eb
125
Biocatalyst
References
Me
1
2
3
4
58
50
48
47
99(?)/92(-)
99(-)/99(-)
76(-)/99(?)
91(-)/99(-)
CDMO
[24]
[35]
[36]
[24]
Allyl
[200
[200
[200
CHMOAcineto
CHMOXantho
CDMO
O
Ph
R
Bn
a
Relative conversion (Conv) of starting material and enantiomeric excess values determined by chiral phase GC; sign of optical rotation is given
in parentheses and assigned on the basis of reference biotransformations; eeS (substrate), eeP (product)
b
Enantiomeric ratio E was estimated using Sih’s equation
123

F. Rudroff et al.
A second tier of substrates for regiodivergent reactions
consisted of optically pure terpenones (8–10a). Previous
studies revealed that CHMO family enzymes preferably
gave n lactones, whereas CPMO-type enzymes formed abn
lactones [10]. With those compounds, the preference of
migration strongly depends on the absolute configuration of
the starting material. We tested three different terpenones
8–10a with CAMO and OTEMO (Table 3). The former
showed poor regioselectivity; in contrast OTEMO was
highly selective in the oxidation of substrates 9a and 10a,
yielding the n lactones 9, 10b exclusively. These results
demonstrated that neither CAMO nor OTEMO behaved
like a classical CHMO- or CPMO-type BVMO.
Scheme 2
In general, fused bicyclic cyclobutanones are good
substrates for BVMO-mediated biooxidations due to the
highly favored alleviation of ring strain by insertion of an
oxygen atom (Table 2). Ketone 5a was fully converted by
CAMO and gave an equal ratio of n and abn lactones 5b/c.
Enantioselectivity for the n lactone was good [ee 88% (-)]
and excellent for the abn lactone [ee 99% (-)]. Again,
CAMO gave comparable results to previously published
data of CHMO-type BVMOs. In contrast, OTEMO gave
5b/c in a 70/30 ratio of n/abn with moderate stereoselec-
tivity for the n lactone [ee 46% (-)] and excellent optical
purity for the abn lactone [ee 99% (-)]. Contrasting results
were obtained for ketone 6a: whereas CAMO gave a 70/30
n/abn ratio with poor [6b: ee 41% (?)] to excellent [6c: ee
98% (?)] enantioselectivities, OTEMO transformations
yielded in a 1/1 ratio of abn/n lactone with perfect optical
purity for both regioisomers. This result is the best obtained
so far for this compound (6a). The last example of this
substrate class (7a) was fully converted by both enzymes,
with slight preference towards the abn lactone 7c. Optical
purities were moderate to poor for the n lactone 7b and
high for the abn lactone 7c for both enzymes.
Desymmetrization reactions
A series of 11 prochiral ketones was biooxidized by
CAMO and OTEMO in desymmetrization reactions
(Scheme 3; Table 4). All 3-substituted cyclobutanones 11–
15a were fully converted to the lactones 11–15b.
3-Vinylcyclobutanone 11a was transformed with perfect
stereoselectivity by both tested BVMOs (both giving (-)-
11b with 99% ee).
CAMO’s stereopreference for cyclobutanones 12–14a
was divergent from other CHMO-type BVMOs; it pro-
duces the enantiomers usually obtained by CPMO-type
transformations, but with much higher selectivity
(Table 4). Lactones 12b and 14b were thus obtained with
97% (?) and 95% (?) ee. The optical purity of lactones
13b, 15b was much higher than previously published
Table 2 Regiodivergent Baeyer–Villiger oxidations of fused and racemic (cis) bicyclic cyclobutanones
Substrate
Comp. no
CAMO
OTEMO
Reference biotransformation
% Conva/
ratiob
% eec
n/abn
% Conva/
ratiob
% eec
n/abn
% Conva/
ratiob
% eec
n/abn
Biocatalyst
References
5
???
88(-)/99(-)
41(?)/98(?)
46(?)/99(?)
???
46(-)/99(-)
97(?)/99(?)
20(?)/95(?)
???
97:03
???
51:49
???
87:13
???
70:30
???
75:25
???
97:3
rac./99(?)
CPMO
[37]
[25]
[37]
[37]
[37]
[37]
53:47
70:30
95(-)/99(-)
14(?)/99(?)
44(?)/99(?)
33(-)/56(-)
12(?)/88(?)
CHMOAcineto
CPMOComa
CHMOAcineto
CPMOComa
CHMOAcineto
6
7
???
???
70:30
50:50
???
???
68:32
89:11
rac. racemic
a
Relative conversion (Conv) of starting material determined by chiral phase GC after 24 h: ???[90%, ??50–90%, ?\50%
Ratio of regioisomers (normal:abnormal)
b
c
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation is given in parenthesis and assigned on the basis of reference
biotransformations
123

First chemo-enzymatic synthesis of the (R)-Taniguchi lactone and substrate profiles of CAMO and…
Table 3 Regiodivergent transformations of optically pure terpenones
Substrate
Comp. no
CAMOc
OTEMOc
Reference biotransformationc
% Conva/
ratiob
% Conva/
ratiob
% Conva/
ratiob
Biocatalyst
References
8
???
???
???
0:100
??
CHMOAcineto
CHMOBrevi1
[22]
[22]
53:47
11:89
51/49
9
???
???
???
100:0
???
0:100
CHMOAcineto
CPMOComa
[22]
[22]
70:30
100:0
10
???
???
???
CHMOAcineto
[22]
68:32
100:0
100:0
a
Relative conversion (Conv) of starting material determined by chiral phase GC after 24 h: ???[90%, ??50–90%, ?\50%
Ratio of regioisomers (normal:abnormal)
b
c
Enantiomeric excess [99%
The latter result again exceeded the best previously
obtained value with wild-type BVMOs.
Scheme 3
Substrates 20a and 21a were transformed with very
good enantioselectivity by both enzymes, as commonly
found with other BVMOs.
Chemo-enzymatic synthesis of (R)-(2)-Taniguchi
lactone
values [13b = 94% (-) vs 63% (-) and 15b = 92% (?)
vs 73% (?)]. These lactones are important intermediates
for the synthesis of tricyclic benzomorphan analogs, (?)-
harzialactone A, chiral tricyclic amines [38–40], and for
the synthesis of lignans such as enterolactone, hinokinin,
arctigenin [41], the synthesis of optically pure gosmin A
and schizandrin [42]. In all cases, OTEMO had the same
stereopreference as CAMO, but was less stereoselective.
Both enzymes fully converted all tested 4-substituted
cyclohexanones (substrates 16–21a). Remarkably, lactone
17b was formed with perfect enantiomeric purity [ee 99%,
(?)] using CAMO, thus surpassing the best known result.
Both optical antipodes of lactone 18b could be obtained by
CAMO and OTEMO, respectively: whereas CAMO gave
the levorotatory isomer with 77% ee, OTEMO yielded in
the antipodal product with 98% ee. A similar trend was
observed for substrate 19a, where CAMO produced (-)-
19b with 95% ee, and OTEMO gave (?)-19b with 96% ee.
After identification of CAMO and OTEMO as excellent
catalysts for the enantioselective synthesis of lactone 11b,
we investigated a new chemo-enzymatic route to Taniguchi
lactone 11b, starting from readily available 1,3-butadiene.
This lactone is a valuable chiral building block for the
synthesis of several natural products (Scheme 4).
We performed
a
[2 ? 2] cycloaddition using
dichloroketone, generated in situ from trichloroacetyl chlo-
ride in the presence of activated zinc [46]. Subsequent
reduction of the dichloroketone led to 3-Vinylcyclobutanone
11a in 44% over two steps. We then screened a library of
cycloketone-converting BVMOs, composed of 13 wild-type
enzymes, plus mutants of CHMOAcineto and CPMOComa, that
had been generated in previous studies [47, 48].
Most wild-type BVMOs gave the desired Taniguchi
lactone with excellent conversion and optical purity
(ee [ 95%), exclusively with (R)-configuration (data not
shown). All mutants of CHMOAcineto that where designed
123

F. Rudroff et al.
Table 4 Desymmetrizations of substituted prochiral cycloketones 11–21a
Structure
R
Comp. no CAMO
OTEMO
Reference biotransformation
Conv (%)a ee (%)b Conv (%)a ee (%)b Conv (%)a ee (%)b Biocatalyst
References
3-Vinyl
Bn
11
12
100
99
99(-)
97(?)
100
98
99(-)
11(-)
n.a.
n.a.
n.a.
This work
[43]
???
???
???
???
???
???
???
44(?)
93(-)
63(-)
24(?)
79(?)
99(-)
64(?)
HAPMO
CHMOBrevi1
CPMOComa
CPMOComa
3-(3-MeOBn)
3-(4-MeOBn)
13
14
100
95
94(-)
95(?)
92(?)
99(-)
100
100
87
82(-)
64(?)
63(?)
58(?)
[43]
[43]
3-(3,4,5-tri-MeOBn) 15
100
100
CHMOBrevi1 [43]
Me
16
100
CDMO
[24]
[44]
CPMOComa
OH
17
100
[99(?) n.a.
n.a.
???
???
???
???
???
99(-)
44(?)
99(-)
99(-)
64(?)
CHMOXantho [24]
CHMOBrevi2
tBu
18
19
98
99
77(-)
95(-)
100
57
98(?)
96(?)
CPDMO
[18]
[18]
[18]
COOEt
CPDMO
CPMOComa
H, H
20
21
100
100
97(-)
96(?)
100
100
96(-)
94(?)
???
???
99(-)
96(?)
CDMO
[24]
H, OH (trans)
CHMOAcineto [45]
n.a. not applicable
a
Relative conversion (Conv) of starting material determined by chiral phase GC after 24 h: ???[90%, ??50–90%, ?\50%
Enantiomeric excess values determined by chiral phase GC; sign of optical rotation is given in parenthesis and assigned on the basis of
reference biotransformations
b
Scheme 4
for enantiodivergence towards cyclobutanones and cyclo-
hexanones, showed a reduced stereospecificity, and no
inversion of the selectivity was observed with any biocat-
alyst. A similar trend was seen using the variants of
CPMOComa. We then performed biotransformation on a
preparative scale (50 mg) with CAMO (57% isolated yield)
and OTEMO (60% isolated yield) to confirm the results
obtained in the screening reactions on the analytical scale.
Overall, we were able to synthesize enantiomerically pure
Taniguchi lactone 11b in 26% yield over three steps
having ee 99% (-) optical purity by biocatalytic
desymmetrization.
123

First chemo-enzymatic synthesis of the (R)-Taniguchi lactone and substrate profiles of CAMO and…
optical density of 0.2–0.6 was reached. Then, 2 cm3 of
Conclusion
fermentation medium was charged to 12-well plates and
IPTG (0.2 mM) was added. Substrates were added as
0.8 M solutions in 1,4-dioxane to a final concentration of
4 mM. The plates were sealed with adhesive film and
incubated at 24 °C and 200 rpm in an orbital shaker for up
to 24 h. Analytical samples were prepared by extraction of
0.5 cm3 of fermentation mixture with 1.0 cm3 of EtOAc
(supplemented with 1 mM methyl benzoate as internal
standard) after centrifugation to separate the biomass.
In this work, we reported the substrate scope and compared
the performance of CAMO and OTEMO as whole-cell
biocatalysts overexpressed in E. coli. We investigated
commercially available and in-house synthesized racemic
2-substituted cyclohexanones, fused bicyclic cyclobu-
tanones,
optically
pure
terpenones,
prochiral
cyclobutanones and cyclohexanones, all either serving as
model substrates or intermediates in the synthesis of nat-
ural compounds. The biocatalysts gave remarkable results
in the desymmetrization of cyclobutanones and -hex-
anones, in some cases improving the enantioselectivity
significantly over prior best values. The other reaction
classes were generally not converted with a performance
equal to reference BVMOs.
3-Vinylcyclobutanone (11a)
1,3-Butadiene (1 g, 18.5 mmol, 49 cm3 of a 15 wt% solu-
tion of 1,3-butadiene in hexane) was added to freshly
prepared and dry Cu/Zn couple [49] (3.5 g, 52 mmol) at 0 °C
under Ar atmosphere. A mixture of 9.5 g trichloroacetyl
chloride (52 mmol) and 7.8 g POCl3 (50 mmol) was added
dropwise at 0 °C under argon within 2–4 h. The mixture was
stirred overnight at r.t., zinc salts were removed by filtration
through a pad of Celite; the filtrate was diluted with 150 cm3
of diethyl ether and carefully diluted with 100 cm3 of cold
water. The organic phase was separated and washed with aq.
NaHCO3 (4 9 50 cm3), then with 100 cm3 brine and dried
over anhydrous Na2SO4. The solvent was removed in vacuo
and the residue was used in the next step without further
purification.
A biocatalytic route for the synthesis of optically pure
(R)-(-)-Taniguchi lactone was established, starting from
1,3-butadiene via biooxidation of 3-Vinylcyclobutanone
with CAMO and OTEMO as recombinant whole-cell cat-
alysts. This synthesis is an interesting alternative approach
to known preparations of the target lactone.
Experimental
The crude residue (2.1 g) was dissolved in 10 cm3 acetic
acid and added dropwise to a suspension of zinc in 30 cm3
acetic acid within 1 h, followed by heating to 70–80 °C for
another hour. The reaction mixture was cooled to r.t.,
filtered through a pad of Celite, diluted with 100 cm3
water, and extracted with dichloromethane (4 9 50 cm3).
The combined organic phases were washed with saturated
aq. NaHCO3 (4 9 50 cm3), then with 100 cm3 brine, dried
over anhydrous Na2SO4, and the solvent was removed in
vacuo to obtain the desired product 11a as a yellow oil.
Yield: 44% (782 mg) over two steps; 1H NMR (200 MHz,
CDCl3): d = 6.02 (ddd, 1H), 5.01–5.36 (m, 2H), 3.39 (s,
1H), 2.89–3.36 (m, 4H) ppm; 13C NMR (50 MHz, CDCl3):
d = 206.7, 140.2, 114.6, 52.6, 26.9 ppm.
All purchased reagents were used without further purifi-
cation. All solvents were distilled prior to use. Substrates
and reference compounds for substrate acceptance screen-
ing were synthesized according to literature-known
protocols. Silica gel 60 was used for column chromatog-
raphy and progress of the reactions was monitored by thin
layer chromatography (TLC). The analysis and identifica-
tion of all molecules were conducted using chiral phase GC
(BGB 173 and BGB 175 Column: 30 m 9 0.25 mm ID,
0.25 lm film). NMR spectra were recorded on a Bruker
AC 200 (200 MHz) spectrometer. Specific rotation was
determined using an Anton Paar Polarimeter MCP500. LB
medium was not buffered and used without pH correction;
ampicillin and kanamycin concentrations were set to
100 mg/cm3 (LBamp and LBkan). Cloning, expression and
characterization of CAMO [26] and OTEMO [27, 28] is
described elsewhere.
rac-4-Vinyldihydrofuran-2(3H)-one (Taniguchi lactone)
(11b)
2-Buten-1,4-diol (6 g, 68 mmol), 22 g triethyl orthoacetate
(136 mmol), and 2.25 g p-hydroquinone (20.4 mmol) were
stirred at 120 °C. Generated ethanol was removed using a
Dean-Stark apparatus. After the condensation of ethanol
had ceased, the temperature was increased to 150 °C for
24 h. The crude residue was isolated via distillation under
reduced pressure (60–70 °C, 36 mbar) and the product was
obtained by column chromatography rac-11b as a yellow
oil to serve as reference material for chiral analytics. Yield:
Substrate acceptance screening reactions
A baffled Erlenmeyer flask was charged with 3 cm3 LBamp
medium, inoculated with a bacterial single colony from an
agar plate, and incubated at 37 °C and 200 rpm in an
orbital shaker overnight. LBamp medium (10 cm3) was
inoculated with 1% (v/v) of the overnight culture and
incubated for 1–2 h under the same conditions until an
1
42% (3.35 g); H NMR (200 MHz, CDCl3): d = 5.76 (m,
123

F. Rudroff et al.
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1H), 5.07–5.27 (m, 2H), 4.42 (m, 1H), 4.00 (m, 1H), 3.21
(m, 1H), 2.65 (m, 1H), 2.36 (m, 1H) ppm; 13C NMR
(50 MHz, CDCl3): d = 176.5, 135.7, 117.4, 72.1, 39.7,
34.1 ppm.
(R)-4-Vinyldihydrofuran-2(3H)-one (Taniguchi lactone)
(11b)
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An overnight culture with LBkan medium (3 cm3), inocu-
lated with a bacterial single colony from an agar plate and
incubated at 37 °C and 200 rpm in an orbital shaker was
prepared. A baffled Erlenmeyer (500 cm3) flask was
charged with 100 cm3 of LBkan medium and inoculated
with 1% (v/v) of the overnight culture and incubated for
1–2 h under the same conditions until an optical density of
0.2–0.6 was reached. IPTG (0.2 mM) and 50 mg ketone
11a (0.52 mmol, dissolved in 200 mm3 of 1,4-dioxane)
was added. The reaction flask was incubated at 24 °C and
200 rpm in an orbital shaker until full conversion was
determined via GC analysis (18–24 h). After completion of
the reaction, the reaction mixture was centrifuged (12000 x
g, 15 min, 4 °C). The supernatant was extracted with
EtOAc (5 9 40 cm3), and the pooled organic phases were
dried over anhydrous Na2SO4, and concentrated. The
product was purified by column chromatography using
light petroleum and EtOAc. Yields: 57% (33 mg) for
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1
CAMO and 60% (35 mg) for OTEMO. H NMR and 13C
NMR were identical to the racemic Taniguchi lactone [32].
25
[a]D = -7.0 (c = 1 in CHCl3), 99% ee.
¨
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Acknowledgements Open access funding provided by Austrian
Science Fund (FWF). This work was funded by the Austrian Scien-
tific Foundation FWF (Grant No. I-723-N17). M. J. F. was funded
within the EU-FP7 program Oxygreen (Grant #212281).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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