Stereoselective Reduction of Prochiral Cyclic 1,3-Diketones Using Different Biocatalysts

Article abstract of DOI:10.1007/s10562-019-03015-y

We have developed biocatalytic methods for the stereoselective reduction of cyclic prochiral 1,3-diketones for the production of optically active β-hydroxyketones and/or 1,3-diols. The recombinant ketoreductase KRED1-Pglu (formulated as purified catalyst)

Full text of DOI:10.1007/s10562-019-03015-y

Catalysis Letters
https://doi.org/10.1007/s10562-019-03015-y
Stereoselective Reduction of Prochiral Cyclic 1,3‑Diketones Using
Diferent Biocatalysts
Martina Letizia Contente1,3 · Federica Dall’Oglio2 · Francesca Annunziata2 · Francesco Molinari3 · Marco Rabufetti3 ·
Diego Romano3 · Lucia Tamborini2 · Dörte Rother4 · Andrea Pinto3
Received: 22 August 2019 / Accepted: 27 October 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
We have developed biocatalytic methods for the stereoselective reduction of cyclic prochiral 1,3-diketones for the production
of optically active β-hydroxyketones and/or 1,3-diols. The recombinant ketoreductase KRED1-Pglu (formulated as purifed
catalyst) and whole cells of wild type Escherichia coli DE3 Star were used as biocatalysts, displaying diferent and some-
times complementary stereoselectivity, thus allowing the preparation of stereochemically pure β-hydroxyketones (12–66%
isolated yields,>99% e.e.) and 1,3-diols (40–60% isolated yields,>99% e.e.).
Graphic Abstract
Keywords Biocatalytic reduction · 1-3 diketones · β-hydroxyketones · 1,3-Diols · Enzymatic · Whole cells
Martina Letizia Contente and Federica Dall’Oglio have
contributed equally to this work.
1 Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10562-019-03015-y) contains
Biocatalytic reduction of prochiral diketones is an attractive
supplementary material, which is available to authorized users.
method for the preparation of optically active hydroxyke-
tones [1–3] and diols [4–6]. Chiral diols have a wide range
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

M. L. Contente et al.
of synthetic applications [7, 8] and can be obtained using
diketoreductases (DKR), which are enzymes able to reduce
both carbonyl groups of diketones [4]; these enzymes have
been used for diferent biotransformations, including the
preparation of (3R,5S)-ethyl-6-(benzyloxy)-3,5-dihydroxy-
hexanoate, the chiral side chain of antihypertensive rosuv-
astatin, using a DKR from Acinetobacter baylyi [4]. Alter-
natively, monoreduction of 1,2-diketones and 1,3-diketones
afords α- and β-hydroxyketones, respectively, which are
highly valuable chiral synthons for the synthesis of natural
products and pharmaceuticals [9–11].
2,2-disubstituted 1,3-cyclohexanediones using a heterolo-
gously expressed ketoreductase (KRED1-Pglu) in a purifed
formulation [22, 23]. KRED1-Pglu has been previously used
as recombinant protein over-expressed in a whole cell system
[24, 25], free enzyme [26], and immobilized one onto aga-
rose beads [27] for the stereoselective reduction of several
aromatic and cyclic mono- and diketones. Whole cells of E.
coli DE3 Star were also used as biocatalyst for the reduc-
tion of the same substrates, exploiting the ketoreductase(s)
expressed in the cells (Scheme 1).
Regio- and stereoselective reductions of diketones aford-
ing diferent enantiopure hydroxy ketones or diols can be
catalyzed by isolated alcohol dehydrogenases (ADHs)
[12–16]. Linear aliphatic diketones are reduced by ADHs
with diferent selectivity leading to ketols or diols depend-
ing on the substrate-binding modes in the active site of
the enzyme [16]. Preparation of chiral 1,4-diaryl-1,4-diols
from sterically hindered 1,4-diaryl-1,4-diketones has been
accomplished using the ADH from Ralstonia sp. [17]; in
this case, diols were the main products with no signifcant
accumulation of the intermediate hydroxyketones. Enzy-
matic reduction of 2,2-disubstituted 1,3-alkanediones (with
diferent substitution patterns on the tetrahedral carbon C2)
is remarkably attractive from a synthetic point of view, since
the biocatalyst may accomplish enantioselective hydrogen
transfer with simultaneous stereo-discrimination between
the two groups bond to the tetrahedral center C2, thus giving
one stereoisomer as major fnal product. Diferent 2,2-dis-
ubstituted 1,3-cycloalkanediones were reduced using Sac-
charomyces cerevisiae giving good yields and sometimes
excellent stereoselectivity, with formation, in all the cases,
of the stereoisomers having (S)-confguration at the carbon
bearing the hydroxy group [18, 19]. An example of indus-
trial relevant application of this type of stereoselectivity is
ofered by the reduction of ethyl secodione, which can be
enzymatically reduced to (13R,17S)-ethyl secol, applied for
the synthesis of contraceptive hormones [20, 21].
2 Materials and Methods
2.1 Chemicals
All reagents and solvents were purchased from
Sigma–Aldrich. Flash column chromatography was per-
formed on Merck Silica Gel (200 and 400 mesh).
2.2 Analytical Methods
1H NMR spectra were recorded with a Varian Mercury 300
(300 MHz) spectrometer. Chemical shifts (δ) are expressed
in ppm, and coupling constants (J) are expressed in Hz.
Gas-chromatographic (GC) analysis were carried out
using a Carlo Erba Fractovap GC (FID detector) equipped
with a fused-silica capillary column MEGA-DEX DMP-Beta
(dimethyl pentyl-β-cyclodextrin; 25 m×0.25 mm i.d.), with
the injector temperature at 200 °C. Diferent temperature
gradients were applied depending on the biotransformation:
Bioreduction of 3: at 90 °C for 10 min, then a temperature
gradient of 4 °C/min to 180 °C;
bioreduction of 4: at 80 °C for 10 min, then a temperature
gradient of 2 °C/min to 180 °C;
bioreduction of 5: at 90 °C for 10 min, then a temperature
gradient of 3 °C/min to 180 °C;
In this work, we have studied the reduction of vari-
ous 2,2-disubstituted 1,3-cyclopentanediones and
bioreduction of 6: at 90 °C for 10 min, then a temperature
gradient of 2 °C/min to 180 °C;
Scheme 1 Aim of the work
R
R
R
O
OH
HO
OH
O
OH
+
+
KRED1-Pglu
(CH2)n
(CH2)n
(CH2)n
R
O
O
(CH2)n
R
O
OH
E.coli BL21DE3 Star
(CH2)n
1 3

Stereoselective Reduction of Prochiral Cyclic 1,3-Diketones Using Diferent Biocatalysts
bioreduction of 7: at 90 °C for 10 min, then a temperature
gradient of 3 °C/min to 180 °C;
4.4 mmol). Rf = 0.55 (7:3 cyclohexane/ethyl acetate);
1H NMR (CDCl3, 300 MHz): δ 0.85 (3 H, t, J = 7 Hz),
1.12 (3 H, s), 1.15-1.30 (2 H, m), 1.55–1.65 (2H, m),
2.70–2.80 (4H, m). Anal. Calcd for C9H14O2: C, 70.10;
H, 9.15. Found C, 69.95; H, 9.25. Chiral GC retention
time: 16.2 min.
bioreduction of 8: at 90 °C for 10 min, then a temperature
gradient of 2 °C/min to 180 °C.
Specifc optical rotation measurements were carried out
using a Jasco P-1010 spectropolarimeter, coupled with a
Haake N3-B thermostat. Elemental analyses were carried
out on a Carlo Erba Model 1106 (Elemental Analyzer for
C, H, and N), and the obtained results are within 0.4% of
theoretical values.
2-Methyl-2-propylcyclohexane-1,3-dione (6) [18]: a
solution of compound 4 (700 mg, 4.2 mmol) was prepared
in MeOH (70 mL) and it was submitted to hydrogena-
tion in a ThalesNano H-Cube Mini (T = 30 °C, P = 1 bar,
CatCart Pd/C 5%, fow rate: 0.5 mL/min). The reaction
was monitored by TLC (7:3 cyclohexane/EtOAc). The
exiting fow stream was collected and MeOH was evapo-
rated. Compound 6 was obtained in 95% yield (671 mg,
3.9 mmol). Rf = 0.55 (7:3 cyclohexane/ethyl acetate);
1H NMR (CDCl3, 300 MHz): δ 0.87 (3H, t, J = 7 Hz),
1.05–1.25 (2H, m), 1.28 (3H, s), 1.70–1.95 (3H, m),
1.95–2.10 (1H, m) 2.58–2.80 (4H, m). Anal. Calcd for
C10H16O2: C, 71.39; H, 9.59. Found C, 71.42; H, 9.48.
Chiral GC retention time: 25.8 min.
2.3 Synthesis and Characterization of Substrates
2-Allyl-2-methylcyclopentane-1,3-dione (3) [18]: to a solu-
tion of compound 1 (673 mg, 6.0 mmol) in 1 M NaOH
(6.0 mL) allyl bromide was added (1.45 g, 12.0 mmol). The
reaction was stirred for 24 h at room temperature, and moni-
tored by TLC (9:1 DCM/MeOH). The mixture was extracted
with dichloromethane (5 × 5 mL) and the organic phase
was dried over Na2SO4, fltered and evaporated. The crude
extract was then purifed by column chromatography (9:1
cyclohexane/ethyl acetate) to obtain compound 3 as a pale-
yellow oil (yield: 50%, 470 mg, 3.0 mmol). Rf =0.55 (7:3
cyclohexane/ethyl acetate); 1H NMR (CDCl3, 300 MHz): δ
1.10 (3H, s), 2.35 (2 H, d, J = 8 Hz), 2.60-2.80 (4 H, m),
5.00–5.10 (2 H, m), 5.50–5.70 (1 H, m). Anal. Calcd for
C9H12O2: C, 71.03; H, 7.95. Found: C, 71.12; H, 7.83. Chiral
GC retention time: 15.3 min.
2-Methyl-2-benzylcyclopentane-1,3-dione (7) [28]: to a
solution of compound 1 (650 mg, 5.8 mmol) in 1 M NaOH
(5.8 mL) benzyl bromide was added (1.98 g, 11.6 mmol).
The reaction was stirred for 36 h at room temperature
and monitored by TLC (7:3 cyclohexane/EtOAc). The
mixture was then extracted with ethyl acetate (3 × 5 mL)
and the organic phase was dried over Na2SO4 and evap-
orated. The crude extract was purifed by column chro-
matography (9:1 cyclohexane/EtOAc). Compound 7 was
obtained as a white solid (yield: 50%, 587 mg, 2.9 mmol);
2-Allyl-2-methylcyclohexane-1,3-dione (4) [18]: to a
solution of compound 2 (757 mg, 6.0 mmol) in 1 M NaOH
(6.0 mL) allyl bromide was added (1.45 g, 12.0 mmol).
The reaction was stirred for 24 h at room temperature, and
monitored by TLC (9:1 DCM/MeOH). The mixture was
extracted with dichloromethane (3×5 mL) and the organic
phase was dried over Na2SO4, fltered and evaporated. The
crude extract was then purifed by column chromatography
(8:2 cyclohexane/EtOAc) to obtain compound 4 as a pale-
yellow oil (yield: 50%, 500 mg, 3.0 mmol). Rf =0.55 (7:3
cyclohexane/ethyl acetate); 1H NMR (CDCl3, 300 MHz): δ
1.25 (3 H, s), 1.80–1.95 (1H, m), 1.95–2.05 (1 H, m), 2.45-
2.55 (2 H, d, J=8 Hz), 2.60–2.68 (4H, m), 5.00–5.10 (2H,
m), 5.50–5.63 (1H, m). Anal. Calcd for C10H14O2: C, 72.26;
H, 8.49. Found: C, 72.34; H, 8.29. Chiral GC retention time:
22.7 min.
1
mp 50 °C; Rf = 0.58 (7:3 cyclohexane/ethyl acetate); H
NMR (CDCl3, 300 MHz): δ 1.20 (3H, s), 2.00–2.10 (2H,
m), 2.50–2.60 (2H, m), 2.95 (2H, s), 7.00–7.05 (2H, m),
7.20–7.30 (3H, m). Anal. Calcd for C13H14O2: C, 77.20;
H, 6.98. Found C, 77.42; H, 7.06., Chiral GC retention
time: 18.1 min.
2-Methyl-2-benzylcyclohexane-1,3-dione (8) [29]: to a
solution of compound 2 (650 mg, 5.2 mmol) in 1 M NaOH
(5.2 mL) benzyl bromide was added (1.78 g, 10.4 mmol).
The reaction was stirred for 36 h at room temperature and
monitored by TLC (7:3 cyclohexane/EtOAc). The mix-
ture was then extracted with ethyl acetate (3 × 5 mL) and
the organic phase was dried on Na2SO4 and evaporated.
The crude extract was purifed by column chromatogra-
phy (9:1 cyclohexane/EtOAc). Compound 8 was obtained
as a white solid (yield: 50%, 562 mg, 2.6 mmol); mp
2-Methyl-2-propylcyclopentane-1,3-dione (5) [18]: a
solution of compound 3 (700 mg, 4.6 mmol) was prepared
in MeOH (70 mL) and it was submitted to hydrogenation
in a ThalesNano H-Cube Mini (T = 30 °C, P = 1 bar, Cat-
Cart Pd/C 5%, fow rate: 0.5 mL/min). The reaction was
monitored by TLC (7:3 cyclohexane/EtOAc). The exit-
ing fow stream was collected and the solvent was evapo-
rated. Compound 5 was obtained in 95% yield (674 mg,
1
43–45 °C; Rf = 0.58 (7:3 cyclohexane/ethyl acetate); H
NMR (CDCl3, 300 MHz): δ 1.28 (3H, s), 1.42–1.55 (1H,
m), 1.67–1.80 (1H, m), 2.25–2.38 (2H, m), 2.50–2.60 (2H,
m), 3.12 (2H, s), 7.00–7.08 (2H, m), 7.18–7.30 (3H, m).
Anal. Calcd for C14H16O2: C, 77.75; H, 7.46. Found C,
77.53; H, 7.28. Chiral GC retention time: 46.7 min.
1 3

M. L. Contente et al.
at 21000 rpm for 30 min at 4 °C. The enzyme was purifed
by afnity chromatography with HIS-Select® Nickel Afn-
ity Gel using an ÄKTӒ system. Briefy, the column was
equilibrated with 50 mM phosphate bufer, 300 mM NaCl,
pH 8.0, 20 mM imidazole and the crude extract loaded. Sub-
sequently, the column was washed with 50 mM phosphate
bufer, 300 mM NaCl, 20 mM imidazole, pH 8.0. Finally, the
adsorbed enzyme was eluted with 50 mM phosphate bufer,
300 mM NaCl, 250 mM imidazole, pH 8.0. Pellet, crude
extract and collected fractions were analyzed by SDS-PAGE.
The fractions showing the presence of a band of the expected
size (27 kDa) were pooled, dialyzed against 50 mM phos-
phate bufer pH 8.0, and stored at −20 °C. After 24 h, frozen
samples were lyophilized. From 4 g of whole cells 600 mg
of lyophilized enzyme were obtained.
2.4 Preparation of Recombinant Escherichia coli
BL21 (DE3) Star Cells Expressing KRED1‑Pglu
The gene sequence of KRED1-Pglu (GenBank: AKP95857)
was previously identifed from the genome of the non-con-
ventional yeast Pichia glucozyma [26] (subsequently re-
classifed as Ogataea glucozyma). Cultures of E. coli BL21
(DE3) Star containing plasmid pKJE7 of Takara Chaperone
plasmid set (for dnaK-dnaJ-grpE expression) transformed
with the plasmid pET26 KRED1-Pglu [30] were pre-inocu-
lated in 100 mL Erlenmeyer bafed fasks containing 20 mL
of LB medium [yeast extract (5 g L−1), tryptone (10 g L−1)
and NaCl (10 g L−1)] supplemented with 25 μg/mL kana-
mycin and 25 μg/mL chloramphenicol. After 16 h of growth
(37 °C at 150 rpm) the starting culture was inoculated in the
200 mL of TB medium [yeast extract (24 g L−1), tryptone
(12 g L−1), glycerol (4 g L−1), 10% of 100 mM phosphate
bufer pH 7.0] supplemented with 25 μg/mL kanamycin,
25 μg/mL chloramphenicol and 0.5 mg/mL L−1 arabinose to
an initial OD600 of 0.1. The new culture was grown at 37 °C,
90 rpm until OD600 of 0.5. The induction was performed
with isopropyl-β-D-thiogalactopyranoside (IPTG) fnal con-
centration of 0.5 mM. After 72 h at 10 °C and 90 rpm cells
were harvested by centrifugation (8000 rpm, 4 °C, 45 min),
washed once with 20 mM phosphate bufer pH 7.0 and
stored at −20 °C.
2.7 KRED1‑Pglu Activity Test
Activity measurements were performed following a protocol
previously reported [26]. The test is based on a spectropho-
tometric assay at 340 nm by determining the consumption
of NAD(P)H at 25 °C in a half-microcuvette (total vol-
ume 1 mL) for 5 min. One unit (U) of activity is defned
as the amount of enzyme which catalyzes the consumption
of 1 μmol of NAD(P)H per minute under reference condi-
tions, namely with 0.25 mM NAD(P)H and 0.47 mM benzyl
as substrate (added as concentrated DMSO solution; fnal
DMSO concentration in cuvette is 0.1% v/v), in 50 mM
Tris–HCl bufer, pH 8.0.
2.5 Preparation of Lyophilized Wild Type E. coli
BL21 DE3 Star cells
2.8 General Procedure for Bioreductions
with Lyophilized Cells
Cultures of E. coli BL21 (DE3) Star containing plasmid
pKJE7 of Takara Chaperone plasmid set (for dnaK-dnaJ-
grpE expression) were pre-inoculated in 100 mL Erlenmeyer
bafed fasks containing 20 mL of LB medium [yeast extract
(5 g L−1), tryptone (10 g L−1) and NaCl (10 g L−1)] sup-
plemented with 25 μg/mL chloramphenicol. After 16 h of
growth (37 °C at 150 rpm) the starting culture was inocu-
lated in the 200 mL of TB medium [yeast extract (24 g L−1),
tryptone (12 g L−1), glycerol (4 g L−1), 10% of 100 mM
phosphate bufer pH 7.0] supplemented with 25 μg/mL chlo-
ramphenicol to an initial OD600 of 0.1. After 16 h at 37 °C,
90 rpm, microbial cells were harvested by centrifugation
(8000 rpm, 4 °C, 45 min), washed once with 20 mM phos-
phate bufer pH 7.0, lyophilized and stored at −20 °C.
A solution of the selected substrate (3–8) (14 mM) was pre-
pared in 20 mL of phosphate bufer 50 mM pH 8.0 with 1%
of CHCl3. In the same solution glucose (40 mM), NADP+
(0.1 mM) and 1 U mL−1 of GDH from Bacillus megate-
rium for cofactor regeneration and lyophilized E. coli cells
BL21 DE3 Star-pKJE7 (5 mg/mL); compound 5 was tested
in the same reaction conditions with E. coli cells BL21 DE3
Star- pKJE7 expressing heterologous KRED1-Pglu. After
72 h, the reactions were acidifed with HCl 2 N, extracted
with ethyl acetate (3 × 10 mL) and the organic phase was
dried over Na2SO4. After removal of the solvent, a chroma-
tographic column was performed to separate the unreacted
substrate from the products (8:2 cyclohexane/ethyl acetate).
2.6 Purifcation of KRED1‑Pglu Enzyme
2.9 General Procedure for Bioreductions
For the expression of KRED1-Pglu enzyme, the hosting cells
of E. coli BL21 DE3 Star-pKJE7 were prepared as described
above. Cells were suspended in 50 mM phosphate bufer
and 300 mM NaCl at pH 8.0, 20 mM imidazole. Proteins
were extracted by sonication (10 cycles of 1 min on and
1 min of) and cell debris were harvested by centrifugation
with Purifed KRED1‑Pglu and Product Recovery
A solution of the selected substrate (3–8) (28 mM) was pre-
pared in 20 mL of bufer TRIS HCl 50 mM pH 8.0 contain-
ing 5% of DMSO. In the same solution were added glucose
1 3

Stereoselective Reduction of Prochiral Cyclic 1,3-Diketones Using Diferent Biocatalysts
(40 mM), NADP+ (0.1 mM) and 1 U mL−1 of GDH from
Bacillus megaterium. 20 mU mL−1 of lyophilized KRED1-
Pglu was used as biocatalyst. The reactions were monitored
at diferent times, acidifed with HCl 2 N, extracted with
ethyl acetate (3 times) and the organic phases were dried
over Na2SO4. After removal of the solvent, a chromato-
graphic column was performed to separate the mono-alco-
hols and the diols (8:2 cyclohexane/ethyl acetate).
(2S,3S)-2-Benzyl-3-hydroxy-2-methylcyclohexanone
(14c) [32]: Rf = 0.42 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =+20.5 (c 1.2, CHCl3); 1H NMR (CDCl3, 300 MHz):
δ 1.08 (3H, s), 1.70–1.90 (3H, m), 2.00–2.15 (2H, m), 2.55
(2H, t, J=6.9 Hz), 2.96 (1H, d, J=13.7 Hz), 3.10 (1H, d,
J=13.7 Hz), 3.75–3.80 (1H, m), 7.15–7.30 (5H, m). Anal.
Calcd for C14H18O2: C, 77.03; H, 8.31. Found: C, 76.80; H,
8.12. Chiral GC retention time: 52.7 min (>99% e.e.).
(2R,3S)-2-Allyl-3-hydroxy-2-methylcyclopentanone (9a)
[18]: Rf =0.40 (7:3 cyclohexane/ethyl acetate); [α]2D0 =‒73.0
(c 0.26, CHCl3); 1H NMR (CDCl3, 300 MHz): δ 1.02 (3H,
s), 1.69 (1H, br s), 1.80-1.92 (1H, m), 2.10–2.30 (4H, m),
2.40–2.52 (1H, m), 4.23 (1H, t, J=6.4 Hz), 5.05–5.15 (2H,
m), 5.68–5.72 (1H, m). Anal. Calcd for C9H14O2: C, 70.10;
H, 9.15. Found: C, 70.06; H, 9.40. Chiral GC retention time:
23.0 min (>99% e.e.).
2.10 Product Characterization
(2S,3S)-2-Allyl-3-hydroxy-2-methylcyclopentanone (9c)
[18]: Rf =0.40 (7:3 cyclohexane/ethyl acetate); [α]2D0 =+84.0
(c 0.4, CHCl3); 1H NMR (CDCl3, 300 MHz): δ 1.00 (3 H, s),
1.80 (1 H, bs), 1.92–2.02 (1 H, m), 2.16–2.52 (5 H, m), 4.15
(1 H, dd, J=4.0, 3.0), 5.10–5.20 (2 H, m), 5.80–5.95 (1 H,
m). Anal. Calcd for C9H14O2: C, 70.10; H, 9.15. Found: C,
70.40; H, 9.43. Chiral GC retention time: 23.6 min (>99%
e.e.).
(2R,3S)-2-Allyl-3-hydroxy-2-methylcyclohexan-1-one
(10a) [18]: Rf = 0.40 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =‒9.4 (c 0.6, CHCl3); 1H NMR (CDCl3, 300 MHz):
δ 1.15 (3H, s), 1.60–1.75 (2H, m), 1.80-1.92 (1H, m),
1.95–2.10 (2H, m), 2.30–2.50 (4H, m), 3.85–3.95 (1H,
m), 5.02–5.18 (2H, m), 5.70–5.82 (1H, m). Anal. Calcd
for C10H16O2: C, 71.39; H, 9.59. Found: C, 71.43; H, 9.48.
Chiral GC retention time: 26.5 min (>99% e.e.).
(2S,3S)-2-Allyl-3-hydroxy-2-methylcyclohexanone (10c)
[18]: Rf =0.40 (7:3 cyclohexane/ethyl acetate); [α]2D0 =+27.5
(c 0.42, CHCl3); 1H NMR (CDCl3, 300 MHz): δ 1.19 (3H, s,
CH3), 1.60–1.80 (1H, m), 1.80–2.10 (4H, m), 2.35–2.45 (4H,
m), 3.78–3.82 (1H, m), 5.05–5.18 (2H, m), 5.70–5.82 (1H,
m). Anal. Calcd for C10H16O2: C, 71.39; H, 9.59. Found: C,
71.41; H, 9.45. Chiral retention time: 27.3 min (>99% e.e.).
(2S,3S)-3-Hydroxy-2-methyl-2-propylcyclopentanone
(11c) [18]: Rf = 0.40 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =+61.0 (c 1.94, CHCl3,); 1H NMR (CDCl3, 300 MHz):
δ 0.94 (3 H, t, J=7.2 Hz, CH,), 1.00 (3 H, s, CH,), 1.25–1.30
(1 H, m), 1.35–1.45 (1 H, m), 1.45-1.50 (2 H, m), 1.61 (1 H,
br s, OH), 1.92–1.97 (1 H, m), 2.16-2.32 (2 H, m), 2.42–2.50
(1 H, m), 4.11 (1 H, t, J=4.5 Hz). Anal. Calcd for C9H16O2:
C, 69.19; H, 10.32. Found: C, 68.97; H, 10.59. Chiral GC
retention time: 24.0 min (>99% e.e.).
(2R,3S)-3-Hydroxy-2-methyl-2-propylcyclopentanone
(11a) [18]: Rf = 0.40 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =‒110 (c 1.5, CHCl3); 1H NMR (CDCl3, 300 MHz):
δ 0.89 (3 H, t, J=7.2 Hz), 1.02 (3 H, s), 1.20–1.40 (4H, m),
1.65 (1 H, br s, OH), 1.85–1.90 (1H, m), 2.16–2.30 (2H, m),
2.41–2.47 (1H, m), 4.22 (1H, t, J=6.0 Hz). Anal. Calcd for
C9H16O2: C, 69.19; H, 10.32. Found: C, 69.39; H, 10.45.
Chiral GC retention time: 23.6 min (>99% e.e.).
(2R,3S)-3-Hydroxy-2-methyl-2-propylcyclohexanone
(12a) [18]: Rf = 0.40 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =‒40.8 (c 0.54, CHCl3); 1H NMR (CDCl3, 300 MHz):
δ 0.85 (3H, t, J=7.5 Hz), 1.00–1.10 (1H, m), 1.12 (3H, s),
1.20–1.35 (1H, m), 1.42–1.55 (1H, m), 1.60–1.70 (2H, m),
1.75–1.85 (2H, m), 2.00–2.20 (2H, m), 2.30–2.45 (3H, m),
3.90 (1H, dd, J = 5.5, 2.5 Hz). Anal. Calcd for C10H18O2:
C, 70.55; H, 10.66. Found: C, 70.48; H, 10.71. Chiral GC
retention time: 27.3 min (>99% e.e.).
(2S,3S)-3-Hydroxy-2-methyl-2-propylcyclohexanone
(12c) [18]: Rf = 0.40 (7:3 cyclohexane/ethyl acetate);
[α]2D0 =+65.0 (c 0.40, CHCl3); 1H NMR (CDCl3, 300 MHz):
δ 0.92 (3H, t, J=7.5 Hz), 1.00–1.10 (1H, m), 1.18 (3H, s),
1.20–1.25 (1H, m), 1.50-1.65 (4H, m), 1.65–180 (1H, m),
1.95-2.05 (2H, m), 2.30–2.42 (2H, m), 3.65 (1H, dd, J=8.5,
4 Hz). Anal. Calcd for C10H18O2: C, 70.55; H, 10.66. Found:
C, 70.67; H, 10.56. Chiral GC retention time: 27.2 min
(>99% e.e.).
2-Allyl-2-methylcyclopentane-1,3-diol (15): Rf = 0.32
(1:1 cyclohexane/ethyl acetate); [α]2D0 = + 69.7 (c 2.5,
CHCl3); 1H NMR (CDCl3, 300 MHz): δ 0.89 (3H, s), 1.42-
1.60 (2H, m), 2.09–2.22 (2H, m), 2.22–2.35 (2H, m), 3.90
(1H, dd, J=4.3 Hz), 4.11 (1H, t, J=6.4 Hz), 5.05–5.20 (2H,
m), 5.90–6.05 (1H, m).6 Anal. Calcd for C9H16O2: C, 69.19;
H, 10.32. Found: C, 69.00; H, 10.09. Chiral GC retention
time: 26.9 min (>99% e.e.).
(2S,3S)-2-Benzyl-3-hydroxy-2-methylcyclopentanone
(13c) [31]: Rf = 0.42 (7:3 cyclohexane/ethyl acetate);
[α]2D0 = + 74.9 (c 1.0, CHCl3); 1H NMR (300 MHz,
CDCl3): δ 0.87 (3H, s), 1.85–1.93 (2H, m); 2.15–2.22
(1H, m), 2.30–2.42 (1H, m), 2.50–2.60 (1H, m), 2.73 (1H,
d, J = 14.0 Hz), 3.06 (1H, d, J = 14.0 Hz), 4.05 (1H, t,
J=3.0 Hz), 7.20–7.35 (5H, m). mp 88‒90 °C; Anal. Calcd
for C13H16O2: C, 76.44; H, 7.90. Found: C, 76.59; H, 8.08.
Chiral GC retention time: 34.4 min (>99% e.e.).
2-Allyl-2-methylcyclohexane-1,3-diol (16): Rf =0.32 (1:1
cyclohexane/ethyl acetate); [α]2D0 =+42.0 (c 2.5, CHCl3); 1H
NMR (CDCl3, 300 MHz): δ 0.94 (3H, s), 1.44–1.80 (6H,
m), 2.32 (2H, d, J=4.8), 3.70–3.75 (1H, m), 3.78–3.83 (1H,
1 3

M. L. Contente et al.
m), 5.08–5.20 (2H, m), 5.90–6.05 (1H, m).7 Anal. Calcd for
C10H18O2: C, 70.55; H, 10.66. Found: C, 70.68; H, 10.80.
Chiral GC retention time: 29.3 min (>99% e.e.).
4.26 (1H, t, J=3.0 Hz), 7.20–7.35 (5H, m). Anal. Calcd for
C13H18O2: C, 75.69; H, 8.80. Found: C, 75.88; H, 8.99.
2-Benzyl-2-methylcyclohexane-1,3-diol (20): Rf =0.37
(1:1 cyclohexane/ethyl acetate); [α]2D0 = + 36.0 (c 3.5,
2-Methyl-2-propylcyclopentane-1,3-diol (17): Rf =0.32
(1:1 cyclohexane/ethyl acetate); [α]2D0 = + 44.6 (c 2.0,
CHCl3); 1H NMR (CDCl3, 300 MHz): δ 0.92 (3H, s), 0.96
(3H, t, J=7.2), 1.22–1.60 (7H, m), 2.05–2.22 (2H, m), 3.90
(1H, t, J = 4.5 Hz), 4.03 (1H, t, J = 6.0). Anal. Calcd for
C9H18O2: C, 68.31; H, 11.47. Found: C, 68.45; H, 11.58.
Chiral GC retention time: 27.1 min (>99% e.e.).
1
CHCl3); H NMR (CDCl3, 300 MHz): δ 0.86 (3H, s),
1.46–1.91 (6H, m), 2.84 (1H, d, J=13.7 Hz), 2.99 (1H, d,
J=13.7 Hz), 3.58–3.62 (1H, m), 3.88 (1H, dd, J=3.0 Hz),
7.18–7.32 (5H, m). Anal. Calcd for C14H20O2 : C, 76.33; H,
9.15. Found: C, 76.56; H, 9.00.
2-Methyl-2-propylcyclohexane-1,3-diol (18): Rf = 0.32
(1:1 cyclohexane/ethyl acetate); [α]2D0 = + 40.6 (c 0.95,
CHCl3); 1H NMR (CDCl3, 300 MHz): δ 0.92 (3H, s), 0.95
(3H, t, J=7.5 Hz), 1.20–1.35 (2H, m), 1.35–1.45 (2H, m),
1.35–1.76 (6H, m), 3.71 (1H, dd, J=8.5, 4.0 Hz), 3.77 (1H,
dd, J=5.5, 2.5 Hz). Anal. Calcd for C10H20O2: C, 69.72; H,
11.70. Found: C, 69.90; H, 11.79. Chiral GC retention time:
29.7 min (>99% e.e.).
3 Results and Discussion
3.1 Synthesis of 2,2‑Disubstituted
1,3‑Cyclopentanediones
and Cyclohexanediones
Substrates (3–8) were synthesized, as previously reported,
starting from commercially available 2-methyl-1,3-cyclo-
pentanedione (1) and 2-methyl-1,3-cyclohexanedione (2)
(Scheme 2) [18, 28, 29].
2-Benzyl-2-methylcyclopentane-1,3-diol (19): Rf =0.37
(1:1 cyclohexane/ethyl acetate); [α]2D0 = + 91.0 (c 1.5,
1
CHCl3); H NMR (300 MHz, CDCl3): δ 0.80 (3H, s),
1.45–1.62 (3H, m); 2.10–2.36 (2H, m), 2.67 (1H, d,
Compounds 3–8 were monoreduced with NaBH4 to
obtain the four possible stereoisomers of the chiral ketols as
J=14.0), 2.91 (1H, d, J=14.0), 3.89 (1H, dd, J=1.5 Hz),
i
iii
ii
O
O
O
O
O
O
O
O
(CH2)n
(CH2)n
(CH2)n
(CH2)n
3
4
5
6
: n = 1
: n = 2
: n = 1
: n = 2
7
8
: n = 1
: n = 2
1
2
: n = 1
: n = 2
i: Allyl bromide (2eq.), 1N NaOH (1eq.), 50% yield; ii: MeOH, H-Cube Mini, CatCart Pd/C 5% T = 30 °C, P = 1 bar,
flow rate: 0.5 mL/min, 95% yield ; iii: Benzyl bromide (2 eq.), 1N NaOH (1 eq.), 50% yield
Scheme 2 Preparation of 2,2-disubstituted 1,3-cycloalkanediones 3-8
R
R
R
R
R
NaBH4
O
OH
O
O
O
OH
O
OH
O
OH
+
+
+
(CH2)n
(CH2)n
3 8
(CH2)n
(CH2)n
(CH2)n
9-14a
-
9-14b
(2R,3R)-
9-14c
(2S,3S)-
9-14d
(2S,3R)-
(2R,3S)-
3, 9
:
n = 1; R = -CH2-CH=CH2
4, 10
5, 11
6, 12
7, 13
8, 14
: n = 2; R = -CH2-CH=CH2
: n = 1; R = -CH2-CH2-CH3
: n = 2; R = -CH2-CH2-CH3
: n = 1; R = -CH2-Ph
: n = 2; R = -CH2-Ph
Scheme 3 Products obtained by monoreduction of 3-8
1 3

Stereoselective Reduction of Prochiral Cyclic 1,3-Diketones Using Diferent Biocatalysts
analytical standards (Scheme 3). Absolute confguration of
the four stereoisomers of 9–14 was assigned on the basis of
chiral GC and 1H NMR spectra in comparison with literature
data [18, 31, 32].
concentrations, carbonyl reduction occurred also on 11c,
afording (1S,3S)-2-methyl-2-propylcyclopentane-1,3-diol
(17), while 11a remained unreacted in the reaction mixture
and could be recovered as pure stereoisomer at the end of the
reaction (entries 3 and 6, Table 1). Absolute confguration
and optical purity of diol 17 were assigned based on the fact
that the recovered diol is chiral and that the (1S,3S)-confg-
uration is formed independently of which of the two ketols
(11a and 11c) is further reduced to diol. In other words,
KRED1-Pglu, when used at low substrate/enzyme ratio, was
able to perform the second reduction with high enantiose-
lectivity, but also with high stereo-recognition of the stereo-
genic center in position 2, thus carrying out the resolution
of the diastereoisomeric mixture composed of 11a/11c. It
should be emphasized that this behavior was only observed
when the initial substrate concentration was up to 28 mM,
and most likely no substrate inhibition was signifcantly rel-
evant on the enzymatic activity.
3.2 Reduction with Isolated KRED1‑Pglu
2-Methyl-2-propylcyclopentane-1,3-dione (5) was initially
chosen as current substrate for the enzymatic reduction
catalyzed by the purified ketoreductase (KRED1-Pglu)
from Ogataea glucozyma. KRED1-Pglu was used as free
enzyme in the presence of a catalytic amount of NADP+ and
a system for the regeneration of the cofactor, composed of
glucose and glucose dehydrogenase (GDH) from Bacillus
megaterium. The biotransformation was frstly carried out
using diferent substrate concentrations (Table 1).
With higher substrate concentrations (>28 mM, entries 7
and 8), monoreduction was the only transformation observed
during a 24 h reaction time, with complete enantioselectiv-
ity of the carbonyl reduction (formation of S-stereocenter
at position 3) and low diastereoselectivity, hence furnish-
ing 11a/11c with a 42/58 ratio. Notably, under these condi-
tions no full conversion could be achieved and no traces of
diol were observed, indicating a possible inhibitory efect
on KRED1-Pglu exerted by an excess of substrate. When
the reaction was carried out in conditions of lower substrate
The results obtained using compound 5 as substrate led
us to perform the biotransformations of the other 2,2-dis-
ubstituted 1,3-cycloalkanediones using an initial substrate
concentration of 28 mM (Table 2).
All the tested substrates showed similar behavior in terms
of general reactivity (Scheme 4): frstly, monoreduction
occurred with high enantioselectivity and low diastereose-
lectivity, giving a (2S,3S)/(2R,3S) stereoisomeric mixture of
Table 1 Reduction of 2-methyl-2-propylcyclopentane-1,3-dione 5 with KRED1-Pglu using diferent initial substrate/enzyme ratios. Stereoiso-
mers 11b and 11d were not detected
Entry
Substrate 5 (μM) Time (min)
Remaining
substratea
(2R,3S)-11aa,b
(2S,3S)-11ca,b
(1S,3S)-17a
1
2
3
4
5
6
7
8
14
14
14
28
28
28
56
56
5
60
240
5
60
480
30
15
0
0
36
0
0
35
42
42
27
42
40
25
26
30
38
0
32
40
0
20
20
58
5
18
60
0
40
36
35
38
1440
0
Reaction conditions: 14–56 mM substrate, 0.1 mM NADP+, KRED1-Pglu (20 mU/mL), GDH (1 U/mL), glucose 40 mM in Tris/HCl bufer pH
8.0 (0.05 M, pH 8.0)
aAs determined by chiral GC
1
bAbsolute confgurations were assigned by comparison with H NMR of the Mosher’s derivatives.18 Results are the mean of three independent
assays performed in triplicate
1 3

M. L. Contente et al.
Table 2 Reduction
Entry
Substrate
Conv. (%)a
(2R, 3S)-ketola
(2S, 3S)-ketola
(1S, 3S)-diol
Time (h)
of 2,2-disubstituted
1,3-cycloalkanediones 3–8
using KRED1-Pglu
1
2
3
4
5
3
4
6
7
8
> 99
> 99
> 99
> 99
> 99
50 (9a)
52 (10a)
42 (12a)
0 (13a)
0 (14a)
0 (9c)
50 (15)
48 (16)
58 (18)
60 (19)
40 (20)
24
36
4
1
24
0 (10c)
0 (12c)
40 (13c)
60 (14c)
Reaction conditions: 28 mM substrate, 0.1 mM NADP+, KRED1-Pglu (20 mU/mL), GDH (1 U/mL), glu-
cose 40 mM in Tris/HCl bufer pH 8.0 (0.05 M, pH 8.0)
aAs determined by chiral GC. Results are the mean of three independent assays performed in triplicate
R
R
R
3, 9, 15
:
n = 1; R = -CH2-CH=CH2
: n = 2; R = -CH2-CH=CH2
: n = 1; R = -CH2-CH2-CH3
: n = 2; R = -CH2-CH2-CH3
KRED1-Pglu
KRED1-Pglu
HO
OH
(CH2)n
15-18
O
O
O
O
OH
4, 10, 16
5, 11, 17
6, 12, 18
+
+
(CH2)n
3 6
(CH2)n
-
9-12a
(2R,3S)-
(1S,3S)-
R
R
R
HO
OH
(CH2)n
19-20
O
O
OH
7, 13, 19
8, 14, 20
: n = 1; R = -CH2-Ph
: n = 2; R = -CH2-Ph
(CH2)n
7 8
(CH2)n
-
13-14c
(2S,3S)-
(1S,3S)-
Scheme 4 Reduction of 2,2-disubstituted-1,3-cyclopentanediones and 1,3-cyclohexanediones with KRED1-Pglu
the corresponding β-hydroxyketones; the second reduction
furnished the diol, leaving one stereoisomer unreacted.
Although similar reactivity was observed, reduction of
2,2-disubstituted cyclohexanediones was generally faster
than cyclopentadiones. Reduction of 2-propyl and 2-allyl
derivatives took place with the same stereobias in all the
cases, allowing for the recovery of the (2R,3S)-stereoisomer
of the ketol. On the other hand, the presence of the bulkier
benzyl substituent in position 2 (Table 2, entries 4 and 5)
had a striking efect on the stereorecognition of the sec-
ond reduction, with preference for the consumption of the
(2R,3S)-stereoisomers and thus fnal accumulation of (2S,
3S)-13c and (2S, 3S)-14c.
reduction of compound 5 [30]; indeed, the use of whole
cells guarantees the supply of cofactors and systems
for their recycling. Surprisingly, enantioselectivity was
generally diferent from what observed with the puri-
fed KRED1-Pglu and strongly variable depending on
the reaction conditions. These observations led us to the
hypothesis that whole cells of E. coli DE3 Star contained
endogenous ketoreductase(s) with activity towards 5, but
diferent enantioselectivity than what displayed by isolated
KRED1-Pglu. Accordingly, we utilized wild-type whole
cells of E. coli DE3 Star that did not express the recombi-
nant ketoreductase for evaluating their intrinsic activity.
Lyophilized cells E. coli DE3 Star (5 mg/mL) lacking
the plasmid coding for KRED1-Pglu (hereinafter simply
called E. coli DE3 Star) were therefore employed for the
reduction of 5, showing the formation of 11c as the only
product of the reaction, although with low yields (24%
after 72 h) and longer reaction time (Table 3, entry 1).
The use of higher amounts of biocatalysts did not give a
signifcant improve of the yields (28% conversion after
72 h using 25 mg/mL of lyophilized cells).
Notably, in all the cases, at the end of the reaction, the
recovered diol was obtained as single, optically active ste-
reoisomer, as determined by optical rotation and 1H NMR.
It should be noted that during the second reduction, the ste-
reogenic center at C2 is lost and all the diols produced had
(1S,3S)-confguration.
3.3 Reduction with Whole Cells of Escherichia coli
DE3 Star
A screening of diferent organic co-solvents was per-
formed. One reason was to potentially increase cell perme-
ability and thus to enhance the yield of the biotransforma-
tion (Table 3) [33]. Further reasons were to determine the
efect of solvent addition to both, conversion and selec-
tivity, as it was seen in frst quick and preliminary trials
With the aim of preparing an easier to use biocatalyst,
containing both recombinant ketoreductase and cofac-
tors, lyophilized whole cells of E. coli DE3 Star express-
ing heterologous KRED1-Pglu were evaluated for the
1 3

Stereoselective Reduction of Prochiral Cyclic 1,3-Diketones Using Diferent Biocatalysts
Table 3 Reduction of 2-methyl-2-propylcyclopentane-1,3-dione
using lyophilized cells of E. coli DE3 Star
5
Table 4 Reduction of 2,2-disubstituted 1,3-cycloalkanediones using
whole cells of E. coli DE3 Star
Entry
Substrate
Conversion (%)a Isolated
(2S,3S)-
ketol
1
2
3
4
5
6
3
4
5
6
7
8
41
34
70
30
74
16
35
30
66
25
70
12
Entry
Co-solvent (% vol/vol)
Remaining sub-
11ca (%)
strate 5a (%)
1
2
3
4
5
6
7
8
No co-solvent
DMSO (5%)
DMSO (10%)
CH2Cl2 (1%)
CH2Cl2 (2%)
Acetone (2%)
Acetone (5%)
CHCl3 (1%)
CHCl3 (2%)
MTBE (2%)
MTBE (5%)
MTBE (10%)
MIBKb (2%)
MIBKb (5%)
76
72
88
84
98
80
85
50
95
48
75
97
75
80
24
28
12
16
< 5
20
15
50
5
42
25
< 5
25
20
Reaction conditions: 14 mM substrate, 5 mg of lyophilized E. coli
DE3 Star, 0.1 mM NADP+, GDH (1 U/mL), glucose 40 mM in Tris/
HCl bufer pH 8.0 (0.05 M, pH 8.0) in the presence of diferent
amounts of co-solvent
aAs determined by GC. Results are the mean of three independent
assays performed in triplicate
9
Notice that, in general, cyclopentanediones produced
the corresponding ketol in higher yields compared to
cyclohexanediones.
10
11
12
13
14
4 Concluding Remarks
Reaction conditions: 14 mM substrate, 5 mg of lyophilized E. coli
DE3 Star, 0.1 mM NADP+, GDH (1 U/mL), glucose 40 mM in Tris/
HCl bufer pH 8.0 (0.05 M, pH 8.0) in the presence of diferent
amounts of co-solvent
In conclusion, we have reported on the stereoselective
reduction of prochiral cyclic 1,3-diketones using difer-
ent biocatalytic systems. Under selected conditions, whole
cells of E. coli DE3 Star furnished only one stereoisomer,
in most of the cases diferent than the one recovered at the
end of the reaction with purifed KRED1-Pglu. KRED1-
Pglu, employed under conditions of low substrate/enzyme
ratio, was also able to give (1S, 3S)-diols with excellent
enantioselectivity (> 99% e.e). It is important to note that
the strain E. coli DE3 Star, commonly used to produce
recombinant proteins, showed relevant ketoreductase
activity towards these particular substrates.
aAs determined by chiral GC. Results are the mean of three independ-
ent assays performed in triplicate
bMethyl Isobutyl Ketone
that reaction conditions strongly infuenced the catalyst
performance.
The addition of diferent co-solvents led to variable molar
conversion (< 5–50%) of 11c while no efect on the stere-
oselectivity was observed. The best results were obtained
with 1% CHCl3 (entry 8, Table 3); under this condition, the
mono-alcohol 11c was obtained as unique product with 50%
of molar conversion, whereas higher concentration of CHCl3
inhibited the enzymatic activity.
Purifed KRED1-Pglu and whole cells of E. coli DE3
Star can be used as alternative biocatalysts to Baker’s
yeast [18] for the reduction of prochiral 2,2-disubstituted-
1,3-cyclopentadiones and 1,3-cyclohexanediones, since
different products and different stereoselectivity were
obtained.
As proof of concept, we checked the reactivity of the
other 2,2-disubstituted 1,3-cycloalkanediones with lyophi-
lized cells of E. coli DE3 Star in the presence of CHCl3 (1%
vol/vol) (Table 4).
Acknowledgements The authors thank the University of Milan for
funding the stay of Federica Dall’Oglio at the Biotechnology Forschun-
gszentrum of Jülich.
All substrates were converted into the corresponding
(2S,3S)-β-hydroxyketones as the only product (Table 4),
although with diferent yields. The ketoreductase(s) in the
cells of E. coli DE3 Star is therefore able to catalyze the
mono reduction of 2,2-disubstituted 1,3-cycloalkanedi-
ones with high enantioselectivity and diastereoselectivity.
Compliance with Ethical Standards
Conflict of interest The authors declare no confict of interest.
1 3

M. L. Contente et al.
20. Kosmol H, Kieslich K, Vossing R, Koch HJ, Petzoldt K, Gibian
H (1967) Justus Liebigs Ann Chem 701:199–205
References
21. Contente ML, Molinari F, Serra I, Pinto A, Romano D (2016) Eur
J Org Chem 2016:1260–1263
1. Nakamura K, Matsuda T (2006) Curr Org Chem 10:1217–1246
2. Hoyos P, Sinisterra JV, Molinari F, Alcantara AR, Dominguez de
Maria P (2010) Acc Chem Res 43:288–299
22. Contente ML, Serra I, Brambilla M, Eberini I, Gianazza E, De
Vitis V, Molinari F, Zambelli P, Romano D (2016) Appl Microbiol
Biotechnol 100:193–201
3. Sehl T, Hailes HC, Ward JM, Menyes U, Pohl M, Rother D (2014)
Green Chem 16:3341–3348
23. Contente ML, Serra I, Molinari F, Gandolf R, Pinto A, Romano
D (2016) Tetrahedron 72:3974–3979
4. Chen Y, Chen C, Wu X (2012) Chem Soc Rev 41:1742–1753
5. Kalaitzakis D, Rozzell JD, Smonou I, Kambourakis S (2006) Adv
Synth Catal 348:1958–1969
24. Contente ML, Molinari F, Zambelli P, De Vitis V, Gandolf R,
Pinto A, Romano D (2014) Tetrahedron Lett 55:7051–7053
25. Contente ML, Zambelli P, Galafassi S, Tamborini L, Pinto A,
Conti P, Molinari F, Romano D (2015) J Mol Catal B 114:7–12
26. Contente ML, Serra I, Palazzolo L, Parravicini C, Gianazza E,
Eberini I, Pinto A, Molinari F, Guidi B, Romano D (2016) Org
Biomol Chem 14:3404–3408
6. Kalaitzakis D, Smonou I (2010) J Org Chem 75:8658–8661
7. Bataille CJR, Donohoe TJ (2011) Chem Soc Rev 40:114–128
8. Nguyen TN, Chen PA, Setthakarn K, May JA (2018) Molecules
23:2317
9. Paterson I, Chen DYK, Acena JL, Franklin AS (2000) Org Lett
2:1513–1516
27. Dall’Oglio F, Contente ML, Conti P, Molinari F, Monfredi D,
Pinto A, Romano D, Ubiali D, Tamborini L (2017) Catal Commun
93:29–32
10. Mukai K, Urabe D, Kasura S, Aoki N, Inoue MA (2013) Angew
Chem Int Ed 52:5300–5304
11. Urabe D, Nakagawa Y, Mukai K, Fukushima K, Aoki N, Itoh H,
Nagatomo M, Inoue M (2018) J Org Chem 83:13888–13910
12. Haberland J, Kriegesmann A, Wolfram E, Hummel W, Liese A
(2002) Appl Microbiol Biotechnol 58:595–599
13. Grau BT, Devine PN, Di Michele LN, Kosjek B (2007) Org Lett
9:4951–4954
28. Manna MS, Mukherjee S (2014) Chem Sci 5:1627–1633
29. Byrne SJ, Fletcher AJ, Hebeisen P, Willis MC (2010) Org Biomol
Chem 8:758–760
30. Oeggl R, Neumann T, Gätgens J, Romano D, Noack S, Rother D
(2018) Front Bioeng Biotechnol 6:196
31. Yeung YY, Chein RJ, Corey EJ (2007) J Am Chem Soc
129:10346–10347
14. Lüdeke S, Richter M, Müller M (2009) Adv Synth Catal
351:253–259
32. Kolakowski RV, Manpadi M, Zhang Y, Emge TJ, Williams LJ
(2009) J Am Chem Soc 131:12910–12911
15. Kurina-Sanz M, Bisogno FR, Lavandera I, Orden AA, Gotor V
(2009) Adv Synth Catal 351:1842–1848
33. Salter GJ, Kell DB (1995) Crit Rev Biotechnol 15:139–177
16. Husain SH, Stillger T, Dünkelmann P, Lödige M, Walter L, Brei-
tling E, Pohl M, Bürchner M, Krossing I, Müller M, Romano D,
Molinari F (2011) Adv Synth Catal 353:2359–2362
17. Mourelle-Insua A, de Gonzalo G, Lavandera I, Gotor-Fernández
V (2018) Catalysts 8:150
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
18. Brooks W, Mazdiyasni M, Grothaus PG (1987) J Org Chem
52:3223–3232
19. Watanabe W, Iwamoto M, Nakada M (2005) J Org Chem
70:4652–4658
Afliations
Martina Letizia Contente1,3 · Federica Dall’Oglio2 · Francesca Annunziata2 · Francesco Molinari3 · Marco Rabufetti3 ·
Diego Romano3 · Lucia Tamborini2 · Dörte Rother4 · Andrea Pinto3
3
* Andrea Pinto
Department of Food, Environmental and Nutritional Sciences
(DeFENS), University of Milan, via Mangiagalli 25,
20133 Milan, Italy
andrea.pinto@unimi.it
1
School of Chemistry, University of Nottingham, University
4
Institute of Bio- and Geosciences, IBG-1: Biotechnology,
Forschungszentrum Jülich GmbH, 52425 Jülich, Germany
Park, Nottingham NG7 2RD, UK
2
Department of Pharmaceutical Sciences (DISFARM),
University of Milan, via Mangiagalli 25, 20133 Milan, Italy
1 3