Chemoenzymatic synthesis of 2-oxabicyclo[3.3.1]nonan-3-one enantiomers via microbial reduction by Absidia coerulea AM 93

Article abstract of DOI:10.1016/j.molcatb.2009.11.005

Microbial enantioselective reduction of (±)-diethyl 2-(3-oxocyclohexyl)malonate (1) has been described. A screening test on twenty-four fungi strains was carried out. Most of the microorganisms preferred bioreduction of (+)-isomer of δ-ketoester (1) to (+

Full text of DOI:10.1016/j.molcatb.2009.11.005

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Chemoenzymatic synthesis of 2-oxabicyclo[3.3.1]nonan-3-one enantiomers via
microbial reduction by Absidia coerulea AM 93
Teresa Olejniczak∗
Department of Chemistry, University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 March 2009
Received in revised form 18 October 2009
Accepted 16 November 2009
Available online 24 November 2009
Microbial enantioselective reduction of ( )-diethyl 2-(3-oxocyclohexyl)malonate (1) has been described.
A screening test on twenty-four fungi strains was carried out. Most of the microorganisms preferred
bioreduction of (+)-isomer of ␦-ketoester (1) to (+)-trans ␦-hydroxy ester (2) with the anti-Prelog
selectivity. Biotransformation conditions using Absidia coerulea AM 93 were optimized with respect
to the growth medium, temperature and pH. An effect of 48 chemical additives on the course of
biotransformation was checked. (−)-Diethyl 2-((S)-3-oxocyclohexyl)malonate ((−)-1) (ee = 98%) and
(+)-diethyl 2-((1R, 3R)-3-hydroxycyclohexyl)malonate ((+)-2) (ee = 99%) were isolated and subjected to
chemical lactonization, leading to (+)-(1R, 5S)-2-oxabicyclo[3.3.1]nonan-3-one ((+)-3) and (−)-(1S, 5R)-
2-oxabicyclo[3.3.1]nonan-3-one ((−)-3). The absolute configuration of product (−)-1 was confirmed by
comparison of its optical rotation with the literature data. The absolute configuration the carbon atom
bearing hydroxyl group in product (+)-2 was determined using the Mosher’s ester.
Keywords:
Biotransformation
Enantioselective reduction
Anti-Prelog selectivity
Absidia coerulea
Lactonization
Inhibitor
Activator
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
In recent years, syntheses of lactones using chemoenzymatic
methods have been extensively investigated. These strategies bond
␦-Lactone ring is a structural fragment of many natural products.
Lactones play important role in food industry as flavour compo-
nents [1–4], in agriculture as sex attractant pheromones [5–8]
and also they are used as chiral building blocks for synthesis of
pharmaceuticals [9]. Their physiological activity depends on con-
figuration of stereogenic centres and on enantiomeric purity of the
compounds [1–8].
Under our project we are interested in synthesis of optically
active lactones with [3.3.1] carbon skeleton as potential antifun-
gal agents. Naturally occurring, biologically active lactones of such
a structure are presented in Fig. 1. They are isolated from stem
bark of Goniothalamus giganteus (Annonaceae) [10–12] and Illicium
floridanum [13,14], and from fruit kernels of Otoba parvifolia [15,16].
Styryl lactones, shown in Fig. 1, exhibit antitumor activity
against several human tumor cells [10–12], whereas sesquiterpene
lactones isolated from Illicium species are known as GABA antago-
nists in central nervous system [14]. The third group—lactones from
Otoba parvifolia show antibiotic activity against such important
microorganisms as Staphylococcus aureus, Staphylococcus epidemis
and Peptococus sp. [15,16].
the most efficient biocatalysis with chemical synthesis. Quick
development of biocatalysis results from the fact, that enzyme-
catalyzed reactions are highly enantioselective and regioselective,
usually more than chemical synthesis using chiral catalysts. Sev-
eral review articles have been published on the use of enzymes
in organic synthesis [17–20]. Particularly two groups of enzymes:
lipases and dehydrogenases are very useful [18–21]. Biological
reductions catalyzed either by isolated enzymes or by whole micro-
bial cells provide an attractive method that may be applied to a
broad range of ketones [22–24] and ketoesters [25,26]. The reports
comprise search for efficient and enentioselective biocatalysts and
optimization of the process with respect to yields and enantiomeric
excess of products [22–26]. Many biotransformations are per-
formed in organic solvents [19] or in hydrophobic ionic liquids [20].
One of the ways to improve the yield of process is to add either
inhibitors or activators to the cultivation mixture [27–33].
Herein we report on the resolution of racemic ( )-diethyl 2-
(3-oxocyclohexyl)malonate (1) by means of microbial reduction,
which could be a good alternative to organocatalytic conjugate
1,4-additions [34,35] of a variety of malonates to ␣,␤-unsaturated
enones. Optimization of the transformation conditions occu-
pies a large part of this work. The aim of this part of the
research was to increase enantiomeric excess of diethyl 2-(3-
oxocyclohexyl)malonate (1). Effects of the additives on activities
of the dehydrogenases were assessed.
∗
Tel.: +48 71 320 52 67; fax: +48 71 328 35 76.
E-mail address: tmolejniczak@yahoo.com.
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.11.005

2
T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
Fig. 1. Natural lactones with [3.3.1] carbon skeleton.
(qt, J = 12.6 Hz, J = 3.6 Hz, 1H, one of 6CH2),), 1.90–1.94 and 1.92–1.97
2. Materials and methods
(two m, 1H, one of 5CH2), 2.05–2.09 (m, 1H, one of 5CH2), 2.26 (ddd,
J = 8.1 Hz, J = 8.7 Hz, J = 12.6 Hz 1H, one of 4CH2), 2.25 (t, J = 12.6 Hz
1H, one of 4CH2), 2.39–2.40 and 2.40–2.41 (two m, 1H, one of 2CH2),
2.43–2.44 and 2.45–2.46 (two m, 1H, one of 2CH2), 2.50–2.56 (m,
1H, HCCH2) 3.29 (d, J = 8.1 Hz, 1H, CH(CO2C2H5)2) 4.20 and 4.21
(two q, J = 7.2 Hz, 4H, 2× CO2CH2CH3); 13C NMR (151 MHz), ı (ppm):
14.05 (2× CO2CH2CH3), 24.51 (CH2), 28.77 (CH2), 38.00 (CH), 41.00
(CH2), 45.09 (CH2), 56.90 (CH(CO2C2H5)2), 61.52 and 61.53 (2×
CO2CH2CH3), 167.73 and 167.82 (C O, ester), 209.62 (C O); IR
(film, cm−1): 1731.13 (s), 1733.47 (s), 1738.58 (s); GC-EIMS: 257
(M+1).
2.1. Analysis
The structures of compounds 1–3 were determined based on
1H NMR, 13C NMR, COSY, DEPT, HMQC, IR and GC-MS. NMR spec-
tra were recorded on Bruker DRX-500 spectrometer. The residual
protic solvent CHCl3 (ıH = 7.26 ppm) in CDCl3 was used as internal
standard. 13C NMR (ıC = 77.0 ppm). Infrared spectra were measured
on a Mattson FTIR-300 Thermo Nicolet spectrometer. Molecular
mass was confirmed on a Varian Chrompack GC-MS CP-3800 Saturn
2000 GC-MS with an ionizing energy of 70 eV, using HP-1 col-
umn (crosslinked methyl silicone gum, 25 m × 0.32 mm, 0.25 ␮m
film thickness). Optical rotation was measured on an Autopol IV
automatic polarimeter (Rudolph).
2.3. Synthesis of trans diethyl 2-(3-hydroxycyclohexyl)malonate
(( )-2)
Compositions of crude biotransformation extracts as well as
purity of isolated products were determined by TLC and GC.
TLC was carried out on silica gel Kieselgel 60 F254 glass plates
(Merck) with petroleum ether:acetone:iso-propanol:ethyl acetate
(40:1:3:1, v/v/v/v) or petroleum ether:acetone (2:1, v/v) as devel-
oping systems. The compounds were visualized by spraying the
plates with 1% Ce(SO4)2, 2% H3[P(Mo3O10)4] in 10% H2SO4, followed
by heating. The same eluents were applied to preparative col-
umn chromatography (silica gel: 60 230–400 mesh) for separation
and purification of final products. Gas chromatography analyses
were performed on a Hewlett Packard 5890A series II instrument,
fitted with a flame ionization detector (FID), using column: Car-
bowax (30 m, 0.53 mm, 0.88 ␮m film thickness). H2 at a flow rate
of 2 cm3/min was used as a carrier gas.
Diethyl
2-(3-oxocyclohexyl)malonate
(( )-1)
(1.250 g,
4.88 mmol) was dissolved in ethanol (25 cm3) and sodium
borohydride (195.5 mg, 4.90 mmol) was added. The reaction
mixture was stirred at room temperature. When the reaction
was complete (TLC, GC, 3 h), ethanol was evaporated off in vacuo.
The reaction mixture was diluted with ethyl ether (75 cm3) and
washed successively with 10% aqueous solution of HCl (5 cm3) and
water (until neutral). Ethereal extract was dried over MgSO4. After
solvent evaporation, the crude product was purified by column
chromatography (eluent:petroleum ether:acetone, 2:1, v/v) to give
1.03 g pure ( )-2 in 82% yield. The physical and spectral data of
(
)-2 are as follows: Colourless oil; Rf = 0.45 (PE:acetone, 2:1); 1
H
NMR (500 MHz, CDCl3): ı (ppm): 1.22 and 1.23 (two t, J = 7.2 Hz, 6H,
2× CO2CH2CH3), 1.61–1.63 and 1.64–1.66 (two m, 2H, 6CH2), 1.73
(quintet, J = 3.5 Hz, 2H, 5CH2), 1.90–1.99 (m, 2H, 4CH2), 2.05–2.09
and 2.07–2.10 (two m, 2H, 2CH2) 2.11–2.14 (m, 1H, HCCH2) 3.15 (d
J = 8.1 Hz, 1H, CH(CO2C2H5)2), 3.55–3.61 (m, 1H, HC(OH)), 4.14 and
4.17 (two q, J = 7.2 Hz, 4H, 2× CO2CH2CH3); 13C NMR (151 MHz),
ı (ppm): 14.06 (2× CO2CH2CH3), 23.43 (CH2), 29.41 (CH2), 35.13
(CH), 36.28 (CH2), 39.65 (CH2), 57.75 (CH(CO2C2H5)2), 61.23 and
61.27 (2× CO2CH2CH3), 70.12 (HCOH), 168.40 and 168.46 (C O,
ester); IR (film, cm−1): 1733.22 (s), 1738.51 (s), 3393.23 (s);
GC-EIMS: 259 (241) (M+1).
Conversion degree and enantiomeric excess were determi-
nated by capillary GC using a chiral column–CP-cyclodextrin
(25 m × 0.25 mm, 0.25 ␮m film thickness).
2.2. Synthesis of diethyl 2-(3-oxocyclohexyl)malonate (( )-1)
The solution of cyclohex-2-en-1-one (3.5 g, 36.4 mmol) in
10 cm3 of dry ethyl ether was added dropwise to a stirred solu-
tion of diethyl malonate (17.9 g, 112.0 mmol) with NaH (0.95 g,
39.6 mmol) and the stirring was continued at room temperature.
When the reaction was complete (TLC, GC, 1 h) the mixture was
diluted with ethyl ether (150 cm3) and washed successively with
aqueous solution of 10% HCl (50 cm3) and water (until neutral).
After solvent evaporation, diethyl malonate was distilled off under
reduced pressure. The crude product was purified by column chro-
matography (eluent:petroleum ether:acetone, 2:1, v/v) to give
8.57 g of ( )-1 in 92% yield. Physical and spectral data of ( )-1
are as follows: Colourless oil; Rf = 0.61 (PE:acetone, 2:1); 1H NMR
(500 MHz, CDCl3): ı (ppm): 1.27 and 1.28 (two t, J = 7.2 Hz, 6H, 2×
CO2CH2CH3), 1.51 (qd, J = 12.6 Hz, J = 3.6 Hz, 1H, one of 6CH2), 1.69
2.4. Synthesis of 2-oxabicyclo[3.3.1]nonan-3-one (( )-3)
Trans diethyl 2-(3-hydroxycyclohexyl)malonate (( )-2)
(0.453 g, 1.76 mmol) was refluxed with 10% ethanolic KOH
solution (30 cm3). When the reaction was complete (TLC, 5 h),
ethanol was evaporated off in vacuo. The reaction mixture was
diluted with ethyl ether (50 cm3) and washed successively with
10% aqueous solution of HCl (10 cm3) and water to pH = 6. Ethereal
extract was dried over MgSO4. After solvent evaporation, the crude

T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
3
product was diluted with benzene (25 cm3) and p-toluenosulfonic
acid (TsOH × H2O) (0.35 g, 1.78 mmol) was added. The reaction
mixture was refluxed for 5 h. Next, benzene was evaporated off
and the product was diluted with ethyl ether (50 cm3) and washed
with 10% aqueous solution of NaOH (5 cm3) and water (until
neutral). The crude product was purified by column chromatogra-
phy (eluent:petroleum ether:acetone:iso-propanol:ethyl acetate,
40:1:3:1, v/v/v/v) to give 150 mg of pure ( )-3 in 61% yield.
35.30 (4CH2), 35.99 (HCCH2), 55.33 (OCH3), 57.34 (HC(CO2Et)2),
61.38 (2× CO2CH2CH3), 75.01 (H3COOC(CF3)), 127.24 (
),
128.32 (
), 129.50 (
), 132.36 (
), 165.65
(COOC(CF3)). 168.11 and 168.17 (2× (CO2C2H5)).
2.4.4. Synthesis of diethyl 2-((1R, 3R)-3-((2ꢀR)-3ꢀ,3ꢀ,3ꢀtrifluoro-2ꢀ-
methoxy-2ꢀ-phenyl-propionyloxy)cyclohexyl)malonate
(2-R-MTPA)
2-R-MTPA: was prepared using 75 mg (0.291 mmol) of
(+)-trans diethyl 2-((1R, 3R)-3-hydroxycyclohexyl)malonate
2.4.1. Synthesis of (−)-(1S, 5R)-2-oxabicyclo[3.3.1]nonan-3-one
((-)-3)
Prepared from 120 mg (0.47 mmol) of (+)-diethyl 2-((1R, 3R)-
3-hydroxycyclohexyl)malonate ((+)-2) according to the procedure
described for preparation of ( )-3, obtained 42 mg. Yield: 64%,
20
˛
= −18.6 (c = 2.05, CHCl3).
[ ]589
((+)-2)
and
59 mg
(0.283 mmol)
of
(S)-2-metoxy-2-
trifluoromethylphenylacetic acid chloride according to the same
procedure as for 2-S-MTPA. 89 mg of the product was obtained
(yield 73%). Physical and spectral data of 2-R-MTPA obtained are
as follows: 1H NMR (ı, ppm): 1.24 and 1.25 (two t, J = 7.0 Hz, 6H,
CO2CH2CH3), 1.30–1.44 (m 2H, 6CH2), 1.70–1.74 and 1.72–1.76
(two m, 1H, one of 5CH2), 1.86 (dt J = 3.5 Hz, J = 13.5 Hz, one of 5CH2),
1.98–2.05 (m, 1H, one of 4CH2), 2.09–2.11 (m, 1H one of 4CH2),
2.12–2.26 (m, 2H, 2CH2), 2.29–2.32 (HCCH2), 3.18 (d, J = 8.5 Hz, 1H,
HC(CO2Et)2), 3.53 (s, 1H, OCH3), 4.16 and 4.20 (two q, J = 7.0 Hz 4H,
2× CO2CH2CH3), 4.96–5.2 (m, 1H, H3COOC(CF3)), 7.50–5.52 (m, 2H
2.4.2. Synthesis of (+)-(1R, 5S)-2-oxabicyclo[3.3.1]nonan-3-one
((+)-3)
Prepared from 143 mg (0.56 mmol) of (−)-diethyl 2-((S)-3-
oxycyclohexyl)malonate ((−)-1), which was dissolved in 5 cm3 of
ethanol and reduced with 25 mg (0.55 mmol) of NaBH4 according to
the procedure given for racemic ( )-1. After flash column purifica-
20
tion 115 mg of product (−)-2 ( ˛
= −1.04 (c = 3.11, CHCl3)) was
[ ]589
obtained with 80% yield.
(−)-Isomer of 2-oxabicyclo[3.3.1]nonan-3-one ((−)-3) was
prepared from 97 mg (0.38 mmol) of (−)-diethyl 2-((1S, 3S)-3-
hydroxycyclohexyl)malonate ((−)-2) according to the procedure
20
), 7.37–7.39 (m, 3H
), 13C NMR (ı, ppm): 13.99,
described for preparation of ( )-3. Yield: 59%,
(c = 1.5, CHCl3) (ee = 93%).
˛
= +17.9
[ ]589
14.01 (2× CO2CH2CH3), 23.20 (6CH2), 29.06 (5CH2), 31.13 (2CH2),
35.09 (4CH2), 36.00 (HCCH2), 55.25 (OCH3), 57.33 (HC(CO2Et)2),
The physical and spectral data of 2-oxabicyclo[3.3.1]nonan-
3-one (3) are as follows: Rf = 0.20 (PE:acetone:iso-propanol:ethyl
acetate, 40:1:3:1) 1H NMR (500 MHz, CDCl3), ı (ppm): 1.74–1.54
(m, 6H, 7CH2, 8CH2, 9CH2), 1.92–2.02 and 2.03–2.08 (two m, 2H,
6CH2), 2.24–2.26 (m, 1H, 5CH), 2.45 (d, J = 18.56 Hz, 1H, one of
4CH2), 2.70 (dd, J = 18.56 Hz, J = 6.64 Hz, 1H one of 4CH2), 4.70–4.72
(m, 1H, H1CO); 13C NMR (151 MHz), ı (ppm): 15.93 (8CH2), 25.98
(7CH2), 30.31(9CH2), 30.96(5CH), 35.99(6CH2, 2CH2), 75.43(H1CO),
171.96 (C O); IR (film, cm−1): 1218.14 (s), 1725.55 (s); GC-EIMS:
141 (M+1).
61.33 (2× CO2CH2CH3), 74.99 (H3COOC(CF3), 127.24 (
),
128.30 (
), 129.48 (
), 132.35 (
),165.62
(COOC(CF3), 168.06 and 168.12 (2× (CO2C2H5)).
2.5. Biocatalysts
2.4.3. Synthesis of diethyl 2-((1R, 3R)-3-((2ꢀS)-3ꢀ,3ꢀ,3ꢀ-trifluoro-
2ꢀ-methoxy-2ꢀ-phenyl-propionyloxy)cyclohexyl)malonate
(2-S-MTPA)
The following microorganisms were obtained from the col-
lection of Institute of Biology and Botany, Medical University in
Wrocław: Absidia coerulea AM 93, A. cylindrospora AM 336, Aphan-
ocladium album AM 417, Ascosphaera aspis AM 496, Aspergillus
ochraceus AM 456, A. wenthi AM 413, Beauveria bassiana AM 278,
B. bassiana AM 446, Circinella muscaes AM 302, Dothiorella ribis AM
273, Fusarium oxysporum AM 13, F. solani AM 203, Fusicoccum amyg-
dali AM 258, Laetiporus sulphurens AM 524, Mortiella vinaceae AM
149, Mucor circinelloides AM 385, Penicillium camemberti AM 83, P.
notatum AM 904, Rhizopus nigricans AM 394, Rhodotorula glutinis
AM 242, R. rubra AM 4, Saccharomyces cerevisiae AM 464, Tricho-
derma viride AM 523, and Yarrowia lipolytica AM 71. Whereas the
strains of Hanseniaspora vinaea 912, Papularia rosea 17, and Penicil-
lium vermiculatum 30 came from the Collection of the University of
Environmental and Life Sciences in Wrocław.
(+)-Trans diethyl 2-((1R, 3R)-3-hydroxycyclohexyl)malonate
((+)-2) (70 mg, 0.271 mmol) was dissolved in dry pyridine (1 cm3),
then (R)-2-metoxy-2-trifluoromethylphenylacetic acid chloride
(57 mg, 0.273 mmol) was slowly added and reaction mixture was
stirred at room temperature. When reaction was completed (TLC,
10 h), the mixture was diluted with ethyl ether (30 cm3) and
washed successively with aqueous solution of 10% HCl (5 cm3)
and water (until neutral). Ethereal extract was dried over MgSO4.
After solvent evaporation, the crude product was purified by col-
umn chromatography (eluent:petroleum ether:acetone, 4:1, v/v)
to give 93 mg of the product (yield 81%). Physical and spectral
data of 2-S-MTPA obtained are as follows: 1H NMR (ı, ppm):
1.25 and 1.26 (two t, J = 7.0 Hz, 6H, CO2CH2CH3), 1.28–1.42 m 2H,
6CH2), 1.68–1.72 and 1.70–1.74 (two m, 1H, one of 5CH2), 1.83
(dt, 1H, J = 13.5 Hz, J = 3.5 Hz, one of 1H 5CH2), 2.01–2.09 (m, 2H,
4CH2), 2.02–2.16 (m, 2H, 2CH2), 2.13–2.17 (m, 1H, HCCH2), 3.20 (d,
J = 8.5 Hz, 1H, HC(CO2Et)2), 3.53 (s, 13H, OCH3), 4.19 (q, J = 7.0 Hz,
4H, CO2CH2CH3), 4.95–5.01 (m, 1H, H3COOC(CF3), 7.49–7.51 (m, 2H
2.5.1. Initial screening
Screening procedure: The microorganisms were cultivated at
25 ◦C in 300 cm3 Erlenmeyer flasks containing 75 cm3 of the follow-
ing nutrients: 1% solution of peptone and glucose (3%). After 3–5
days of growth, 20 mg of a substrate in 0.5 cm3 of acetone was added
to the shaken cultures. The transformation was being continued for
3, 6, 9, 12, 21 or 81 h. The products were extracted with diethyl
ether and analysed by TLC and GC. Enantiomeric excesses were
determined by GC (CP-cyclodextrin, 25 m × 0.25 mm, 0.25 ␮m film
), 7.36–7.38 (m, 3H
). 13C NMR (ı, ppm): 14.04;
14.06 (2× CO2CH2CH3), 23.14 (6CH2), 29.09 (5CH2), 30.84 (2CH2),

4
T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
thickness) under the following conditions: injector 200 ◦C; detec-
tor 250 ◦C; flow 2 ml/min (carrier gas–H2); temperature program:
120 ◦C for 1 min, 0.4 ◦C/min to 160 ◦C.
2.5.6. Effect of inhibitors/activators addition
Portions of 0.5 cm3 of the inoculum (spore concentration of
107 cm−3) were transferred to 300 cm3 Erlenmeyer flasks, each
containing 75 cm3 of growth medium P. The cultures were incu-
bated at 28 ◦C with shaking at 150 rpm. After 5 days of growth,
specified amounts of either an inhibitor or an activator (10, 30
or 50 mg) were added to each flask and the cultures were incu-
bated for the next 1 h. Then, 20 mg of 2-(3-oxocyclohexyl)malonate
(1) in 0.5 cm3 of acetone was added. The biotransformation was
stopped after 1.5 h. The conversion and enantiomeric excesses
were measured. Dry mass of A. coerulea AM 93 was established
after washing and lyophilization. Simultaneously, under the same
conditions, transformations without the additives were run as a
control.
2.5.2. Effect of growth medium
Portions of 0.5 cm3 of the inoculum (spore concentration of
107 cm−3) were transferred to 300 cm3 Erlenmeyer flasks, each
containing 75 cm3 of one of six sterile growth media. The following
media were used: C: (3 g NaNO3, 1 g KH2PO4, 0.5 g KCl, 0.01 g FeSO4,
0.5 g MgSO4 × 7H2O, 30 g saccharose/1000 ml H2O);) E: (4 g yeast
extract, 10 g starch, 0.1 g K2HPO4, 0.05 g MgSO4 × 7H2O/1000 ml
H2O). G: (10 g glucose, 0.5 g K2HPO4, 0.5 g, asparagine/1000 ml
H2O); M: (40 g glucose, 2 g asparagine, 0.5 g KH2PO4, 0.25 g
MgSO4 × 7H2O, 0.5 g tiamine/1000 ml H2O); P: 30 g glucose, 10 g
peptone/1000 ml H2O S: (2.5 mg geistageine, 2.5 g NaCl, 2.5 g
K2HPO4, 10 g glucose/1000 ml H2O). The cultures were incubated
at 26 ◦C with shaking at 150 rpm. After 5 days of cultivation, 20 mg
of diethyl 2-(3-oxocyclohexyl)malonate (1) in 0.5 cm3 of acetone
was added to each flask. The conversion and enantiomeric excesses
were measured after 9 h of the transformation, followed by the
extraction with diethyl ether (25 cm3). Dry mass of A. coerulea AM
93 in different media was established after washing and lyophiliza-
tion.
2.5.7. Preparative resolution of racemic diethyl
2-(3-oxocyclohexyl)malonate-( )-1
Absidia coerulea AM 93 was cultivated at 28 ◦C in ten 300 cm3
Erlenmeyer flasks, each containing 75 cm3 of solution of pep-
tone (1%) and glucose (3%) in deionized water. After 5 days
of growth, the cultures of microorganism were incubated with
15 mg of the Eschenmoser salt for 1 h. Next, racemic diethyl
2-(3-oxocyclohexyl) malonate (1), 75 mg in 0.5 cm3 of acetone
for each flask (initial concentration of 1 mg/cm3), was added
to the shaken cultures. After 12 h the products were extracted
with diethyl ether (750 cm3) and dried (MgSO4). The solvent
was evaporated off and the crude product mixture (720 mg;
49% of molar conversion of 1 by GC) was separated by col-
umn chromatography (silica gel, petroleum ether:acetone, 2:1)
2.5.3. Effect of temperature
Portions of 0.5 cm3 of the inoculum (spore concentration of
107 cm−3) were transferred to 300 cm3 Erlenmeyer flasks, each
containing 75 cm3 of growth medium P. The cultures were incu-
bated at one of the following temperatures: 21, 25, 28, 30, or 32 ◦C,
with shaking at 150 rpm. After 5 days of cultivation, 20 mg of diethyl
2-(3-oxocyclohexyl)malonate (1) in 0.5 cm3 of acetone was added
to each flask. The conversion and enantiomeric excesses were mea-
sured after 9 h of the transformation followed by the extraction
with diethyl ether (25 cm3). Dry mass of A. coerulea AM 93 in dif-
ferent media was established after washing and lyophilization.
to give 310 mg of (−)-diethyl 2-((S)-3-oxocyclohexyl)malonate
20
((−)-1) (ee = 98%),{ ˛
= −3.4 (c = 4.25, CHCl3)}; and 290 mg
[ ]589
of (+)-diethyl 2-((1R, 3R)-3-hydroxycyclohexyl)malonate ((+)-2)
20
(ee = 99%) { ˛
= +1.2 (c = 3.7, CHCl3)}. Overall yield of isolated
[ ]589
products was 80%.
3. Results and discussion
2.5.4. Effect of pH
3.1. Initial screening
Portions of 0.5 cm3 of the inoculum (spore concentration of
107 cm−3) were transferred to 300 cm3 Erlenmeyer flasks, each
containing 75 cm3 of growth medium P. The cultures were incu-
bated at 28 ◦C with shaking at 150 rpm. After 5 days of cultivation,
the biomass was washed under sterile conditions and transferred
to Erlenmeyer flasks, each containing 75 cm3 of one of four phos-
phate buffer solutions of different pH values (4.5; 6.2; 7.2; 8.5).
Next 20 mg of diethyl 2-(3-oxocyclohexyl)malonate (1) in 0.5 cm3
of acetone was added to each flask. The conversion and enan-
tiomeric excesses were measured after 9 h of the transformation
followed by the extraction with diethyl ether (25 cm3). Dry mass of
A. coerulea AM 93 in different media was established after washing
and lyophilization.
In the initial screening we searched for microorganisms capa-
ble of resolution of racemic diethyl 2-(3-oxocyclohexyl)malonate
(1) by enantioselective reduction to the corresponding cis or trans
alcohols. We examined twenty-four fungi strains belonging to
twenty different species. In general, the tested microorganisms,
except for Laetiporus sulphurens AM 524, preferred reduction of
(+)-R-enantiomer of the racemic ␦-ketoester (1) to (+)-trans diethyl
2-(3-hydroxycyclohexyl)malonate with moderate both conversion
and enantiomeric excess (Scheme 1).
The trans-diastereoisomer obtained is a product of the anti-
Prelog’s reduction. The cis-isomer was the major product in
biotransformations with Ascosphaera aspis AM 496 (37.7%),
Aspergillus ochraceus AM 456 (58.0%), Fusicoccum amygdale AM 258
(70.1%), and Rhodotorula glutinis AM 242 (45.1%)—unfortunately, it
has never been isolated in a pure form.
2.5.5. Effect of substrate concentration
Portions of 0.5 cm3 of the inoculum (spore concentration of
107 cm−3) were transferred to 300 cm3 Erlenmeyer flasks, each
containing 75 cm3 of growth medium P. The cultures were incu-
bated at 28 ◦C with shaking at 150 rpm. After 5 days of cultivation,
one of the following amounts of 2-(3-oxocyclohexyl)malonate
(1) were added to each flask: 20 mg (1 mmol/dm3), 40 mg
(2 mmol/dm3), 60 mg (3 mmol/dm3), 80 mg (4 mmol/dm3), 100 mg
(5 mmol/dm3) or 150 mg (7 mmol/dm3). The conversion and
enantiomeric excesses were measured after 9 and 12 h of the trans-
formation followed by the extraction with diethyl ether (25 cm3).
Dry mass of A. coerulea AM 93 was established after washing and
lyophilization.
We
planned
to
obtain
(+)-
and
(−)-2-
oxabicyclo[3.3.1]nonan-3-one (3) from two enantiomers of
2-(3-hydroxycyclohexyl)malonate (2). One enantiomer of 2 can
be obtained by biotransformation and the second one may be
obtained by chemical reduction of the unreacted substrate (1).
Unfortunately, there is no chemical method for reduction of
1,3-substituted ketones to cis diastereoisomers as the major prod-
ucts. This is the reason why we concentrated on microorganisms
performing reduction to trans products with a high diastereos-
electivity. The selected results of this transformation (leading
preferentially to the trans alcohol) are presented in Table 1.

T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
5
Scheme 1.
Table 1
Microbial reduction of racemic diethyl 2-(3-oxocyclohexyl)malonate ( )-1)—composition of product mixtures (by GC).
Entry
1
Microorganism
Time [h]
Conversion of 1 [%]
Ee of 1 [%]
Ee of trans-2 [%]
De of trans-2 [%]
3
12
47
58
(−) 82
(−) 99
(+) 99
(+) 89
95
92
Absidia coerulea AM 93
2
21
81
24
79
(−) 14
(−) 42
(+) 22
(+) 62
94
95
Aphanocladium album AM 417
Beauveria bassiana AM 278
Beauveria bassiana AM 446
Fusarium solani AM 203
3
3
6
42
65
(−) 72
(−) 62
(+) 89
(+) 57
76
73
4
6
9
29
47
(−) 9
(−) 8
(+) 5
(+) 10
71
71
5
6
12
37
53
(−) 29
(−) 27
(+) 99
(+) 76
55
63
6
6
12
44
74
(−) 39
(−) 19
(+) 33
(+) 44
77
69
Penicillium camemberti AM 83
Penicillium notatum 904
7
3
6
35
51
(−) 42
(−) 82
(+) 20
(+) 32
81
83
8
9
81
22
80
(−) 21
(−) 51
(−) 63
(−) 35
62
79
Rhizopus nigricans AM 394
9
6
21
35
75
(−) 29
(−) 45
(−) 47
(−) 12
86
83
Saccharomyces cerevisiae AM
464
10
11
3
6
67
72
(−) 62
(−) 79
(+) 69
(+) 67
61
61
Rhodotorula rubra AM 4
9
21
36
83
(−) 22
(−) 32
(+) 19
(+) 31
58
75
Yarrowia lipolytica AM 71
Four of the tested microorganisms: Absidia coerulea AM 93
(ee = 99%), Beauveria bassiana AM 278 (ee = 72%), Penicillium nota-
tum AM 904 (ee = 99%), and Rhodotorula rubra AM 4 (ee = 79%)
transformed substrate 1 with a high enantioselectivity.
3.2. Optimization of biotransformation conditions
3.2.1. Effect of growth medium
The cultures of Absidia coerulea AM 93 were cultivated in the
six growth media of different composition (see Section 2.5.2). A
degree of conversion of diethyl 2-(3-oxocyclohexyl)malonate (1)
as well as enantioselectivity of the process and dry mass of the
culture were measured. The results obtained are presented in
Table 2.
Such big differences in enantioselectivity of the process run in
different growth media suggest that the ingredients of the cultiva-
tion media may affect the dehydrogenases activity. The best growth
medium with respect to the conversion degree and enantiomeric
excess of the substrate was the mixture of peptone and glucose
(growth medium P).
In the initial screening A. coerulea AM 93 [36–38] transformed
racemic diethyl 2-(3-oxocyclohexyl)malonate (( )-1) to (+) iso-
mer of trans diethyl 2-(3-hydroxycyclohexyl)malonate ((+)-2) in
3 h with 99% of enantiomeric excess and 44% of conversion. Dur-
ing the first 3 h the process of reduction was fast (44%) and highly
enantioselective (ee = 99%), whereas later on it proceeded much
slower (8.9% of progress in 9 h). Therefore, not only did we opti-
mized biotransformation conditions with respect to the growth
medium, temperature and pH, but also we have noticed that the
added compound changed activity of dehydrogenases present in
the microorganism.
Table 2
Effect of growth medium on dry mass, enzyme activity, degree of conversion and enantiomeric excesses of (−)-1 and (+)-2.
Entry
Growth medium
Dry mass [g]
Enzymatic activitya
Conversion of 1 [%]
Ee of 1 [%]
Ee of trans-2 [%]
1
2
3
4
5
6
C
E
G
M
S
0.49
0.44
0.46
0.49
0.47
0.60
7.9
5.4
6.8
7.8
10.4
7.5
45
15
36
45
57
52
(−) 62
(−) 8
(+) 93
(+) 99
(+) 99
(+) 95
(+) 92
(+) 95
(−) 40
(−) 52
(−) 13
(−) 92
P
a
Enzymatic activity A = a b h−1 g−1 (a, amount of substrate [␮mol]; b, percent of conversion/100; h, time [h]; g, mass of lyophilized mycelium [g]) AA, enzymatic activity of
the microorganisms with additives; AC, enzymatic activity of the microorganism-control.

6
T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
Fig. 2. Effect of temperature on substrate conversion and enantiomeric excesses of
(−)-1 and (+)-2.
We have also examined growth process of Absidia coerulea
AM 93 in the cultivation medium P. We observed that the initial
pH of the medium (6.5) gradually changed with the progress of
growth.
Fig. 4. Effect of substrate concentration on conversion and enantiomeric excesses
of (−)-1 and (+)-2.
conversion degree and on enantiomeric excess of the products were
determined. The results are presented in Fig. 4.
3.2.2. Effect of temperature
In the next experiment we determined optimal temperature
for the microbial activity. In general, conversion of diethyl 2-(3-
oxocyclohexyl)malonate (1) is directly proportional to temperature
within the range of 21–32 ◦C (about 1 percent of conversion
increase for 1 ◦C) (Fig. 2).
Degree of conversion of ( )-1 depends on substrate concen-
tration. For substrate concentration of from 1 to 4 mmol/dm3 the
conversion degree decreases along with an increase of concen-
tration by about 3% per 1 mmol/dm3. Whereas, for concentration
of ( )-1 of above 4 mmol/dm3 it drops as much as 10% per
1 mmol/dm3.
If the degree of conversion is less than 50% then the enan-
tantiomeric excess of product 2 is above 93%. These data suggest
that it is a single dehydrogenase that is responsible for reduction
(+)-diethyl 2-((R)-3-oxocyclohexyl)malonate ((+)-1) to (+)-diethyl
2-((1R, 3R)-3-hydroxycyclohexyl)malonate.
Within the range of 25–28 ◦C the ee of 2-(3-
hydroxycyclohexyl)malonate (2) is high and the ee of (−)-1
grows. When the temperature reached 30–32 ◦C, we observed a
decrease of the enantioselectivity of dehydrogenases. Therefore,
28 ◦C was chosen as the optimal temperature of the process.
3.2.3. Effect of pH
An activity of the dehydrogenase is proportional to the amount
of (+)-2, calculated from concentration of the substrate and the
degree of conversion. Concentration of the racemic ketone 1 affects
the enzyme activity. Initially, an increase in substrate concentration
activates the dehydrogenase, but when the concentration of ketone
1 is above 4 mmol/dm3, it begins to inhibit it.
A degree of conversion of diethyl 2-(3-oxocyclohexyl)malonate
(1) and an enantioselectivity of the biotransformation process were
evaluated in different pH values. The results are presented in Fig. 3.
We observed, that bioreduction of the substrate is more effi-
cient and more enantioselective under acidic conditions than under
neutral or alkaline ones.
In summary, at this stage of the study it is only (+)-
diethyl 2-(3-hydroxycyclohexyl)malonate (2) that is obtained
3.2.4. Effect of substrate concentration
as
a
pure enantiomer, whereas the (−) isomer of diethyl
The aim of this experiment was to find out what is the maximal
amount of the substrate that may be successfully transformed by
a 5-day Absidia coerulea AM 93 culture, cultivated in 75 cm3 of the
growth medium P, without the loss of enantioselectivity compared
to the screening test. An impact of the substrate amount on the
2-(3-oxocyclohexyl)malonate (1) is obtained only with some enan-
tiomeric excess. Longer reaction time (12 h instead of 9 h) does not
improve the results.
3.2.5. Effect of additives on activity of dehydrogenases
Incubation of the microorganism culture with some additives
is a known method of changing of activity and enantioselectivity
of dehydrogenases [27–33]. For the experiments we employed 48
compounds, the structures of which are presented in Fig. 5.
Activity of dehydrogenases (calculated with respect to dry
lyophilized biomass of the microorganism [27]) and differences
between enantiomeric excesses of biotransformation carried out
with additives and a control sample are presented in a graph (Fig. 6).
We divided the tested compounds into the following groups: A:
acryl, propionic, and crotonic acid or their derivatives and 5 or 6-
member ring compounds with oxygen or nitrogen as heteroatoms;
B: compounds containing bromine atom; C: compounds with a car-
bonyl group; E: inorganic salts. Most of the compounds are of low
molecular weightandcontain adouble bond along witha functional
group in the allylic position. In the last group of compounds, marked
as D, we included the compounds of different structures—it com-
Fig. 3. Effect of pH on conversion and enantiomeric excesses of (−)-1 and (+)-2.

T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
7
Fig. 5. Chemical structures and symbols of the additives used. ꢀA = AA − AC × 100%/Ac, A = a b h−1 g−1 (a, amount of substrate [␮mol]; b, percent of conversion/100; h, time
[h]; g, mass of lyophilized mycelium [g]) AA, enzymatic activity of the microorganisms with additives; AC, enzymatic activity of the microorganism-control. ꢀee = eeA − eeC;
eeA, enantiomeric excesses of the microorganisms with additives; eeC, enantiomeric excesses of the microorganism-control.
prises natural compounds like ergosterol (D-14) or myo-inozytol
(D-13), and organic salts like Eschenmoser salt (D-3), cinchonine
sulphate (D-7) or zircocene dichloride (D-10).
occurred to be an inhibitor. Compound C-2 considerably affects the
diastereoselectivity of the reaction. It activates the cis-reduction
dehydrogenases, but unfortunately, it does not inhibit the dehy-
drogenase responsible for the reduction to trans alcohol.
All the examinated inorganic salts inhibited activity and enan-
tioselectivity of dehydrogenases, whereas the salts with organic
ligands, like the Eschenmoser salt (D-3), cinchonine sulphate (D-
7) or zircocene dichloride (D-10) expressed a high activating and
enantioselectivity-improving effect. The Eschenmoser salt – N,N-
dimethylmethylene-ammonium iodide – is a small molecule, but
such compounds as cinchonine sulphate or zircocene dichloride
have large, spatial structures. Similarly high activity was observed
for HMPA (D-12) (hexamethylphosphoramide).
As we expected, the compounds with a carbonyl group act as
activators for dehydrogenases. Four cyclic ␣,␤-unsaturated ketones
were checked (C-1, C-2, C-3 and C-4). The first three compounds
increased the dehydrogenases activity by 40–50% and the enen-
tiomeric excesses by 17–43%, whereas the fourth one—toluchinon,
Crotonaldehyde (C-6), an aliphatic aldehyde with an ␣,␤-
unsaturated bond, inhibits the bioreduction, whereas the similar
compound – 6-methylhex-5-en-2-one (C-7) – has the opposite
effect. The shift in a double bond position in relation to the carbonyl
group changes ꢀA from −83% for C-6 to +103% for C-7, whereas
ꢀee changes from −32% for C-6 to +42% for C-7. The similar results
were observed for bromine derivatives: the compounds B-1 and B-3
have ␤-unsaturated bonds and both show inhibiting activity, which
is lost for compound B-2 with a double bond in the ␣-position and
for B-4 with no double bond at all. Interestingly, an introduction of
the second bromine atom to 1-bromopropane (B-4) increases the
activity of the dehydrogenase by +60%, and the enantioselectivity
by 59% (compound B-5).

8
T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
Fig. 6. Effect of additives on differences in enzymatic activity and enantiomeric excesses calculated for samples with additives and for control samples. ꢀA = AA − AC × 100%/Ac,
A = a b h−1 g−1 (a, amount of substrate [␮mol]; b, percent of conversion/100; h, time [h]; g, mass of lyophilized mycelium [g]) AA, enzymatic activity of the microorganisms
with additives; AC, enzymatic activity of the microorganism-control. ꢀee = eeA − eeC; eeA, enantiomeric excesses of the microorganisms with additives; eeC, enantiomeric
excesses of the microorganism-control.
In general, we have observed, that the tested organic acids
and their derivatives, except for n-butyl acrylate (A-6; ꢀA = 27%;
ꢀee = 15%) and glutaric anhydride (A-12; ꢀA = 29%; ꢀee = 16%),
inhibit the activity of the dehydrogenases.
Allylic alcohol (D-3; ꢀA = 38%; ꢀee = 17%) is an activator for
oxidoreductases, whereas the replacement of the hydroxyl group
with CN (D-2) or Br (B-3) inhibits the bioreduction. The aromatic
compounds: acenaphthenequinone (C-5), benzyl bromide (B-6), 3-
chloroperbenzoic acid (D-15) act as inhibitors.
3.3. Determination of absolute configuration
Absolute configuration of diethyl 2-(3-oxocyclohexyl)malonate
(1) was confirmed by a comparison of the sign of optical rotation
with the literature data [38–40]. Absolute configuration of the car-
bon bearing hydroxyl group in (+)-2 was determined by the Mosher
ester method [41]. Single enantiomers of the alcohol were treated
separately with two enantiomers of the Mosher’s acid chloride
(Scheme 2).
To sum up, 19 out of the 48 tested additives proved to be strong
dehydrogenase inhibitors. We have selected 5 compounds which
increase the activity and the enantioselectivity of oxidoreductases
in the examined bioreduction process using Absidia coerulea AM
93: C-7 (ꢀA = 103%; ꢀee = 42%), D-7 (ꢀA = 106%; ꢀee = 50%), D-
9 (ꢀA = 100%; ꢀee = 32%), D-10 (ꢀA = 89%; ꢀee = 53%), and D-12
(ꢀA = 130%; ꢀee = 50%).
The observed strongly activating effect of the molecules D-
7, D-9, D-10, and D-12 on the dehydrogenases comes from the
fact that these compounds can be easily attachable by micro-
bial enzymes. Probably, the studied dehydrogenase is an allosteric
enzyme, because it is activated by small compounds (C-7, D-
9), and also by large, spatial molecules (D-7, D-9) which force
change of conformation. Compounds D-7 and D-9 have ionic
bond with an organic ligand. Zyrcone as a transition metal may
form coordination bonds, as well as the highly polarized HMPA
molecule. However, the most important may be the presence of
a nitrogen atom that could be coordinated to metal in the active
site.
The resulting pair of diastereomeric esters (2-S-MTPA, 2-R-
MTPA) was analyzed by high field 1H MNR, 13C NMR, COSY, DEPT
and HMQC, in CDCl3 as a solvent.
The absolute configuration of alcohol (+)-2 was determined by
interpretation of the sign of ꢀıSR value, using empirical models
[41]. This method is based on the anisotropic effect between the
phenyl group of the chiral auxiliary MTPA with the substituents
L1 and L2 of the alcohol. Mosher assumed that the R- and S-
MTPA esters of secondary alcohols exist in a conformation in which
CH(OR), the carbonyl, and the CF3 groups are situated in the same
plane. When ligand L1 (2CH2) is more bulky than L2 (4CH2), it
results in a negative value of ꢀıSR (in both 1H NMR and 13C
NMR). In the case of MTPA-derivative of (+)-2 we observed ꢀıSR < 0,
which means that the absolute configuration of the carbon bearing
hydroxyl group in (+)-2 alcohol is R (Fig. 7).
For further research we used the Eschenmoser salt as an acti-
vator, due to its small molecule which is not troublesome in the
product mixture.
The final optimized biotransformation conditions are as fol-
lows: temperature: 28 ◦C; growth medium: 1% peptone and
3% glucose in deionized water; pH: 3.4–3.8. The microorgan-
ism was incubated with the Eschenmoser salt for 1 h before
adding the substrate in concentration of 1 g/dm3. After 12 h of
the reaction we observed almost ideal resolution (GC analysis
of the crude reaction mixture). After chromatographic separation
we obtained (−)-diethyl 2-((1S)-3-oxocyclohexyl)malonate ((-)-1)
20
{ ˛
= −3.4 (c = 4.25, CHCl3) (ee = 98%)} and (+)-diethyl 2-((1R,
[ ]589
20
3R)-3-hydroxycyclohexyl)malonate ((+)-2) { ˛
= +1.2 (c = 3.7,
[ ]589
CHCl3) (ee = 99%)}.
Scheme 2.

T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
9
Fig. 7. ꢀı values for 2-S-MTPA and 2-R-MTPA calculated from 1H NMR and 13C NMR spectra. *−ꢀı for 13C NMR.
Scheme 3.
3.4. Lactonization of (+)- and (−)-isomer of diethyl
2-(3-hydroxycyclohexyl)malonate (2)
lactone 3. Therefore, at first we hydrolyzed trans diethyl 2-(3-
hydrocyclohexyl)malonate (1) to the corresponding mono acid, and
in next step, we performed lactonization by refluxing the acid with
TsOH × H2O in benzene [44]. As a result, the desired cis lactones
were obtained.
Chemical reduction of diethyl 2-(3-oxocyclohexyl)malonate
(1) with borohydride derivatives led to
a diastereoisomeric
mixture of trans and cis alcohols (ratio 9:1). Under acidic con-
ditions [15% BF3 in diethyl eter or THF:H2O:HClO4 (pH = 1),
or TsOH × H2O in benzene or toluene] racemic cis diethyl 2-
(3-hydroxycyclohexyl)malonate cyclized to the corresponding
bicyclic lactone: 2-oxabicyclo[3.3.1]nonan-3-one ( )-3 [42,43].
Under the same conditions the trans-diastereoisomer of 2 did
not undergo this process. Refluxing of trans diethyl 2-(3-
hydroxyclohexyl)malonate (trans-( )-2) with p-toluenosulfonic
acid in benzene led to the unsaturated diester as the main prod-
uct (80%). The product of dehydratation was more stabile than
The inversion of configuration is the result of formation
of a tosylate, which is an easy-leaving group. The carboca-
tion formed is then attacked by
a water molecule, which
leads to mixture of cis and trans-diastereoisomers of 2-
a
(3-hydroxycyclohexyl)propionic acid. The cis isomer closes to
cis lactone (−)-(1S, 5R)-2-oxabicyclo[3.3.1]nonan-3-one ((−)-3)
20
{ ˛
= −18.6 (c = 2.05, CHCl3), ee = 95%}, whereas the trans one
[ ]589
is tosylated again (Scheme 3).
The unreacted diethyl (−)-2-(3-oxocyclohexyl)malonate ((−)-
1) was isolated from the biotransformation mixture and reduced
Scheme 4.

10
T. Olejniczak / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 1–10
with sodium borohydride to the corresponding trans hydroxy
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20
diester (−)-2 { ˛
= −1.04 (c = 3.11, CHCl3)} with 95% ee.
[ ]589
(+)-(1R, 5S)-2-oxabicyclo[3.3.1]nonan-3-one ((+)-3) was pre-
pared from (−)-diethyl 2-((1R, 3S)-3-hydroxycyclohexyl)malonate
20
(−)-2 in the same way as the opposite isomer { ˛
= +17.9
[ ]589
(c = 1.5, CHCl3), ee = 93%} (Scheme 4).
4. Conclusions
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oxabicyclo[3.3.1]nonan-3-one (3) from the corresponding two
enantiomers of trans diethyl 2-(3-hydroxycyclohexyl)malonate (2).
Four fungal strains: Absidia coerulea AM 93, Beauveria bassiana
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Highly enantioselective biotransformation by Absidia
coerulea AM 93 leading to formation of (−)-diethyl 2-((S)-3-
oxocyclohexyl)malonate (1) (ee = 98%) and (+)-diethyl 2-((1R,
3R)-3-hydroxycyclohex-1-yl)malonate (2) (ee = 99%) can be an
interesting alternative for the enantioselective Michael addition.
The biotransformation conditions were optimized. We have
selected 5 compounds which considerably increase both the activ-
ity and enantioselectivity of the oxidoreductases of Absidia coerulea
AM 93. These are: acetonylacetone C-7 (ꢀA = 103%; ꢀee = 42%),
cinchonine sulphate D-7 (ꢀA = 106%; ꢀee = 50%), Eschenmoser
salt D-9 (ꢀA = 100%; ꢀee = 32%) (ꢀA = 100%; ꢀee = 32%), zircocene
dichloride D-10 (89%), and HMPA D-12 (ꢀA = 130%; ꢀee = 50%).
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Acknowledgement
This work was supported by the Polish State Committee for Sci-
entific Research, Grant No 2200/B/P01/2007/33.
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