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),