56
T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 55–61
stevioside, glycyrrhizin and osladin are also commonly
applied [35,36].
in vacuo and analyzed by GC. All the experiments were repeated
three times.
2.4. Preparative biotransformation
2. Experimental
The same transformations were performed on the preparative
scale in 2000 ml flasks, each containing 500 ml of the cultivation
medium. The cultures were incubated under the same conditions
and then 200 mg of substrates dissolved in 10 ml of acetone were
added to the grown cultures. After incubation the mixtures were
extracted with CHCl3 (3 × 300 ml), dried (MgSO4) and concentrated
in vacuo. The transformation products were separated by column
chromatography and analyzed (TLC, GC, 1H NMR and also con-
firmed by 13C NMR and correlation spectroscopy).
2.1. Materials
The substrates: chalcone (1) and 2ꢀ-hydroxychalcone (4)
were obtained following the method by Yadav et al. [11]; (E)-
4-phenyl-but-3-en-2-one (6) was purchased from Fluka. The
microorganisms, including four strains of yeast: Yarrowia lipolyt-
ica KCh 71, Rhodotorula glutinis KCh 242, Rhodotorula rubra KCh 4,
and Saccharomyces cerevisiae KCh 464 and five filamentous fungi:
Syncephalastrum racemosum KCh 105, Chaetomium sp. KCh 6651,
Didymosphaeria igniaria KCh 6670, Absidia coerulea KCh 93, and
Fusarium culmorum KCh 10 were obtained from the Department
of Chemistry of Wrocław University of Environmental and Life Sci-
ences. All the strains were cultivated on a Sabouraud agar consisting
of aminobac (5 g), peptone K (5 g), glucose (40 g) and agar (15 g) dis-
solved in 1 l of distilled water, at 28 ◦C and pH 5.5 and stored in a
refrigerator at 4 ◦C.
2.5. Spectral data of isolated metabolites
2.5.1. 1,3-diphenylpropan-1-one (2)
Seven-hour transformation of chalcone (1) (200 mg) in the cul-
ture Y. lipolytica KCh 71 yielded 188 mg of compound 2 (colorless
crystals); 1H NMR (600 MHz) (CDCl3) ı (ppm): 3.09 (t, 2H, J = 7.7 Hz,
H-2); 3.29 (t, 2H, J = 7.7 Hz, H-3); 7.22 (t, 1H, J = 7.2 Hz, H-4ꢀꢀ), 7.27
(t, 2H, J = 7.1 Hz, H-2ꢀꢀ, H-6ꢀꢀ), 7.31 (t, 2H, J = 7.5 Hz, H-3ꢀꢀ, H-5ꢀꢀ), 7.46
(t, 2H, J = 7.7 Hz, H-3ꢀ, H-5ꢀ), 7.56 (t, 1H, J = 7.4 Hz, H-4ꢀ), and 7.97 (d,
2H, J = 7.7 Hz, H-2ꢀ, H-6ꢀ).
2.2. Analytical methods
The course of biotransformation was controlled by means of
TLC. Analytical TLC was carried out on silica gel G 60 F254 plates
(Merck). Chromatograms were developed using hexane/acetone
mixture (3:1, v/v) as the eluent. Compounds were detected by
spraying the plates with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in
10% H2SO4. The products were separated by column chromatogra-
phy using silica gel (SiO2, Kieselgel 60, 230–400 mesh, 40–63 m,
Merck) and hexane/acetone mixture (3:1, v/v) as the developing
system. Composition of biotransformation mixtures was estab-
lished by gas chromatography (GC) on Agilent Technologies 7890
A GC instrument, fitted with a flame ionization detector (FID)
and a chiral column Chirasil-Dex CB 25 m × 0.25 mm × 0.25 m. To
determinate the composition and enantiomeric excesses of prod-
uct mixtures the following temperature programs were used: the
temperature program for 1: 90 ◦C/0 min, gradient 3 ◦C min−1 to
200 ◦C/5 min; the retention times: 2 – 24.12 min, (S)-3 – 25.64 min,
(R)-3 – 25.87 min, 1 – 29.69 min; the temperature program for 4:
90 ◦C/0 min, gradient 3 ◦C min−1 to 200 ◦C/10 min; the retention
times: 5 – 30.16 min, 4 – 35.35 min; and the temperature pro-
gram for 6: 90 ◦C/0 min, gradient 5 ◦C min−1 to 140 ◦C, gradient
20 ◦C min−1 to 200 ◦C/5 min; the retention times: 7 – 6.97 min, (S)-
8 – 8.84 min, (R)-8 – 9.01 min, 6 – 9.54 min. Reference samples of
the racemic alcohols were prepared by reducing the ketones with
sodium borohydride in methanol. NMR spectra were recorded on a
DRX 600 MHz Bruker spectrometer and measured in CDCl3. Optical
rotations were measured with an Autopol IV automatic polarimeter
(Rudolph). Absolute configurations of the products were deter-
mined by comparison of their optical rotation values with literature
data.
13C NMR (151 MHz) ı (ppm): 30.18 (C-2), 40.49 (C-3), ꢀ126.19
(C-4ꢀꢀ), 128.09 (C-3ꢀꢀ, C-5ꢀꢀ), 128.48 (C-2ꢀꢀ, C-6ꢀꢀ), 128.58 (C-3 , C-5ꢀ),
128.66 (C-2ꢀ, C-6ꢀ), 133.12 (C-4ꢀ), 136.91, (C-1ꢀ), 141.35 (C-1ꢀꢀ), and
199.27(C-1).
One-day transformation of chalcone (1) (200 mg) in the cul-
ture R. rubra KCh 4 yielded 146 mg of compound 3 (pale yellow
oil); [˛]2D3 = +23.6◦ (c = 1.2, CHCl3) (78% ee) (lit. [˛]25 = +28◦ (c = 1,
D
CH2Cl2), 86% ee [37]. 1H NMR (600 MHz) (CDCl3) ı (ppm): 1.54
(s, 1H, OH), 2.09 (m, 1H, Wh = 36.9 Hz, one of H-2), 2.19 (m, 1H,
Wh = 36.9 Hz, one of H-2), 2.73 (ddd, 1H, J = 16.0, 9.6, 6.4 Hz, one of
H-3), 2.82 (ddd, 1H, J = 15.1, 9.3, 5.1 Hz, one of H-3), 4.73 (dd, 1H,
J = 7.7, 5.5 Hz, H-1), 7.24–7.28 (m, 3H, H-3ꢀ, H-5ꢀ, H-4ꢀꢀ), 7.33–7.36
(m, 3H, H-4ꢀ, H-2ꢀꢀ, H-4ꢀ), and 7.39–7.43 (m, 4H, H-2ꢀ, H-6ꢀ, H-3ꢀꢀ,
H-5ꢀꢀ).
13C NMR (151 MHz) ı (ppm); 32.10 (C-3), 40.50 (C-2), 73.93 (C-
1), 125.92 (C-4ꢀꢀ), 126.04 (C-3ꢀꢀ, C-5ꢀꢀ), 127.68 (C-4ꢀ), 128.46 (C-2ꢀꢀ,
C-6ꢀꢀ), 128.52 (C-3ꢀ, C-5ꢀ), 128.56 (C-2ꢀ, C-6ꢀ), 141.87 (C-1ꢀꢀ), 144.57
(C-1ꢀ).
2.5.3. 1-(2-hydroxyphenyl)-3-phenylpropan-1-one (5)
Three-day transformation of 2ꢀ-hydroxychalcone (4) (200 mg)
in the culture Y. lipolytica KCh 71 yielded 172 mg of compound 5
(colorless crystals); 1H NMR (600 MHz) (CDCl3) ı (ppm): 3.09 (t,
2H, J = 7.7 Hz, H-2), 3.33 (t, 1H, J = 7.7 Hz, H-2), 6.90 (td, 1H, J = 7.6,
1.0 Hz, H-5ꢀ), 7.01 (dd, 1H, J = 8.5, 0.7 Hz, H-3ꢀ), 7.25 (t, 1H, J = 7.3 Hz,
H-4ꢀꢀ), 7.28 (d, 2H, J = 7.2 Hz, H-2ꢀꢀ, H-6ꢀꢀ), 7.34 (t, 2H, J = 7.5 Hz, H-3ꢀꢀ,
H-5ꢀꢀ), 7.47 (ddd, 1H, J = 8.3, 7.3, 1.6 Hz, H-4ꢀ), 7.75 (dd, 1H, J = 8.1,
1.6 Hz, H-6ꢀ), and 12.36 (s, 1H, OH).
2.3. Screening procedure
13C NMR (151 MHz) ı (ppm): 30.04 (C-2), 40.07 (C-3), 118.61
(C-3ꢀ), 119.00 (C-5ꢀ), 119.36 (C-1ꢀ), 126.41 (C-4ꢀꢀ), 128.50 (C-2ꢀꢀ, C-
6ꢀꢀ), 128.70 (C-3ꢀꢀ, C-5ꢀꢀ), 129.94 (C-6ꢀ), 136.41 (C-4ꢀ), 140.84 (C-1ꢀꢀ),
162.55 (C-2ꢀ), 205.45 (C-1).
Erlenmeyer flasks (300 ml), each containing 100 ml of the
medium consisting of 3 g glucose and 1 g aminobac dissolved in
water, were inoculated with a suspension of microorganisms and
then incubated for 3–7 days at 25 ◦C on a rotary shaker (190 rpm).
After full growth of the culture 20 mg of a substrate dissolved in
1 ml of acetone was added. After 1, 3, 7, 12 h and 1, 3, 6, 9 days
of incubation under the above conditions, portions of 10 ml of the
transformation mixture were taken out and extracted with CHCl3
(3 × 10 ml). The extracts were dried over MgSO4, concentrated
2.5.4. 4-phenylbutan-2-one (7)
Seven-hour transformation of (E)-4-phenyl-but-3-en-2-one (6)
(200 mg) in the culture R. rubra KCh 4 yielded 178 mg of compound
7 (pale yellow oil); 1H NMR (600 MHz) (CDCl3) ı (ppm): 2.09 (s,
3H, CH3), 2.71 (t, 2H, J = 7.3 Hz, H-2), 2.84 (t, 2H, J = 7.3 Hz, H-1),
7,11-7,16 (m, 3H, H-2ꢀ, H-4ꢀ, H-6ꢀ), and 7.20–7.25 (m, 2H, H-3ꢀ, H-5ꢀ).