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materials and medium were sterilized in an autoclave at 121 ◦C
before use and the yeast was manipulated in a laminar flow cabinet.
with sodium sulfate, the solvent evaporated, and the products were
purified by column chromatography (hexane/ethyl acetate 9:1).
2.3. Procedure for the P. stipitis mediated bioreduction of
trans-4-phenyl-3-buten-2-one
2.7. General procedure for the epoxide ring closure from 4a and
4b
trans-4-Phenyl-3-buten-2-one (20 mg) dissolved in 0.5 mL of
ethanol was added to an Erlenmeyer containing 20 mL of slurry
of growing P. stipitis cells. The reaction mixture was incu-
bated in an orbital shaker (200 rpm, 30 ◦C) for 72 h. Then,
the product was extracted with dichloromethane. The solvent
was dried with sodium sulfate, evaporated, and the products
were analyzed by GC/MS giving 24.3% of 4-phenylbutan-2-one
(retention time = 5.9 min), 1.6% of 4-phenylbutan-2-ol (retention
time = 6.1 min) and 73.9% of trans-4-phenyl-3-buten-2-one (reten-
tion time = 7.2 min).
An aqueous solution of NaOH (2 M, 1.2 mL) was added drop wise
to a round-bottom flask containing a solution of 4a or 4b (4 mmol)
in diethyl ether (5 mL). The reaction mixture was maintained under
magnetic stirring for 6 h. After that, the organic layer was sepa-
rated, the aqueous layer was extracted with dichloromethane and
the combined organic layers were dried under sodium sulfate. The
solvent was evaporated and the product was purified by column
chromatography (hexane/ethyl acetate 9:1).
2.8. (Z)-3-Chloro-4-phenyl-3-buten-2-one (1a)
2.4. General procedure for the preparation of ˛-haloenones 1a
and 1b [36]
Following the general procedure for preparation of ␣-
haloenones, compound 1a was obtained in 20% yield as pale yellow
oil; retention time on GC/MS(A): 7.68 min, GC/MS(B): 8.57 min EI-
MS m/z (relative intensity): 51 (14), 63 (6), 75 (15), 102 (55), 115
(5), 129 (20), 137 (24), 145 (28), 165 (25), 179 (100), 180 (M+, 81),
181 (42), 182 (27); IR (film) 3353, 3055, 2928, 1686, 1596, 1573,
1491, 1447, 1359, 756, 690; 1H NMR (250 MHz, CDCl3): ı 2.56 (s,
3H), 7.41–7.43 (m, 3H), 7.75 (s, 1H), 7.83–7.87 (m, 2H); 13C NMR
(62.5 MHz, CDCl3): ı 26.81, 128.65, 130.19, 130.39, 130.86, 132.74,
135.62; 193.37.
Oxone® (25.2 g, 40 mmol) and NaCl (2.4 g, 40 mmol) or NaBr
(4.1 g, 40 mmol) were added to an ice bath cooled mixture of
dichloromethane (100 mL) and water (20 mL) in a 250 mL in
a round-bottom flask. The mixture was stirred for 40 minutes,
and after that trans-4-phenyl-3-buten-2-one (3.0 g, 20 mmol) was
added and the mixture was stirred for 4 h. After that, triethylamine
(8.6 mL, 30 mmol) was added with a dropping funnel, and the reac-
tion mixture was stirred for 1 h. After that, the reaction mixture
was neutralized with a 1 M HCl solution, the organic layer was sepa-
rated, and the aqueous phase was extracted with dichloromethane.
The combined organic fractions were dried with sodium sulfate and
the solvent was evaporated. The product 1a was purified by prepar-
ative TLC (silica gel containing 10% AgNO3 in mass; elution solvent
as hexane/ethyl acetate 9:1) and the 1b product was purified by
preparative TLC (silica gel and hexane/ethyl acetate 9:1).
2.9. (Z)-3-Bromo-4-phenyl-3-buten-2-one (1b)
Following the general procedure for preparation of ␣-
haloenones, compound 1a was obtained in 78% yield as yellow oil;
retention time on GC/MS(A): 8.21 min, GC/MS(B): 9.19 min, EI-MS
m/z (relative intensity): 51 (23), 63 (13), 77 (23), 102 (98), 145 (100),
181 (10), 183 (9), 210 (9), 212 (8), 224 (M+, 53), 226 (52); IR (film):
3102, 30,29, 2922, 2855, 1685, 1602, 1480, 1446, 1356, 1219, 1171,
1073; 1H NMR (500 MHz, CDCl3): ı 2.60 (s, 3H), 7.43–7.45 (m, 3H),
7.85–7.87 (m, 2H), 8.03 (s, 1H); 13C NMR (125 MHz, CDCl3): ı 27.02,
123.25, 128.50, 130.42, 130.47, 133.71, 139.92, 193.13.
2.5. General procedure for the P. stipitis mediated bioreduction of
˛-haloenones 1a and 1b
Procedure A (to collect data shown in Figs. 1 and 2): compound
1a or 1b (10 mg) dissolved in 1 mL of ethanol was added in each of
8 Erlenmeyer flasks containing 10 mL of slurry of growing P. stipi-
(200 rpm at 30 ◦C). After the appropriate intervals, one Erlenmeyer
was collected and the products were extracted with ethyl acetate,
dried with sodium sulfate, the solvent was evaporated and the
products analyzed by GC/MS. The results are shown in Fig. 1 for
1a and Fig. 2 for 1b.
Procedure B (for isolation the reaction product): compound 1a
or 1b (100 mg) dissolved in 1 mL of ethanol was added in an Erlen-
meyer flask containing 200 mL of slurry of growing P. stipitis cells.
The reaction mixture was incubated in an orbital shaker (200 rpm
at 30 ◦C) for 72 h. Then, the product was extracted in a continuous
extractor with dichloromethane for 2 days. The solvent was dried
with sodium sulfate, evaporated, and the products were purified
by column chromatography (hexane/ethyl acetate 9:1).
2.10. 4-Phenyl-2-butanone (2)
Following the general procedure for bioreduction of haloenones
1a and 1b mediated by P. stipitis (Procedure B), after 72 h at
30 ◦C compound 2 was obtained as colorless oil; retention time on
GC/MS(A): 7.30 min, GC/MS(B): 6.79 min, EI-MS m/z (relative inten-
sity): 51 (12), 65 (9), 77 (23), 91 (64), 105, (92), 133 (19), 148 (M+,
100); IV (film): 3085, 3062, 3027, 3002, 2922, 1718, 1602, 1583,
1496, 1453, 1408, 1357, 1283, 1228, 1161, 1080, 749, 699; 1H NMR
(250 MHz, CDCl3): ı 2.14 (s, 3H), 2.77 (t, 2H, J = 8 Hz), 2.91 (t, 2H,
J = 8 Hz), 7.16–7.31 (m, 5H); 13C NMR (62.5 MHz, CDCl3): ı 29.72,
30.07, 45.16, 126.10, 128.49, 148.29, 140.97, 207.95.
2.6. General procedure for the P. stipitis mediated bioreduction of
˛-haloenones 1a and 1b with addition of m-dinitrobenzene
Following the general procedure for bioreduction of haloenone
1a mediated by P. stipitis (Procedure A), compounds 1a, 2 and 3a
were detected by GC/MS in each Erlenmeyer flasks (data shown
in Fig. 1). Retention time of compound 3a on GC/MS(A): 7.78 min,
GC/MS(B): 8.63 min, EI-MS m/z (relative intensity): 43 (100), 63
(14), 77 (25), 91 (4), 103 (22), 115 (5), 129 (28), 145 (21), 182 (5),
184 (2).
DNB (1 mg) was added to an Erlenmeyer flask containing 200 mL
of slurry of growing P. stipitis cells and the mixture was stirred for
40 min in an orbital shaker (200 rpm, 30 ◦C). Thus, 100 mg 1a or 1b
dissolved in 1 mL of ethanol were added. The stirring was continued
for 24 h. Then, the products were extracted in a continuous extrac-
tor with dichloromethane for 2 days. The organic phase was dried