G. Hasnaoui et al. / Tetrahedron: Asymmetry 16 (2005) 1685–1692
1691
pH = 8.5) was added to the mixture, which was subse-
quently extracted twice with ether. The organic phase
was dried over MgSO and the solvent removed by a
rotary evaporator yielding an orange oil. This oil was
˚
loaded on a silica column (60 A) and eluted using a mix-
(3 mM), 2 (2 mM), 3 (5 mM), 4 (2 mM), 5 (2 mM) or
6 (2 mM) and 2% DMSO to facilitate the solubilization
of the substrate, purified enzyme was added to a final
concentration of (10 or 35 lM). Samples of 1 mL were
taken at regular times, extracted with 1.5 mL of diethyl
ether or hexane, containing an internal standard. Sam-
4
ture of heptane and ethyl acetate (7:3). Fractions were
analyzed using chiral HPLC.
ples were dried over MgSO prior to analysis chiral
4
HPLC or GLC.
4
.2.3. Racemic 2-hydroxy-2-(para-nitrophenyl)-nitro-
ethane d. To a solution of para-nitro benzaldehyde
0.5 g, 3.3 mmol) and nitromethane (2 g, 33 mmol) in
Conditions and retention times were as follows:
(
ethanol (50 mL), potassium tert-butoxide (2 mg) was
added. The mixture was stirred for 4 h at room temper-
ature and monitored by TLC (eluent: heptane, ethyl ace-
tate 7:3). After complete conversion, water (50 mL) was
added and the mixture extracted twice with ether. After
separation, the organicphase was washed with water
Epoxide 1, col. I (heptane/isopropanol 90:10), (R)-1
t = 9.0 min, (S)-1 t = 14.0 min; 2-(para-nitrophenyl)-
R
R
1,2-ethanediol c, 2-hydroxy-2-(para-nitrophenyl)-ethyl-
nitrite ester b and 2-hydroxy-2-(para-nitrophenyl)-
nitroethane d: col. II, (heptane/isopropanol 90:10),
respectively: (R)-c t = 24.5; (S)-c t = 30.4 min; (R,
R
R
and dried over MgSO . The solvent was then removed
4
S)-b t = 11.9 min; (R)-d t = 34.3 min; (S)-d t =
R
R
R
by a rotary evaporator yielding 0.6 g of an orange oil.
Recrystallization in ethanol yielded 0.4 g of crystals of
pure d. H NMR in DMSO: d = 3.29, m, 1H; d = 3.60,
dd, 1H; d = 4.08, t, 1H; d = 5.07, d, 1H (OH);
d = 6.38, d, 2H; d = 6.88, d, 2H.
46.5 min.
1
Epoxide 2, col. IV (temperature program: 7 min at
120 °C followed by an increase by 5 °C/min to 150 °C):
(R)-2 t = 11.5 min; (S)-2 t = 13.5 min.
R
R
4.2.4. Racemic para-nitrophenylethane-1,2-diol c. To a
solution of epoxide 1 (200 mg, 1.2 mmol) in 10 mL of
Epoxide 3, col. III (heptane/isopropanol 95:5): (S)-3
t = 20.5 min; (R)-3 t = 22.1 min.
R
R
acetonitrile, 10 mL of 1 M H SO was added. The mix-
2
4
ture was then stirred overnight. The solution was then
neutralized by the addition of 5 mL of a solution of
Epoxide 4, col. V (110 °C): (R)-4 t = 12.4 min, (S)-4
R
t = 14.8 min.
R
1
the organicphase was dried over MgSO and the solvent
M NaOH and extracted with ether. After separation,
Epoxide 5, col. V (100 °C): (R)-5 t = 35.0 min, (S)-5
4
R
removed by rotary evaporator yielding 130 mg of a
white powder. This powder was dissolved in warm ethyl
acetate after which the slow addition of cold hexane
t = 38.5 min.
R
Epoxide 6, col. I (heptane/isopropanol 99:1): (R)-6
t = 7.1 min, (S)-6 t = 8.3 min.
1
yielded pure crystals of c. H NMR in DMSO:
R
R
d = 3.50, m, 1H; d = 4.35, m, 1H; d = 4.79, t, 1H;
d = 5.34, m, 1H (OH); d = 5.70, d, 1H (OH); d = 7.55,
d, 2H; d = 8.12, d, 2H.
Acknowledgements
This work was supported by the Nederlandse Organisa-
tie voor Wetenschappelijk Onderzoek (NWO).
4
.3. Kinetic resolution of epoxides
4.3.1. NMR monitoring of kinetic resolution of 1. Seven
hundred microlitres of a near-saturated solution of
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(
100 mM, pH = 7.5), containing either epoxide
1