After complete consumption of the substrate, the reaction mixture
was saturated with solid NaCl, extracted with EtOAc (340 mL).
The combined organic layers were dried over anhydrous Na2SO4,
filtered and concentrated in vacuo to afford the optically pure
product. The yields and ee values are presented in Table 2.
Experimental Section
General: Carbonyl reductase from Sporobolomyces salmonicolor
AKU4429 (SSCR),[18] GCY1, Ymr226c and Gre3 from Saccharomyces
cerevisiae,[19] PFADH from Pyrococcus furiosus,[20] carbonyl reductase
from Candida magnolia (CMCR) and d-glucose dehydrogenase
(GDH)[21] were prepared as described previously. Substituted a-hy-
droxy acetophenones and 1-hydroxy-2-pentanone were prepared
and purified according to the literature.[22] (S)-1-Phenyl-1,2-ethane-
diol, (R)-1-phenyl-1,2-ethanediol and all other ketones were pur-
chased from commercial sources, and the cofactors were obtained
from F. Hoffmann-La Roche AG. The racemic alcohol standard sam-
ples used in HPLC analysis were prepared by reduction of the cor-
responding ketones with NaBH4. NaBH4 (2 mmol) was added into
the ketone (1 mmol) solution in MeOH (10 mL). The reaction mix-
ture was stirred for 30 min at rt. After the solvent was removed, sa-
turated NH4Cl solution (10 mL) was added, and the aqueous phase
was extracted with ethyl acetate to give the solution of the prod-
uct. The substrate conversion and product ee values were deter-
mined by gas chromatography (GC) analysis using a CP-Chirasil-
DEX CB (Varian, USA) after trimethylsilyl (TMS) derivatization (car-
ried out by addition of anhydrous EtOAc (700 mL) and bis(trime-
thylsilyl)trifluoroacetamide (90 mL)+trimethyl chlorosilane (10 mL)
at 60 8C for 1 h, or HPLC analysis using a Chiralcel OD-H column
(4.6250 mm; Daicel Co., Japan). Enzyme activities toward the re-
duction of ketones were assayed using a SpectraMax M2 micro-
plate reader (Molecular Devices). The 1H NMR spectra were mea-
sured on a Brucker Avance 600 spectrophotometer using CDCl3 as
the solvent. When the reaction was conducted, NADP+ not NADPH
was added, because NADPH could be regenerated by GDH and
d-glucose.
1
(S)-1-Phenyl-1,2-ethanediol [(S)-2a]: H NMR (600 MHz, CDCl3): d=
3.61 (dd, J=8.4, 11.4.0 Hz, 1H), 3.71 (dd, J=3.0, 11.4 Hz, 1H), 4.78
(dd, J=3.6, 8.4 Hz, 1H), 7.26–7.47 ppm (m, 5H); Analytical HPLC: n-
hexane/2-propanol=97:3, flow rate=1.0 mLminÀ1, T=308C, UV
detection: 230 nm; tR: 33.6 min for (R)-2a and 36.4 min (S)-2a.
1
(S)-1-(4-Methylphenyl)-1,2-ethanediol [(S)-2b]: H NMR (600 MHz,
CDCl3): d=2.34 (s, 3H), 3.61 (dd, J=5.4, 10.2 Hz, 1H), 3.71 (dd, J=
6.6, 9.0 Hz, 1H), 4.75 (dd, J=5.4, 11.4 Hz, 1H), 7.16–7.27 ppm (m,
4H); Analytical HPLC: n-hexane/2-propanol=96:4, flow rate=
0.9 mLminÀ1, T=308C, UV detection: 230 nm; tR: 25.3 min for
(R)-2b and 28.4 min for (S)-2b.
(S)-1-(4-Cyanophenyl)-1,2-ethanediol [(S)-2c]: 1H NMR (600 MHz,
CDCl3): d=3.61 (dd, J=3.6, 9.6 Hz, 1H), 3.74 (dd, J=4.2, 7.2 Hz,
1H), 4.75 (dd, J=6.6, 9.0 Hz, 1H), 7.16–7.27 ppm (m, 4H); Analytical
HPLC: n-hexane/2-propanol=92:8, flow rate=0.9 mLminÀ1, T=
308C, UV detection: 254 nm; tR: 31.9 min for (R)-2c and 35.9 min
for (S)-2c.
(S)-1-(4-Fluorophenyl)-1,2-ethanediol [(S)-2d]: 1H NMR (600 MHz,
CDCl3): d=3.57 (dd, J=8.4, 11.4 Hz, 1H), 3.69 (dd, J=3.6, 12.0 Hz,
1H), 4.76 (dd, J=3.6, 8.4 Hz, 1H), 7.01–7.32 ppm (m, 4H); Analytical
HPLC: n-hexane/2-propanol=96:4, flow rate=0.9 mLminÀ1, T=
308C, UV detection: 230 nm; tR: 27.4 min for (R)-2d and 30.3 min
for (S)-2d.
(S)-1-(4-Chlorophenyl)-1,2-ethanediol [(S)-2e]: 1H NMR (600 MHz,
CDCl3): d=3.57 (dd, J=8.4, 11.4 Hz, 1H), 3.70 (dd, J=3.0, 11.4 Hz,
1H), 4.76 (dd, J=3.6, 8.4 Hz, 1H), 7.26–7.32 ppm (m, 4H); Analytical
HPLC: n-hexane/2-propanol=97:3, flow rate=1.0 mLminÀ1, T=
308C, UV detection: 230 nm; tR: 34.8 min for (R)-2e and 38.5 min
for (S)-2e.
Activity assay of carbonyl reductase for the reduction of a-hy-
droxy acetophenone: The specific activity of purified carbonyl re-
ductase SSCR, GCY1, Ymr226c, Gre3, PFADH, CMCR toward the re-
duction of a-hydroxy acetophenone in Table 1 and Table 2 were
determined by spectrophotometrically measuring the oxidation of
NADPH at 340 nm (e=6.22 mmÀ1 cmÀ1) in the presence of an
excess amount of ketone. The activity was measured at room tem-
perature in a 96-well plate, in which each well contained ketone
(6.25 mm), NADPH (0.40 mm) and 10% v/v DMSO in sodium phos-
phate buffer (100 mm, 190 mL). The reaction was initiated by the
addition of the carbonyl reductase (10 mL solution containing 0.5–
20 mg of enzyme). The specific activity (UmgÀ1) was defined as the
number of micromoles of NADPH converted in 1 min by 1 mg of
enzyme (mmol·minÀ1 mgÀ1).
(S)-1-(4-Bromophenyl)-1,2-ethanediol [(S)-2 f]: 1H NMR (600 MHz,
CDCl3): d=3.58 (dd, J=8.4, 11.4 Hz, 1H), 3.72 (dd, J=3.0, 10.8 Hz,
1H), 4.77 (dd, J=3.6, 7.8 Hz, 1H), 7.01–7.32 ppm (m, 4H); Analytical
HPLC: n-hexane/2-propanol=97:3, flow rate=1.0 mLminÀ1, T=
308C, UV detection: 230 nm; tR: 38.6 min for (R)-2 f and 42.1 min
for (S)-2 f.
(S)-1-(4-Methoxyphenyl)-1,2-ethanediol
[(S)-2g]:
1H NMR
(600 MHz, CDCl3): d=3.61 (dd, J=7.8, 10.8 Hz, 1H), 3.69 (dd, J=
3.6, 11.4 Hz, 1H), 3.80 (s, 3H), 4.74 (dd, J=3.6, 8.4 Hz, 1H), 6.88–
7.32 ppm (m, 4H); Analytical HPLC: n-hexane/2-propanol=95:5,
Screening of the cosolvent: Sodium phosphate buffer (1 mL,
100 mm, pH 6.5) containing 10% v/v organic solvent (methanol,
ethanol, isopropanol, THF, 1,4-dioxane, DMSO, DMF, EtOAc, butyl
acetate, acetone, toluene), substrate (50 mm), glucose (100 mm),
NADP+ (1.0 mm), CMCR (3.5 U, 1 U was defined as the enzyme con-
verting 1 mmol of NADPH to NADP+ per min with a-hydroxyaceto-
phenone as the substrate) and GDH (4 U, 1 U was defined as the
enzyme converting 1 mmol of NADP+ to NADPH per min with
d-glucose as the substrate) was shaken at rt. The process of the re-
action was monitor by TLC every 2 h.
flow rate=0.8 mLminÀ1
, T=308C, UV detection: 230 nm; tR:
39.8 min for (R)-2g and 42.2 min for (S)-2g.
(S)-1-(2-Chlorophenyl)-1,2-ethanediol [(S)-2h]: Analytical HPLC: n-
hexane/2-propanol=95:5, flow rate=0.8 mLminÀ1, T=308C, UV
detection: 230 nm; tR: 18.0 min for (S)-2h and 20.5 min for (R)-2h.
(S)-1-(3-Chlorophenyl)-1,2-ethanediol [(S)-2i]: 1H NMR (600 MHz,
CDCl3): d=3.61 (dd, J=7.8, 11.4 Hz, 1H), 3.75 (dd, J=3.6, 11.4 Hz,
1H), 4.79 (dd, J=3.6, 7.8 Hz, 1H), 7.23–7.39 ppm (m, 4H); Analytical
HPLC: n-hexane/2-propanol=95:5, flow rate=0.8 mLminÀ1, T=
308C, UV detection: 230 nm; tR: 23.5 min for (R)-2i and 27.2 min
for (S)-2i.
Enzymatic reduction of a-hydroxy ketones: A typical procedure
for the enzymatic reduction of a-hydroxy ketones was as follows:
a solution of a-hydroxy acetophenone in DMSO (5 mL, 0.5m) was
added to sodium phosphate buffer (100 mm, pH 6.5, 45 mL) con-
taining CMCR (70 U), GDH (80 U), NADP+ (5 mg), and d-glucose
(1.0 g). The reaction mixture was stirred at 308C with TLC monitor-
ing from time to time. All the reactions were finished within 6 h.
1
(S)-Pentane-1,2-diol [(S)-2j]: H NMR (600 MHz, CDCl3): d=0.94 (t,
J=7.2 Hz, 3H), 1.35–1.51 (m, 4H), 3.42 (dd, J=7.8, 10.8 Hz, 1H),
3.64 (dd, J=3.0, 10.8 Hz, 1H), 3.71—3.75 (m, 1H); Analytical GC:
ChemistryOpen 2015, 4, 483 – 488
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