1888
P. D. Gennaro et al. / Tetrahedron: Asymmetry 21 (2010) 1885–1889
from our stocks by usual cultivation procedures10 and used as rest-
ing cells.
4.3.4. (2S)-Hydroxy(phenyl)ethanoic acid 7 one pot preparation
At first, 6.58 mM of phenylglyoxal 1, directly suspended in the
medium, was allowed to react with E. coli JM109 (1 g/L CDW) in
50 mL of M9 medium, at 30 °C. After 2 h, the product was allowed
to react with L. bacillus ADH (25 U) in the same medium, containing
MgSO4 (1 mM), iso-propanol (3 mL), and NADP (3 mg), at 30 °C.
After 22 h, the product was allowed to react with E. coli JM109
(pVL2028) (1 g/L CDW) in the same medium, at 30 °C. After 20 h,
the cells were separated by centrifugation (10,000 rpm, 4 °C); the
supernatant was acidified with HCl 3 M, and extracted using AcOEt
(three 30 mL portions); the organic phases were collected, washed
with water, dried over Na2SO4, and evaporated at reduced pres-
sure. The crude product (46 mg, 92%) contained exclusively (2S)-
hydroxy(phenyl)ethanoic acid 7.
4.2. Analytical methods
Substrates and products were monitored by analyzing the
water phase by HPLC, Hitachi-Merck, UV–visible detector at
220 nm, reverse phase column C18 (Hibar LICHROSORB 50334,
10 lm, 25 cm), H2O/CH3CN 1:1 eluent, 1 mL/min flow, Hitachi
D2500 integrator. The absolute (S)-configuration of biocatalytically
prepared (2S)-hydroxy(phenyl)ethanoic acid was proven via com-
parison with commercially available enantiopure (2S)-hydroxy
(phenyl)ethanoic acid (Aldrich). The absolute (R)-configuration
of biocatalytically prepared 3-[(1R)-1-hydroxyethyl]benzoic acid
was presumed considering the known selectivity of Lactobacillus
brevis ADH. Enantiomeric excesses were measured by GLC using
a Chrompack ChiralDex-CB column. 1H NMR and 13C NMR spectra
were obtained in CDCl3 or DMSO-d6 (Merck) using Bruker AC-200
instrument. All signals are expressed as ppm downfield from tetra-
methylsilane. Optical rotation was obtained in CHCl3 or CH3OH
using JASCO P-1030 polarimeter.
4.3.5. (2S)-Hydroxy(phenyl)ethanoic acid 7 one-pot preparation
on a 250 mg scale
At first, 6.6 mM of phenylglyoxal 1, directly suspended in the
medium, was allowed to react with E. coli JM109 (1 g/L CDW) in
250 mL of M9 medium, at 30 °C. After 2 h, the product was allowed
to react with L. bacillus ADH (125 U) in the same medium, contain-
ing MgSO4 (1 mM), iso-propanol (15 mL), and NADP (15 mg) at
30 °C. After 24 h, the product was allowed to react with E. coli
JM109 (pVL2028) (1 g/L CDW) in the same medium, at 30 °C. After
20 h, the cells were separated by centrifugation (10,000 rpm, 4 °C);
the supernatant was acidified with HCl 3 M and extracted using
AcOEt (three 100 mL portions); the organic phases were collected,
washed with water, dried over Na2SO4, and evaporated at reduced
pressure. The crude product (225 mg, 90%) contained exclusively
(2S)-hydroxy(phenyl)ethanoic acid 7.
4.3. Preparation procedures
4.3.1. 2-Hydroxy-1-phenylethanone 3 preparation
At first, 6.58 mM of phenylglyoxal 1, directly suspended in the
medium, was allowed to react with E. coli JM109 (1 g/L CDW) in
50 mL of M9 medium, at 30 °C. After 2 h, the cells were separated
by centrifugation (10,000 rpm, 4 °C); the supernatant was ex-
tracted using AcOEt (three 30 mL portions), and the organic phases
were collected, dried over Na2SO4, and evaporated at reduced pres-
sure. The crude product (43 mg, 99%) exclusively contained 2-hy-
droxy-1-phenylethanone 3 (42.8 mg, 99.6%). Oil; dH (200 MHz,
CDCl3) 4.92 (2H, s), 7.5–7.7 (3H, m), 7.94 (2H, d, J = 7.5 Hz). dC
(75.5 MHz, CDCl3) 65.5 (t), 127.3 (d), 128.6 (d), 133.2 (d), 134.2
(s), 198.2 (s).
4.3.6. 3-(1-Hydroxyethyl)benzaldehyde 4 preparation
At first, 6.75 mM of 3-acetylbenzaldehyde 2, directly suspended
in the medium, was allowed to react with L. bacillus ADH (25 U) in
50 mL of M9 medium, containing MgSO4 (1 mM), iso-propanol
(3 mL), and NADP (3 mg), at 30 °C. After 20 h, the solution was ex-
tracted using AcOEt (three 30 mL portions), and the organic phases
were collected, dried over Na2SO4, and evaporated at reduced pres-
sure. The crude product (44 mg, 88%) contained 3-(1-hydroxy-
ethyl)benzaldehyde 4 with trace amounts of 8. Oil; dH (200 MHz,
CDCl3) 5.0 (2H, s), 7.5 (2H, t, J = 7.5 Hz), 7.63 (1H, d, J = 7.5 Hz),
7.83 (2H, d, J = 7.5 Hz), 10.1 (1H, s). dC (75.5 MHz, CDCl3) 26.1 (q),
70.5 (d), 127.1 (d), 129.6 (d), 129.9 (d), 132.2 (d), 136.8 (s), 147.1
4.3.2. 1,2-Dihydroxy-1-phenylethanol 6 preparation
At first, 6.47 mM of 2-hydroxy-1-phenylethanone 3 was al-
lowed to react with L. bacillus ADH (25 U) in 50 mL of M9 medium,
containing MgSO4(1 mM), iso-propanol (3 mL), and NADP (3 mg),
at 30 °C. After 20 h. the solution was extracted using AcOEt (three
30 mL portions), and the organic phases were collected, dried over
Na2SO4, and evaporated at reduced pressure. The crude product
(44.5 mg, 99.7%) contained exclusively 1,2-dihydroxy-1-phenyl-
ethanol 6. Oil; dH (200 MHz, CDCl3) 3.68 (1H, dd, J = 7.7, 11.3),
3.81 (1H, dd, J = 3.8, 7.7), 4.86 (1H, dd, J = 3.8, 11.3), 7.3–7.7 (5H,
m). dC (75.5 MHz, CDCl3) 68.3 (t), 74.9 (d), 126.3 (d), 128.2 (d),
(s), 193.0 (d). [a]D = 36.84 (c 9.1 mg, CHCl3). Ee >95%. Retention
times in chiral GLC (t0 = 80 °C for 0 min, tf = 180 °C, 5 °C/min,
PHe = 0.8 atm) of this enantiomer: 10.9 min.
4.3.7. 1-[3-(Hydroxymethyl)phenyl]ethanone 5 preparation
At first, 6.75 mM of 3-acetylbenzaldehyde 2, directly suspended
in the medium, was allowed to react with E. coli JM109 (1 g/L CDW)
in 50 mL of M9 medium, at 30 °C. After 30 min, the cells were sep-
arated by centrifugation (10,000 rpm, 4 °C); the supernatant was
extracted using AcOEt (three 30 mL portions), and the organic
phases were collected, dried over Na2SO4, and evaporated at re-
duced pressure. The crude product (50 mg, 99%) contained exclu-
sively 1-[3-(hydroxymethyl)phenyl]ethanone 5. Oil; dH (200 MHz,
CDCl3) 2.1 (1H, br s), 2.6 (3H, s), 4.8 (2H, s), 7.5 (1H, t, J = 7.5),
7.6 (1H, d, J = 7.5 Hz), 7.9 (2H, d, J = 7.5 Hz), 9.0 (1H, s). dC
(75.5 MHz, CDCl3) 26.9 (q), 65 (t), 126.8 (d), 127.8 (d), 129.0 (d),
131.7 (d), 137.6 (s), 143.9 (s), 198.3 (s).
129.4 (d), 131.3 (d), 140.7 (s). [a]D = +68.23 (c 5.8 mg, CHCl3). Ee
>95%. Retention times in chiral GLC (t0 = 80 °C for 0 min,
tf = 180 °C, 5 °C/min, PHe = 0.8 atm) of this enantiomer: 10.9 min.
4.3.3. (2S)-Hydroxy(phenyl)ethanoic acid 7 preparation
At first, 6.38 mM of 1,2-dihydroxy-1-phenylethanol 6 was al-
lowed to react with E. coli JM109 (pVL2028) (1 g/L CDW) in
50 mL of M9 medium, at 30 °C. After 20 h, the cells were separated
by centrifugation (10,000 rpm, 4 °C); the supernatant was acidified
with HCl 3 M and extracted using AcOEt (three 30 mL portions);
the organic phases were collected, washed with water, dried over
Na2SO4, and evaporated at reduced pressure. The crude product
(45.5 mg, 94%) contained exclusively (2S)-hydroxy(phenyl)etha-
noic acid 7. Oil; dH (200 MHz, DMSO-d6) 5.0 (1H, s), 7.3–7.5 (5H,
m). dC (75.5 MHz, DMSO-d6) 72.3 (d), 126.6 (d), 127.4 (d), 128.4
4.3.8. (1R)1-[3-(Hydroxymethyl)phenyl]ethanol 8 preparation
At first, 5.80 mM of 3-(1-hydroxyethyl)benzaldehyde 4, directly
suspended in the medium, was allowed to react with E. coli
JM109 (1 g/L CDW) in 50 mL of M9 medium, at 30 °C. After 5 h,
the cells were separated by centrifugation (10,000 rpm, 4 °C); the
(d), 140.3 (s), 174.2 (s). [a]D = +153.4 (c 8.8 mg, H2O). Ee >95%.
Retention times in chiral GLC (t0 = 80 °C for 0 min, tf = 180 °C,
5 °C/min, PHe = 0.8 atm) of this enantiomer: 21.4 min.