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65
60
55
50
45
40
35
30
25
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15
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5
2.6. Growth conditions of C. albicans CCT 0776
C. albicans CCT 0776 was grown in a yeast malt broth medium
(1 L) in a 2 L Erlenmeyer flask incubated at 30 ◦C, and 180 rpm for
24 h. The cells were harvested by centrifuging the culture broth at
1844 × g for 30 min and were subsequently washed with distilled
water. The wet cells were used for biotransformation. In total, 20 g
of wet cells was obtained per 1 L medium.
oxygen atmosphere
argon atmosphere
2.7. General procedure for deracemization of 1,2-diols
To a 25 mL Erlenmeyer flask containing 3 g of wet cells of C.
albicans CCT 0776 suspended in 10 mL of distilled water, 50 mg of
1,2-diols dissolved in 0.5 mL of ethanol as co-solvent was added and
incubated at 30 ◦C, and 180 rpm in an orbital shaker, until complete
deracemization was achieved.
In a preparative scale, 200 mg substrate (3, 4 and 5) was added
to a 150 mL Erlenmeyer flask containing a slurry of 12 g of yeast
(wet weight) in 40 mL of water. The crude reaction was extracted
with ethyl acetate (three times), and the organic layer was dried
over anhydrous sodium sulfate. The solvent was removed by rotary
evaporator, and enantiomerically pure (S)-diols were obtained after
purification with silica gel chromatography using hexane/ethyl
acetate as the mobile phase. For ( )-1-phenyl-1,2-ethanediol 1,
was used 500 mg of substrate in 500 mL flask containing 30 g of
0
0
20
40
60
80
100
120
140
160
Time (h)
Fig. 2. Monitoring the deracemization of 1-phenyl-1,2-ethanediol 1 in oxygen and
argon atmosphere.
(0.9), 120 (100), 106 (0.9), 92 (30.1), 77 (1.8), 65 (22.1). HRMS calcd.
for C8H9NO2: [M+] = 151.0633, found [M+] 151.0626.
2.7.1. (S)-1-Phenyl-1,2-ethanediol (S)-1
Colorless solid, mp 63–65 ◦C; [␣]D +66.0 (c 1.0 CHCl3) 99% ee
The deracemization of racemic ( )-1-phenyl-1,2-ethanediol 1
in an aqueous cells suspension of C. albicans in an orbital shaker at
30 ◦C, was monitored by measuring the optical rotation as shown in
20
{lit. [34] [␣]D20 = +66.9 (c 1.0, CHCl3)} 99% ee. 1H NMR (250 MHz,
CDCl3): ı 3.76 (dd, J = 3.5, 11.25, 1H), 3.66 (dd, J = 8.0, 11.25, 1H),
4.81 (dd, J = 3.5, 8.0 Hz, 1H), 7.26–7.37 (m, 5H). 13C NMR (62.5 MHz,
CDCl3): ı 68.0, 74.6, 126.0, 128.0, 128.5, 140.4.
20
Fig. 1. After 48 h, the specific rotation [␣]D stabilized at +66 indi-
cating a quantitative conversion to (S)-1-phenyl-1.2-ethanediol
(S)-1, which was confirmed by chiral GC. Through a preparative
scale reaction, 500 mg of ( )-1 was incubated with C. albicans
for 48 h to produce (S)-1 in 80% yield and 99% ee. In an argon
atmosphere, the reaction was slow, and only a partial deracem-
2.7.2. (S)-1-(4-methylphenyl)-1,2-ethanediol (S)-5
Colorless solid, mp 65–67 ◦C; [␣]D +64.0 (c 1.8, CHCl3) 99% ee
20
{lit. [34] [␣]D20 = +68.5 (c 1.12, CHCl3), 99% ee}. 1H NMR (250 MHz,
CDCl3): ı 2.34 (s, 3H), 3.65 (dd, J = 8.0, 11.25, 1H) 3.74 (dd, J = 3.75,
11.25, 1H), 4.78 (dd, J = 3.75, 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H),
7.25 (d, J = 8.0 Hz, 2H). 13C NMR (62.5 MHz, CDCl3): ı 21.1, 68.0,
74.5, 125.9, 129.2, 137.4, 137.7. The enantiomeric excess was deter-
mined by HPLC analysis using a Chiracel OB-H column (eluent
[(R)-isomer]; tR = 102.534 min [(S)-isomer].
20
ization occurred, because after 48 h, the [␣]D was only +30 and
after 7 days it reached not more than +35 (see Fig. 2). Monitor-
ing the reaction under saturated oxygen atmosphere by chiral GC,
we observed after 1 h the presence of ␣-hydroxyacetophenone 2
in 3%, reached 10% after 7 h and then decreased to 3% after 48 h.
The ketone 2 was isolated in 3–5% when the reaction was per-
formed on a preparative scale, suggesting a deracemization process
through a stereoinversion. It is known that the oxidation of the
alcohol by some fungus strain occurs because of the presence of
NADH-dependent stereoselective ADH(s) and NADH oxidase(s),
which regenerated NAD+ at the expense of molecular oxygen [16].
To obtain more mechanistic information, we studied the behav-
ior of the enantiomer (R)-1 in the presence of C. albicans, which
was incubated with the same conditions used before for the der-
acemization of racemic 1. When monitoring the reaction by GC, an
immediate formation of ␣-hydroxyacetophenone 2 was observed,
which was then promptly reduced to (S)-1 after 2 days giving 99%
ee. The cited stereoisomer (R)-1 was prepared by performing the
reduction of ␣-hydroxyacetophenone 2 with S. cerevisae to produce
(R)-1 in 90% yield and 92% ee after 20 h. In a separate reaction, we
incubated ketone 2 with C. albicans and produced, after 22 h, (S)-1
in 86% yield and 99% ee. Leaving that reaction for a long period,
we observed that (S)-1 was completely stable, indicating no fur-
ther oxidation and conversion to its antipode. Considering all of
the above facts, we propose a two-step one-pot deracemization
of ( )-1-phenyl-1,2-ethanediol 1 by resting cells of C. albicans, in
which an enantioselective oxidation of the transient enantiomer
(R)-1 to ␣-hydroxyacetophenone 2 initially occurred by an alcohol
(R)-specific oxidase, and 2 was then enantioselectively reduced to
2.7.3. (S)-1-(4-chlorophenyl)-1,2-ethanediol (S)-3
Colorless solid, mp 79–80 ◦C; [␣]D +55.0 (c 1.6 CHCl3) 99% ee
20
{lit. [34] [␣]D20 = +52.0 (c 1.6, CHCl3), 99% ee}. 1H NMR (250 MHz,
CDCl3): ı 3.61–3.72 (m, 2H), 4.78 (m, 1H), 7.26–7.36 (m, 4H).
The enantiomeric excess was determined by HPLC analysis using
a Chiracel OB-H column (eluent hexane/2-propanol 90:10, flow
rate 0.5 mL/min) tR = 76.024 min [(R)-isomer]; tR = 78.761 min [(S)-
isomer].
2.7.4. (S)-1-(4-methoxyphenyl)-1,2-ethanediol (S)-4
Colorless solid, mp 76–77 ◦C; [␣]D +61.0 (c 0.5 CHCl3) 99% ee
20
{lit. [34] [␣]D20 = +60.3 (c 0.5, CHCl3), 99% ee}. The enantiomeric
excess was determined by HPLC analysis using a Chiracel OB-H
column (eluent hexane/2-propanol 90:10, flow rate 0.5 mL/min)
tR = 145.086 min [(S)-isomer]; tR = 149.029 min [(R)-isomer].
2.7.5. 1-(4-aminophenyl)-2-hydroxyethanone 10
Brown solid, mp 113–115 ◦C (decomposition). 1H NMR
(400 MHz, DMSO-d6): ı 4.62 (s, 2H), 6.58 (d, J = 8.8 Hz, 2H), 7.66 (d,
J = 8.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): ı 64.7, 113.1, 122.4,
130.4, 154.4, 196.3. MS m/z (rel. intensity %): 151 [M+] (11.5), 135