General Method of Hydrolysis of Esters with Enzymes.
To a weighed amount of enzyme (1 to 5 mg) was added 1
mL of 100 mM phosphate buffer, followed by 20 µL
(approximately 15 mg) of the ester. The mixtures were stirred
for 20 h at room temperature. The reaction mixture was
extracted with 2 mL of MTBE. A portion (1 mL) of the
MTBE extract was evaporated, dissolved in 1 mL of
acetonitrile, and analyzed by HPLC method 1 to determine
the extent of hydrolysis. The remainder of the MTBE extract
was evaporated and dissolved in hexane:IPA (1:1, 1 mL)
and analyzed by HPLC method 3 to determine the ee of the
alcohol produced.
Esterification of (R,S)-Alcohol 4 with Succinic Anhy-
dride Catalyzed by Lipase PS/PP. To a solution of (R,S)-
alcohol 4 (2.4 g) in toluene (120 mL) in a 500-mL, round-
bottomed flask fitted with a Drierite guard tube was added
succinic anhydride (600 mg), and the mixture was stirred
by magnetic stirrer. Lipase PS/PP (60 mg) was added.
Samples were withdrawn at various intervals and analyzed
by HPLC methods 2 and 3 to determine the conversion and
ee. At 5 h the AP ratio showed unreacted alcohol 67% and
hemisuccinate 33%, and 100 mL (equivalent to 2 g alcohol)
of the mixture was taken out and filtered to remove the
insoluble enzyme. Further work on the filtrate from the 5-h
reaction is described below.
Hydrolysis of Esters by Lipase PS. To 40 mL of 100
mM phosphate buffer, pH 7 (in a constant pH-maintaining
system (Dosimat, Brinkman)), maintained at 40 °C, was
added 10 mg of lipase PS-30, followed by 200 mg of
n-butyrate ester. The pH was maintained at 7, and the
progress of the hydrolysis reaction was monitored by
following the consumption of 0.1 N NaOH solution. Con-
sumption of NaOH started immediately, and the rate of
addition was initially very fast. At 30 min intervals (1 h at
the end), 4 mL of sample was withdrawn and extracted with
8 mL of MTBE. The MTBE extract was analyzed by HPLC
to determine the conversion and ee as described before.
Hydrolysis of Acetate by Lipase PS. A solution of lipase
PS-30 (50 mg) in 500 mL of 100 mM phosphate buffer, pH
7, was placed in a jacketed, three-necked, 1-L reaction vessel.
The mixture was stirred magnetically, and the temperature
was maintained at 40 °C throughout the reaction. (R,S)-
acetate 5 (2.5 g) was added, and the pH was maintained at
7 by automatic addition of 0.1 N NaOH. The progress of
the reaction was monitored by following the consumption
of the NaOH solution. The theoretical amounts of 0.1 N
NaOH for 100% and 50% hydrolysis are 97.28 and 48.64
mL, respectively. Consumption of NaOH started immedi-
ately. The reaction was terminated at 4.75 h when 41.585
mL of NaOH was consumed suggesting 43% conversion.
The reaction mixture was extracted with MTBE (2 × 500
mL). Samples from the MTBE extract were evaporated and
analyzed by HPLC as before. HPLC showed 44% alcohol
with an ee of 87.5% for the S-alcohol. The MTBE extract
was evaporated, and the residual oil was crystallized from
hexane to provide 340 mg of solid S-alcohol 1 (16% yield
from acetate) with ee > 95%.
Acetylation of (R,S)-Alcohol with Lipase PS/PP and
Vinyl Acetate. In a vial, (R,S)-alcohol 4 (500 mg) was
dissolved in 10 mL of toluene. Lipase PS/PP (10 mg) was
added to the vial. Vinyl acetate (250 µL) was added to each
vial. The contents were gently stirred by magnetic stirrer at
room temperature. At 0.5 1, 1.5, 2, 2.5, 3, and 4 h, samples
(50 µL each) were withdrawn and analyzed by HPLC as
described before. After 4 h, the mixture was filtered to
separate the enzyme, and the toluene solvent was evaporated
in a rotary evaporator. The residue was crystallized from
hexane. The R-alcohol 7 was obtained as a white solid (135
mg). The mother liquor was analyzed by HPLC and found
to contain both the alcohol and acetate.
The remainder of the reaction was allowed to continue
to 7 h and then was filtered. The AP ratio showed unreacted
alcohol 59% and hemisuccinate 41%. Further work on the
filtrate from the 7-h reaction is described below.
A. Filtrate from the 5-h Reaction. The filtrate (100 mL)
from the 5-hour reaction was extracted with 5% NaHCO3
(2 × 50 mL). The toluene layer containing the unreacted
alcohol was washed with water (3 × 100 mL). HPLC showed
that the ee of the unreacted R-alcohol was 50.4%.
The aqueous layer was washed with toluene (3 × 100
mL). HPLC showed that the 5% NaHCO3 extract contained
only the product hemisuccinate. To regenerate the S-alcohol
from the hemisuccinate, 100 mL of 2 N NaOH was added
to the 5% NaHCO3 solution, and the mixture was stirred at
room temperature for 30 min. The aqueous layer was
extracted with toluene (2 × 50 mL). The toluene extract was
washed with water (3 × 50 mL). HPLC showed that the
hemisuccinate was completely hydrolyzed, and only S-
alcohol with an ee of 95% was present. Removal of the
toluene at the rotary evaporator provided 600 mg of oil (30%
yield from 2 g of alcohol). This was dissolved in hexane (5
mL) and was left in the freezer overnight to crystallize, giving
the S-alcohol (463 mg, 23% yield from 2 g of alcohol) with
ee 95.8%.
B. Filtrate from the 7-h Reaction. The filtrate from the
7-h reaction was worked up in the same way to provide the
S-alcohol (73.4 mg, 18% yield from 400 mg alcohol) with
ee 94.9%. The ee of the unreacted R-alcohol was 72.7%.
Isolation of Hemisuccinate 6. To a solution of (R,S)-
alcohol 4 (2 g) in toluene (20 mL) were added succinic
anhydride (1 g) and lipase PS/PP (50 mg), and the mixture
was stirred by magnetic stirrer at room temperature for 16
h. The reaction mixture was filtered to separate the insoluble
enzyme and succinic anhydride. The filtrate was extracted
with 5% NaHCO3 (2 × 20 mL). The 5% NaHCO3 layer was
washed with toluene (3 × 10 mL) to remove any unreacted
alcohol 7. HPLC showed that the 5% NaHCO3 layer
contained only the hemisuccinate 6.
To isolate the hemisuccinate 6, the 5% NaHCO3 layer
was carefully adjusted to pH 4.5 by slow addition of 1 N
HCl. After acidification, the aqueous layer was extracted with
MTBE (3 × 10 mL). The MTBE extract was washed with
10% NaCl (3 × 10 mL) until the washing was neutral.
Removal of MTBE gave the hemisuccinate ester as an oil.
On mixing with 2-propanol-heptane and scratching, the oil
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Goswami, Animesh
