Organic Process Research & Development
Article
with 98% conversion and is isolated as a stable O-phenylacetyl
derivative in 80% yield based on the furfuryl alcohol used for
transformation. When commercially available immobilized
penicillin G acylase is employed, kinetic resolution can be
performed to obtain both (R)- and (S)-enantiomers with high
enantiomeric excess (>99%) and high yields. The immobilized
enzyme works well in suspension of diisopropyl ether and can
be recycled several times.
(dd, 1H, J = 2.3, 18.8 Hz). 13C NMR (CDCl3): δ 204.5, 170.7,
158.6, 136.8, 133.1, 128.9, 128.4, 127.0, 72.1, 40.8, 40.6. IR
(neat): νmax 2937, 1724, 1660 cm−1. MS (ESI) m/e 217 (M +
1). HRMS [ESI, (M + H)+]: m/z calcd for C13H13O3 217.0864;
found 217.0859.
Enzymatic Reaction. Polymer beads bearing penicillin G
acylase (10 g, 2000 units, water content 67% w/w determined
by Karl Fischer titration) were washed with phosphate buffer
(0.05 M, pH 7.5) several times to remove the preservatives and
dried by tapping with a filter paper. The moist polymer beads
were taken in a glass kettle (250 mL), and Celite (3 g) and
substrate solution (5 g, 23 mmol) in diisopropyl ether (100
mL) were added and the contents were stirred mechanically at
100 rpm using an overhead stirrer. The reaction was found to
be inhibited by product when enantiomeric excess of the
unreacted ester reached 40% (24 h). At this stage, the enzyme
was removed, washed with diisopropyl ether (2 × 25 mL), and
the washings were combined with the reaction mixture. The
reaction mixture was then concentrated on a rotavapor to 100
mL, and fresh enzyme (10 g) and Celite (3 g) were added and
stirring was continued. After one more repetition with fresh
enzyme, the reaction was complete (ee of the unreacted ester
reached >99%) in 3 days. The reaction mixture was separated
from the enzyme and dried over anhydrous magnesium sulfate
(caution: it is important that the reaction mixture is free of
water for the preparation of TBDMS derivative), and the
solvent was removed by rotary evaporation. The residue
consisted of an equimolar mixture of (S)-1 and the
unhydrolyzed (R)-2.
Product Separation. The residue obtained as described
above was dissolved in dry THF (50 mL) and cooled in ice.
Et3N (5.7 mL, 0.039 mol) and DMAP (300 mg, 0.25 mmol)
were added. A solution of tert-butyldimethylsilyl chloride
(TBDMS) (4.6 g, 30 mmol) in dry THF (20 mL) was added
dropwise over 20 min under nitrogen atmosphere. The mixture
was stirred for 30 min in cold and then allowed to warm to
room temperature. After stirring for 3 h, the solvent was
removed on a rotavapor, distilled water (50 mL) was added to
the residue, and the product was extracted in ethylacetate (3 ×
25 mL). The combined organic fractions were dried over
anhydrous Mg2SO4 and concentrated in vacuum. The resulting
mixture of products (R)-2 and (S)-3 was transferred to the top
of the silica gel column and the TBDMS derivative was eluted
with hexane/ethylacetate (95:5). The phenylacetyl derivative
remaining on the column was eluted with ethylacetate. On
removal of the solvents, 3 was obtained as a pale brown oil (4.8
g) while 2 was obtained as a pale yellow solid (2.3 g). Both of
the products were obtained in 45−46% yield (90−92%
theoretical) and >99% ee. The O-protected products were
stable and could be stored in a refrigerator for months.
HPLC Analysis. The rearrangement of furfuryl alcohol was
followed by reverse-phase HPLC column RP-8 (250 × 4.6
mm), Merck KGa, Germany. Mobile phase 30% MeOH/water;
flow rate 0.5 mL/min; detection wavelength 230 nm. Retention
times: 4-hydroxy-2-cyclopentenone 1, 6.9 min; N-methyl-
pyrrolidinone (NMP), 8.4 min; furfuryl alcohol (FA), 10.4
min. NMP served as the internal standard.
EXPERIMENTAL SECTION
■
General. HPLC analysis was carried out on Varian Pro Star
HPLC unit. All other reagents and solvents used were of
analytical grade obtained from Hi Media and Qualigens, India.
SS tube of 0.5 mm diameter was obtained from M/s Swagelok,
Germany. HPLC pump LC-10AT (Shimadzu, Japan) was used
for pumping. The reactor chamber was placed in a Shimadzu
GC oven model GC-2010 for temperature control. Immobi-
lized CAL B (Addzyme CAL B 10 P, activity 1000 tributyrin
units/g) was a generous gift from M/s Advanced Enzyme
Technologies Ltd., Mumbai, India. The enzyme penicillin G
acylase immobilized on a polymeric support with an average
particle size of 150 μm (200 units/g, wet) was a generous gift
from M/s KDL Biotech Limited, Savroli, India.
Conversion of Furfuryl Alcohol to 4-Hydroxy-2-cyclo-
pentenone 1. A solution of furfuryl alcohol in water (39.2 g/
L, 0.4 M) containing NMP (100 g/L, 1 M) and acetic acid (210
mg/L, 0.03M) was passed through the microreactor channel of
0.5 mm diameter and 1.5 m length with a feed rate of 0.2 mL/
min at 240 °C and 200 bar pressure. The product stream was
collected in a two-necked round-bottom flask containing NMP
(10 mL) that was maintained at 60 °C and also connected to a
vacuum pump (50 mmHg pressure). At intervals of 1 h, the
pumping of reactants was stopped for 10 min; the inlet valve to
the round-bottom flask was closed, and vacuum pump was
switched on to remove water from the product stream. After 11
h reaction, a solution of product in NMP was obtained (29 g).
For immediate use, NMP was removed from this solution by
further decreasing the pressure to 10 mmHg and increasing the
temperature to 80 °C. The product obtained as a pale yellow
residue (5.1 g, 98% yield, 98% HPLC purity) was collected.
For long-term storage and enzymatic resolution, the product
in NMP was directly converted to its phenylacetate derivative 2
without isolation.
( )-4-Oxocyclopent-2-enyl 2-Phenylacetate 2. The
solution of ( )-4-hydroxycyclopent-2-en-1-one 1 (28 mL
product solution in NMP containing 4.9 g, 50 mmol) was
diluted with dichloromethane (100 mL). Phenylacetic acid (7.4
g, 55 mmol) and 4-N,N-dimethylaminopyridine (DMAP, 0.6 g,
0.5 mmol) were added, and the solution was cooled in an ice
bath. A solution of dicyclohexylcarbodiimide (DCC, 10.2 g, 50
mmol) in DCM (50 mL) was added to the reaction mixture,
and after being stirred for 20 min, the ice bath was removed.
The dark brown reaction mixture was stirred for 3 h at room
temperature and filtered. The filtrate was washed with
hydrochloric acid (0.5 N, 2 × 10 mL) and then with sodium
carbonate solution (10%, 2 × 10 mL). The organic layer was
then dried over anhydrous magnesium sulfate and evaporated
on a rotavapor. The dark brown residue was extracted with
diisopropyl ether. Removal of the solvent gave 4-oxocyclopent-
2-enyl 2-phenylacetate as a pale yellow low melting powder
Enzymatic hydrolysis of phenylacetyl derivative 2 was
followed by reverse-phase HPLC column C-8 (250 × 5 mm),
Chrompack, The Netherlands. Mobile phase, 50% MeOH/
water containing 0.1% perchloric acid; flow rate, 0.7 mL/min;
detection wavelength, 230 nm. Retention times: 4-hydroxy-2-
1
(8.7 g, 81%). Mp 55−56.2 °C. H NMR (CDCl3): δ 7.51 (dd,
1H, J= 5.6, 4.8 Hz), 7.29−7.22 (m, 5H), 6.3 (d, IH, J = 6 Hz),
5.8 (m, 1H), 3.6 (s, 2H), 2.8 (dd, 1H, J = 6.0, 18.1 Hz), 2.28
1529
dx.doi.org/10.1021/op400266k | Org. Process Res. Dev. 2013, 17, 1526−1530