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unless otherwise indicated. GC analyses were carried out
with DB-17 (0.25 mm · 25 m) or Chirasil-Dex CB
(0.25 mm · 25 m) columns for normal and chiral separa-
tions, respectively. GC samples from the bioconversion
were prepared by vortex mixing 0.50 mL portions of
the reaction mixture and EtOAc for 10 s followed by
brief centrifugation to remove insoluble cell debris.
The conversion of 7 to 2 was monitored by reversed-
phase HPLC (4.6 · 250 mm C18 column) using a
water–CH3CN solvent system (both solvents containing
0.1% trifluoracetic acid). Glucose concentrations during
the bioconversion were assayed by a commercialkit
using the Trinder reagent. Non-aqueous reactions were
carried out in dried glassware under an argon atmo-
sphere. b-Keto ester 4 was prepared using the method
described by Chowdhury et al.27
the reaction was monitored by GC and the consumption
of both 3 and glucose slowed significantly after 7 h.
After 24 h, the reaction product was gently extracted
with CH2Cl2 (2 · 300 mL) to avoid forming an emul-
sion.29 The combined organics were dried over MgSO4,
concentrated in vacuo, and the residue subjected to silica
gelchromatography (cycol hexane:Et 2O, 85:15) to afford
1.1 g of 5 as a colorless oil (82% yield). [a]D = +24 (c 0.7,
1
CHCl3). H NMR: (C6D6) d 7.06 (m, 5H), 4.24 (ddd,
1H, J1 = 3.3 Hz, J2 = 6.6 Hz, J3 = 7.1 Hz), 4.05 (d, 1H
J = 3.3 Hz), 3.78 (q, 2H, J = 7.2 Hz), 2.82 (dd, 1H,
J1 = 7.1 Hz, J2 = 13.8 Hz), 2.72 (dd, 1H, J1 = 6.6 Hz,
J2 = 13.8 Hz), 2.4 (br s, 1H), 0.79 (t, 3H, J =
7.2 Hz). 13C NMR: (CDCl3) d 169.1, 137.1, 129.8,
129.2, 127.4, 73.4, 63.0, 61.2, 40.4, 14.4. IR (film): m
(cmÀ1): 3483, 1740, 1300, 1183, 1025. Anal. Calcd for
C12H15ClO3: C, 59.39; H, 6.23. Found: C, 59.75; H, 6.72.
4.1.1. Ethyl 2-chloro-3-oxo-4-phenylbutyrate 3. Sulfu-
ryl chloride (5.7 mL, 60 mmol, 0.60 equiv) was added
over a 15 min period to a solution of b-keto ester 4
(15.5 g, 75 mmol) in CHCl3 (170 mL) at room tempera-
ture. After stirring for an additional3 h (or untilno fur-
ther reaction occurred according to TLC), water
(100 mL) was added and the mixture extracted with
CH2Cl2 (3 · 50 mL). The combined organics were dried
over MgSO4, concentrated in vacuo, and the residue
purified by flash chromatography on silica gel (hex-
anes:toluene, 1:1) to yield 13.2 g of 3 as a pink oilin
addition to unreacted starting material(3.4 g) (92% yield
of 3 based on recovered SM). 1H NMR: (CDCl3) d 7.32
(m, 5H), 4.87 (s, 1H), 4.22 (q, 2H, J = 6.2 Hz), 4.01 (d,
2H, J = 4.5 Hz), 1.27 (t, 3H, J = 6.2 Hz). 13C NMR:
(CDCl3) d 196.5, 165.1, 132.7, 129.9, 129.0, 127.7,
63.4, 60.6, 45.9, 14.1. IR (film): m (cmÀ1): 2987, 1764,
1692, 1269, 1185.
4.1.3. Ethyl (2S,3S)-cis-4-phenyl-2,3-oxiranebutanoate
6. K2CO3 (1.7 g, 12.4 mmol, 3 equiv) and water
(390 lL) were added to a solution of chlorohydrin 5
(1.0 g, 4.1 mmol) in DMF (19 mL). After stirring for
5 h at rt, the mixture was diluted with water (50 mL)
and the aqueous layer extracted with Et2O
(3 · 50 mL). The combined organic layers were washed
with water to remove residualDMF (6 · 5 mL), dried
over MgSO4, concentrated in vacuo, and purified by
flash chromatography on silica gel (cyclohexane:Et2O,
85:15) to afford 0.84 g of 6 as a colorless oil (99% yield).
[a]D = +37, (c 3.0, CHCl3). Anal. Calcd for C12H14O3:
C, 69.89; H, 6.84. Found: C, 70.10; H, 6.91. Spectral
data matched those reported for the racemic material.32
4.1.4.
(4S,5R)-4,5-Dihydro-2-phenyl-4-carboethoxy-5-
benzyl-1,3-oxazole 7. BF3ÆOEt2 (62 lL, 0.49 mmol,
1 equiv) was added over 10 min to a solution of glycidic
ester 6 (100 mg, 0.49 mmol) in benzonitrile (900 lL)
cooled to 0 ꢂC. The reaction was allowed to warm to
rt over a 3 h period. Saturated NaHCO3 (2 mL) was
added and the mixture was stirred for an additional
2 h before being diluted with water (10 mL). After
extracting with CH2Cl2 (3 · 15 mL), the combined
organics were dried over MgSO4, concentrated in vacuo,
and purified by silica gel chromatography (cyclohex-
ane:Et2O, 9:1) to yield 0.12 g of 7 as a colorless oil
(78% yield). [a]D = À57, (c 2.0, CHCl3). 1H NMR:
(C6D6) d 8.37 (m, 2H), 7.28 (m, 8H), 4.90 (ddd, 1H,
J1 = 6.3 Hz, J2 = 6.3 Hz, J3 = 6.9 Hz), 4.78 (d, 1H,
J = 6.3 Hz), 3.93 (m, 2H), 3.18 (dd, 1H, J1 = 6.3 Hz,
J2 = 13.8 Hz), 2.92 (dd, 1H, J1 = 6.9 Hz, J2 =
13.8 Hz), 0.91 (t, 3H, J = 7.2 Hz). 13C NMR: (C6D6) d
170.2, 163.2, 131.8 (other aromatic signals obscured by
solvent) 80.3, 73.7, 61.2, 42.1, 14.0. IR (film): m (cmÀ1):
3029, 2982, 1752, 1655, 1206, 1027, 695. Anal. Calcd
for C19H19O3N: C, 73.77; H, 6.19; N, 4.53. Found: C,
73.63; H, 6.14; N, 4.56.
4.1.2. Ethyl (2R,3S)-2-chloro-3-hydroxy-4-phenylbuty-
rate 5. A 45 mL portion of LB medium supplemented
with 30 lg/mL kanamycin was inoculated with a single
colony of E. coli BL21(DE3)(pIK4) and the culture sha-
ken overnight at 37 ꢂC. The preculture was diluted 1:100
into 4 L of the same medium in a New Brunswick M19
fermenter. The culture was grown for 2 h at 37 ꢂC with a
stir rate of 800 rpm and an air flow of 0.5 vesselvolumes
per min (vvm) untilreaching an OD 600 = 0.6. After
cooling to 28 ꢂC over 15 min, reductase overexpression
was induced by adding isopropylthio-b-D-galactoside
to a finalconcentration of 100 lM and the culture was
maintained under the same conditions for an additional
6 h. Cells were collected by centrifugation (6000g for
10 min at 4 ꢂC), then half of the biomass (25 g wet
weight) was resuspended in 1 L of 10 mM KPi
(pH 5.6) containing 4 g/L glucose. The bioconversion
was carried out in a Braun Biostat B fermenter with
the temperature, pH, and dissolved oxygen maintained
at 30 ꢂC, 5.6 (3 M NaOH titrant) and 75% saturation
(fixed air flow of 0.25 vvm and variable stirring rate),
respectively. Solid XAD-4 resin (0.5 g) was added, fol-
lowed by portions of neat 3 (0.2 mL) approximately
every hour over a totalof 12 h to provide a finaladded
substrate concentration of 5 mM. A glucose stock solu-
tion was also added after 3 and 6 h to keep the glucose
concentration at approximately 4 g/L. The progress of
4.1.5. (2S,3R)-2-Hydroxy-3-amino-4-phenylbutyric acid 2
hydrochloride. To a solution of oxazoline 7 (323 mg,
1.04 mmol) in a mixture of EtOH (8 mL) and water
(6 mL) was added concentrated HCl(15 mL). The mix-
ture was held at reflux for 8 h before being concentrated
under reduced pressure. After resuspending the residue