Z.-Y. Wang et al. / Tetrahedron: Asymmetry 23 (2012) 1653–1656
1655
results in high yielding production of optically active amino acid
without the loss of substrate, again reducing production costs.
over 10 min. During the addition of bromine, the mixture heated
spontaneously to the boiling temperature of approximately 65°C.
The mixture was heated at reflux for 2 h until consumption of bro-
mine was complete. The reaction mixture was then evaporated to
3
. Conclusion
dryness, 228.2 g of white needle-like crystals was obtained (yield:
1
9
9%). mp 71–72 °C (decomp). H NMR (400 MHz, D
2
O): d 4.01 (d,
O): 178.3 (CO),
O: C 15.56, H
.20, N 6.10, Br 69.30. Found: C 15.58, H 2.16, N 6.06, Br 69.26.
Optically active O-methyl-L-serine 5 was prepared chemoenzy-
1
3
J = 4.4 Hz, 2H), 4.65 (t, 1H), C NMR (100 MHz, D
7.1 (CH2), 37.5 (CH). Anal. Calcd for C H NBr
3 5 2
2
matically by starting from inexpensive acrylamide 1. The double-
enzyme catalyst system, consisting of -amino- -caprolactam rac-
5
2
a
e
emase (ACL racemase, Locus, E01594) and peptidase B (pepB, Lo-
cus, D84499), was successfully applied as an enantiopurity-
producing step via kinetic resolution of the racemic amino acid
amide precursor. The product was obtained with excellent enan-
tiopurity (>99.9%) and in high yields (>99.7%). This method avoids
the 50% waste associated with traditional resolution methods. The
overall yield of the whole chemoenzymatic process was 82.4%.
ESI-MS (m/z): 232.1328 [M+H]+.
.3. Preparation of -bromo-b-methoxy-propionamide 3
The above mixture was cooled to about 20°C and 360 ml of a so-
4
a
dium methoxide solution, formed by reacting 26.5 g (1.15 g atomic
weight) of sodium with methanol, was added over 30 min. During,
and for 3 h after, the addition of the sodium methoxide, the mix-
ture was stirred and cooled to maintain it between 20 and 0 °C
in a bath of ice. Afterward the reaction mixture was distilled to a
quarter of the volume and NaBr (reaction by-product) was re-
4
4
4
. Experimental part
.1. General
moved by filtration. The filtrate was evaporated to dryness to give
.1.1. Reagents and instruments
1
1
56.7 g of white powder (yield: 87%). mp 83–85 °C (decomp). H
All chemicals were of analytical grade and purchased from the
NMR (400 MHz, D
H), 4.32 (dd, J = 6.0, 10.1 Hz, 1H), 4.51–4.75 (m, 1H), C NMR
(100 MHz, D O): 177.3 (CO), 74.8 (CH ), 53.1 (CH), 55.6 (OCH ).
Anal. Calcd for C NBrO : C 26.37, H 4.40, N 7.69, Br 43.96. Found:
C 26.35, H 4.39, N 7.71, Br 43.93. ESI-MS (m/z): 183.1245 [M+H] .
2
O): d 3.25 (s, 3H), 4.16 (dd, J = 3.5, 10.1 Hz,
China Medicine (Group) Shanghai Chemical Reagent Corporation
or other recognized commercial suppliers. Melting points were re-
13
1
1
13
2
2
3
corded on a WRR melt apparatus. H NMR and C NMR were re-
corded on a Bruker DRX500 (500 MHz). Mass spectra were
recorded on a Mariner ESI-TOF mass spectrometer. Rotations were
recorded on a WZZ-2B polarimeter. The enantiomeric composi-
tions of residual substrates were determined as described by
4
H
8
2
+
4
.4. Preparation of a-amino-b-methoxy-propionamide 4
1
9
Zheng.
(
Enantiomeric excess (ee) is defined as (A1 ꢀ A2)/
Compound 3 (150 g) was placed in a stainless steel bomb and
A1 + A2) ꢁ 100, where A1 and A2 are peak areas of the enantio-
3
40 g (20 mol) of anhydrous ammonia was added. The bomb was
mers; element composition was measured by trace element auto-
analyzer of EA 3000 type.
closed and heated, while rotating between 60 and 70 °C for 3 h.
The conversion was determined to be 96% by HPLC detection.
The bomb was then cooled, opened to release the pressure, and
the charge removed. Afterward the reaction products were dis-
solved in 200 ml of water and the solution was extracted with
4
.1.2. Enzymes
ACL racemase (Locus, E01594) was obtained from Toyobo Co. Ltd
(
Osaka, Japan). The pepB gene from E. coli K-12 MG1655 was cloned
into E. coli BL21 (DE3) using plasmid pET32a as the vector in our lab-
oratory. The strain was cultured in LB broth containing 50 g ampi-
4
00 ml of N-butanol repeatedly. The N-butanol phase continued
to be evaporated to dryness and 90.4 g of light yellow oil was ob-
l
1
tained. H NMR (400 MHz, D
2
O): d 3.28 (s, 3H), 3.77 (dd, J = 3.6,
cillin/ml for 4 h and induced with 0.4 mM IPTG at 30 °C. After further
incubation for 8 h, the cells were harvested by centrifugation at
1
0.3 Hz, 1H), 3.89 (dd, J = 5.8, 10.3 Hz, 1H), 3.92–4.13 (m, 1H),
13
C NMR (100 MHz, D
OCH ). Anal. Calcd for C
C 40.70, H 8.45, N 23.77, ESI-MS (m/z): 119.1321 [M+H] .
2
O): 176.9 (CO), 76.2 (CH
2
), 60.7 (CH), 56.6
8
2
000ꢁg for 20 min at 4 °C. Harvested cells were suspended in
(
3
4 10 2 2
H N O
: C 40.68, H 8.47, N 23.73. Found:
0 ml of 20 mM Tris–HCl (pH 7.5) and then ultrasonicated at 4 °C.
+
The cell-free extracts were fractionated with ammonium sulfate
30–70%). The precipitate obtained from ammonium sulfate frac-
tionation was dissolved in 50 mM of Tris–HCl (pH 7.5) containing
mM MnSO , dialyzed against the same buffer, and applied to a
(
4
.5. Preparation of O-methyl-L-serine 5 by the double-enzyme
system
1
4
DEAE-Sepharose CL-6B column (1.5 ꢁ 55 cm) equilibrated with
the same buffer. The column was washed with the same buffer,
and then the enzyme was eluted with a gradient formed between
the buffer and that containing 0.4 M NaCl. The active fractions were
concentrated by ammonium sulfate precipitation and then applied
to a Cellulofine GC-700 m column (2.1 ꢁ 94 cm) (Seikagaku Kogyo,
Japan) equilibrated with 50 mM Tris–HCl (pH 7.5) containing
In a typical small-scale experiment, ACL racemase (Locus,
E01594) and PepB (Locus, D84499) were added to a solution of
the
mixture (20 ml), containing 2 mM of pyridoxal 5’-phosphate
PLP), 0.1 M of potassium phosphate buffer (KPB) (pH 9.0),
.5 mol/l of -amino-b-methoxy-propionamide 4, ACL racemase
Locus, E01594) (3.0 mg), and pep B (Locus, D84499) (1.2 mg)
was incubated at 38 °C for 18 h. The progress of the reaction was
followed by taking samples at intervals. The amounts of -ami-
no-b-methoxy-propionamide 4 and O-methyl- -serine 5 were
a-amino-b-methoxy-propionamide 4 in water. The reaction
(
0
a
(
1
4
mM MnSO . The enzyme was eluted with the same buffer. The final
sample was homogeneous by SDS–polyacrylamide gel electropho-
resis. Ammonium sulfate was added to the final enzyme sample to
D/L-a
L
7
0% saturation, and the sample was stored at 4 °C until it was used.
determined with an HPLC equipped with a Crownpak CR (+) col-
umn at a flow rate of 0.5 ml/min, using the solvent system of
In this case, the buffer used throughout the purification procedures
20
contained 1 mM MnSO
4
.
5
0 mM HClO
4
. Absorbance of the eluate was monitored at
-serine 5 was formed in the mixture and iso-
2
10 nm. O-Methyl-
L
4
.2. Preparation of ,b-dibromopropionamide 2
a
lated by a procedure involving deproteinization by trichloroacetic
acid and column chromatography on a Dowex-X8 (H+) column.
To a slurry of 71 g (1 mol) of crystalline acrylamide in 450 ml
methanol, 160 g (1 mol) of bromine was added with stirring for
The isolated O-methyl-
tallized from water–methanol–isopropyl alcohol–ether. The enan-
L-serine 5 (7.98 mmol, 950 mg) was recrys-