6
720 Musa
Asian J. Chem.
analyzed by a GC equipped with a chiral column. The percent
conversion to ketone was 45.8 % and the optical purity of
EXPERIMENTAL
Capillary gas chromatographic measurements were
(
R)-2 was 87.1 % (E-value = 190).
Calculation of E-value: E-value was calculated from the
performed on a GC equipped with a flame ionization detector
and HP chiral-20B column (30 m, 0.32 mm [i.d.], 0.25 µm
film thickness) using Helium as the carrier gas. Nuclear Magnetic
Resonance spectra were recorded on 500 MHz spectrometer
formula E = ln[(1-c)(1-ee
s
)]/ln[(1-c)(1 + ee
percentage conversion of (rac)-2 to 1 and ee
s
)], where c is the
is the enantio-
s
meric excess of the slow reacting enantiomer, (R)-2, in W110A
TeSADH-catalyzed KR.
1
13
at 500 MHz ( H) and at 125 MHz ( C) at room temperature
using the solvent peak as an internal standard. Commercial
grade solvents were used without further purification. 4-(4'-
RESULTS AND DISCUSSION
+
Hydroxyphenyl)-2-butanone, NaBH
4
and NADP were used
By using W110A TeSADH, the enantioselective reduction
of raspberry ketone 1 produced (S)-2 in 61 % conversion and
as purchased from commercial suppliers. All buffer solutions
were adjusted to pH 8.0 at room temperature.
9
7.2 % ee (Scheme -I). The reaction was conducted in a Tris-
Gene expression and purification of W110A TeSADH:
W110A TeSADH was expressed in recombinant Escherichia
HCl buffer solution (50 mM, pH 8.0) containing 2-propanol
(30 %, v/v). 2-Propanol serves as a cosubstrate by delivering
+
a hydride to the oxidized form of the coenzyme NADP and,
13
coli BL21(DE3) cells and purified as reported .
Synthesis of (S)-rhododendrol [(S)-2]:A mixture of Tris-
HCl buffer solution (7 mL, 50 mM, pH 8), 2-propanol (3 mL),
therefore, regenerates the reduced form of the coenzyme,
1
5
+
NADP (1 mg), W110A TeSADH [0.7 mg in 100 µL of Tris-
NADPH. This approach, called "substrate-coupling ", has
been used successfully in ADH-catalyzed transformations in
which ADHs with high tolerance to organic solvents, such as
HCl buffer (50 mM, pH 8.0)] and 1 (0.365 mmol) was added
in the same sequence to a round-bottomed flask equipped with
a magnetic stirrer and a condenser. The reaction mixture was
stirred for 24 h at 50 °C. It was then extracted with ethyl acetate
16
TeSADH and Rhodococcus ruber DSM 44541, are employed .
Using a high percentage of 2-propanol not only enhances the
solubility of hydrophobic substrate 1, but also shifts the
equilibrium to the reduction pathway. The produced alcohol
has an S configuration, which is consistent with Prelog's rule.
The same stereochemical results were obtained for theW110A
TeSADH-catalyzed reduction of a series of phenyl-ring-
(
3 × 5 mL). The combined organic layers were washed with
brine solution (5 mL), dried with sodium sulfate and then
concentrated under vacuum.A fraction of the remaining residue
was treated with pyridine and acetic anhydride as reported
14
prior to its analysis by a GC equipped with a chiral column .
The percent conversion was 61 % and the optical purity was
17
containing ketones with similar structures to 1 .
9
7.2 %. The remaining underivatized residue was purified with
20
O
OH
silica gel using hexane/ethyl acetate. [α]
D
+14.1 (c 0.667,
4b
24
D
1
ethyl acetate), lit. , [α]
NMR (CD OD), δ: 1.18 (d, 3H, J = 6.1 Hz), 1.60-1.75 (m,
H), 2.50-2.59 (m, 1H), 2.60-2.71 (m, 1H), 3.68-3.76 (m, 1H),
+ 16.9 (c 1.06, EtOH) 99% ee. H
W110A TeSADH
3
HO
HO
+
NADPH
NADP
(S)-2
2
6
1
reduction product
conversion= 61%
ee= 97.2 %
13
O
OH
.70 (d, 2H, J = 8.25 Hz), 7.01 (d, 2H, J = 8.25 Hz); C NMR
OD), δ: 23.5, 32.3, 42.4, 67.9, 116.1, 130.2, 134.4, 156.3.
Synthesis of (rac)-rhododendrol [(rac)-2]: A mixture
of 1 (1 g, 6.2 mmol) and methanol (25 mL) was placed in a
00 mL round-bottomed flask equipped with a magnetic stirrer
then placed in an ice bath. A solution of NaBH (0.26 g, 7
W110A TeSADH
(CD
3
Scheme-I:Synthesis of (S)-2 via W110A TeSADH-catalyzed asymmetric
reduction
1
By taking advantage of the reversibility ofADH-catalyzed
asymmetric transformations, anti-Prelog alcohols can be
produced through enantiospecific oxidation of their racemates
through KR. (rac)-Rhododendrol was produced by reducing
4
mmol) in distilled water (20 mL) was added slowly to the
reaction flask. The mixture was stirred at room temperature
for 10 h. It was then filtered and concentrated under vacuum
until 20 mL remains. The remaining was extracted with ethyl
acetate (3 × 20 mL), dried with sodium sulfate and then concen-
trated under vacuum to produce oil that soon became white
4
1 with NaBH . The enantiospecific oxidation of (rac)-2,
through KR resulted in 45.8 % conversion to 1, leaving (R)-2,
the slow-reacting enantiomer, in 87.1 % ee (Scheme- II). This
reaction was conducted in a Tris-HCl buffer solution (50 mM,
pH 8) containing acetone (10 %, v/v).Acetone was used as both
a cosubstrate to regenerate the oxidized form of the coenzyme,
1
13
solid in 94 % yield. H NMR and C NMR are similar to
those for (S)-2.
Synthesis of (R)-rhododendrol [(R)-2]: A mixture of
Tris-HCl buffer solution (4.5 mL, 50 mM, pH 8), acetone (0.5
+
mL), NADP (1 mg), W110A TeSADH [0.7 mg in 100 µL of
+
NADP , and as a cosolvent to enhance the solubility of the
substrate. Although the ee of (R)-2 in the W110A TeSADH-
catalyzed KR is 87.1 % ee, the process is still stereospecific
and the E-value is 190. This stereospecific KR method can be
used to produce (R)-2 from (rac)-2 and generate raspberry
ketone 1, which is a crucial ketone for the flavor industry.
The stereochemical identity of (S)-2 was confirmed by
comparing its sign of optical rotation with reported values.
The stereochemical identity of (R)-2 was confirmed by co-
injecting (R)-2 with (S)-2 in a GC equipped with a chiral
Tris-HCl buffer (50 mM, pH 8)] and (rac)-2 (0.365 mmol)
was added in the same sequence to a round-bottomed flask
equipped with a magnetic stirrer and a condenser. The reaction
mixture was stirred for 24 h at 50 °C. It was then extracted
with ethyl acetate (3 × 5 mL). The combined organic layers
were washed with brine solution (5 mL), dried with sodium
sulfate and then concentrated under vacuum. The remaining
residue was treated with pyridine and acetic anhydride then
Musa, Musa M.
