108
Letter / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 106–109
(c 1.35, 1 M HCl) [lit.[1] [˛]D −26.1 (c 2, H2O) for HCl salt]. 1H
20
To the broth was added 2b (250 mg, 0.76 mmol) and the mixture
was stirred at 30 ◦C for 76 h. The progress of the reduction was
monitored by a TLC analysis: Rf for 2b: 0.42; 4a: 0.35 (developed
with hexane/AcOEt = 1:5). The broth was centrifuged (5000 rpm,
1200 g) for 15 min. The precipitated cells were extracted three
times with THF. The supernatant in the step of centrifugation
was saturated with NaCl and extracted with AcOEt four times.
The combined extract was concentrated in vacuo. The residue
was diluted with brine and extracted with AcOEt three times.
The extract was dried over anhydrous Na2SO4 and concentrated
in vacuo. The conversion was estimated by comparing the area of
signals in 1H NMR: ı 4.68 for 2b and 3.63 and 3.75 for 4a of this
crude product. The residue (300 mg) was charged on a silica gel
column (12 g). Elution with hexane/AcOEt = 1:5 afforded (R)-4a
(203 mg, 81%) as white solid. HPLC analysis was performed as in
Section 2.4: tR (min) = 21.7 [>99.2%, (R)-4a], 30.1 [<0.8%, (S)-4a].
Due to the presence of unknown contaminant with tR = 30.1 min in
a small amount in HPLC analysis, the ee was temporarily estimated
to be over 98.4% at this stage. Recrystallization of the small portion
from a hot mixture of CHCl3 and tert-butyl methyl ether to afford
NMR (400 MHz, CDCl3): ı 1.13 (s, 9H), 2.65 (dd, J = 8.8,12.0 Hz,1H),
2.94 (dd, J = 3.5,12.0 Hz, 1H), 2.99 (s, 6H),3.06 (s, 6H), 4.68 (dd,
J = 3.5, 8.8 Hz, 1H), 6.88 (t, J = 2.1 Hz, 1H), 7.00 (d, J = 2.1 Hz, 2H);
13C NMR (CDCl3, 125 MHz): ı 28·7 (x3), 36.3 (x2), 36.5 (x2), 49.8,
50.8, 71.2, 114.5 (x2), 115.8 (x2), 145.3, 151.6 (x2), 154.5; IR: 694,
756, 818, 887, 955, 1030, 1153, 1267, 1379, 1443, 1616, 1708,
2360, 2964, 3433 cm−1. HRMS (FAB+): m/z 368.2171 (M)+; calc. for
C18H30N3O5: 368.2185.
3. Results and discussion
To realize the synthesis of (R)-bambuterol, dihydroxyketone 3a
was considered for the starting material, but we decided the use of
less expensive (2/5 price of 3a) diacetylated form (3b). In the ini-
as methanolysis in the presence of K2CO3, the color of the reaction
mixture quickly turned dark, accompanied by formation of polar
byproducts. In contrast, lipase-catalyzed transesterification with 2-
propanol as nucleophile [8] was really advantageous. In this case, B.
cepacia lipase (Amano PS-IM) was superior to C. antarctica lipase B
(Novozymes, Novozym 435), which had been applied successfully
to the transformation of 2-chloroarylethanone 2e to 2d [6]. B. cepa-
cia lipase-catalyzed deacetylation proceeded in quantitative yield
and the recovered lipase was able to be re-used in another batch.
Carbamoylation of 3a, subsequent bromination and substitution
with chloride provided the substrate 2b. Simple recrystallization
high melting point (126.0–127.0 ◦C). However, the highly crys-
talline substrates were minimally soluble in the incubation broth of
whole-cell yeasts, which caused the reduction to occur very slowly.
Trying to promote the reduction by addition of organic solvent led
to considerable cell damage (Scheme 1).
To overcome the low solubility while preventing cell damage,
glycerol was used as a carbon source for recycling the reduced form
of cofactors upon asymmetric reduction of the carbonyl compounds
with W. californica. Thus, the W. californica cells were pre-incubated
on glycerol. In a previous synthesis involving the reduction of 2c,
2% glycerol was applied and resulted in the desired (R)-alcohol with
98.4% ee in 80% yield [6]. In the present synthesis, concentrations
of glycerol between 2% and 30% were examined to determine the
effect on the solubility of crystalline 2b. The results are summarized
in Table 1. Among the entries, results indicated that reduction at
10% glycerol was optimal for providing (−)-4a (81% isolated yield)
at an ee greater than 98.4%. The ee of the product was temporarily
an analytical sample as colorless fine needles, mp 105.6–106.3 ◦C;
27.7
[˛]D
−25.2 (c 1.10, CHCl3). Its 1H NMR spectrum was identical
with that of ( )-4a. 13C NMR (CDCl3, 125 MHz): ı 36.4 (x2), 36.6
(x2), 50.3, 73.2, 115.3 (x2), 116.1 (x2), 142.3, 151.8 (x2), 154.3;
IR: 756, 818, 887, 955, 1030, 1153, 1261, 1292, 1379, 1443, 1616,
1705, 2341, 2360, 2933, 3419 cm−1. HRMS (FAB+): m/z 331.1074
(M)+; calc. for C14H19ClN2O5: 331.1061.
2.7. (R)-[3,5-Bis(dimethylcarbamoyloxy)phenyl]oxirane (5)
To a solution of (R)-4a (162 mg, 0.49 mmol, the sample obtained
after chromatographic purification as in Section 2.6, without any
crystallization) in EtOH (1.5 mL) were added aqueous NaOH solu-
tion (2 M, 1.5 mL). The mixture was stirred for 10 min at room
temperature and quenched with saturated aqueous NH4Cl solu-
tion. The organic layer was separated and the aqueous layer
was extracted with AcOEt three times. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4,
and concentrated in vacuo to give crude product (144 mg). The
residue was charged on a silica gel column (1 g). Elution with
27.3
hexane/AcOEt = 1:1 afforded (R)-5 (131 mg, 91%). [˛]D
−10.9 (c
1.39, CHCl3); HPLC [column, CHIRALCEL® OD-H, 0.46 cm × 25 cm;
hexane-2-propanol (5:1); flow rate 0.5 mL/min; detected at
226 nm]: tR (min) = 28.4 (single peak). The peak for (S)-5 appeared
at tR 34.4 min, which was confirmed by the co-injection with an
authentic racemic sample. 1H NMR (500 MHz, CDCl3): ı 2.74 (dd,
J = 2.5, 5.6 Hz, 1H), 2.98 (s, 6H), 3.05 (s, 6H), 3.09 (dd, J = 4.0, 5.6 Hz,
1H), 3.82 (dd, J = 2.5, 4.0 Hz, 1H), 6.90 (br-s, 3H). Its 1H NMR spec-
trum was reasonable, but all the chemical shifts were parallelly
shifted from those reported previously [1]. We assume that the
previous authors misread the three protons [6.90 (br-s, 3H)] as the
signal of CHCl3 involved in CDCl3. 13C NMR (CDCl3, 125 MHz): ı
36.4 (x2), 36.6 (x2), 51.2, 51.8, 115.3 (x2), 115.4 (x2), 139.8, 152.0
(x2), 154.3; IR: 687, 756, 843, 887, 935, 1035, 1149, 1250, 1298,
1373, 1443, 1616, 1716, 2333, 2364, 2933 cm−1. HRMS (FAB+): m/z
295.1318 (M)+; calc. for C14H19N2O5: 295.1294.
its absolute configuration was elucidated using lipase-catalyzed
kinetic resolution. The 2-halo-arylethanols and corresponding
[9,10]. Hydrolysis of acetate ( )-4b was attempted using B. cepacia
and C. antarctica lipase. In both cases, the products (S)-4a with high
ee values but varying E values were obtained as shown in Table 2.
This authentic (S)-4a coincided with the slow eluting peak from
CHIRALCEL® OD-H analysis (see Section 2.5), which strongly sup-
ported an absolute configuration of (R) for (−)-4a, which eluted
2.8. (R)-2-(N-tert-Butylamino)-1-[3,5-bis(dimethyl-
carbamoyloxy)phenyl]ethanol (1a)
Table 2
Lipase-catalyzed resolution of ( )-4b, for the preparation of an authentic sample of
(S)-4a.a
To the epoxide (R)-5 (82.8 mg, 0.282 mmol) was added tert-
butylamine (2.00 mL, 19.0 mmol, 67 equiv.). The mixture was
stirred for 48 h at reflux. The mixture was concentrated in vacuo,
and the residue was purified by silica gel column chromatography
(2.0 g). Elution with CHCl3/MeOH = 33:1 afforded 1a (90.2 mg, 88%)
Origin of lipase
Conversion (%)
Ee of (S)-4a (%)
E value
B. cepacia
C. antarctica
45.1
39.7
96.5
99.8
120
40
28.1
27.3
a
For the detailed information, see Section 2.5.
as slightly yellow oil. [˛]D
−28.7 (c 1.46, CHCl3); [˛]D
−21.4