Asymmetric reduction of substituted α- and β-ketoesters by Bacillus pumilus Phe-C3

Article abstract of DOI:10.1016/j.tetasy.2008.05.003

The enantioselective reduction of substituted α- and β-ketoesters using resting cells of Bacillus pumilus Phe-C3 was investigated. Effects of substrate concentration on the catalytic efficiency of the microorganism were studied. Preparative scale productions were carried out under the optimized conditions with 62.4-91.0% yields and 90.2-97.1% ee. The cells retained 80% of initial activity after recycling for six times.

Full text of DOI:10.1016/j.tetasy.2008.05.003

Tetrahedron: Asymmetry 19 (2008) 1347–1351
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Tetrahedron: Asymmetry
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Asymmetric reduction of substituted a- and b-ketoesters by Bacillus pumilus
Phe-C3
c
Chunmao He a,b, Dongliang Chang a,c, , Jie Zhang
*
a Guangzhou Institute of Chemistry, Chinese Academy of Sciences, Guangzhou 510650, PR China
b Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China
c Jiaxing Research Center for Applied Chemistry, Chinese Academy of Sciences, Jiaxing 314006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The enantioselective reduction of substituted a- and b-ketoesters using resting cells of Bacillus pumilus
Phe-C3 was investigated. Effects of substrate concentration on the catalytic efficiency of the microorgan-
ism were studied. Preparative scale productions were carried out under the optimized conditions with
62.4–91.0% yields and 90.2–97.1% ee. The cells retained 80% of initial activity after recycling for six times.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 3 April 2008
Accepted 1 May 2008
Available online 10 June 2008
1. Introduction
and maximum substrate concentration burden. The strain Bacillus
pumilus Phe-C3 was reported to be capable of reducing b-ketoxy-
Optically active secondary alcohols are important building
blocks for the production of flavors, pharmaceuticals, and
agrochemicals.1 Ethyl (R)-3-hydroxybutanoate and ethyl (S)-4-
halo-3-hydroxybutanoate are key intermediates of inhibitors of
angiotensin-converting enzyme,2 and HMG-CoA reductase,3,4
respectively. They are also valuable synthons for the production
esters to optically active secondary alcohols. It was also identified
that NADPH-dependent alcohol dehydrogenase was responsible
for the reduction.45 Herein, we have extended the substrate
diversity of this strain for the biocatalytic reduction of series
substituted prochiral a- and b-ketoesters to the corresponding
hydroxyl esters.
of pharmaceuticals such as
L
-carnitine,5 and (R)-4-amino-
3-hydroxybutanoic acid (GABOB).6
2. Results and discussion
Methods for the production of a- and b-hydroxy ester include
reduction of the corresponding ketoesters using either metal cata-
lysts7–16 or biocatalysts.17–28 Moreover, enzymatic deracemiza-
tion29–38 and resolution39–44 of racemic hydroxy esters have also
been employed in some cases. Among these synthetic strategies
problems exist with, either less than 50% yields presented for
resolution methods or low overall yield for chemical methods with
tedious synthetic routes. Instead of these methods, the asymmetric
bioreduction of prochiral ketones with dehydrogenase is a superior
way for producing secondary alcohol building blocks. Bacterial
whole cells have the advantage over isolated enzymes since they
contain all the necessary cofactors and the metabolic pathway
for their regeneration. Moreover, all the enzymes and cofactors
are well protected within their natural cellular environment.17
We are interested in expanding the scope of the enzymatic
reduction of ketoesters in practical syntheses by developing highly
active and easy handle bacterial catalysts with new substrate spec-
ificity. Ideal catalyst for this conversion could offer broad substrate
acceptance, high enantioselectivity, rapid reaction rates, low cost,
2.1. Reduction of b-ketoesters with B. pumilus Phe-C3
Bioreductions of b-ketoesters were carried out with the cells of
B. pumilus Phe-C3 on a 10-mL scale in the exploratory stage.45 Sub-
strate tolerance of biocatalyst is a crucial factor for its potential
industrial application. Higher substrate tolerance of the biocatalyst
can increase its production capability in industrial process. There-
fore, different substrate concentrations with the same cell density
of 3 g cdw/L were explored for the determination of catalytic
capacity of the strain. The bioconversions were followed by GC or
HPLC analyses.
As shown in Table 1, reduction of b-ketoesters 1a–6a gave the
corresponding b-hydroxyesters 1b–6b. High activities and high
conversions were observed in the bioreduction of 2a, 4a, and 5a
with 19.3, 23.4, and 27.8 U/g cdw (U: lmol/min, cdw: cell dry
weight), respectively. While low to moderate activities were ob-
tained for 1a, 3a, and 6a with 9.1, 6.7, and 11.8 U/g cdw, respec-
tively. It was shown that the bioreduction of 10 mM of 2a
formed 2b with a lower conversion of 85.3%, compared to 96.3%
conversion with a substrate concentration of 15 mM. This is
probably due to the further degradation of the product 2b by other
enzymes inside the strain with lower 2b concentration.
* Corresponding author. Tel.: +86 573 82585235; fax: +86 573 82585233.
E-mail address: chang@gic.ac.cn (D. Chang).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.05.003

1348
C. He et al. / Tetrahedron: Asymmetry 19 (2008) 1347–1351
Table 1
Reduction of b-ketoesters with B. pumilus Phe-C3 (cells 3.0 g cdw/L)
O
O
OH
O
OH
O
B.pumilus Phe-C3
or
R
OEt
R
OEt
OEt
CF3
1a-6a
1b-5b
6b
1: R = CH3
4: R = Ph
2: R = CH2Cl
3: R = CH2Br
5: R = CH2CN
6: R = CF3
Substrate (mM)
Activitya (U/g cdw)
Conv. and eeb,c (%)
9 h
0.5 h
2 h
5 h
12 h
16 h
19 h
1a
1a
2a
2a
3a
3a
5
7
10
15
3
9.1
8.8
18.1
19.3
4.8
16.4(92.6)
11.3(90.8)
16.3(93.5)
14.1(95.9)
14.4(91.4)
12.1(90.3)
23.3(94.0)
21.1(95.7)
18.8(94.5)
16.7(97.0)
21.3(90.2)
12.6(90.2)
29.8
25.3
50.2
48.1
17.2
20.8
50.4
66.6
41.3
39.9
40.5
39.6
73.0
40.1
74.7
77.7
41.3
40.3
76.3
72.4
64.1
62.7
61.3
52.8
90.5
51.9
85.3(93.5)
86.5
50.7
52.5
91.1
86.7
73.2
99(94.5)
62.4
—
92.1
75.6
66.3
98.7(94.0)
96.9(95.7)
85.3
—
77.3
—
96.3(95.9)
82.3
71.5
—
—
79.5(90.8)
—
—
88.1(91.4)
85.4(90.3)
—
5
6.7
4ad
4ad
5a
5
10
10
15
5
12.9
23.4
20.9
27.8
11.8
9.8
—
—
91.2
90.5
87.2
80.9
96.7(94.5)
95.1(97.0)
93.9(90.2)
85.3(90.2)
5a
6a
6a
70.1
74.5
60.4
85.0
80.3
76.1
7
a
b
c
The activity was determined from the product formation over the first 30 min.
ee was given in brackets.
Conversion and ee were determined by GC analysis; error limit: 2% of the stated values.
Conversion and ee were determined by HPLC analysis; error limit: 2% of the stated values.
d
2.2. Reduction of a-ketoesters with B. pumilus Phe-C3
Ethyl (R)-2-hydroxy-4-phenylbutyrate (R-EHPB) 9b is an impor-
tant intermediate for the synthesis of ACE inhibitors such as cila-
zapril, benazepril, and enalapril.46 Regarding its practical
preparation, the reaction capacity of B. pumilus Phe-C3 with 9a
was further investigated. Therefore, 20–40 mM of 9a with a cell
density of 12.0 g cdw/L was used to test its influence on the cata-
lytic efficiency of B. pumilus Phe-C3. Higher product concentration
could be easily achieved by use of a higher cell density and sub-
strate concentration. A concentration up to 30 mM was still well
tolerated by B. pumilus Phe-C3 at the cell density of 12.0 g cdw/L,
with 95.3% conversion at 19 h with 97.1% ee, whereas 40 mM of
In order to further explore the substrate versatility of B. pumilus
Phe-C3, substituted a-ketoesters were used as precursors. Reduc-
tion of 7a–9a gave the corresponding a-hydroxyesters 7b–9b. As
shown in Table 2, high activities and high conversions were ob-
tained for all of a-ketoesters tested. Extremely high activity
(40.4 U/g cdw) was observed in bioreduction of 7a at substrate
concentration of 5 mM with cell density of 3 g cdw/L. With the
same cell density, high activity was also obtained for 8a and 9a
as 17.8 and 13.9 U/g cdw, respectively.
Table 2
Reduction of a-ketoesters with B. pumilus Phe-C3
O
OH
B.pumilus Phe-C3
R
COOEt
R
COOEt
7b-9b
7a-9a
7: R = CH3; 8: R = Ph; 9: R = CH2CH2Ph
Substrate (mM)
Cell density (g cdw/L)
Activitya (U/g cdw)
Conv. and eeb,c (%)
0.5 h
2 h
5 h
9 h
12 h
16 h
19 h
7ad
5
7
10
15
5
20
30
40
3
3
3
3
3
12
12
12
40.4
23.0
15.1
17.8
13.9
14.6
17.3
7.4
72.8(94.1)
29.6(94.1)
13.6(93.9)
10.7(96.6)
25.1(94.5)
26.3(96.7)
20.7(97.1)
5.6(87.3)
83.9
30.1
41.9
33.4
62.3
63.5
60.1
—
92.6
41.1
70.7
56.5
76.3
75.3
69.8
—
>99(94.1)
66.6
81.3
72.7
85.4
80.2
77.2
11.5(86.9)
—
—
—
—
—
7ad
8a
8a
9a
9a
9a
9a
76.5
90.1
84.5
88.7
86.3
81.2
—
82.4(94.1)
95.7(93.9)
90.6
93.1
91.0
94.8(96.6)
99.0(94.5)
97.9(96.7)
95.3(97.1)
29.7(86.9)
89.9
—
a
b
c
The activity was determined from the product formation over the first 30 min.
ee was given in brackets.
Conversion and ee were determined by HPLC analysis; error limit: 2% of the stated values.
Conversion and ee were determined by GC analysis; error limit: 2% of the stated values.
d

C. He et al. / Tetrahedron: Asymmetry 19 (2008) 1347–1351
1349
the substrate inhibited the catalytic activity severely; the maxi-
mum conversion obtained was only 29.7%.
penicillins, cephalosporins, and antiobesity agents.51–53 Recently,
the strain Geotrichum candidum CIOC2.61654 was reported to be
capable of catalyzing the biosynthesis of (R)-methyl mandelate
with high yield (92%) and ee (95%). In the present study, B. pumilus
Phe-C3 is able to catalyze the synthesis of (R)-ethyl mandelate 8b
with 96.6% ee and 85.4% yield.
2.3. The enantioselectivity and stereoselectivity of the
bioreduction
The enantiomeric excess of the product was measured by chiral
GC and HPLC after the first 30 min and the end of the bioreduction.
b-Hydroxyesters 1b–6b were obtained with a high ee of 94.5%,
95.9%, 91.4%, 95.7%, 97.0%, and 90.2%, respectively. For a-hydroxy-
esters 7b–9b, high ee was also achieved with ees of 94.1%, 96.6%,
and 97.1%, respectively. In terms of stereoselectivity, 1b and 5b–
9b were obtained with the (R)-configuration while 2b–4b formed
with an (S)-configuration.
The stereoselectivity of the reaction indicates that the dehydro-
genases inside the cell deliver hydride ion from the same side of
prochiral ketoesters for all of the substrates except 6a. This phe-
nomenon is probably due to the CF3 functional group in 6a, com-
pared with other substituted substrates, which interferes with
the binding configuration strongly inside the active pocket of the
dehydrogenase.
Finally, preparative scale production of (R)-EHPB 9b was carried
out by the reduction of 30 mM EOPB 9a using B. pumilus Phe-C3
(12.0 g cdw/L) at 30 °C, 220 rpm (Table 3, entry 9). 1.7 g (R)-9b
was obtained in 26 h after purification; the yield and the ee of
(R)-9b were 91.0% and 97.1%, respectively. The previous reported
plant cells of Daucus carota could also be used to catalyze the syn-
thesis of (R)-9b with high yield (90%) and ee (>99%).35 However,
the reaction required a very long time of 10 days, and the sub-
strate/catalyst ratio (0.1/10, w/w) was moderate. For the present
study, the catalytic synthesis of (R)-9b with B. pumilus Phe-C3
was performed within 26 h while the substrate/catalyst ratio
increased to 5.7/12 (w/w).
2.5. Reuse of B. pumilus Phe-C3 for the biotransformation
With 30 mM 9a used as substrates each time, the cells of B.
pumilus Phe-C3 could be reused for the bioreduction. In each run,
the cells were collected by centrifugation at 9000 rpm, 4 °C for
15 min after the reaction. The cells were then washed by
phosphate buffer (pH 7.0) and reused under the same conditions.
As shown in Figure 1, each cycle of the biotransformation
resulted in a slight loss of the catalytic activity of the strain.
2.4. Preparative bioreduction of 1a–9a
Preparative biotransformations were performed on a 300-mL
scale in a 1000-mL shaking flask (Table 3). The yields of the prod-
ucts were calculated after purification via column chromatogra-
phy. Bioreduction of 1a–8a with resting cells (3.0 g cdw/L)
afforded 1b–8b with 87.3%, 90.1%, 62.4%, 88.4%, 83.1%, 82.7%,
79.7%, 85.4%, and 91.0%, respectively.
Reduction of ketoesters, such as 1a, 2a, and 5a, were efficiently
accompolished by B. pumilus Phe-C3 within 24 h, and gave the cor-
responding hydroxyesters which are important synthetic interme-
diates for L
-carnitine5 and inhibitors of HMG-CoA reductase.3,4
Interestingly, for ketoesters 2a and 5a, the conversion, activity,
and enantioselectivity are not very much dependent on the start-
ing concentration of substrate, which is advantageous for practical
production. In the present study, the substrate concentration of 2a
and 5a was easily increased without losing of the catalytic effi-
ciency. These results were competitive with other reports, such
as Davignon from Codexis47 and Li et al.,48 in terms of high product
concentration and ee.
Optically active ethyl (S)-3-hydroxy-3-phenylpropionate 4b,
the precursor of the anti-depressant tomoxetine,49 was obtained
with 95.7% ee and a production concentration of 2.7 g/L. The result
is comparable with that of reported by Salvi et al.50 recently.
Mandelic acid and its derivatives are key intermediates for the
production of various pharmaceuticals, such as semi-synthetic
Figure 1. Reuse of Bacillus pumilus Phe-C3 for the biotransformation of 9a. Reaction
conditions: substrate (30 mM), phosphate buffer pH 7.0 (10 mL), glucose (2%) and
cells (12 g cdw Lꢀ1) were incubated with shaking at 220 rpm for 24 h of each cycle.
(j)—isolation yield, (h)—ee.
Table 3
Preparation of 1b-9b by bioreduction with B. pumilus Phe-C3
Entry
Substrate (mM)
Cell density (g cdw/L)
Time (h)
Conv.a (%)
ee (%)
Yieldb
Config.c
(%)
(mg)
172.9
1
2
3
4
5
6
7
8
9
1a
2a
5
15
3
10
15
5
5
15
30
3
3
3
3
3
3
3
3
12
12
12
24
12
24
24
9
99.0
99.0
86.0
99.0
99.0
95.2
99.0
99.0
98.5
94.5
95.9
91.4
95.7
97.0
90.2
94.1
96.6
97.1
87.3
90.1
62.4
88.4
83.1
82.7
79.7
85.4
91.0
(R)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(R)
675.1
118.5
514.5
587.1
193.5
141.1
464.2
1703.5
3a
4ad
5a
6a
7a
8ad
9ad
24
26
a
Conversion and ee were determined by GC analysis; error limit: 2% of the stated values.
Yield of the isolated pure product.
The absolute configuration was determined by comparison of the specific rotation with the literature value.
Conversion and ee were determined by HPLC analysis; error limit: 2% of the stated values.
b
c
d

1350
C. He et al. / Tetrahedron: Asymmetry 19 (2008) 1347–1351
However, the cells still retained more than 80% of the initial activ-
ity after six reaction cycles. It was also observed that there was no
significant change in the enantioselectivity of the bioreduction
after each cycle. From a practical point of view, the reuse of bio-
catalysts is an effective means for cost-effective production.
4.3. General procedure for preparative bioreduction
Ketoesters 1a–9a (3–30 mM) were added to a suspension of B.
pumilus Phe-C3 cells (3.0 or 12.0 g cdw/L) in 300 mL of phosphate
buffer (pH 7.0) containing glucose (2%). The mixture was incubated
in a 1000-mL shaking flask at 220 rpm and 30 °C. The reaction was
stopped by centrifugation, and the product was extracted with
CHCl3. The organic phase was dried over anhydrous Na2SO4, fil-
tered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel to afford the cor-
responding hydroxyester.
3. Conclusions
The enantioselective reduction of various ketoesters by the
strain B. pumilus Phe-C3 was investigated. The wide substrate spec-
trum and high chemical yield with short reaction time made B.
pumilus Phe-C3 a promising biocatalyst in the construction of some
important optically active hydroxyesters. Meanwhile, compared
with previous reported plant cells of Daucus carota, a more efficient
synthesis of (R)-EHPB was carried out on a preparative scale with
high yield and ee. Moreover, the reuse of the cells further increased
their potential for large-scale production process.
4.3.1. Ethyl (R)-3-hydroxybutyrate 1b
25
25
½aꢁD ¼ ꢀ43:1 (c 1.0, CHCl3), lit.57 for (R)-1b ½aꢁD ¼ ꢀ44:5 (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.25 (d, 3H), 1.27 (t,
3H), 2.48 (m, 2H), 3.06 (br, 1H), 4.16 (q, 2H); 13C NMR (75 MHz,
CDCl3): d (ppm) 14.2, 22.7, 43.1, 60.6, 64.5, 173.1.
4.3.2. Ethyl (S)-4-chloro-3-hydroxybutyrate 2b
25
25
½aꢁD ¼ ꢀ21:7 (c 7.0, CHCl3), lit.58 for (R)-2b, ½aꢁD ¼ þ20:9 (c 7.7,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.27 (t, 3H), 2.65 (d,
2H), 3.60 (d, 2H), 4.19 (q, 2H), 4.28 (m, 1H); 13C NMR (75 MHz,
CDCl3): d (ppm) 14.4, 38.8, 48.4, 61.1, 68.5, 172.0.
4. Experimental
b-Ketoesters 3a55 and 5a56 were prepared based on the
reported procedures in our laboratory, other ketoesters were
obtained from commercial suppliers and used without further
purification. ( )-Ethyl hydroxyesters were synthesized by reducing
the corresponding ethyl oxobutyrates with sodium borohydride.35
All other chemicals were of analytic grade. B. pumilus Phe-C3 was
obtained from Institute of Biotechnology, ETH Zurich. Column
chromatography was carried out using silica gel. GC analyses were
performed using a chiral CP-Chirasil Dex CB column, HPLC analy-
ses were performed using a Chiralcel OB-H column. The absolute
configurations were determined using a Perkin Elmer Model 343
polarimeter in CHCl3, and assigned by comparison of the specific
rotations with the literature values. The NMR spectra were mea-
sured on a Bruker AM 300 spectrometer or a Bruker AM 500
spectrometer.
4.3.3. Ethyl (S)-4-bromo-3-hydroxybutyrate 3b
25
25
½aꢁD ¼ ꢀ10:9 (c 1.0, CHCl3), lit.59 for (S)-3b ½aꢁD ¼ ꢀ9:1 (c 10.4,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.38 (t, 3H), 2.74 (d,
2H), 3.28 (br, 1H), 3.51 (m, 2H), 4.18 (q, 2H), 4.25 (m, 1H); 13C
NMR (75 MHz, CDCl3): d (ppm) 14.4, 37.6, 39.6, 61.3, 67.8, 172.1.
4.3.4. Ethyl (S)-3-hydroxy-3-phenylpropionate 4b
25
25
½aꢁD ¼ ꢀ33:1 (c 1.0, CHCl3), lit.60 for (S)-4b ½aꢁD ¼ ꢀ25:8 (c 1.3,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.35 (t, 3H), 2.72 (d,
2H), 3.32 (br, 1H), 4.18 (q, 2H), 7.26–7.37 (m, 5H). 13C NMR
(75 MHz, CDCl3): 14.1, 43.3, 60.8, 70.4, 125.7, 127.8, 128.5, 142.5,
172.4.
4.1. Cultivation and preparation of the biocatalyst
4.3.5. Ethyl (R)-4-cyano-3-hydroxybutyrate 5b
25
25
½aꢁD ¼ ꢀ32:1 (c 1.0, CHCl3), lit.61 for (R)-5b ½aꢁD ¼ ꢀ33:1 (c 1.2,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.25 (t, 3H), 2.65 (d,
2H), 3.13 (m, 1H), 3.75 (m, 2H), 4.15 (q, 2H); 13C NMR (75 MHz,
CDCl3): d (ppm) 14.4, 25.2, 40.1, 61.5, 64.2, 117.3, 171.2.
B. pumilus Phe-C3 was grown on LB-agar plate for 5 days, and
then inoculated into 100 mL M9 medium containing 2% (w/v) glu-
cose in a 500-mL shaking flask. The cells were grown at 30 °C and
220 rpm for 38 h, then harvested by centrifugation at 4 °C and
washed twice, stored at ꢀ80 °C in a refrigerator. M9 medium com-
position: Na2HPO4 6.78‰; KH2PO4 3.0‰; NaCl 0.5‰; NH4Cl 1.0‰;
MgSO4 0.24‰; CaCl2 0.01‰, pH 7.0.
4.3.6. Ethyl (R)-4,4,4-trifluoro-3-hydroxybutyrate 6b
25
25
½aꢁD ¼ þ20:1 (c 1.0, CHCl3), lit.45 for (R)-6b ½aꢁD ¼ þ18:7 (c 1.2,
CHCl3); 1H NMR (500 MHz, CDCl3): d (ppm) 1.28 (t, 3H), 2.72–2.64
(m, 2H), 3.58 (m, 1H), 4.22 (q, 2H), 4.42 (m, 1H); 13C NMR (75 MHz,
CDCl3): d (ppm) 14.1, 34.7, 61.7, 66.8, 125.4, 170.8.
4.2. General procedure for small scale bioreduction
The cells of B. pumilus Phe-C3 were suspended to a cell density
of 3.0 g cdw/L in 10 mL of phosphate buffer (pH 7.0) containing
glucose (2% w/v) in a 100-mL Erlenmeyer flask. Substrates were
added as a solution in 10% MeOH to a final concentration of 3–
15 mM. The mixture was shaken at 200 rpm at 30 °C. The reactions
were then followed by GC or HPLC analysis. Then 300 lL aliquots
were taken at predetermined time points each time for analysis.
Samples for GC analysis were prepared by centrifugation to remove
the cell debris, mixing of 100 lL supernatant with 400 lL phos-
phate buffer (pH 7.0), extracting with 500 lL chloroform and dry-
ing with anhydrous Na2SO4. For HPLC analysis, samples were
prepared by mixing the aliquots with an equal volume of MeOH
followed by centrifugation.
4.3.7. Ethyl (R)-2-hydroxy-propionate 7b
25
25
½aꢁD ¼ þ14:3 (c 1.0, CHCl3), lit.62 for (R)-7b ½aꢁD ¼ þ12:8 (c 2.0,
CHCl3); 1H NMR (300 MHz, CDCl3): d (ppm) 1.26 (t, 3H), 1.38 (d,
2H), 4.22 (m, 4H); 13C NMR (75 MHz, CDCl3): d (ppm) 14.1, 20.9,
62.3, 66.9, 176.0.
4.3.8. Ethyl (R)-a-hydroxybenzeneacetate 8b
25
25
½aꢁD ¼ ꢀ99:3 (c 1.0, CHCl3), lit.63 for (R)-8b ½aꢁD ¼ ꢀ104:4 (c 1.0,
EtOH); 1H NMR (500 MHz, CDCl3): d (ppm) 1.20 (t, 3H), 3.63 (s, 1H),
4.15–4.25 (m, 2H), 5.20 (d, 1H), 7.33–7.42 (m, 5H); 13C NMR
(75 MHz, CDCl3): d (ppm) 13.9, 62.1, 72.8, 126.3, 128.2, 128.5,
138.3, 173.8.
The specific activity of B. pumilus Phe-C3 was examined during
the first 30 min of each reaction; one unit (U) of the activity was
defined as the amount biocatalyst capable of catalyzing the reduc-
tion of 1 lmol of substrate per minute.
4.3.9. Ethyl (R)-2-hydroxy-4-phenylbutyrate 9b
25
25
½aꢁD ¼ ꢀ19:5 (c 1.0, CHCl3), lit.27 for (R)-9b ½aꢁD ¼ ꢀ18:1 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): d (ppm) 1.28 (t, 3H), 1.89–2.18
(m, 2H), 2.79 (m, 2H), 4.20 (q, 2H), 7.15–7.35 (m, 5H); 13C NMR

C. He et al. / Tetrahedron: Asymmetry 19 (2008) 1347–1351
1351
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