Enantioselective hydrolysis of styrene oxide with the epoxide hydrolase of Sphingomonas sp. HXN-200

Article abstract of DOI:10.1016/j.tetasy.2005.11.018

The soluble bacterial epoxide hydrolase (EH) from Sphingomonas sp. HXN-200 catalyzed the enantioselective hydrolysis of racemic styrene oxide to give (S)-styrene oxide with an enantiomeric ratio (E) of 21-23 in aqueous buffer, better than any reported native EHs. The ring opening of the styrene oxide with this EH was only at the terminal position for the (S)-enantiomer and at the terminal and benzylic position in an 87:13 ratio for the (R)-enantiomer. Enzymatic hydrolysis of the styrene oxide in a two-liquid phase system significantly reduced autohydrolysis, thus improving the E to 26-29. Hydrolysis of 160 mM styrene oxide with cell-free extract (CFE) of Sphingomonas sp. HXN-200 (10 mg protein/mL) in aqueous buffer and n-hexane (1:1) for 30.7 h afforded 39.2% (62.7 mM) of (S)-styrene oxide in >99.9% ee. The lyophilized CFE was proven to be stable, while the rehydrated lyophilized CFE powder was successfully used for the hydrolysis of 320 mM styrene oxide in the two-liquid phase system, yielding 40.2% (128.6 mM) of (S)-styrene oxide in >99.9% ee after 13.8 h. No inhibitory effect of the diol product on the hydrolysis was observed when the diol concentration was lower than 476 mM, suggesting a straightforward process for the hydrolysis of up to 1 M styrene oxide.

Full text of DOI:10.1016/j.tetasy.2005.11.018

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 17 (2006) 47–52
Enantioselective hydrolysis of styrene oxide with the
epoxide hydrolase of Sphingomonas sp. HXN-200
Zeya Liu, Johannes Michel, Zunsheng Wang, Bernard Witholt and Zhi Li*
Institute of Biotechnology, ETH Zurich, CH-8093 Zurich, Switzerland
Received 6 October 2005; accepted 17 November 2005
Available online 9 January 2006
Abstract—The soluble bacterial epoxide hydrolase (EH) from Sphingomonas sp. HXN-200 catalyzed the enantioselective hydrolysis
of racemic styrene oxide to give (S)-styrene oxide with an enantiomeric ratio (E) of 21–23 in aqueous buffer, better than any reported
native EHs. The ring opening of the styrene oxide with this EH was only at the terminal position for the (S)-enantiomer and at the
terminal and benzylic position in an 87:13 ratio for the (R)-enantiomer. Enzymatic hydrolysis of the styrene oxide in a two-liquid
phase system significantly reduced autohydrolysis, thus improving the E to 26–29. Hydrolysis of 160 mM styrene oxide with cell-
free extract (CFE) of Sphingomonas sp. HXN-200 (10 mg protein/mL) in aqueous buffer and n-hexane (1:1) for 30.7 h afforded
3
9.2% (62.7 mM) of (S)-styrene oxide in >99.9% ee. The lyophilized CFE was proven to be stable, while the rehydrated lyophilized
CFE powder was successfully used for the hydrolysis of 320 mM styrene oxide in the two-liquid phase system, yielding 40.2%
128.6 mM) of (S)-styrene oxide in >99.9% ee after 13.8 h. No inhibitory effect of the diol product on the hydrolysis was observed
(
when the diol concentration was lower than 476 mM, suggesting a straightforward process for the hydrolysis of up to 1 M styrene
oxide.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
oxide in high ee. A higher enantioselectivity was
observed for the latter EH with an enantiomeric ratio
(E) of 31, but the activity was low. On the other hand,
some EHs showed an (R)-enantioselectivity for the
hydrolysis affording enantiopure (S)-styrene oxide
(Table 1). These include the EHs from microsome (rab-
5
Epoxide hydrolase (EH) catalyzes the enantioselective
hydrolysis of either racemic or meso-epoxides, provid-
ing a useful and green method for the preparation of
enantiopure epoxides or vicinal diols that are useful syn-
thons and pharmaceutical intermediates. EH is cofactor
independent and relatively stable, and many EHs have
been discovered from various microorganisms for syn-
6
7
bit liver mEH ), yeasts (Rhodotorula glutinis CIMW147
8
and Rhodosporidium kratochvilovae SYU-08 ), fungi
(Aspergillus niger LCP521 and new Aspergillus niger
isolate ), and a bacterium (Agrobacterium radiobacter
AD1 ). The bacterial EH of A. radiobacter AD1
showed the best enantioselectivity, with an E of 14–16.
Methods for improving the enantioselectivity of the
existing EHs have also been developed: the EH of A.
radiobacter AD1 was engineered to create the Y215F
4
1
9
thetic applications. A very interesting target reaction
1
0
for EH is the enantioselective hydrolysis of racemic sty-
rene oxide, since the product, enantiopure styrene oxide,
is a valuable and very useful synthon for the prepara-
tion of different types of enantiopure compounds.
Moreover, (S)-styrene oxide is a useful intermediate
for the synthesis of Levamisole, a nematocide and
Several EHs have so far been
reported for the hydrolysis of styrene oxide.
2
3
a
11
mutant, which gave an E of 30–32; and immobiliza-
3
b
anticancer agent.
tion of the multimeric EH of A. niger LCP521 onto
4
–12
12
Among
Eupergit C/CDE increased also the enantioselectivity.
4
them, EHs from Beauvaria sulfurescens ATCC7159 and
Streptomyces Antibioticus T u¨ 4 demonstrated (S)-
enantioselectivity in the hydrolysis giving (R)-styrene
These approaches could have great synthetic potential.
Herein, we report a novel soluble bacterial EH from
5
1
3
Sphingomonas sp. HXN-200 for the hydrolysis of sty-
rene oxide with higher (R)-enantioselectivity than any
other known native EHs and the application of this
EH in a two-liquid phase system to prepare enantiopure
(S)-styrene oxide.
*
Corresponding author. Tel.: +41 1 633 3811; fax +41 1 633 1051;
e-mail: zhi@biotech.biol.ethz.ch
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.11.018

48
Z. Liu et al. / Tetrahedron: Asymmetry 17 (2006) 47–52
Table 1. Enantioselective hydrolysis of styrene oxide with known native epoxide hydrolases for the preparation of (S)-styrene oxide
a
Epoxide hydrolase
Ref. Cat. form Cat. conc.
(± )-1 conc. Temp. pH Time (S)-1 ee Yield/ Conv.
E
b
(
g/L)
(mM)
(ꢁC)
(h)
(%)
(%)
c
Rabbit liver mEH
6
Enzyme
5
5
10
526
80
8
37
35
4
35
27
30
30
7.4 n.i.
7.5
8.0 16
98
98
98
99
99
22
18
36
19
28
32
33
7
Rhodotorula glutinis CIMW147
Rhodotorula glutinis CIMW147
Rhodosporidium kratochvilovae SYU-08
Aspergillus niger LCP521
Asperginus niger isolate
Agrobacterium radiobacter AD1
7a Cells
7b Cells
8
4
9
50–75
21
n.i.
8
75
0.8
6
15
7
10
13
16
c
Cells
Cells
Cells
8.0
8.0
8.0
9.0
2.7
2
6
d
e
10
5
100
99
10a Enzyme
0.025–0.125
0.8
a
b
c
15
s s
)], c: conversion, ee : ee of substrate.
Enantiomeric ratio E was calculated by E = ln[(1 Àc)(1 À ee
s
)]/ln[(1 Àc)(1 + ee
g cell dry weight/L and g protein/L for whole cells and enzyme preparation, respectively.
No information.
g wet cell weight/L.
Calculated with an ee of 99%.
d
e
2
. Results and discussion
umn and a chiralpark AS column, respectively. In the
hydrolysis of epoxide (R)-1, the reaction was complete
after 2 h, with a specific activity of 9.3 U/g protein
(U = lmol/min) over the first hour. This gave 83% of
(R)-diol 2 and 17% of (S)-diol 2. For the hydrolysis of
epoxide (S)-1, the reaction rate was slower, with an
activity of 5.6 U/g protein. Styrene oxide (S)-1 was com-
pletely converted to diol 2 after 4 h. After 2 h, a conver-
sion of 72% was reached with the formation of diol (S)-2
and (R)-2 in a 94:6 ratio. To examine non-enzymatic
hydrolysis, 10 mL CFE (9.8 mg/mL) in buffer (pH =
7.5) was first boiled for 45 min and then used for the
hydrolysis of 11.9 mM epoxide (R)-1. This gave diol
(S)-2 as the sole product, confirming the ring opening
of styrene oxide at the benzylic position for non-enzy-
matic hydrolysis; 4.2% of diol was formed after 2 h.
The non-enzymatic hydrolysis suggested that (S)-epox-
ide 1 was enzymatically opened only at the terminal
position, while (R)-epoxide 1 was enzymatically opened
at the terminal and benzylic position in a 87:13 ratio
(Scheme 1).
We recently discovered that Sphingomonas sp. HXN-
00 contains a novel soluble EH, which catalyzes the
enantioselective hydrolysis of meso-alicyclic epoxides
as well as racemic alicyclic epoxides.
nation of hydrolysis of styrene oxide with this EH, the
cells of Sphingomonas sp. HXN-200 were prepared by
growing on n-octane in E2 medium. The cell free
extract (CFE) was prepared by passing the cell suspen-
sion in 50 mM Tris–HCl buffer (pH = 7.5) through a
French press and removing the cell debris by ultracentri-
fugation. The resulting CFE was used in different
2
1
3a
1
3b
For the exami-
1
4
1
6
concentrations, determined by Bradford protein assay,
for the hydrolysis experiment.
2.1. Hydrolysis of styrene oxides (S)-1, (R)-1, and
racemic 1 with CFE of Sphingomonas sp. HXN-200
in aqueous buffer
To examine the selectivity for the hydrolysis, pure sty-
rene oxide (R)-1 (9.5 mM) and (S)-1 (9.4 mM) were used
as the substrates. Biotransformations were performed
with CFE (9.8 g protein/L) in 10 mL 50 mM Tris–HCl
buffer (pH = 7.5) at 25 ꢁC and 300 rpm. The conversion
and ee of diol 2 were analyzed by HPLC with a C18 col-
To explore the enzymatic resolution of styrene oxide
with this EH, 20 mM racemic 1 was hydrolyzed with
CFE (10 g protein/L) under the same conditions as
described above. As shown in Figure 1, the hydrolysis
OH
O
*
H
O
*
OH
Epoxide hydrolase of
+
+
H
2
O
+
Sphingomonas sp. HXN-200
(
±)-1
2
(S)-1
Autohydrolysis
(
(
R)-1
R)-1
H O
2
(S)-2
1
00%
Pure enzymatic hydrolysis
Pure enzymatic hydrolysis
+
+
H O
(S)-2
+
(R)-2
2
1
3%
87%
(
S)-1
H O
2
(S)-2
00%
1
Scheme 1.

Z. Liu et al. / Tetrahedron: Asymmetry 17 (2006) 47–52
49
1
2
0
8
6
4
2
0
2.2. Hydrolysis of racemic styrene oxide 1 with CFE of
Sphingomonas sp. HXN-200 in a two-phase system
1
To reduce the rate of autohydrolysis, a two-liquid phase
system containing buffer and n-hexane (1:1) was applied
for the biotransformation. The non-enzymatic hydroly-
sis in this system was first examined. Tris–HCl buffer
(50 mM; pH = 7.5) containing CFE (10 g protein/L)
was boiled for 45 min. The solution was then mixed with
an equal volume of n-hexane containing 160 mM race-
mic styrene oxide 1, followed by shaking at 25 ꢁC and
300 rpm. After 68.5 h, 7.0% of diol 2 was formed, which
corresponds to an autohydrolysis rate of 0.1% per hour.
This is a significantly reduced rate compared to that in
buffer. Hydrolysis of 160 mM racemic epoxide 1 was
performed with 10 mL buffer containing CFE (10 g pro-
tein/L) and 10 mL n-hexane. As shown in Figure 2, the
reaction rates for both enantiomers were slightly slower
in the two-phase system than in the aqueous buffer. This
was possibly due to the decreased autohydrolysis rates.
The enantioselectivity in the two-phase system was
much higher (E = 26–29) than that in buffer (E = 13).
Finally epoxide (S)-1 was formed in >99.9% ee and
39.2% (62.7 mM).
(
(
R)-1
S)-1
0
50
100
Time (min)
150
200
Figure 1. Enantioselective hydrolysis of 20 mM racemic styrene oxide
with a cell-free extract of Sphingomonas sp. HXN-200 (10 g protein/
L) in 50 mM Tris–HCl buffer (pH = 7.5).
1
of the (R)-enantiomer is much faster than that of the
S)-enantiomer, leaving 40% of the unreacted epoxide
S)-1 in 98.4% ee at 3 h and 34.6% in >99.9% ee at
(
(
3
.5 h. From these data, an E of 23 was calculated, which
is higher than that of any native EHs for this reaction
reported thus far. As described above, the difference
between the reaction rates for the hydrolysis of pure
epoxide (R)-1 and (S)-1 is not very big. Thus, Km of
9
8
7
0
0
0
(
R)-1 should be much smaller than that of (S)-1.
To further explore the synthetic potential of this EH,
higher substrate concentration was examined. As listed
in Table 2, a similar enantioselectivity (E = 21) was
obtained with 40 mM (± )-1, leading to the formation
of 38.4% of epoxide (S)-1 in 98.8% ee after 5.8 h reac-
tion. Increasing the substrate concentration to 160 mM
resulted in a significant decrease in enantioselectivity.
After 23.7 h, 37.2% of epoxide (S)-1 was obtained in
60
(
(
(
R)-1, 2 phase
S)-1, 2 phase
R)-1, 1 phase
5
4
3
2
0
0
0
0
(S)-1, 1 phase
10
0
9
3.8% ee, corresponding to an E of 13. The decrease
0
5
10
15
Time (h)
20
25
30
in the enantioselectivity with higher substrate concentra-
tion at a given CFE concentration was expected while
the velocity of real enzymatic hydrolysis remains the
same. The velocity of the autohydrolysis increased with
substrate concentration.
Figure 2. Enantioselective hydrolysis of 160 mM racemic styrene oxide
with a cell-free extract of Sphingomonas sp. HXN-200 (10 g protein/
L) in 50 mM Tris–HCl buffer (pH = 7.5) and in buffer/n-hexane (1:1),
1
respectively.
Table 2. Preparation of (S)-styrene oxide 1 by enantioselective hydrolysis of racemic styrene oxide (± )-1 with EH of Sphingomonas sp. HXN-200
a
(
± )-1 (mM) CFE (g prot./L) Lyophilized CFE (g powder/L) Phase Activity (U/g p.) Time (h) (S)-1 ee (%) (S)-1 Conv. (%)
E
b
2
4
60
0
0
10
10
10
One
One
One
6.2
6.2
7.4
3.0
5.8
23.4
98.4
98.8
92.8
93.8
96.6
97.9
98.6
>99.9
87.7
96.2
98.8
>99.9
40.0
38.4
37.9
37.2
43.3
42.6
40.9
39.2
47.4
44.9
41.6
40.2
23
21
13
13
29
29
26
b
b
1
2
3.7
c
1
60
10
Two
5.7
28.4
2
3
3
9.5
0.1
0.7
d
3
20
60
Two
5.0
10.7
52
38
29
1
1
1
2.1
3.1
3.8
a
b
c
Average activity during the first hour; in U/g protein.
In 10 mL cell-free extract in 50 mM Tris–HCl (pH = 7.5).
In 10 mL buffer and 10 mL n-hexane.
d
In 2 mL buffer and 2 mL n-hexane.

50
Z. Liu et al. / Tetrahedron: Asymmetry 17 (2006) 47–52
2.3. Hydrolysis of racemic styrene oxide 1 with lyophi-
lized/rehydrated CFE of Sphingomonas sp. HXN-200
in a two-phase system
4.5
4
3
.5
3
The soluble EH of Sphingomonas sp. HXN-200 was pro-
ven to be stable. Lyophilization of the CFE of this strain
resulted in a catalyst powder, which could be easily
stored and transported. The lyophilized powder was
rehydrated and then used for the hydrolysis of styrene
oxide in a higher concentration: 2 mL buffer containing
2
.5
2
1.5
1
0.5
1
20 mg lyophilized CFE was mixed with 2 mL n-hexane
0
containing 320 mM (± )-1. This gave a hydrolysis activ-
ity of 5.0 U/g powder, similar to the activity of 5.7 U/g
protein obtained with CFE in the same two-phase sys-
tem. As shown in Figure 3, the reaction velocities for
0
362
467
521
718
904
Diol conc. (mM)
Figure 4. Effect of the concentration of diol 2 on the activity of
enantioselective hydrolysis of 15 mM racemic styrene oxide 1 with a
cell-free extract (15 g protein/L) of Sphingomonas sp. HXN-200 in
(
R)- and (S)-enantiomers were nearly constant and the
former is much bigger than the latter during 11 h, indi-
cating a great enantioselectivity (E = 52 at 10.7 h). Sim-
ilar to the hydrolysis with CFE, as described before,
hydrolysis became less enantioselective at the later
stages. Nevertheless, styrene oxide (S)-1 was obtained
in 41.6% and in 98.8% ee after 13.1 h with an E of 29.
Continuing the reaction for 13.8 h afforded (S)-1 in
5
0 mM Tris–HCl buffer (pH = 7.5) in the presence of diol 2. Activity
is given as average activity in the first hour.
necessary to remove diol 2 from the enzyme-containing
aqueous phase for retaining high activity.
>99.9% ee and 40.2% yield (128.6 mM). This provided
us with a useful method for the preparation of (S)-sty-
3. Conclusion
rene oxide.
The soluble bacterial epoxide hydrolase (EH) from
Sphingomonas sp. HXN-200 demonstrated the highest
enantioselectivity among the known native EHs for the
enantioselective hydrolysis of racemic styrene oxide giv-
ing (S)-styrene oxide, with an enantiomeric ratio (E) of
21–23 in aqueous buffer and 26–29 in a two-liquid phase
system. Hydrolysis of 320 mM styrene oxide with rehy-
drated lyophilized CFE of Sphingomonas sp. HXN-200
in buffer/n-hexane (1:1) gave 40.2% (128.6 mM) of
1
1
1
1
1
80
60
40
20
00
(
(
R)-1
S)-1
8
6
4
2
0
0
0
0
0
(
S)-styrene oxide in >99.9% ee, providing a practical
synthesis of this useful and valuable pharmaceutical
intermediate. Further improvement of the productivity
could be achieved by the development of recombinant
strain overexpressing this novel EH.
0
2
4
6
8
10
12
14
Time (h)
Figure 3. Enantioselective hydrolysis of 320 mM racemic styrene oxide
1
with a lyophilized cell-free extract of Sphingomonas sp. HXN-200
4. Experimental
(
(
60 g powder/L) in 50 mM Tris–HCl buffer (pH = 7.5) and n-hexane
1:1).
4
.1. Analytical methods
2.4. Inhibitory effect of diol 2 on the enzymatic hydrolysis
of racemic styrene oxide 1 with CFE of Sphingomonas sp.
HXN-200
Concentrations of styrene oxide 1 and 1-phenyl-1,2-
ethanediol 2 in an aqueous sample were analyzed by
high performance liquid chromatography (HPLC)
with a Hewlett Packard 1040M Series II instrument.
Column: Hypersil BDS-C18 (4 · 125 mm). Eluent:
10 mM KH PO (pH = 7)/acetonitrile (80:20). Flow
rate: 1 mL/min. DAD detection: 210 nm. Retention
time: 1.9 min for 2; 2.7 min for benzyl alcohol (internal
standard); 9.7 min for 1.
It was known that hydrolysis product diol 2 could inhibit
the enzymatic hydrolysis of epoxide 1. To investigate this
effect, racemic diol 2 at different concentrations was
added to a CFE suspension (15 g protein/L) in 50 mM
Tris–HCl buffer (pH = 7.5). The mixture was then used
for the hydrolysis of 15 mM racemic epoxide 1. As
shown in Figure 4, there was no inhibitory effect of the
diol on the hydrolysis activity when the concentration
of diol 2 was lower than 476 mM. Increasing diol concen-
tration to 521 mM decreased the hydrolysis activity by
one third. Thus, it was possible to use as high as 1 M sty-
rene oxide for the enzymatic resolution without diol inhi-
bition. Beyond this substrate concentration, it was
2
4
Concentrations and ees of styrene oxide 1 and 1-phenyl-
1,2-ethanediol 2 in an organic sample were analyzed
with a Merck Hitachi L-500 HPLC on a Chiralpak AS
column. Eluents: n-hexane/isopropanol (95:5). Flow
rate: 1.0 mL/min. UV detection: 210 nm. Retention
times: 5.3 min for (R)-1; 5.8 min for (S)-1; 20.2 min for
(S)-2; 20.6 min for (R)-2.

Z. Liu et al. / Tetrahedron: Asymmetry 17 (2006) 47–52
51
The enantiomeric excess of 1-phenyl-1,2-ethanediol 2 in
organic sample was also analyzed with a Merck Hitachi
D-7000 HPLC on a Chiralpak AS column. Eluents:
n-hexane/isopropanol (98:2). Flow rate: 0.6 mL/min.
DAD detection: 210 nm. Retention times: 77.8 min
rated, dried, and mixed with n-hexane in 1:4, and the
sample was analyzed by HPLC on Chiralpak AS col-
umn with n-hexane/isopropanol (98:2) to determine
the ee of diol 2.
(
S)-2, 86.4 min for (R)-2.
4.6. General procedure for hydrolysis of racemic styrene
oxide 1 with CFE of Sphingomonas sp. HXN-200 in
aqueous buffer
4
.2. Materials
Racemic styrene oxide 1 (97%) was purchased from
Janssen. Styrene oxide (R)-1 (P98%) and (S)-1 (P98%)
were obtained from Fluka. 1-Phenyl-1,2-ethanediol
racemic-2 (97%), (S)-2 (99%) and (R)-2 (99%) were
purchased from Aldrich.
To a 10 mL 50 mM Tris–HCl buffer (pH = 7.5) contain-
ing CFE (10 g protein/L) of Sphingomonas sp., HXN-
200 was added racemic 1 (20–160 mM), and the mix-
tures shaken at 25 ꢁC and 300 rpm. Aliquots (200 lL)
were taken at different time points, mixed with 800 lL
ethyl acetate, and centrifuged. The organic phase was
4
.3. Growth of Sphingomonas sp. HXN-200
dried over MgSO and mixed with n-hexane in a 1:1–
4
19 ratio depending on the substrate concentration. Sam-
Sphingomonas sp. HXN-200 was grown on E2-medium
agar plate under n-octane vapor. It was then inoculated
into 20 mL LB-medium (1 L LB-medium contains
ples were analyzed by HPLC on Chiralpak AS column
with n-hexane/isopropanol (95:5) to determine the
concentration and ee of styrene oxide 1 and diol 2.
The activity was calculated as the following: average
activity at 1 h (U/g cdw) = product concentration (lM)/
[60 (min) Ã cell density (g cdw/L)].
1
0.0 g tryptone, 5.0 g yeast extract, and 10.0 g NaCl;
pH = 7.0) and incubated at 30 ꢁC and 300 rpm to an
optic density at 450 nm (OD ) of 6.0. The culture was
4
50
transferred into a 100 mL LB-medium and shaken at
0 ꢁC and 300 rpm to an OD of 6.0. The preculture
3
4.7. Hydrolysis of racemic styrene oxide 1 with CFE of
Sphingomonas sp. HXN-200 in a two-liquid phase system
4
50
1
7
was inoculated into 2 L E2 medium containing 2 mL
1
2
1
2
1
M MgSO , 100 lL polypropylene glycol P2000, and
4
mL standard 1000*MT solution (2.78 g FeSO *7H O,
To a 10 mL 50 mM Tris–HCl buffer (pH = 7.5) contain-
ing CFE (10 g protein/L) of Sphingomonas sp. HXN-200
was added 10 mL n-hexane containing racemic 1
(160 mM), and the mixtures shaken at 25 ꢁC and
300 rpm. Aliquots (200 lL) were taken at different time
points, centrifuged, and separated into aqueous and
organic phase. Analytic samples from aqueous phase
were prepared by extraction with ethyl acetate, as
described above. An analytic sample from the organic
phase was prepared by mixing with n-hexane in a 1:79
ratio. All samples were analyzed by HPLC on Chiralpak
AS column with n-hexane/isopropanol (95:5) to deter-
mine the concentration and ee of styrene oxide 1 and
diol 2. The reaction was stopped at 30.7 h, giving
39.2% (62.7 mM) of (S)-styrene oxide 1 in >99.9% ee.
4
2
.98 g MnCl 4H O, 2.81 g CoSO 7H O, 1.47 g CaCl
2
*
2
4
*
2
2
*
H O, 0.17 g CuCl *2H O, and 0.29 g ZnSO *7H O in
2
2
2
4
2
L 1 N HCl) in a 3-L bioreactor. The bioreactor was stir-
red at 1500 rpm and at 30 ꢁC, and air saturated with
n-octane vapor (at 20 ꢁC) was introduced at 1 L/min.
The pH was maintained at 7.0–7.2 by using 25% NH OH
4
and 25% phosphoric acid. Cells were harvested at OD450
of 24 at 62 h. The cell pellet was washed with 5 mM
KH PO buffer (pH = 7.5) and stored at À80 ꢁC.
2
4
4.4. Preparation of the cell free extract (CFE) of
Sphingomonas sp. HXN-200
Frozen cells of Sphingomonas sp. HXN-200 were thawed
and then suspended in 50 mM Tris–HCl buffer
(
pH = 7.5) to an OD450 of 140. Cells were broken by
4.8. Hydrolysis of racemic styrene oxide 1 with
rehydrated lyophilized CFE of Sphingomonas sp.
HXN-200 in a two-liquid phase system
passing three times through a French press. Ultracentri-
fugation at 245,000g and 4 ꢁC for 45 min gave the super-
natant as the CFE. The protein concentration of the
1
6
CFE was determined by a Bradford protein assay.
To 2 mL of 50 mM Tris–HCl buffer (pH = 7.5) was
added the lyophilized CFE powder (120 mg) of Sphingo-
monas sp. HXN-200 and 2 mL n-hexane containing
racemic-1 (320 mM). The mixtures were shaken at
4.5. Hydrolysis of styrene oxide (R)-1 or (S)-1 with CFE
of Sphingomonas sp. HXN-200 in aqueous buffer
25 ꢁC and 300 rpm, and the aliquots (200 lL) taken at
To a 10 mL 50 mM Tris–HCl buffer (pH = 7.5) contain-
ing CFE (10 g protein/L) of Sphingomonas sp. HXN-
different time points for the analysis of the concentra-
tion and ee of styrene oxide 1 and diol 2, as described
above. After 13.8 h, (S)-styrene oxide 1 was formed in
40.2% (128.6 mM) and in >99.9% ee.
2
00 was added either enantiopure (R)-1 (9.5 mM) or
(
S)-1 (9.4 mM). The biotransformation was performed
at 25 ꢁC and 300 rpm, and samples (500 lL) were taken
at different time points for the analysis. After centrifuga-
tion, 200 lL supernatant was mixed with 800 lL MeOH
containing 4 mM benzyl alcohol as internal standard for
the analysis of the conversion of diol 2 by HPLC on C-
4.9. General procedure for hydrolysis of racemic styrene
oxide 1 with CFE of Sphingomonas sp. HXN-200 in the
presence of diol 2 in aqueous buffer
18 column; another 200 lL supernatant was extracted
with 800 lL ethyl acetate, the organic phase was sepa-
To 5 mL 50 mM Tris–HCl buffer (pH = 7.5) containing
CFE (16 g protein/L) of Sphingomonas sp. HXN-200

52
Z. Liu et al. / Tetrahedron: Asymmetry 17 (2006) 47–52
was added 1-phenyl-1,2-ethanediol 2 (0–904 mM). Race-
mic styrene oxide 1 was added to a concentration of
Wickerham, D. L.; Wolmark, N. Oncology 1997, 11, 44–
7.
. (a) Pedragosa-Moreau, S.; Archelas, A.; Furstoss, R. J.
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1
5.0 mM, and the mixtures shaken at 25 ꢁC and
3
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6
09–616.
fuged. The organic phase was dried over MgSO and
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2
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