414
X. Jia et al. / Tetrahedron: Asymmetry 19 (2008) 407–415
4
.7. General procedure for enzymatic hydrolysis of 2-, 3-,
mixed and shaken at 300 rpm and 25 °C. For following
the reaction, 300 lL liquid samples were taken at 0, 21,
28, 41, 55, and 75 h, respectively, and analytical samples
were prepared by centrifugation, separation, and dilution
of 20 lL organic layer with 1.580 mL 1 mM (R)-styrene
oxide as an internal standard. The ee and concentration
of the remaining epoxide 1 were quantified by chiral
HPLC. The reaction was stopped at 75 h with an ee of
(S)-1 >98%. The product was recovered according to the
same procedure described above for (S)-2. Purification by
flash chromatography on a silica gel column with n-hexane
and 4-chlorostyrene oxides (±)-1–3 with cell-free extracts
of Sphingomonas sp. HXN-200 in a two-phase system
containing aqueous buffer/n-hexane (1:1)
Frozen cells of Sphingomonas sp. HXN-200 were thawed
and then suspended in 50 mM Tris–HCl buffer (pH 7.5)
to a cell density of 18 g/L. The cells were disrupted with
a homogenizer (Constant Cell Disruption System ) at
3
high-speed refrigerated centrifuge followed by the removal
of cell debris gave the cell-free extracts. All the operations
were carried out at 4 °C to avoid enzyme deactivation. The
protein concentration was determined to be 7.0 g protein/L
TM
0 psi. Centrifugation at 25,200g for 0.5 h in a Hitachi
(R = 0.46) gave 0.109 g (S)-1 in 35.7% yield and 98.5% ee
f
2
D
0
16
with an ½aꢂ ¼ þ58:2 (c 0.20, CHCl ) {lit.: +61.5, (c 1.62,
3
CHCl )}. The yield was corrected as 37% after deducing
3
1
5
with Bradford method.
the sampling volume during the biotransformation.
Then, 10 mL of freshly prepared CFE (7.0 g protein/L) was
mixed with an equal volume of n-hexane containing
4
.8.3. (S)-4-Chlorostyrene oxide 3. In a procedure similar
to the synthesis of (S)-2, a mixture of 50 mL n-hexane con-
taining (± )-3 (0.618 g, 4.00 mMol, 79.9 mM) and 50 mL
5
2
the bioconversion, 300 lL samples were taken at 0, 5, 16,
2
HPLC analysis were prepared by centrifugation and sepa-
ration of organic layer followed by diluting 20 lL organic
layer with 0.620 mL 1.032 mM benzyl alcohol as the inter-
nal standard. The reaction was stopped after 61 h when ee
of (S)-3 reached >98%, and the product was recovered
according to the same procedure described above for (S)-
5
3
.0 mM (± )-1, 2, or 3, and the mixture was incubated at
00 rpm and 25 °C. At different time points, 500 lL sample
0 mM Tris–HCl buffer (pH 7.5) with cell density of
4 g cdw/L was shaken at 300 rpm and 25 °C. To follow
was taken out and centrifuged for phase separation, 100 lL
of organic phase was diluted with equal volume of n-hex-
ane containing 2.0 mM benzyl alcohol as the internal stan-
dard, and the sample was analyzed by chiral HPLC to
determine the ee and concentration of the epoxide. The
results are shown in Figure 3.
7, 39, 45, 52, and 61 h, respectively. Samples for chiral
4
2
.8. Enzymatic preparation of (S)-1–3 by the hydrolysis of
-, 3-, and 4-chlorostyrene oxides (±)-1–3 with frozen/thawed
cells of Sphingomonas sp. HXN-200 in a two-phase system
containing aqueous buffer/n-hexane (1:1)
2
. After workup and flash chromatography on a silica gel
column with n-hexane (R = 0.29), 0.117 g of (S)-3 was
f
2
0
obtained in 18.9% yield and 99.1% ee {½aꢂ ¼ þ24:5 (c
D
1
3 3
6
4
.8.1. (S)-3-Chlorostyrene oxide 2. A suspension of
1.37, CHCl ); lit.: +24.9, (c 1.50, CHCl )}. The corrected
frozen/thawed cells (16 g cdw/L) of Sphingomonas sp.
HXN-200 in 50 mL 50 mM Tris–HCl buffer (pH 7.5) was
mixed with an equal volume of n-hexane containing 3-
chlorostyrene oxide (± )-2 (0.656 g, 4.24 mMol, 84.8 mM)
in a 250 mL shaking flask. The mixture was incubated at
yield was 20% after the deduction of analytic samples taken
during the biotransformation.
References
3
00 rpm and 25 °C, and 300 lL samples of liquid were
taken at 0, 24, 43, 79, 90, and 100 h, respectively, to follow
the reaction. Samples were prepared by centrifugation, sep-
aration, dilution of 20 lL organic layer into 1.580 mL
1
. For reviews, see: (a) Archer, I. V. J. Tetrahedron 1997, 53,
15617–15662; (b) Archelas, A.; Furstoss, R. Annu. Rev.
Microbiol. 1997, 51, 491–525; (c) Archelas, A.; Furstoss, R.
Tibtech 1998, 16, 108–116; (d) Archelas, A.; Furstoss, R.
Tibtech 1998, 16, 108–116; (e) Swaving, J.; Bont, J. A. M.
Enzyme Microb. Technol. 1998, 22, 19–26; (f) Orru, R. V. A.;
Faber, K. Curr. Opin. Chem. Biol. 1999, 3, 16–21; (g)
Weijiers, C. A. G. M.; Bont, J. A. M. J. Mol. Catal. B:
Enzym. 1999, 6, 199–214; (h) Steinreiber, A.; Faber, K. Curr.
Opin. Biotechnol. 2001, 12, 552–558; (i) Archelas, A.; Furs-
toss, R. Curr. Opin. Chem. Biol. 2001, 5, 112–119; (j) de Vries,
E. J.; Janssen, D. B. Curr. Opin. Biotechnol. 2003, 14, 414–
420.
1
mM benzyl alcohol (internal standard), and analyzed
by chiral HPLC to determine the ee and the concentration
of the remaining epoxide 2. After 100 h, the ee of epoxide 2
exceeded 98%, and the reaction was stopped. The mixture
was centrifuged at 10,000 rpm for 30 min at 4 °C, and the
organic phase was collected. The aqueous phase was
extracted with n-hexane (2 ꢁ 50 mL), and all organic phase
was combined. After drying over MgSO , the solvent was
4
removed by evaporation. The crude product was then
purified by flash chromatography on a silica gel column
2. Schnute, E.; Anderson, J.; PCT. Int. Appl., WO 2005003140,
2005.
with n-hexane (R = 0.31). 0.254 g (S)-2 was obtained in
f
20
3
. Wittman, M.; Carboni, J.; Attar, R.; Balasubramanian, B.;
Balimane, P.; Brassil, P.; Beaulie, F.; Chang, C.; Clarke, W.;
Dell, J.; Eummer, J.; Frennesson, D.; Gottardis, M.; Greer,
A.; Hansel, S.; Hurlburt, W.; Jacobson, B.; Krishnananthan,
S.; Lee, F.; Li, A.; Lin, T.; Liu, P.; Ouellet, C.; Sang, X.;
Saulnier, M.; Stoffan, K.; Sun, Y.; Vlaparthi, U.; Wong, H.;
Yang, Z.; Zimmermann, K.; Zoeckler, M.; Vyas, D. J. Med.
Chem. 2005, 48, 5639–5643.
3
8.7% yield and 99.1% ee, with an ½aꢂ ¼ þ11:5 (c 1.61,
D
1
6
CHCl ) {lit.: ꢀ10.9, (c 1.80, CHCl ) for (R)-2}. Deducing
3
3
the volume of sampling during the biotransformation gave
a corrected yield of 41%.
4
.8.2. (S)-2-Chlorostyrene oxide 1. In a procedure similar
to the synthesis of (S)-2, 50 mL n-hexane containing (± )-1
0.305 g, 1.97 mMol, 39.4 mM) and 50 mL 50 mM Tris–
HCl buffer (pH 7.5) with a cell density of 32 g cdw/L was
(
4. Spelberg, L. J.; Rink, R.; Kellogg, M. R.; Janssen, D.
Tetrahedron: Asymmetry 1998, 9, 459–466.