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Table 3 Epoxidation of alkenes and hydroxylation of 1-bromo-4-ethylbenzene
with resting cells of E. coli (P450tol-GDH) in an n-hexadecane–water
biphasic system
b
c
Activity
Prod. Conc.
a
À1
d
Entry Sub. Time (h) Prod. (U g CDW) (mM)
ee (%)
1
2
3
4
5
6
1
3
5
7
13
21
12
12
12
12
12
12
(R)-2 6.9
(R)-4 5.7
(R)-6 5.7
(R)-8 2.6
(S)-14 3.2
(S)-22 8.0
17
18
17
6.4
6.5
21
97.6
91.6
99.7
99.1
90.2
99.0
e
Scheme 2 Asymmetric hydroxylation of ethylbenzene and its derivatives
with E. coli (P450tol-GDH) to prepare the corresponding enantiopure
a
Reactions were conducted in a mixture of 2 mL of cell suspension
(10 g CDW per L) of E. coli (P450tol-GDH) in 100 mM KP buffer (pH 8.0)
(S)-benzylic alcohols.
containing 1 wt% glucose with 1 mL n-hexadecane containing 40 mM
substrate (refers to aqueous volume) at 30 1C and 250 rpm. Deter-
mined for the first 1 h. Determined by HPLC analysis (refers to
aqueous volume). Determined by chiral HPLC analysis. Reaction
was conducted using 70 mM substrate in 5 mL of cell suspension
b
c
d
e
Table 2 Hydroxylation of ethylbenzene and its derivatives with resting
cells of E. coli (P450tol-GDH)
(
10 g CDW per L) of E. coli (P450tol-GDH) in 100 mM KP buffer (pH 8.0)
a
b
c
d
Entry Sub.
Prod. Time (h) Conv. (%) Selectivity
ee (%) containing 0.21 g resin XAD16 and 1 wt% glucose at 30 1C and 250 rpm.
1
2
3
4
5
17
19
21
23
25
(S)-18
(S)-20
(S)-22
(S)-24
8
5
5
5
90
98
97
50
25
85 : 15
95 : 5
95 : 5
95 : 5
499 : 1
97.5
99.0
99.0
93.7
97.1
4-substituted styrenes. P450tol was also the first enzyme with high
S)-enantioselectivity for the hydroxylations of ethylbenzenes 17, 19,
1, 23, and 25, producing the corresponding (S)-benzylic alcohols
8, 20, 22, and 26 in 97.1–99.0% ee and (S)-benzylic alcohols 24 in
3.7% ee. Preliminary work on the epoxidation and hydroxylation
with resting cells of E. coli (P450tol-GDH) in an n-hexadecane–water
buffer (pH 8.0) containing 1 wt% glucose at 30 1C and 250 rpm. or resin–water biphasic system showed the potential of using this
(
2
1
9
(S)-26 10
a
Reactions were conducted with different substrate concentrations
(
5 mM for 19, 3 mM for 21 and 23, 2 mM for 23 and 25) in 4 mL of
cell suspension (10 g CDW per L) of E. coli (P450tol-GDH) in 100 mM KP
b
c
Determined by HPLC analysis. Selectivity is defined as the molar
enzyme to prepare the epoxides and benzylic alcohols that are
useful pharmaceutical intermediates.
ratio of the chiral alcohol product to the corresponding ketone.
d
Determined by chiral HPLC analysis.
This work was supported by the GlaxoSmithKline and the
While the chemical selectivity to (S)-26 is 499%, the selec- Singapore Economic Development Board through a Green and
tivities to (S)-20, (S)-22, (S)-24 are 95% with the formation of 5% Sustainable Manufacturing grant (project No. 279-000-331-592).
of a-ketone. 85% selectivity was observed for the hydroxylation
to (S)-18. The conversions for ethylbenzene 17 and its 4-substituted Notes and references
derivatives 19 and 21 were much higher than that for 2-substituted
ethylbenzene 25.
1
(a) F. Hollman, I. W. C. E. Arends, K. Buehler, A. Schallmey and
B. B u¨ hler, Green Chem., 2011, 13, 226; (b) Z. Li, J. B. van Beilen, W. A
Duetz, A. Schmid, A. de Raadt, H. Griengl and B. Witholt, Curr. Opin.
Chem. Biol., 2002, 6, 136.
The epoxidation was then investigated with resting cells
of E. coli (P450tol-GDH) in an n-hexadecane–water biphasic
system, to avoid substrate and product inhibition. The results
are listed in Table 3. Epoxidation of 1, 3, and 5 reached the
2
V. Farina, J. T. Reeves, C. H. Senanayake and J. J. Song, Chem. Rev.,
2006, 106, 2734.
3 T. Katsuki and K. B. Sharpless, J. Am. Chem. Soc., 1980, 102, 7932.
4 W. Zhang, J. L. Loebach, S. R. Wilson and E. N. Jacobsen, J. Am.
Chem. Soc., 1990, 112, 2801.
À1
specific activity of 5.7–6.9 U g CDW and produced (R)-2, 4, 6
in 17–18 mM. The hydroxylation of 21 was performed in
another system, a resin–water biphasic system, to avoid the
substrate and product inhibition as well. It gave a specific
5
(a) S. Panke, B. Witholt, A. Schmid and M. G. Wubbolts, Appl.
Environ. Microbiol., 1998, 64, 2032; (b) A. Schmid, K. Hofstetter,
H. J. Feiten, F. Hollmann and B. Witholt, Adv. Synth. Catal., 2001,
3
43, 732; (c) S. Bernasconi, F. Orsini, G. Selloa and P. D. Gennaro,
À1
activity of 8.0 U g CDW and produced (S)-22 in 21 mM.
Tetrahedron: Asymmetry, 2004, 15, 1603.
In conclusion, P450tol monooxygenase from Rhodococcus copro-
philus TC-2 was discovered as a unique and highly enantioselective
enzyme for asymmetric epoxidation of alkenes and benzylic hydroxyl-
ations. P450tol was the first enzyme with excellent enantioselectivity
and high conversion for the (R)-epoxidation of 2- and 3-substituted
styrenes 1, 5, 7, and 9, (S)-epoxidation of 4-substituted styrenes 11 and
6 B. B u¨ hler, B. Witholt, B. Hauer and A. Schmid, Appl. Environ.
Microbiol., 2002, 68, 560.
7
A. F. Dexter, F. J. Lakner, R. A. Campbell and L. P. Hager, J. Am.
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8 (a) C. A. Martinez and J. D. Stewart, Curr. Org. Chem., 2000, 4, 263;
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(
4
Chem. – Eur. J., 2006, 12, 1216; (d) E. T. Farinas, M. Alcalde and F. H.
Arnold, Tetrahedron, 2004, 60, 525; (e) A. T. Li, J. Liu, S. Q. Pham and
Z. Li, Chem. Commun., 2013, 49, 11572.
1
3, and (R)-epoxidation of unconjugated terminal alkene 15. The
epoxidations provide with simple syntheses of the corresponding
R)-styrene oxides 2, 6, 8 and 10 in 97.5–99.7% ee, (S)-styrene oxides
1 and 13 in 90.0–90.2% ee, and (R)-epoxide 16 in 90.5% ee,
9
(a) M. Palucki, G. J. McCormick and E. N. Jacobsen, Tetrahedron Lett.,
(
1
1995, 36, 5457; (b) Y. Naruta, F. Tani, N. Ishihara and K. Maruyama,
J. Am. Chem. Soc., 1991, 113, 6865; (c) R. Zhang, W. Y. Yu, K. Y. Wong
and C. M. Che, J. Org. Chem., 2001, 66, 8145.
0 Q. S. Li, J. Ogawa, R. D. Schmid and S. Shimizu, FEBS Lett., 2001, 508, 249.
1 J. A. Peterson, J. Y. Lu, J. Geisselsoder, S. Graham-Lorence, C. Carmona,
F. Witney and M. C. Lorence, J. Biol. Chem., 1992, 267, 14193.
respectively. P450tol shares 46% sequence identity with P450terp,
but it is much more enantioselective for the epoxidations and also
shows the opposite enantioselectivity for the epoxidation of
1
1
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Chem. Commun., 2014, 50, 8771--8774 | 8773