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ChemComm
DOI: 10.1039/C3CC46675B
Table 3. Epoxidation of alkenes with the resting cells of E. coli
enantioselectivity as the best catalyst for these reactions.
Molecular docking provides with insight into the understanding
55 of the structural basis for the enantioselectivity of P450pyrTM
and P450pyr, being useful for further engineering of this type of
enzymes for other enantioselective transformations. The
epoxidations with resting cells in a resin/water biphasic system
enhanced the productivity. Further optimization of the
60 biotransformations and processes is underway.
This work was supported by GlaxoSmithKline (GSK) and
Singapore Economic Development Board (EDB) through a Green
and Sustainable Manufacturing grant (project No. 279ꢀ000ꢀ331ꢀ
592). We thank Dr. Joseph P. Adams and Dr. Radka Snajdrova
65 from GSK for helpful discussion.
(P450pyrTM) in a resin/water biphasic system a
Time
(h)
12
12
12
12
12
12
Activity
Conc.
ee
Entry Sub.
Prod.
(U/g cdw) b (mM) c
(%) d
98.5
99.1
99.1
98.7
99.5
90.2
1
2
3
4
5
6
1
3
5
7
9
(R)ꢀ2
(R)ꢀ4
(R)ꢀ6
(R)ꢀ8
(R)ꢀ10
(S)ꢀ22
18
13
15
12
5.4
11
24
18
22
18
11
17
21
a
Reactions were conducted with 70 mM substrate in 5 mL cell
suspension (10 g cdw/L) of E. coli (P450pyrTM) in 100 mM KP buffer
(pH 8.0) containing 0.21 g resin XAD16 and 1 wt% glucose at 30oC and
250 rpm for 12 h. b Determined over the first 1 h. c Determined by HPLC
analysis. d Determined by chiral HPLC analysis.
5
enzyme was reported for the epoxidation of 17. Enantiopure (S)ꢀ
orthoꢀsubstituted styrene oxides are useful intermediates as well.
10 For example, (S)ꢀ20 is useful for synthesizing a NMDA receptor
antagonist for the treatment of Parkinson’s disease.17
Notes and references
1
V. Farina, J. T. Reeves, C. H. Senanayake and J. J. Song, Chem. Rev.
2006, 106, 2734.
2
3
T. Katsuki and K. B. Sharpless, J. Am. Chem. Soc. 1980, 102, 7932.
W. Zhang, J. L. Loebach, S. R.Wilson and E. N. Jacobsen, J. Am.
Chem. Soc. 1990, 112, 2801.
The epoxidation of 1,1ꢀdisubstituted unconjugated alkenes 21
with P450pyrTM gave (S)ꢀ22 in 90.4% ee and 96% conversion
(Table 2, entry 5). (S)ꢀ22 is useful for the synthesis of (R)ꢀ
15 mevalonolactone, a precursor of steroids, terpenoids, carotenoids,
and opentanoids.18 For this epoxidation, P450pyrTM is much
better than other known enzymes such as SMO [13% ee (S), 6%
yield]5c and CPO [70% ee (R), 41% yield].7 There is also no
chemical catalysts reported for this reaction. P450pyr catalysed
20 also the epoxidation of 21, but with lower product ee. On the
other hand, P450pyr showed higher enantioselectivity than
P450pyrTM for the epoxidation of 1, 1ꢀdisubstituted conjugated
alkene 23, giving (S)ꢀ24 in 92.6% ee (Table 2, entry 13).
Finally, P450pyrTM was examined for the epoxidation of
25 cyclic alkene 25. It gave the corresponding epoxide (+)ꢀ26 in
91.0% ee with >99% conversion (Table 2, entry 7). No other
enzyme or chemical catalyst was reported for this reaction. (+)ꢀ26
can be easily transformed to (3R,4R)ꢀ3,4ꢀdihydroxyꢀpiperidine,
an useful intermediate for synthesizing xylanase inhibitor
30 isofagomine for the treatment of diabetes and gaucher disease.19
The epoxidations with resting cells of E. coli (P450pyrTM) was
then performed in a resin/water biphasic system to avoid the
substrate and product inhibition by keeping a low concentration
of substrate and product in aqueous phase (for details, see ESI).
35 The results are listed in Table 3. Better productivity was achieved
than that with single aqueous system: 22 mM vs 1.9 mM of (R)ꢀ6
(70 mM substrate 5, Fig. S29 & S31). (R)ꢀ2, 4, 6, 8, and 10 were
all obtained in >98% ee, meeting the ee requirement for chiral
intermediates in pharmaceutical manufacturing.
40 In conclusion, P450pyrTM was discovered as the first enzyme
with excellent (R)ꢀselectivity and high conversion (82ꢀ97%) for
the epoxidation of several paraꢀsubstituted styrenes. It worked
well also with the substrates containing electronꢀwithdrawing
groups. These epoxidations provides with a simple access to the
45 corresponding (R)ꢀparaꢀsubstituted styrene oxides 2, 4, 6, 8, 10,
and 12 in 98.5ꢀ99.5% ee that are useful pharmaceutical
intermediates and cannot be prepared by using other enzymatic or
chemical epoxidation systems. For the first time, a P450
monooxygenaseꢀcatalysed epoxidation with very high product ee
50 was demonstrated. P450pyrTM showed also a broad substrate
range, catalysing the epoxidation of unconjugated 1,1ꢀ
disubstituted terminal alkene 21 and cyclic alkene 25 with high
70
75
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