Bioproduction of chiral epoxyalkanes using styrene monooxygenase from rhodococcus sp. ST-10 (RhSMO)

Article abstract of DOI:10.1002/adsc.201400383

We describe the enantioselective epoxidation of straight-chain aliphatic alkenes using a biocatalytic system containing styrene monooxygenase from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749. The biocatalyzed enantiomeric epoxidation of 1-hexene to (S)-1,2-epoxyhexane (<44.6 mM) using 2-propanol as the hydrogen donor was achieved under optimized conditions. The biocatalyst had broad substrate specificity for various aliphatic alkenes, including terminal, internal, unfunctionalized, and di- and tri-substituted alkenes. Here, we demonstrate that this biocatalytic system is suitable for the efficient production of enantioenriched (S)-epoxyalkanes.

Full text of DOI:10.1002/adsc.201400383

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DOI: 10.1002/adsc.201400383
Bioproduction of Chiral Epoxyalkanes using Styrene
Monooxygenase from Rhodococcus sp. ST-10 (RhSMO)
Hiroshi Toda,a Ryouta Imae,a and Nobuya Itoha,*
a
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa,
Imizu, Toyama 939-0398, Japan
Fax : (+81)-766-56-2498; phone: (+81)-766-56-7500 ext. 560; e-mail: nbito@pu-toyama.ac.jp
Received: April 17, 2014; Revised: June 6, 2014; Published online: September 24, 2014
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400383.
Abstract: We describe the enantioselective epoxida- for various aliphatic alkenes, including terminal, in-
tion of straight-chain aliphatic alkenes using a biocat- ternal, unfunctionalized, and di- and tri-substituted
alytic system containing styrene monooxygenase alkenes. Here, we demonstrate that this biocatalytic
from Rhodococcus sp. ST-10 and alcohol dehydro- system is suitable for the efficient production of
genase from Leifsonia sp. S749. The biocatalyzed enantioenriched (S)-epoxyalkanes.
enantiomeric epoxidation of 1-hexene to (S)-1,2-ep-
oxyhexane (>44.6 mM) using 2-propanol as the hy-
drogen donor was achieved under optimized condi- Keywords: alkenes; asymmetric epoxidation; biosyn-
tions. The biocatalyst had broad substrate specificity thesis; enzyme catalysis; styrene monooxygenase
Introduction
various styrene derivatives to the corresponding (S)-
Chiral epoxides are important building blocks for the epoxides with an excellent enantiomeric excess
synthesis of chiral target compounds, such as agro- (ee),[13–15] and has been studied for the bioproduction
chemicals and pharmaceuticals, because they can of enantioenriched epoxides.[16–18]
react with a variety of nucleophiles in a highly stereo-
Recently, we have reported the isolation of an
selective fashion. Asymmetric epoxidation of prochi- SMO coding gene from Rhodococcus sp. ST-10[8,19]
ral alkenes is an extremely powerful tool for produc- and characterized recombinant SMO from Rhodococ-
ing optically pure epoxides. Examples of useful ap- cus sp. ST-10 (RhSMO) expressed in Escherichia
proaches for synthesizing enantiomerically enriched coli.[20] Furthermore, we have developed a biocatalytic
epoxides include Sharpless epoxidation and chiral process for producing enantiopure epoxides using
metal catalysis using metal-salen complexes and fruc- RhSMO and alcohol dehydrogenase (LSADH) from
tose-derived dioxiranes.[1–6] However, further im- Leifsonia sp. S749.[21,22] The biocatalyst can catalyze
provements are necessary in the production of chiral the epoxidation of various alkenes to the correspond-
epoxides. For example, direct enantioselective epoxi- ing (S)-epoxides in the presence of 2-propanol as a hy-
dation of unfunctionalized terminal alkenes is difficult drogen donor. Moreover, the biocatalyst converts
due to their low reactivity toward electrophilic oxida- straight-chain terminal alkenes (e.g., 1-hexene and 1-
tion, and controlling the prochiral faces of the simply octene) as well as arylalkenes to (S)-epoxides. There
monosubstituted double bond is also challenging. are some reports of enantioselective epoxidation of
Therefore, hydrolytic kinetic resolution of racemic ep- terminal aliphatic alkenes to primarily (R)-epoxyal-
oxides using Co
ACHTUNGTRENNUNG
duce enantioenriched terminal epoxides.[7]
Styrene monooxygenase (SMO) catalyzes the epox- monooxygenase activity,[23–25] whereas epoxidation of
idation of styrene and is involved in styrene metabo- these substrates by SMO has not been reported.
lism in microorganisms.[8–10] Several SMO coding AMO from Rhodococcus rhodochrous (formerly No-
genes have been isolated from microorganisms and cardia corallina) B-276 is a three-component enzyme
metagenomes, and the enzymatic properties of SMO system that is completely different from SMO.[26] To
have been examined.[10–12] This enzyme can convert the best of our knowledge, RhSMO is the only SMO
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that catalyzes the epoxidation of terminal aliphatic al- ited the highest StyA and StyB activities but showed
kenes to give (S)-epoxyalkanes. the lowest LSADH activity. In contrast, the StyA ac-
In this study, we have systematically evaluated the tivity of entry 4 was an order of magnitude lower than
substrate specificity and enantioselectivity of RhSMO that of the other transformants, and produced
for various aliphatic alkenes. We demonstrate that the 16.7 mM of (S)-1,2-epoxyhexane, which was half the
RhSMO-LSADH co-expression system is effective value of entry 2. The results suggested that the epox-
for asymmetric epoxidation of various straight-chain ide productivity is strongly affected by the StyB activ-
alkenes. The biocatalyst exhibited broad substrate ity rather than the StyA and LSADH activities. In
specificity for functionalized, unfunctionalized, termi- fact, (S)-1,2-epoxyhexane production level responded
nal, and di- or tri-substituted alkenes, and converted linearly to StyB activity (Table 1). StyB catalyzes the
them into (S)-epoxides with fair to excellent ee (64– reduction of FAD to FADH2, and supplies FADH2 as
99%).
an electron donor for the epoxidation reaction of
StyA. We found that SMO activity increased with in-
creasing StyB activity and FAD concentration in the
reaction mixture (data not shown). Furthermore, the
amounts of 2-propanol consumed and (S)-1,2-epoxy-
hexane produced did not agree. Presumably, epoxide
production is limited by the supply of FADH2 because
Results and Discussion
Biocatalyst Development
RhSMO requires NADH as a hydrogen donor to con- the majority of FADH2 generates H2O2 through its
vert alkene substrates to the corresponding epoxides. auto-oxidative reaction with oxygen.[27–29] Therefore,
Thus, we employed LSADH, which reduces NAD+ to the biocatalyst in entry 2 was used for further biocon-
NADH using 2-propanol as a hydrogen donor, to re- version experiments.
cycle NADH (Scheme 1). We constructed several bio-
catalysts with different RhSMO- and LSADH-ex-
pressing plasmids, and evaluated their productivity for Effect of Organic Solvent on Epoxide Productivity
converting
1-hexene
to
(S)-1,2-epoxyhexane
(Table 1). All the biocatalysts catalyzed the epoxida- The bioproduction of organic compounds often suf-
tion of 1-hexene, and the E. coli transformant cells fers from low productivity, because of the low solubil-
that carried the pET-ST10 styAB and pRSFDuet- ity of substrates and products in the aqueous phase
lsadh plasmids (entry 2) showed the highest produc- and their toxicity for the biocatalysts. To overcome
tion of (S)-1,2-epoxyhexane. This transformant exhib- these problems, biphasic reaction systems using water
and an organic solvent are often employed for organic
synthesis with biocatalysts. Biphasic reaction systems
are essential for epoxide production by biocatalysis
because epoxides often readily decompose in aqueous
solution. We have reported that the productivity of
(S)-styrene oxide with RhSMO in biphasic reaction
systems was strictly dependent on the organic solvents
used. When acetate esters (octyl, hexyl, and pentyl
acetate) were used as organic solvents, the RhSMO
biocatalyst exhibited good productivity of (S)-styrene
Scheme 1. Epoxidation of aliphatic alkenes by the RhSMO-
LSADH co-expressing system using 2-propanol as a hydro-
gen donor.
oxide.[22] Figure 1 shows that the productivity was
strongly affected by the organic solvent in the bipha-
Table 1. Comparison of (S)-1,2-epoxyhexane production by biocatalyst systems.
Entry Plasmid
StyA activity
[mU/mg][a]
StyB activity
[mU/mg][a]
LSADH activity
[U/mg][a]
(S)-1,2-Epoxyhexane
[mM][b]
1
2
3
pETDuet-ST10styAB-lsadh
pET-ST10styAB, pRSFDuet-lsadh 45.4
pET-ST10styAB, pRSFDuet-
ST10styAB-lsadh
15.3
526
1170
980
2.73
0.78
1.02
19.8
30.9
25.1
22.9
4
pRSFDuet-ST10styAB-lsadh
0.9
222
1.39
16.7
[a]
[b]
Enzyme activities are reported in units (U) per milligram of protein in cell-free extract.
The amount of product in the organic phase [mM] (hexyl acetate plus extract with ethyl acetate) was used to calculate
the product concentration in 1 mL of the organic phase.
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Bioproduction of Chiral Epoxyalkanes using Styrene Monooxygenase
various alkenes as a substrate (Table 2). The E. coli
biocatalyst possessing RhSMO and LSADH had
broad substrate specificity and produced epoxides
with fair to excellent ee (64–99%). When simple ter-
minal alkene substrates 1–5 were used, the biocatalyst
exhibited the highest production level and enantiose-
lectivity for 1-hexene 3, followed by 1-pentene 2, 1-
heptene 4, propylene 1, and 1-octene 5. Likewise, 1,5-
hexadiene 6 was a better substrate than 1,7-octadiene
7, and only traces of product were observed for
dienes consisting of chains longer than C9 (data not
shown). These results indicate that the reactivity and
enantioselectivity of RhSMO may be affected by the
size of the substrate, which could limit the incorpora-
tion of the substrate into the enzymeꢁs active site. In
the case of 1,5-hexadiene 6, (2S,5S)-diepoxyhexane 6b
was the major product, the minor product was (S)-
1,2-epoxy-5-hexene 6a, and unreacted 6 was also de-
tected. We speculated that 6a is more soluble in the
water phase than 6, and thus the biocatalyst may be
more accessible to 6a and produce diepoxy compound
6b. Consequently, the second epoxidation of 6a may
proceed faster than the first epoxidation of 6.
Figure 1. Effect of organic solvent on the production of (S)-
1,2-epoxyhexane and (S)-styrene oxide in a biphasic reac-
tion system. Each black bar [(S)-1,2-epoxyhexane] and
white bar [(S)-styrene oxide] indicates the product concen-
tration in the organic phase. The data for styrene are repro-
duced from the literature.[22]
Disubstituted alkenes 8–11 were generally good
substrates for RhSMO, with the exception of trans-3-
heptene 10. The relatively low yields from non-substi-
tuted linear alkenes were attributed to the lower reac-
tivity of these substrates compared with substituted
linear or arylalkenes for RhSMO, in other words, the
lower reactivity of the active peroxo-/hydroxyoxofla-
sic system. In this study, we also investigated the vin intermediates in electrophilic epoxidation by
effect of the organic solvent on the production of (S)- SMO.[27,28] However, RhSMO exhibited the lowest
epoxyhexane for comparison with (S)-styrene oxide. enantioselectivity for cis-2-heptene (64.1% ee) of the
The highest production level of (S)-1,2-epoxyhexane substrates tested. A similar trend was observed for
was achieved with pentyl acetate and hexyl acetate, styrene derivatives; the enzyme showed slightly lower
and was similar to the production of styrene. When reactivity and enantioselectivity for cis-b-methylstyr-
more polar methyl, ethyl, and propyl acetate solvents ene than for a-methyl-/trans-b-methylstyrene.[22] Gen-
were used, a negligible amount of the product was ob- erally, the asymmetric epoxidation of unfunctional-
tained. However, octyl acetate, which showed the ized and unconjugated disubstituted alkenes by chem-
highest production level for styrene oxide, produced ical synthesis is difficult because of their electron defi-
less (S)-1,2-epoxyhexane than did pentyl and hexyl ciency and lack of stereochemical features. Therefore,
acetate. Bis(ethylhexyl) phthalate (DEHP) was a suit- there are few reports of chemical asymmetric epoxi-
able solvent for (S)-1,2-epoxyhexane, whereas it re- dation of non-activated cis-alkenes, such as cis-2-hep-
sulted in low production of (S)-styrene oxide. These tene 9.[32] Katsuki and co-workers assumed that the
results suggest that the suitable organic solvent for enantioselectivity of the epoxidation of cis-alkenes
the efficient bioproduction of epoxides depends on depends on the stereochemistry and p-electron inter-
the properties and behavior of the substrate or prod- action between the catalyst and substrates.[33] Like-
uct in the reaction system, including its solubility in wise, Page and co-workers reported that an iminium
the biphasic system in the case of aryl or aliphatic salt catalyst catalyzed the epoxidation of non-aryl cis-
substrates; and on substrate or product inhibition of alkenes, although its enantioselectivity was low.[34]
the biocatalyst.
These studies strongly suggest that chemical reactions
with synthetic catalysts are not suitable for the asym-
metric epoxidation of unfunctionalized non-aryl cis-
alkenes. On the other hand, the enantiomeric epoxi-
dation of non-activated cis-alkenes using biocatalysts
Bioproduction of Various Chiral Epoxides
To investigate the substrate specificity of the biocata- has been reported.[35–37] Thus, fine tuning of RhSMOꢁs
lyst, bioconversion experiments were performed using active site through protein engineering may increase
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Table 2. Bioconversion of aliphatic alkenes and sulfide by SMO biocatalyst.
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Bioproduction of Chiral Epoxyalkanes using Styrene Monooxygenase
the productivity and stereoselectivity of the epoxida- dant and 2-propanol as a hydrogen donor. The enzy-
tion of non-aryl cis-alkenes. matic activities of RhSMO and LSADH and the or-
1,1-Di- and tri-substituted alkenes 11 and 12 were ganic solvent in the biphasic reaction system were op-
also converted to the corresponding epoxides by timized for the production of epoxyalkanes. The bio-
RhSMO with good enantioselectivity. In addition to catalytic system catalyzed the epoxidation of various
unfunctionalized alkenes, RhSMO converted various straight-chain aliphatic alkenes (less than C9), includ-
functionalized alkenes (13–19) with good enantiose- ing those substituted with functional groups, with
lectivity. 5-Hexen-1-ol 15 exhibited the highest reac- good enantioselectivity. Further improvement of the
tivity and enantioselectivity for RhSMO. In contrast, process would make it suitable for producing various
halogenated alkenes 13 and 14 showed slightly lower aliphatic (S)-epoxyalkanes.
reactivities and enantioselectivities. Furthermore,
RhSMO showed negligible reactions with 5-hexenyl
acetate 17 and butyl allyl ether 18, presumably be- Experimental Section
cause these compounds did not fit into the active site
of RhSMO. From these results, we surmised that ali- Chemicals
phatic alkenes with electronegative, polar, and large
substituents affect the reactivity and enantioselectivity
of RhSMO. Interestingly, RhSMO also catalyzed the
Propylene was purchased from Sumitomo Seika. rac-Propyl-
ene oxide was purchased from Nacalai. 1,2-Epoxyhexane
was purchased from Merck. 1-Pentene, rac-1,2-epoxypen-
enantioselective sulfoxidation of sulfide 20 to (S)-sulf-
oxide, although the ee of the product was lower than
that of the epoxyalkanes.
tane, 1,5-hexadiene, 1,7-octadiene, 2-methyl-1-hexene, cis-2-
heptene, trans-3-heptene, 2-methyl-2-hexene, 6-bromo-1-
hexene, 5-hexenyl acetate, allyl ether, meta-chloroperbenzo-
ic acid, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA),
chlorotrimethylsilane, and Chirabite-AR were purchased
from Tokyo Chemical Industry. (R)-1,2-Epoxyhexane, (S)-
1,2-epoxyoctane, (S,S)-(À)-N,N’-bis(3,5-di-tert-butylsalicyli-
dene)-1,2-cyclohexanediaminocobalt(II), and (R,R)-(À)-
N,N’-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediami-
nocobalt(II) were purchased from Sigma. 1-Hexene, 1-hep-
tene, 1-octene, 1,2-epoxyoctane, rac-1,2-epoxy-5-hexene, rac-
1,2,5,6-diepoxyhexane, rac-1,2-epoxy-7-heptene, rac-1,2,7,8-
diepoxyoctane, trans-2-heptene, 6-chloro-1-hexene, 5-hexe-
nenitrile, 5-hexen-1-ol, amyl methyl sulfide, allyl butyl ether,
n-butyl glycidyl ether, allyl glycidyl ether, and all other
chemicals were purchased from Wako Pure Chemical Indus-
tries.
Compared with previously reported biocatalysts,
the RhSMO system shows remarkable versatility in
converting straight-chain alkenes to enantioenriched
(S)-epoxyalkanes with high yields. To our knowledge,
there are no reports of asymmetric epoxidation of ali-
phatic alkenes by SMO to the corresponding (S)-ep-
oxides. Therefore, RhSMO shows superior catalytic
properties to previously reported SMOs. We have
demonstrated that the biocatalytic RhSMO system is
a powerful tool for producing various enantiopure
(S)-epoxyalkanes. Moreover, our biocatalytic system
is environmentally friendly because it requires molec-
ular oxygen as an oxidant and 2-propanol as a hydro-
gen donor instead of metals and oxidizing agents,
such as peroxides, which are generally used in the
chemical synthesis of epoxides.
HPLC Analysis
To determine the absolute configuration of the epoxidation
products of 1-pentene and 1,5-hexadiene, the purified prod-
ucts were ring-opened with 2-naphthalenethiol by using the
method reported by Kulkarni et al.[38] Chiral HPLC analysis
was performed on an LC-10 HPLC system (Shimadzu)
equipped with a Chiracel OD-H column (Diacel). The
mobile phase was hexane/2-propanol with a flow rate of
Conclusions
We have successfully developed a biocatalytic system
for synthesizing enantioenriched (S)-epoxides from
aliphatic alkenes using molecular oxygen as an oxi- 1 mLminÀ1 (see the Supporting Information). The column
[a]
Bioconversion was performed in a biphasic reaction system with hexyl acetate. The
amount of product in the organic phase [mM] (hexyl acetate plus extract with ethyl ace-
tate) was used to calculate the product concentration in 1 mL of the organic phase.
Conversion [%]=[mol (product in organic phase)/mol (substrate)]ꢂ100.
Yield [%]=[mol (purified product)/mol (substrate)]ꢂ100.
Absolute configurations and ee were estimated by HPLC analysis of the 2-naphthyl sul-
[b]
[c]
[d]
fide derivatives of product. ND: not determined.
[e]
Products were reduced by LiAlH4 and absolute configurations were determined by the
comparison with the corresponding 2- or 3-heptanol.[30]
[f]
Absolute configurations were determined by comparing the optical rotations with re-
ported values.
[g]
Enantiomeric excess values were estimated from NMR spectra in the presence of the
chiral shift reagent Chirabite-AR.[31]
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Hiroshi Toda et al.
was kept at 408C and the products were monitored at
254 nm.
cording to our previously reported method.[22] Propylene
was dissolved in the organic solvent by blowing, and used as
a substrate. After the reaction ended, the organic phase was
isolated by centrifugation and the remaining product in the
water phase was extracted with ethyl acetate. The organic
phase and extracted product were collected and dried with
anhydrous Na2SO4. The amounts of product and substrate
were measured by GC or HPLC.
GC-MS Analysis
GC-MS analysis was performed on a QP-5000 mass spec-
trometer (Shimadzu) equipped with a GC-17 A GC system
(Shimadzu). The sample was injected into a TC-70 capillary
column (0.25 mmꢂ60 m, 0.25 mm film, GL Science). The
carrier gas was He at a flow rate of 1.7 mLminÀ1 (14.5 psi),
the split ratio was 26, and the linear velocity was 47.4 cmsÀ1.
Analytical Data for Purified Products
NMR and Optical Rotation Analysis
After the biotransformation, the recovered product was pu-
rified by silica gel chromatography (0.9ꢂ10 cm), and the re-
spective eluates concentrated under vacuum. The products
1H NMR spectra were recorded on an AV400 spectrometer
(Bruker). All signals are expressed as parts per million
(ppm) with TMS or residual undeuterated solvent as refer-
ences. Optical rotations were measured with a P-1030 polar-
imeter (JASCO).
1
were analyzed by GC-EI-MS, H NMR, and polarimetry.
(S)-1,2-Epoxypentane (2a): Colorless liquid, (77.8% ee);
[a]D24:5: À9.6 (c 1.00, CHCl3); 1H NMR (CDCl3, 400 MHz):
d=2.94–2.89 (m, 1H), 2.75 (dd, J=4.0, 5.0 Hz, 1H), 2.47
(dd, J=2.8, 5.0 Hz, 1H), 1.57–1.42 (m, 4H), 0.97 (t, J=
7.2 Hz, 3H); GC-EI-MS: m/z (%)=85 (2), 71 (49), 56 (16),
41 (100), 29 (53).
(S)-1,2-Epoxyhexane (3a): Colorless liquid, (94.8% ee);
[a]D24:5: À9.1 (c 1.00, CHCl3); 1H NMR (CDCl3, 400 MHz):
d=2.91 (ddt, J=2.8, 3.4, 5.8 Hz, 1H), 2.75 (dd, J=4.0,
5.0 Hz, 1H), 2.47 (dd, J=2.8, 5.0 Hz, 1H), 1.57–1.33 (m,
6H), 0.92 (t, J=7.2 Hz, 3H); GC-EI-MS m/z (%)=99 (1),
85 (5), 71 (80), 55 (48), 42 (100), 29 (83).
Synthesis of Authentic Compounds
All racemic epoxides used for product identification were
synthesized using meta-chloroperbenzoic acid (m-CPBA)
through the method reported by Sharma et al.[39] Chiral ep-
oxides used for identifying the absolute configuration of the
bioproduced epoxides were prepared as previously report-
ed.[6,7] The authentic epoxides were purified by silica gel
chromatography and used for the following analyses.
(S)-1,2-Epoxyheptane (4a): Colorless liquid, (84.0% ee);
[a]D24:8: À8.3 (c 1.01, CHCl3); 1H NMR (CDCl3, 400 MHz):
d=2.93–2.89 (m, 1H), 2.75 (dd, J=4.0, 5.0 Hz, 1H), 2.46
(dd, J=2.8, 5.0 Hz, 1H), 1.56–1.28 (m, 8H), 0.90 (m, 3H);
(S)-1,2-Epoxyoctane (5a): Colorless liquid, (86.5% ee);
[a]D23:7: À8.9 (c 1.00, CHCl3); 1H NMR (CDCl3, 400 MHz):
d=2.93–2.89 (m, 1H), 2.75 (dd, J=4.0, 5.0 Hz, 1H), 2.47
(dd, J=2.8, 5.0 Hz, 1H), 1.56–1.27 (m, 10H), 0.89 (t, J=
6.9 Hz, 3H); GC-EI-MS m/z (%)=127 (1), 113 (1), 99 (3),
71 (58), 55 (52), 41 (100), 29 (74).
Preparation of the Biocatalyst
The genes coding RhSMO (StyA and StyB) and LSADH
were amplified by PCR and treated with restriction endonu-
cleases. Each DNA fragment was purified by agarose gel
electrophoresis and cloned into its expression vector. The
resulting plasmids were introduced into E. coli BL21ACTHUNTGRNEUNG(DE3),
which contained the chaperone carrying plasmid pG-KJE8
(TaKaRa Bio Inc.), and the E. coli biocatalyst was prepared
according to our previously reported method.[22]
(2S)-1,2-Epoxy-5-hexene (6a): Colorless liquid, (98.9%
ee); [a]2D3:9
:
À6.5 (c 1.00, CHCl3); 1H NMR (CDCl3,
Measurement of Enzyme Activities
400 MHz): d=5.85 (ddt, J=6.7, 10.3, 17.0 Hz, 1H), 5.07 (dq,
J=1.7, 17.1 Hz, 1H), 5.00 (ddt, J=1.3, 1.8, 10.2 Hz, 1H),
2.95 (m, 1H), 2.76 (dd, J=4.1, 4.9 Hz, 1H), 2.50 (dd, J=2.7,
5.0 Hz, 1H), 2.23 (m, 2H), 1.65 (m, 2H); GC-EI-MS: m/z
(%)=97 (2), 79 (4), 67 (36), 54 (48), 39 (83), 29 (100).
Recombinant E. coli cells were harvested by centrifugation
at 33,800 g for 10 min. Cell pellets were resuspended in
50 mM potassium phosphate buffer (KPB, pH 7.5) and dis-
rupted by sonication for 10 min on ice. The cell lysates were
centrifuged at 33,800 g for 10 min, and cell-free extracts
were recovered. Cell-free extracts were used for measuring
enzyme activities. StyA and StyB activities were measured
according to our previously reported methods.[20] One unit
(U) of StyA or StyB activities were defined as the amount
of the enzyme that converted 1 mmol of styrene or NADH
to (S)-styrene oxide or NAD+ per minute at 308C.
AHCTUNGTERG(NNNU 2S,5S)-1,2,5,6-Diepoxyhexane (6b): Colorless liquid,
1
(97.6% ee); [a]2D4:3: À26.1 (c 1.00, CHCl3); H NMR (CDCl3,
400 MHz): d=3.00–2.97 (m, 2H), 2.78 (dd, J=4.0, 4.9 Hz,
2H), 2.50 (dd, J=2.7, 5.0 Hz, 2H), 1.85–1.59 (m, 4H); GC-
EI-MS: m/z (%)=114 (1), 95 (1), 83 (13), 69 (12), 55 (96),
41(90), 27 (100).
(2S)-1,2-Epoxy-7-octene (7a): Colorless liquid, (91.9%
ee); [a]2D4:5
:
À10.4 (c 1.00, CHCl3); 1H NMR (CDCl3,
400 MHz): d=5.81 (ddt, J=6.7, 10.3, 17.0 Hz, 1H), 5.01
(ddd, J=1.6, 1.9, 17.1 Hz, 1H), 4.95 (m, 1H), 2.91 (m, 1H),
2.75 (dd, J=4.0, 5.0 Hz, 1H), 2.47 (dd, J=2.8, 5.0 Hz, 1H),
2.08 (m, 2H), 1.49 (m, 6H); GC-EI-MS: m/z (%)=107 (1),
93 (11), 79 (15), 67 (54), 54 (91), 41 (100), 29 (93).
Biotransformation Experiment
The biocatalysts were resuspended (20% wet w/v) in 50 mM
KPB (1 mL, pH 7.5) containing 1 mM NAD+ and 0.2 mM
FAD. The cell suspension (1 mL), a 200 mM organic solvent
solution of the substrate (1 mL), and 2-propanol (0.12 mL)
were transferred to a 50-mL tube. The tube was filled with
pure oxygen gas and tightly sealed. The reaction was per-
formed with vertical shaking (300 rpm) at 258C for 24 h, ac-
AHCTUNGTERG(NNUN 2S,3S)-trans-2,3-Epoxyheptane (8a): Colorless liquid, (>
1
99.9% ee); [a]2D4:5: À41.5 (c 1.00, CHCl3); H NMR (CDCl3,
400 MHz): d=2.74 (dq, J=2.3, 5.2 Hz, 1H), 2.63 (dt, J=2.3,
5.6 Hz, 1H), 1.55–1.50 (m, 2H), 1.48–1.32 (m, 4H), 1.29 (d,
3448
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Bioproduction of Chiral Epoxyalkanes using Styrene Monooxygenase
J=5.2 Hz, 3H), 0.91 (t, J=7.1 Hz, 3H); GC-EI-MS: m/z
(%)=114 (1), 99 (7), 85 (34), 71 (20), 55 (58), 43 (100).
7.4 Hz, 3H); GC-EI-MS: m/z (%)=99 (1), 87 (1), 73 (5), 57
(46), 41 (37), 29 (100).
(S)-Allyl glycidyl ether (19a): Colorless liquid, (91.0%
ACHTUNGTRENNUNG(2R,3S)- cis-2,3-Epoxyheptane (9a): Colorless liquid,
ee); [a]2D4:7
:
À10.5 (c 1.00, EtOH), 1H NMR (CDCl3,
1
(51.7% ee); [a]2D4:5: 0.41 (c 1.00, CHCl3); H NMR (CDCl3,
400 MHz): d=3.07–0.02 (m, 1H), 2.92–2.88 (m, 1H), 1.57–
1.36 (m, 6H), 1.26 (d, J=5.6 Hz, 3H), 0.93 (t, J=6.9 Hz,
3H); GC-EI-MS: m/z (%)=114 (1), 99 (6), 85 (33), 71 (20),
55 (59), 43 (100).
400 MHz): d=5.92 (ddt, J=5.7, 10.4, 17.2 Hz, 1H), 5.30 (dq,
J=1.6, 17.2 Hz, 1H), 5.20 (ddt, J=1.3, 1.6, 10.4 Hz, 1H),
4.05 (dddt, J=1.4, 5.8, 12.8, 20.1 Hz, 2H), 3.73 (dd, J=3.1,
11.4 Hz, 1H), 3.41 (dd, J=5.8, 11.4 Hz, 1H), 3.17 (dquin,
J=2.9, 4.2 Hz, 1H), 2.81 (dd, J=4.2, 5.0 Hz, 1H), 2.63 (dd,
J=2.7, 5.0 Hz, 1H); GC-EI-MS: m/z (%)=83 (1), 71 (2), 57
(61), 41 (93), 29 (100).
ACHTUNGTRENNUNG(3S,4S)-trans-3,4-Epoxyheptane (10a): Colorless liquid,
(37.5% ee); [a]D24:3
:
À24.49 (c 1.00, CHCl3); 1H NMR
(CDCl3, 400 MHz): d=2.70–2.63 (m, 2H), 1.58–1.40 (m,
6H), 0.98 (t, J=7.5 Hz, 3H), 0.96 (t, J=7.1 Hz, 3H); GC-
EI-MS: m/z (%)=114 (1), 99 (2), 85 (8), 72 (27), 57 (58), 41
(100).
(S)-Amyl methyl sulfoxide (20a): Yellow liquid, (21.0%
ee); [a]2D4:9
:
+22.6 (c 1.00, EtOH); 1H NMR (CDCl3,
400 MHz): d=2.79–2.62 (m, 2H), 2.57 (s, 3H), 1.81–1.73 (m,
2H), 1.53–1.33 (m, 4H), 0.93 (t, J=7.1 Hz, 3H); GC-EI-
MS: m/z (%)=117 (10), 78 (1), 64 (20), 55 (10), 43 (100), 29
(35).
(S)-2-Methyl-1,2-epoxyhexane (11a): Colorless liquid,
1
(90.2% ee); [a]2D4:3: 7.88 (c 1.00, CHCl3); H NMR (CDCl3,
400 MHz): d=2.61 (dd, J=0.3, 4.8 Hz, 1H), 2.57 (dd, J=
0.7, 4.9 Hz, 1H), 1.63–1.27 (m, 6H), 1.31 (s, 3H), 0.91 (t, J=
7.1 Hz, 3H); GC-EI-MS: m/z (%)=114 (1), 99 (5), 85 (75),
72 (36), 55 (100), 41 (97).
2,2-Dimethyl-3-propyloxirane (12a): Colorless liquid,
(90.4% ee); 1H NMR (CDCl3, 400 MHz): d=2.73 (t, J=
5.9 Hz, 1H), 1.57–1.41 (m, 4H), 1.32 (s, 3H), 1.27 (s, 3H),
0.97 (t, J=7.2 Hz, 3H); GC-EI-MS m/z (%)=99 (4), 85
(32), 72 (9), 59 (76), 41 (100), 27 (59).
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1
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3450
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Adv. Synth. Catal. 2014, 356, 3443 – 3450