Selective enzymatic epoxidation of dienes: Generation of functional enantiomerically enriched diene monoepoxy monomers

Article abstract of DOI:10.1016/S0040-4039(02)01519-8

Enantiomerically enriched diene monoepoxides were selectively synthesized using oxidases from Pseudomonas sp. and chloroperoxidase from Caldariomyces fumago. These monoepoxides are useful monomers for generating functional chiral polymeric materials.

Full text of DOI:10.1016/S0040-4039(02)01519-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6763–6766
Selective enzymatic epoxidation of dienes: generation of
functional enantiomerically enriched diene monoepoxy monomers
Shanghui Hu,a Pankaj Gupta,b Ashok K. Prasad,b Richard A. Grossa,* and Virinder S. Parmara,b,
*
aNSF Center for Biocatalysis and Bioprocessing of Macromolecules, Department of Chemistry, Polytechnic University,
06 Metrotech Center, Brooklyn, NY 11201, USA
bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
Received 20 June 2002; revised 12 July 2002; accepted 19 July 2002
Abstract—Enantiomerically enriched diene monoepoxides were selectively synthesized using oxidases from Pseudomonas sp. and
chloroperoxidase from Caldariomyces fumago. These monoepoxides are useful monomers for generating functional chiral
polymeric materials. © 2002 Elsevier Science Ltd. All rights reserved.
Enantiomerically enriched synthetic polymers are of
special interest due to their potential applications as
chromatographic supports for the separation of enan-
tiomers.1 Enantiomerically enriched polyacrylamide
and polymethacrylate gels have been used to resolve
racemic drugs on a preparative scale.1,2 Selective poly-
merization of enantiomerically enriched monomers hav-
ing two polymerizable groups may afford structurally
well-defined functional polymeric materials. Enan-
tiomerically enriched diene monoepoxides, such as
divinylbenzene monoepoxide can be promising
monomers in the synthesis of various optically active
polymers having structurally defined groups in either
the side chain or the main chain. However, there are
few reports on the preparation of enantiomerically
enriched/pure diene monoepoxides from the diene pre-
cursors. Biocatalysis seems to be a straightforward
approach for the generation of this type of optically
active functional monomer.
Herein, we have investigated the selective epoxidation
of aryl dienes catalyzed by oxidases from Pseudomonas
putida and the epoxidation of unsaturated acrylate
derivatives catalyzed by CPO.
Commercially available divinylbenzene 1, a mixture of
isomers5 was first used as a model substrate for the
investigation of selective epoxidation catalyzed by oxi-
dases from P. putida. The oxidases were produced and
directly used as an n-octane-based two-phase fermenta-
tion system, which allowed us to reduce substrate and
product toxicity to the cells and accumulate the prod-
ucts in the organic phase. In a typical enzymatic-epoxi-
dation reaction, divinylbenzene (1, 0.25 g) in n-octane
(20 mL) was added into 1 L of a culture medium of P.
putida6 and the progress of the reaction was monitored
by TLC and GC analysis. After 72 h of incubation, the
culture was extracted with ether, the organic layer was
separated, dried and evaporated to afford the crude
product, which was purified by flash-column chro-
matography using a pentane–ether mixture to afford
the pure epoxy products 2 and 3 in about 30% yield
(based on the individual isomers in the mixture). For
para- and meta-1, the oxidases from P. putida demon-
strated two types of selectivities: (i) the reaction
stopped at the monoepoxide stage, hardly any diepox-
ide and other products were detected or isolated from
the reaction medium as shown in Scheme 1; (ii) the
epoxidation reactions showed excellent enantioselectivi-
ties (ee 95% for para-2, ee 94% for meta-3).7 Very
surprisingly, ortho-1 is not a substrate for the oxidases
from P. putida. These epoxidation reactions were also
carried out with pure para- and meta-divinylbenzenes
The oxidase systems from the bacteria Pseudomonas
sp., such as xylene oxygenases catalyze the epoxidation
of styrene to styrene oxide with high enantioselectivity.3
Chloroperoxidase (CPO) is a versatile and efficient
biocatalyst that catalyzes a variety of reactions, particu-
larly asymmetric epoxidation and hydroxylation.4
Keywords: enzymatic epoxidation; Pseudomonas putida; Caldari-
omyces fumago; chloroperoxidase; diene monoepoxide; enantioselec-
tive.
* Corresponding authors. Tel.: +91-11-7666 555; fax: +91-11-7667
206; e-mail: virparmar@yahoo.co.in
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01519-8

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S. Hu et al. / Tetrahedron Letters 43 (2002) 6763–6766
as substrates.8 In these cases, the oxidases gave very
similar enantioselectivities for the para- and meta-
divinylbenzene monoepoxides (ee 95% for para-isomer,
ee 94% for meta-isomer), and the mono-epoxides were
also detected as the unique products by TLC and GC
analysis (38% and 25% yields for para- and meta-iso-
mers, respectively). The physical and spectral data of
divinylbenzene monoepoxides 2 and 3 were found to be
identical with the data reported in the literature.9 In
contrast to para- and meta-divinylbenzenes, para- and
meta-allylstyrenes8 are not substrates for the oxidases
from P. putida or P. oleovorans, which suggested that
these enzymes showed very high substrate specificities.
In a typical CPO6 catalyzed epoxidation reaction,
methacrylate 4,10 acrylate 610 or dimethylhexadiene 810
(0.3 mmol, in each case) was stirred with t-BuOOH
(0.60 mmol) or H2O2 (3%, 1.2 equiv.) in 2 ml of 10 mM
sodium citrate buffer (pH 5.5) and 200 mL acetone.
CPO (2.5 mg, 0.06 mmol) was added and the reaction
mixture was stirred at room temperature for 2 h, after
which Na2SO3 was added and the mixture was
extracted twice with ether. The combined organic por-
tions were dried over MgSO4, the ether removed and
the crude product was purified by flash-column chro-
matography using dichloromethane as eluting solvent
to afford the pure epoxides 5, 7 and 9 in 73–87% yields
Scheme 1. Selective epoxidation of aryl dienes by oxidases from P. putida.
Scheme 2. Selective epoxidation of dienes by CPO from C. fumago.

S. Hu et al. / Tetrahedron Letters 43 (2002) 6763–6766
6765
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types of selectivity: (i) only the isolated double bond was
11
epoxidized to produce monoepoxide 5 in 73% yield,
and the conjugated a,b-unsaturated bond of the
methacrylic acid moiety was untouched as shown in
Scheme 2, (ii) the enantioselectivity was high (ee 91%).
It suggested that conjugated terminal olefins might have
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derivative in which the active heme site is N-alkylated.12
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711 (Scheme 2) in high yield and excellent enantioselec-
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symmetrical dienes. This indeed was the case; when
dimethylhexadiene 8 was used as a model substrate,
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epoxide 914 as a unique product (Scheme 2). The yield
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conversion, 81% ee). No diepoxide and other oxidized
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literature.11,14
5. Divinylbenzene, a mixture of meta-, para- and ortho-iso-
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Chemical Co., USA.
6. P. putida ATCC and P. oleovorans ATCC 29347 were
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220) in the following media, respectively. Media A:
(NH4)2HPO4, 3.0 g; KH2PO4, 1.2 g; NaCl, 5.0 g;
MgSO4·7H2O, 0.2 g; yeast extract 0.5 g; sodium benzoate
(filter-sterilized), 3.0 g; distilled water, 1.0 L. Autoclaving
was conducted at 120°C for 15 min with all ingredients
except sodium benzoate. Media B: (NH4)2HPO4, 10.0 g;
K2HPO4, 5.0 g; Na2SO4, 0.5 g; distilled water 1 L. The
mineral medium was autoclaved at 120°C for 15 min and
at the time of culture, filter-sterilized octane (2.5% v/v)
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fumago was obtained as a 20 mg/ml phosphate buffer (0.1
M, pH 4.5) from Chirazyme (Urbana, IL).
7. The enantiomeric excess (ee) values of chiral epoxides
were determined by HPLC analysis on a Chiralcel OJ
column (25×0.46 cm) using hexane/iso-propanol as eluent
with a flow rate of 1.0 mL/min or by GC analysis on an
ALPHA DEX capillary column (30 m×0.32 mm).
8. para- and meta-Divinylbenzenes, and para- and meta-
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See: Frisch, K. C. J. Polym. Sci. 1959, 41, 359.
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7.36 (dt, J=7.5 and 3.0 Hz, 2H, para), 7.32 (m, 4H,
meta), 7.18 (dt, J=7.5 and 3.0 Hz, 2H, para), 6.71 (dd,
J=18.0 and 12.0 Hz, 2H, meta and para), 5.76 (dd,
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omyces fumago, chemical epoxidation of divinylbenzene
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stereoselectivity. These results further demonstrate that
the enzymatic epoxidation of dienes is a more powerful
method for the production of optically active diene
monoepoxides.
Enantiomerically enriched epoxy aryl monomers 2 and
3 can be chemically polymerized either through the vinyl
group or oxide functional groups, which may generate
functional polymers containing the chiral groups on the
side chains and main chains, respectively. Well-defined
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polymerization of compounds 5 and 7, respectively.15
In conclusion, this work demonstrates that enzymatic
epoxidations are efficient methods for the preparation of
chiral diene monoepoxides from dienes with high enan-
tioselectivity. As the oxidases from P. putida and CPO
from C. fumago are readily available, their use in the
synthesis of other optically active epoxide monomers,
which are difficult to obtain by chemical methods might
be a promising approach. Furthermore, directed evolu-
tion of xylene oxidase from P. putida and CPO from C.
fumago would provide an alternative to improve enzy-
matic activity and broaden the substrate specificity

6766
S. Hu et al. / Tetrahedron Letters 43 (2002) 6763–6766
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