Synthesis of chiral epichlorohydrin by chloroperoxidase-catalyzed epoxidation of 3-chloropropene in the presence of an ionic liquid as co-solvent

Article abstract of DOI:10.1016/j.catcom.2010.02.003

Asymmetric epoxidation of 3-chloropropene can be catalyzed by chloroperoxidase (CPO) from Caldariomyces fumago to prepare (R)-epichlorohydrin (ECH) in homogenous phosphate buffer/ionic liquid mixtures using t-butyl hydroperoxide (TBHP) as O₂ donor. Reaction conditions were optimized by the investigation of the choice of oxidants, the presence of ionic liquids (ILs), pH effect and CPO consumption. The best ECH yield reached 88.8% within a duration of 60 min with high enantiomeric excesses (e.e. 97.1%) at pH 5.5 and room temperature, using 1-ethyl-3-methylimidazolium [EMIM][Br] as co-solvent. The ILs with shorter carbon chain was more efficient on chiral ECH preparation.

Full text of DOI:10.1016/j.catcom.2010.02.003

Catalysis Communications 11 (2010) 727–731
Contents lists available at ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Synthesis of chiral epichlorohydrin by chloroperoxidase-catalyzed epoxidation
of 3-chloropropene in the presence of an ionic liquid as co-solvent
Jinyue Wu a, Chen Liu a, Yucheng Jiang a,b, , Mancheng Hu a,b, Shuni Li a,b, Quanguo Zhai a,b
*
a School of Chemistry and Materials Science, Shaanxi Normal University, Xi’an 710062, PR China
b Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi’an 710062, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2009
Received in revised form 4 February 2010
Accepted 4 February 2010
Available online 11 February 2010
Asymmetric epoxidation of 3-chloropropene can be catalyzed by chloroperoxidase (CPO) from
Caldariomyces fumago to prepare (R)-epichlorohydrin (ECH) in homogenous phosphate buffer/ionic liquid
mixtures using t-butyl hydroperoxide (TBHP) as O2 donor.
Reaction conditions were optimized by the investigation of the choice of oxidants, the presence of ionic
liquids (ILs), pH effect and CPO consumption. The best ECH yield reached 88.8% within a duration of
60 min with high enantiomeric excesses (e.e. 97.1%) at pH 5.5 and room temperature, using 1-ethyl-3-
methylimidazolium [EMIM][Br] as co-solvent. The ILs with shorter carbon chain was more efficient on
chiral ECH preparation.
Keywords:
Chloroperoxidase
Asymmetric epoxidation
Chiral epichlorohydrin
Ionic liquids
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
However, the use of CPO was limited due to its poor activity and
stability in solvents with low water content. Recent years, ionic liq-
The synthesis of small chiral fragments attracted considerable
interest due to their potential application. Chiral epichlorohydrin
(ECH) was used widely in organic synthesis and medicinal chemis-
try. For example, it is very easy to use chiral epichlorohydrin to
generate optically active compounds, such as antihypertensive
and antianginal agents.
The original chemical preparation of chiral ECH was reported in
1978 [1], but the synthetic process was considered cumbersome
and impractical. Unlike the traditional chemistry method, biocatal-
ysis presents a straightforward and environment-friendly ap-
proach for the generation of this chiral monomer. However, the
efforts on the asymmetric synthesis of ECH catalyzed by whole
cells, such as Rhodotorula glutinis, Aspergillus niger and Arthrobacter
erithii H10A, were less successful due to low product yields were
observed [2–4].
uids have been investigated as alternative solvents for biocatalysis
in lots of studies. Remarkable results were obtained for lipase and
other peroxidases catalysis in pure ionic liquid or homogeneous io-
nic liquid/water mixtures [7–9].
In this work, we describe the strategies of chiral ECH prepara-
tion by CPO-catalyzed epoxidation of 3-chloropropene in the pres-
ence of an ionic liquid as co-solvent. So far as we know, this is the
first report of the chiral ECH preparation by biocatalysis in ionic li-
quid–water mixtures.
2. Materials and methods
2.1. Materials
Chloroperoxidase was isolated from the growth medium of Cal-
dariomyces fumago according to the method established by Morris
and co-workers [10] with minor modifications, using acetone
rather than ethanol in the solvent fractionation step. The enzyme
had a specific activity of 4800 U/mL based on the standard mono-
chlorodimedone (MCD) assay (Rz = 1.05).
tert-Butyl hydrogen peroxide (TBHP), potassium hydrogen
phosphate, potassium dihydrogen phosphate, hydrogen peroxide
(30% in aqueous solution), dimethylsulfoxide (DMSO), N,N-dimeth-
ylformamide (DMF), methanol (CH3OH), acetonitrile (CH3CN)
and acetone (CH3OCH3) were obtained from Xi0an Chemical Co.
Ltd. 3-Chloropropene, 1-ethyl-3-methylimidazolium [EMIM][Br],
Chloroperoxidase (CPO) is the most synthetically useful perox-
idase due to its flexibility to a wide range of organic substrates as
well as its ability of catalyzing a variety of different reactions. More
importantly, chloroperoxidase is able to catalyze a broad spectrum
of enantioselective reactions, such as epoxidations of olefins,
hydroxylations of benzylic or allylic carbons, oxidations of alco-
hols, sulfides and indole [5,6].
* Corresponding author. Address: School of Chemistry and Materials Science,
Shaanxi Normal University, Chang-an South Road, 199, X’ian 710062, PR China. Tel.:
+86 29 85307765; fax: +86 29 85307774.
E-mail address: jyc@snnu.edu.cn (Y. Jiang).
1566-7367/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2010.02.003

728
J. Wu et al. / Catalysis Communications 11 (2010) 727–731
1-propyl-3-methylimidazolium [PMIM][Br], 1-butyl-3-methylimi-
dazolium [BMIM][Br], and 1-amyl-3-methylimidazolium [AMIM]
[Br] were purchased from Aldrich. All chemicals are of analytical
grade unless otherwise indicated. The standard epichlorohydrin
was obtained from Aldrich at a stated purity of 99.0%.
the other hand, CPO has a catalase activity, which would cause
spontaneous consumption of H2O2. Therefore, it was important to
keep the H2O2 concentration as low as possible in reaction solution
to suppress catalase activity and inactivation of CPO. A H2O2-con-
trolled reaction model was often employed to improve the enzyme
performance, where a prolonged reaction time (even up to 20 h)
was required [13].
2.2. Procedure for enzymatic epoxidation of 3-chloropropene
In this work, TBHP was chosen as the O2 donor instead of H2O2.
TBHP could be introduced into the reaction system directly.
Accordingly, the reaction time was dramatically decreased (from
over 4 h to about 60 min). Moreover, CPO was able to generate
O2 from H2O2 in a catalase-type side reaction, causing foaming
and potentially sweeping away more volatile substrates [14]. How-
ever, the reaction, using TBHP as oxidant instead, can be performed
in a sealed vessel without pressure buildup. In fact, we found that a
change of oxygen source did not affect the high enantioselectivity,
and moreover, a higher ECH yield was achieved even in the pres-
ence of excess oxidant.
3-Chloropropene (0.3 mmol) and CPO (0.04 lmol) were mag-
netically stirred in 3.0 mL 0.1 M aqueous phosphate buffer at room
temperature, pH 5.5. Then, TBHP (0.6 mmol) was added directly.
The reaction was quenched after 60 min with a saturated sodium
sulfite solution and extracted three times by anhydrous ether.
Combined organic extracts can be purified by evaporation, and
was collected at 88 °C and dried with magnesium sulfate.
The epoxidation using ionic liquids as co-solvent was per-
formed under the same conditions as above.
2.3. GC analysis and determination of product yield and enantiomeric
excess
3.3. Effect of organic solvent
Chiral gas chromatography analyses were performed on a Agi-
lent 6890N gas chromatograph equipped with a b-DEX 120
The increasing interest in the use of enzymes in synthesis iden-
tified advantages of enzymatic catalysis in organic media from
those displayed in aqueous media. These advantages included en-
hanced solubility of hydrophobic substrates, improved substrate
specificity and product enantioselectivity [15]. In this work, the ef-
fect of some widely used organic solvents on epoxidation of 3-
chloropropene using CPO was investigated, such as DMSO, DMF,
CH3OH, CH3CN and CH3OCH3. However, it was found that these or-
ganic solvents were not suitable because CPO epoxidation activity
was inhibited in their presence (Fig. 1). This was consistent with
the conclusion of Ref. [16], in which the authors reported that
the chlorination rates of monochlorodimedon (MCD) using CPO
in the presence of 20% DMSO, DMF, methanol or acetonitrile were
only 58% of the rate in pure buffer (pH 2.8) at the same reactant
concentrations. The presence of such organic solvents was found
to inhibit CPO catalysis by altering the protein conformation and
the local environment around the active site [16].
(30 m ꢀ 0.25 mm ꢀ 0.25
lm) chiral column.
GC standard (decane) was added prior to injection. Both chem-
ical yields and enantiomeric excesses of ECH were determined in a
single chromatogram based on their consistent elution order dur-
ing GC analysis compared with the standard ECH. In all cases the
predominant enantiomer produced was R-configuration.
3. Results and discussion
3.1. Optimization of conditions for CPO-catalyzed epoxidation of 3-
chloropropene
The synthetic strategy (described as Scheme 1) is straightfor-
ward and applicable to large scale preparation of chiral ECH. An
increasing–decreasing pattern versus reaction time was found for
product accumulation (expressed as ECH yield) in aqueous phos-
phate buffer at room temperature. The yield reached maximum
within 60 min before it started dropping, probably due to enzyme
inactivation as well as spontaneous hydrolytic epoxide ring open-
ing and aggregation of product. The reaction was rather enantiose-
lective (e.e. > 93.9%), indicating that 3-chloropropene was a good
substrate to CPO. In order to improve the chemical and optical
yield, we investigated the influence of several reaction conditions,
including the choice of oxidants, the presence of ionic liquids, pH
effect and CPO consumption.
3.4. Effect of ionic liquids
Recent years, ionic liquids (ILs) have gained increased attention
as new solvents for non-conventional biocatalysis. Remarkable re-
sults have been obtained for CPO catalysis with respect to the yield,
3.2. Effect of oxidants on CPO-catalyzed epoxidation of 3-
chloropropene
While many CPO-mediated reactions involved H2O2 as the ter-
minal oxidant, this work utilized TBHP instead.
CPO can be inactivated by excess H2O2 in the reaction mixture.
This inactivation, which generally occured with heme proteins
such as cytochrome P450, horseradish peroxidase and CPO, proba-
bly involved internal oxidation of the porphyrin moiety [11,12]. On
Fig. 1. Chiral ECH preparation in the presence of different organic solvent and ILs as
Scheme 1. Synthesis of chiral epichlorohydrin by CPO-catalyzed epoxidation of 3-
co-solvent or in pure buffer. Reaction conditions: 0.04
lmol CPO, 0.3 mmol 3-
chloropropene
chloropropene, 0.6 mmol TBHP, 1.6% (v/v) of ILs or organic solvent, pH 5.5.

J. Wu et al. / Catalysis Communications 11 (2010) 727–731
729
Table 1
enantioselectivity or enzyme stability [17,18]. In this work,
[EMIM][Br], [PMIM][Br], [BMIM][Br], and [AMIM][Br] were consid-
ered due to that the substrate was entirely soluble in them. In addi-
tion, these ILs were miscible with water, but not with the common
organic solvents, thereby permitting the recovery of products by
selective extraction procedure.
Initially, the reaction was carried out with pure ionic liquid as
solvent. However, a longer period was required for the product
to appear. Meanwhile, an enzyme inactivation was observed. So
the reaction was performed in a homogeneous reaction media
formed by phosphate buffer containing 1.6% (v/v) of hydrophilic
ILs as co-solvent.
As expected, all the investigated ionic liquids have positive ef-
fect. The ECH yields were enhanced to 80.4–88.8%, accompanying
an improved enantiomeric excess (95.6–97.1%), compared to opti-
mum 41.6% yield and 93.9% e.e. in pure phosphate buffer (Figs. 1
and 2). The ILs with shorter carbon chain was more efficient on chi-
ral ECH preparation.
Successively, the ionic liquids content was extended gradually
to 45% (VILs/Vbuffer). Higher concentrations of ILs were not taken
into account because an undesirable pH variation was observed,
ECH chemical yields and enantiomeric excesses of the CPO-catalyzed epoxidation of
3-chloropropene with ILs as co-solvent at optimum reaction condition.
ILs
VILs/Vbuffer (%)
ECH yield (%)
e.e. (%)
[EMIM][Br]
[PMIM][Br]
[BMIM][Br]
[AMIM][Br]
1.6
1.6
1.6
1.6
88.8
84.4
82.5
80.4
97.1
96.3
96.0
95.6
which was reported to be detrimental to the activity of CPO [19].
A ‘‘bell-shaped” dependence of ECH yields on ILs concentration
was observed (Fig. 3), where [EMIM][Br] was taken as an example.
Table 1 reported the optimum ILs concentrations and the corre-
sponding ECH yields and enantiomeric excesses values.
Obviously, the detrimental effect on epoxidation of 3-chloro-
propene in organic media using CPO was favorably absent in the
ionic liquids considered. The product yield was higher than that
in pure buffer in a wide concentration range of ILs. CPO-tolerated
ILs concentration was 38.7% (VILs/Vbuffer). UV–visible spectrum of
CPO in the presence of ILs showed that the absorption of CPO Soret
band at kmax = 398 nm was increased. No blue-shift of absorption
peak was observed (Fig. 4). This phenomenon indicated that the
heme was more exposure, so it was easier for CPO to bind with
substrates. But a decrease of kmax appeared gradually, accompany-
ing an increase of k = 280 nm when VILS/Vbuffer was higher than 10%.
Circular dichroism (CD) spectroscopy was employed to investigate
the structure and conformation of CPO in this process (Fig. 5). In
‘‘far-UV” spectral region (190–250 nm) of CPO, there was a large
negative absorption peak around 206 nm, which indicated CPO
was a typical alpha-helix protein. This negative absorption became
stronger when the concentration of ILs increased. This increase was
due to the strengthening of alpha-helix structure of CPO. However,
a loose of this alpha-helix structure was observed at higher ILs con-
tent, which was not favorable for CPO catalytic performance. In
‘‘near-UV” spectral region (250–350 nm), the increase of CD signal
around 276 nm in the presence of ILs showed the corresponding
change of tertiary structure, which was favorable for stability and
activity of enzyme. No obvious change of CD signal at 350–700 nm
was observed, meaning that the microenvironment around heme
was not changed in the presence of ILs.
Fig. 2. Comparison of chemical yield and enantiomeric excesses of ECH in pure
buffer or in the presence of 1.6% (v/v) of [EMIM][Br] as co-solvent. Reaction
conditions: 0.04 lmol CPO, 0.3 mmol 3-chloropropene, 0.6 mmol TBHP, pH 5.5.
3.5. Effect of pH
The epoxidation of 3-chloropropene was highly pH-dependent.
But it only had influence on the yield. No great changes on e.e. val-
Fig. 3. Effect of [EMIM][Br] concentration on the epoxidation of 3-chloropropene
catalyzed by CPO. Reaction conditions: 0.04
0.6 mmol TBHP, pH 5.5.
lmol CPO, 0.3 mmol 3-chloropropene,
Fig. 4. UV–vis spectrum of CPO in the presence of different ILs from 1.6% to 10%
VILS/Vbuffer
.

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J. Wu et al. / Catalysis Communications 11 (2010) 727–731
Fig. 5. CD spectrum of CPO in the presence of different ILs from 1.6% to 10% VILs/Vbuffer
.
Fig. 6. Effect of pH on CPO-catalyzed epoxidation of 3-chloropropene. Reaction
conditions: 0.3 mmol 3-chloropropene, 0.6 mmol TBHP, 0.04 mol CPO.
l
Fig. 7. Effect of enzyme consumption on CPO-catalyzed epoxidation of 3-chloro-
propene. Reaction conditions: 0.3 mmol 3-chloropropene, 0.6 mmol TBHP, pH 5.5.
ues were observed under different pH. The highest ECH yield was
obtained at pH 5.5, as shown in Fig. 6. We did not test when
pH > 7 because under alkaline conditions, CPO was extremely
unstable. It would lose its activity completely in a short time. On
the other hand, some side reactions will probably arise under alka-
line conditions. At the range of pH 3.0–7.0, the ECH yield signifi-
cantly increased with the increase of pH from 3 to 5.5, and then,
dropped due to the CPO inactivation. Strong acidic condition was
also not chosen because of the poor stability of enzyme.
To ensure both the completion of reaction and fewer consump-
tion of enzyme, a CPO concentration of 0.04 lmol was used in all
experiments, indicating a very small enzyme amount was required
in this reaction.
4. Conclusion
Chiral epichlorohydrin with high yields and enantioselectivity
was prepared in this work. Under the optimum conditions, the
ECH yield reached 88.8% with e.e. of 97.1%. This work shows that
the enzymatic epoxidation of 3-chloropropene is a very powerful
method for the production of optically active epichlorohydrin,
especially with imidazole ionic liquids as co-solvent.
As compared to the behavior observed in conventional organic
solvents, CPO in ILs presented enhanced activity, stability and
selectivity. Moreover, the presence of ILs improved substrate solu-
bility in the reaction medium. The ionic liquid having shorter car-
bon chain was more efficient on chiral ECH preparation.
3.6. Effect of enzyme consumption
Enzyme amount ranging from 0.01 to 0.06 lmol was employed
to determine the effect of protein dosage on the ECH yield (Fig. 7).
An enzyme concentration-dependent model of product yield was
found. The yield firstly increased linearly with the increase of
CPO amount, thereafter the increase slowed down and reached a
plateau with high enantioselectivity maintained. Here, TBHP was
used at least 2-fold molar excess over substrate concentration,
but no inactivation of CPO was observed.

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Acknowledgment
This work is supported by the National Natural Science Founda-
tion of China (20876094).
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