Application of various ionic liquids as cosolvents for chloroperoxidase- catalysed biotransformations

Article abstract of DOI:10.1007/s00706-008-0081-7

Chloroperoxidase from Caldariomyces fumago catalyses oxidation of indole and thioanisole in reaction mixtures containing up to 40% (v/v) of different ionic liquids (ILs). Results indicate that ILs containing tosylate, trifluoroacetate, chloride, and methylsulfate anions are suitable cosolvents for these transformations, yielding high enantiomeric excess and good conversion rates.

Full text of DOI:10.1007/s00706-008-0081-7

Monatsh Chem (2009) 140:509–512
DOI 10.1007/s00706-008-0081-7
ORIGINAL PAPER
Application of various ionic liquids as cosolvents
for chloroperoxidase-catalysed biotransformations
Roman J. Lichtenecker Æ Walther Schmid
Received: 3 October 2008 / Accepted: 7 October 2008 / Published online: 1 November 2008
Ó Springer-Verlag 2008
Abstract Chloroperoxidase from Caldariomyces fumago
catalyses oxidation of indole and thioanisole in reaction
mixtures containing up to 40% (v/v) of different ionic
liquids (ILs). Results indicate that ILs containing tosylate,
trifluoroacetate, chloride, and methylsulfate anions are
suitable cosolvents for these transformations, yielding high
enantiomeric excess and good conversion rates.
in the reactions catalysed [15, 16]. However, because of the
low solubility of organic substrates in aqueous solution,
many applications suffer from low yields and make the use
of cosolvents necessary.
It has been demonstrated, that CPO exhibits good con-
version rates in the oxidation of 1,2-dihydronaphthalene in
the presence of up to 30% (v/v) 1-butyl-3-methylimidazo-
lium methylsulfate [17]. Recently, the stability of CPO in
the presence of different ILs has been confirmed using
other substrate compounds [18]. These results inspired us
to investigate the effect of various ILs as cosolvents in
CPO-catalysed transformations, with regard to conversion
rates and enantiomeric excess values. As model reactions,
we chose the oxidation of indole (1) to oxindole (2), and
the conversion of thioanisole (3) to the corresponding
sulfoxide (4) using H O and tert-butyl hydroperoxide
Keywords Caldariomyces fumago Á Sulfoxides Á
Chirality Á Oxidation Á Ionic liquids
Introduction
Ionic liquids (ILs) have gained increasing interest in recent
years [1–4]. Their properties, such as viscosity, polarity,
and miscibility with various solvents, can be tuned by
appropriate variation of the cation and anion [5–8]. This
feature, together with their lack of vapour pressure and the
possibility of recycling, makes ILs excellent candidates for
cosolvents in biocatalytic transformation reactions [9–11].
Applications of ILs as reaction media for enzyme catalysis
have so far focussed on lipases, proteases, and glycosi-
dases. Very few examples of oxidase or peroxidase
reactions in ILs have been described in the literature [12–
2
2
(tert-BuOOH) as oxidants (Fig. 1). Both transformations
are of interest in organic chemistry; in particular, the
synthesis of enantioselective sulfoxides still poses a syn-
thetic challenge [19].
Results and discussion
To examine the applicability of ILs in CPO-catalysed
biotransformation we synthesized and purified a variety of
these cosolvents following established methods [20–22].
Chloroperoxidase-catalysed indole oxidation in the
presence of these ILs (20% v/v) using slow addition of
H O was performed as described in the Experimental
1
4].
In our studies we used chloroperoxidase (CPO) from
Caldariomyces fumago [E.C. 1.11.1.10] as biocatalyst.
This enzyme has proved to be of great value in chemical
synthesis, because it tolerates a wide variety of substrates
2
2
section. The conversion rates of these experiments are
summarized in Table 1. The results indicate that, in par-
ticular, ILs containing tosylate, trifluoroacetate, chloride,
or methylsulfate anions are suitable cosolvents in this
biotransformation reaction.
R. J. Lichtenecker Á W. Schmid (&)
Institute of Organic Chemistry, University of Vienna,
W a¨ hringerstrasse 38, 1090 Vienna, Austria
e-mail: walther.schmid@univie.ac.at
123

5
10
R. J. Lichtenecker, W. Schmid
Chloroperoxidase,
100
H O or tert-BuOOH
2
2
9
0
O
Ionic liquid
(20-40% v/v)
80
70
N
H
N
H
1
3
2
4
6
5
4
0
0
0
O
[
BMIM][BF4]
S
S
30
20
10
0
Chloroperoxidase,
H2O2 or tert-BuOOH
[BMIM][tf]
[BMIM][NO3]
[MMIM][ms]
(R)
Ionic liquid
20-40% v/v)
(
0
10
20
30
40
IL/buffer/% (v/v)
Fig. 1 Model reactions used to investigate the effect of various ionic
liquids as cosolvents on CPO-catalysed transformations, with regard
to conversion rates and enantiomeric excess values
1
00
9
8
7
6
5
0
0
0
0
0
2 2
Table 1 Conversion rates for indole oxidation using H O as oxidant
Entry
Ionic liquid
Conversion (%)
[
[
BMPY][ac]
BMIM][ts]
1
2
3
4
5
6
7
8
[BMIM][ts]
[BMPY][ta]
[BMIM][ta]
[BMIM][Cl]
[BMPY][tf]
[MMIM][ms]
100
98
97
94
90
83
71
48
44
16
15
12
9
40
30
20
10
0
[BMIM][ac]
[BMIM][Cl]
[BMPY][tf]
0
10
20
30
40
IL/buffer/% (v/v)
4
[BMIM][BF ]
Fig. 2 Conversion rate as a function of amount of ionic liquid in the
reaction mixture for CPO-catalysed oxidation of indole
[BMIM][NO
[BMIM][tf]
3
]
9
a
10
11
12
13
[BMPY][bta]
[BMIM][PF
[Oct NMe][NO
[BMIM][bta]
a
a
a
In general, more than 40% IL in the reaction medium
always led to poor conversions with the single exception of
[MMIM][ms], which still gave 58% yield. When pure ILs
were used as cosolvents for CPO-catalysed indole oxida-
tion no reaction products were obtained.
6
]
3
3
]
Reactions were performed in acetate buffer (pH 4.5) containing 20%
(
v/v) ionic liquid
To gain further insight into the applicability of ILs for
CPO-catalysed oxidation reactions, we investigated the
transformation of thioanisole (3) to its corresponding sulf-
oxide 4 as a model reaction. To obtain a general picture, we
also included the most common organic cosolvents for
CPO-catalysed biotransformation, tert-butanol and acetone,
in our investigations. The results obtained from the oxida-
tions in various ILs are summarized in Figs. 3 and 4 for
use of H O and tert-BuOOH, respectively, as oxidizing
[
BMIM], 1-butyl-3-methylimidazolium; [BMPY], 1-butyl-1-meth-
ylpyrrolidinium; [MMIM], 1,3-dimethylimidazolium; [ts], tosylate;
bta], bis(trifluoromethylsulfonyl)imide; [Oct], octyl; [ta], trifluoro-
acetate; [tf], trifluoromethylsulfonate; [ms], methylsulfate
[
a
IL not miscible with aqueous buffer
As expected, application of water-immiscible ILs led to
a significant decrease in conversion rates (entries 10–13 in
Table 1). The latter results led us to examine the tolerance
of the ILs by CPO in respect of increasing amounts of the
ILs in the reaction media. The correlation of conversion
rates with IL content of the reaction mixture is summarized
in Fig. 2.
2
2
reagents.
As can be concluded from Fig. 3, conversion rates for
this biotransformation significantly increased when ILs
containing trifluoroacetate, chloride, methylsulfate, and
tosylate anions were added to the pure buffer solutions.
Additionally, the higher conversion rates could be reached
at a lower IL content (20%) than was the case for the
usually applied organic cosolvents acetone and tert-butanol
(40%).
The data suggest that the cosolvents applied in this
reaction system can be divided into two different groups:
1
2
the first showed a significant decrease in conversion
between 30 and 40% (v/v) cosolvent, whereas
the second group of ILs exhibited a rather linear decay
in the conversion rates.
Changing the oxidant from H
O to tert-BuOOH yielded
2 2
slightly different results, summarized in Fig. 4. In the case
1
23

Cosolvents for chloroperoxidase catalysed biotransformations
511
Table 2 Enantiomeric excess values for thioanisole oxidation in
acetate buffer, containing 20 or 40% cosolvent
Cosolvent
ee (%)
0%/40% (v/v)
2
2
H O
2
oxidation
tert-BuOOH oxidation
Acetate buffer
98
99
99/95
97/92
48/81
98/95
98/95
91/83
89/83
95/57
92/42
99/98
99/98
[
[
[
[
[
[
[
[
[
MMIM][ms]
BMIM][ac]
BMIM][Cl]
BMPY][tf]
99/99
97/98
48/90
83/99
65/92
65/65
68/60
96/94
95/70
99/99
99/99
2 2
Fig. 3 Conversion rate for thioanisole oxidation with H O . Reaction
mixtures contain 20% (v/v) (black) or 40% (v/v) cosolvent (grey)
4
BMIM][BF ]
BMIM][NO
BMIM][tf]
BMIM][ts]
BMPY][ac]
3
]
Acetone
tert-Butanol
The results of our model reactions demonstrate that several
ILs are suitable cosolvents in biotransformations catalysed
by CPO from C. fumago. Reaction mixtures, especially
those containing chloride, trifluoroacetate, methylsulfate, and
tosylate ILs show high conversion rates in thioanisole and
indole oxidation. The applicability of aqueous IL reaction
mixtures for CPO-catalysed bioconversions was supported
by the following observations:
Fig. 4 Conversion rate for thioanisole oxidation with tert-BuOOH.
Reaction mixtures contain 20% (v/v) (black) or 40% (v/v) of cosolvent
(
grey)
1
2
the enzyme had high activity in mixtures containing 20
and 40% of the ILs mentioned above;
of a 20% (v/v) IL content in the solvent mixture the con-
version rate was increased when changing from H O to
2
2
conversion rates of thioanisole oxidation with H O
2
2
tert-BuOOH. However, the overall reaction time was also
significantly raised. This fact may be responsible for the
significant drop in the conversion rate observed when
increasing the IL content to 40%. Obviously, longer reac-
tion times led to lower conversion rates because of reduced
enzyme stability in solvent mixtures containing larger
amounts of IL. Notable exceptions to this behaviour are
represented by [BMIM][Cl] and [MMIM][ms], for which,
even in the presence of 40% (v/v) IL, comparable rates could
be observed. All efforts to perform CPO-catalysed oxidation
in pure ILs failed. The enantiomeric excess values obtained
from the enzyme reaction by applying both oxidants in
different solvent mixtures are summarized in Table 2.
Usually the ee values do not differ when the IL content
of the solvent mixture is increased. One interesting
exception is found when [BMIM][Cl] is applied as cosol-
vent. In this case 40% (v/v) IL led to significantly higher ee
values than was observed for 20% (v/v) content, irrespec-
tive of the oxidant used. At present, we do not have a good
explanation for this behaviour, which needs further inves-
tigation of solvent structure and function of the ILs and the
mixtures thereof.
could be increased from 40% in pure acetate buffer to
over 80% by addition of ILs, proving the feasibility of
enhancing CPO-catalysed reaction yields by increasing
the solubility of the hydrophobic reaction substrates;
and, finally
3
ee values of thioanisole oxidation products provide
evidence that CPO retains high enantioselectivity in the
presence of most of the tested ILs in the reaction
mixture.
Further investigation of synthetic applications of other
heme peroxidases in ILs are currently in progress in our
laboratories.
Experimental
Chloroperoxidase from C. fumago [E.C. 1.11.1.10] was
purchased from Fluka (CAS: 9055-20-3) as a brown sus-
pension in 0.1 M sodium phosphate (pH = 4). ILs were
synthesized using literature protocols [20–22]. The result-
ing products were identified and checked for organic
123

5
12
R. J. Lichtenecker, W. Schmid
1
13
3
impurities using H and C NMR spectroscopy. Inorganic
salt content was qualitatively determined by addition of
organic phases were combined and washed with 3 cm
water. The mixture was diluted with diethyl ether to a total
3
volume of 50 cm . An aliquot of this solution (20 mm )
3
AgNO and precipitation of Ag halides. Purification of the
3
ILs was accomplished by using extraction with water
was injected to a HPLC column and eluted with n-hexane–
3
isopropanol 8:2 using a flow rate of 0.8 cm /min
(
water-immiscible ILs), crystallization of inorganic impu-
rities from dichloromethane–IL mixtures, with subsequent
filtration, or filtration of IL solutions in dichloromethane
over silica-gel, if necessary. CPO reactions were run in a
Carousel 12 Reaction Station from Radleys and oxidant
addition was controlled by an Orion M362 Sage pump.
HPLC analysis was performed using a Chiralcel OD-H
column (0.46 cm 9 25 cm).
The eluent was monitored at 256 nm at a flow rate
3
of 0.8 cm /min. Retention times were: thioanisole (3)
5.5 min; acetophenone 5.7 min; and methylphenylsulfox-
ide (R)-4 8.8 min and (S)-4 10.1 min. The enantiomeric
excess was assigned as reported elsewhere [23]. Conver-
sion rates were determined via peak-area integration. The
values were corrected using a factor for recovery of the
internal standard and substance.
Indole oxidation
References
In a typical procedure for indole oxidation, 10 mg substrate
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1
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3
2
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was dissolved in the appropriate reaction mixture con-
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1
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added (1.1 equiv. dissolved in 150 mm water) over a
period of 94 min using a syringe pump. After a total
1
2
3
reaction time of 4 h, 2 cm acetophenone standard solution
3
498.8 mg in 50 cm diethyl ether) was added and the
2
2
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(
reaction mixture was extracted with diethyl ether
3
2 9 20 cm ). When tert-BuOOH was used as an oxidant,
(
addition was accomplished in the same way as with H O ,
2
2
but the total reaction time was extended to 24 h. The
1
23