Ionic liquids as a new reaction medium for oxidase-peroxidase-catalyzed sulfoxidation

Article abstract of DOI:10.1016/S0957-4166(03)00578-0

The possibility of the application of oxidase and peroxidase as catalysts in ionic liquids is demonstrated in the chemo- and stereoselective oxidation of sulfides. The high operational stability of these enzymes in ionic liquids is reported. The substrate

Full text of DOI:10.1016/S0957-4166(03)00578-0

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 2487–2490
Ionic liquids as a new reaction medium for
oxidase–peroxidase-catalyzed sulfoxidation
Krzysztof Okrasa,a,b Eryka Guibe´-Jampelb and Michel Therisodb,*
aZTiBSL, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-662 Warsaw, Poland
bLab. Chimie Bioorganique et Bioinorganique, ICMMO, Bat. 420, Universite´ Paris-Sud, F-91405 Orsay cedex, France
Received 26 May 2003; accepted 11 July 2003
Abstract—The possibility of the application of oxidase and peroxidase as catalysts in ionic liquids is demonstrated in the chemo-
and stereoselective oxidation of sulfides. The high operational stability of these enzymes in ionic liquids is reported. The substrate
of glucose oxidase (glucose) and the substrate of peroxidase (sulfide) are perfectly soluble.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
enzymes by stabilizing the highly charged iron-peroxo
or -oxo intermediate. The activities of heme-derived
catalysts (hemine, MP-11) are markedly higher in ILs
than in methanol or DMSO.12 Moreover, reactions in
Ils have potential environmental advantages since ILs
are non-volatile and can usually be recycled in good
yield and reused.3,4
The use of non-aqueous solvents as a medium for
enzymatic reactions has been a major breakthrough for
bioconversions. However, the insolubility of polar sub-
strates in the apolar organic solvents, which are used
normally, is one disadvantage of non-aqueous tech-
niques. Ambient-temperature ionic liquids (ILs) are
very promising reaction media for chemical and bio-
chemical transformations. They have been known for a
long time, but their applications as a solvent for
organic synthesis were reported in the literature only
within the past few years.1–4 ILs are good solvents for a
wide range of both inorganic and organic compounds.
Some ILs are water-immiscible and have polarities
comparable to short-chain primary alcohols.5–8 ILs
have been already used as reaction media for lipase-cat-
alyzed esterifications, ammonolysis of esters and perhy-
drolysis.9–11 Oxidoreductases have also been the subject
of some studies.12,13 Results on biocatalysis in ILs have
been reported in the recent reviews.1,14 A different or
increased selectivity was often observed by comparison
to conventional organic solvents. On the other hand,
ILs can enhance the catalytic activity of transition
metal complexes, due to their combination of high
polarity and low coordination power for the cata-
lyst.15,16 This non-coordinating nature might be inter-
esting for oxidations catalyzed by heme-containing
Having devised oxidation reactions catalyzed by bi-
enzymatic systems in water (which failed in organic
solvents) we were interested in the study of the activity
of such systems in ionic liquids. Thus, we wish to report
our preliminary results on the oxidation of thioanisoles
by a bi-enzymatic system glucose oxidase–peroxidase in
ionic liquids.
Peroxidases are very attractive biocatalysts for selective
oxidative transformations, but their low operational
stability limits their application.
Peroxidases are inactivated by an excess of hydrogen
peroxide, their primary substrate. Only a slow flux of
hydrogen peroxide can insure the stereoselectivity of
reactions catalyzed by these enzymes. In our previous
work, hydrogen peroxide was progressively generated
in situ at the expense of glucose and oxygen (air) by
glucose oxidase and used by a peroxidase to oxidise
sulfides to chiral sulfoxides.17 Co-immobilization of
peroxidase with an oxidase18 and employment of perox-
idase suspended in methanol19 and chloroform20 were
also experimented by other authors to allow recovering
of the expensive POD.
* Corresponding author. Tel.: +33-169156311; fax: +33-169157281;
e-mail: therisod@icmo.u-psud.fr
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0957-4166(03)00578-0

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K. Okrasa et al. / Tetrahedron: Asymmetry 14 (2003) 2487–2490
Scheme 1.
Since oxygen and organic substrates are more soluble in
ILs than in water,2,21 glucose oxidase–peroxidase-cata-
lyzed enantioselective sulfoxidation of thioanisoles in
IL as the reaction medium appeared to be a promising
alternative to aqueous solution (Scheme 1).
Figure 1. GOD-catalyzed oxidation of glucose in [BMIM]PF6
as a function of concentration of water. Conditions: 3 mg
GOD and 0.6 mmol glucose in 3 ml IL, rt.
We used for our investigations two commercially avail-
able and inexpensive enzymes: peroxidase from Copri-
nus cinereus (Cip)22 and glucose oxidase (GOD) from
Aspergilius niger.
We have chosen as a solvent 1-butyl-3-methyl imida-
zolium hexafluorophosphate ([BMIM]PF6) a hydropho-
bic ionic liquid, immiscible with water and diethyl
ether, but easily miscible with organic solvents such as
AcOEt or CH2Cl2.
These properties facilitated us an easy recycling of the
ionic liquids with or without enzymes as well as the
extraction of sulfides and sulfoxides. Synthesis and
purification of the IL was proceeded according to a
modified Kaslauskas’ method.23 The use of expensive
silver salts and column chromatography were avoided.
Figure 2. Conversion of thioanisole to phenyl methyl sulfox-
ide and of glucose to gluconic acid after 16 h of reaction
catalysed by GOD/Cip at rt. Conditions: 0.3 mmol thioanisole
in 3 ml of IL (0.1 M), 3 mg GOD, 0.6 mmol glucose, 48 mg
Cip.
Our attempts on the direct oxidation of thioanisole by
a slow addition of hydrogen peroxide to a suspension
of Cip in IL failed. H2O2 was rapidly decomposed by
the enzyme acting as a catalase under these conditions
and no sulfoxide was formed.
Efficiency of enzyme-catalyzed reactions in non-
aqueous media (organic solvents or ionic liquids), as it
has been demonstrated in literature,24,25 is under the
control of water activity rather than water concentra-
tion. Thus, we found that the rate of production of
hydrogen peroxide by GOD in IL was influenced by the
amount of water added (Fig. 1). It was estimated by
measuring spectrophotometrically the disappearance of
glucose.26
It appears that a 10% v/v content of water in IL is the
best choice for Cip and GOD, while 5% v/v is sufficient
to achieve an optimum activity of GOD. Glucose
oxidase is markedly active even at 1% of water. In the
optimum conditions for peroxidase, GOD activity in IL
is about 20 times lower than in water.
The yield of sulfoxide relative to the hydrogen peroxide
produced does not exceed 50%. Production of hydrogen
peroxide by GOD increased dramatically at 40°C (Fig.
3) but resulted in low yield and low enantiomeric excess
of the formed sulfoxide (Table 1, entry 4).
The influence of the concentration of water in
[BMIM]PF6 on the conversion of thioanisole to sulfox-
ide catalyzed by the bi-enzymatic system GOD/Cip is
illustrated in Figure 2.

K. Okrasa et al. / Tetrahedron: Asymmetry 14 (2003) 2487–2490
2489
ionic liquid–water mixture. Owing to the good solubil-
ity of sulfide and sulfoxide in IL, the reaction mixture
was stirred in open vessel under air. No evaporation of
substrate or product which often occurred with suspen-
sions in water was observed. One equivalent of sodium
bicarbonate, added in three portions, was required to
neutralize the formed gluconic acid. Sulfide and sulfox-
ide were extracted by diethyl ether and enzymes in IL
were recycled directly. The enantiomeric excess of the
obtained sulfoxides is comparable to those in water and
stay constant during the reaction. No further oxidation
to sulfone was observed. The obtained chiral aryl-
methyl sulfoxides have the S absolute configuration as
in water.17
In water, methyl-2-naphthyl sulfide 1b (water insoluble,
solid compound) was oxidized significantly slower than
thioanisole.17 On the contrary, in IL, the rate of oxida-
tion of 1a and 1b were comparable (Table 1, entry 9),
probably because both compounds have similar solubil-
ity in IL. Methyl-2-naphthyl sulfoxide 2b was obtained
with ee=92%. After 48 h of peroxidase pre-incubation
in [BMIM]PF6 its activity was unchanged (Table 1,
entry 7). Due to the high operational stability of
enzymes in [BMIM]PF6, after 32 h sulfoxide and its
substrate could be extracted from the reaction mixture
and a new portion of sulfide could be added. This
second run proceeded with the same rate and with the
same stereoselectivity (Table 1, entries 2 and 10), indi-
cating that the enzymes were fully active in IL after 64
h. Thus, the reduced rate of sulfoxidation in compari-
son to that in water is not a consequence of the weak
stability of enzymes but rather of a lower value of the
kinetic constant kcat. In IL 0.1 mmol of formed sulfox-
ide inhibits Cip and extraction of this product is indis-
pensable to obtain complete conversion. Pure
[BMIM]PF6 can be easily recovered after enzymes,
glucose and gluconic acid extraction with water.27
Figure 3. Rates of formation of phenyl methyl sulfoxide and
hydrogen peroxide (consumption of glucose) during oxidation
by GOD/Cip in [BMIM]PF6. Conditions: 0.3 mmol thioan-
isole in 3 ml of IL (0.1 M), 0.3 ml water, 3 mg GOD, 0.6
mmol glucose, 48 mg Cip.
This probably indicates an inhibition of peroxidase by
excess of hydrogen peroxide and the partial decomposi-
tion of H2O2 by Cip, due to its catalase activity. The
low enantiomeric excess of sulfoxide obtained when an
excess of GOD was used (Table 1, entry 5) shows that
spontaneous oxidation of sulfide by hydrogen peroxide
is in competition with that catalyzed by Cip.
Two arylmethyl sulfides: thioanisole 1a and methyl-2-
naphthyl sulfide 1b were tested as substrates of the
peroxidase. Peroxidase and glucose oxidase were added
at once to the solution of thioanisole and glucose in
Table 1. Asymmetric oxidation of arylmethyl sulfides 1a and 1b catalysed by GOD/Cip in [BMIM]PF6
Entry
Sulfide
1a
Glucose (mmol) GOD (mg)
Water added to IL (%)
Temp. (°C)
Time (h)
Conv. (%)
Ee (%)
1
0.6
0.6
3
3
10
10
rt
rt
16
32
16
32
16
16
32
16
32
16
32
16
16
16
32
32
13
32
16
28
7
66
68
nd
67
69
46
nd
nd
13
67
nd
66
63
92
91
91
2a
1a
3
4
1a
1a
0.6
0.6
1.5
3
10
10
rt
40
3
5
5
6
1a
1a
0.6
0.3
6
3
10
10
rt
rt
11
25
14
15
14
6
14
36
34
7b
8
9
1a
1a
1b
0.6
0.6
0.6
3
3
3
10
5
10
rt
rt
rt
10a
1b
0.6
3
10
rt
Reaction conditions: sulfide 1a, 1b: 0.3 mmol, peroxidase: 48 mg, [BMIM]PF6: 3 ml.
a Second run with enzymes–ionic liquid mixture directly recycled.
b Peroxidase pre-incubated in [BMIM]PF6 for 48 h at rt.

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K. Okrasa et al. / Tetrahedron: Asymmetry 14 (2003) 2487–2490
2. Preparation of 1-butyl-3-methyl imidazolium
hexafluorophosphate ([BMIM]PF6)
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1.11.1.7; specific activity: 231 kU/ml) having broad pH
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activity: 1.2 U/mg) from Aspergilius niger with pH
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sodium bicarbonate, were added every 8 hours. After 32
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similar to that obtained in water. All substrates of
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sulfides and sulfoxides are perfectly soluble in IL. Thus,
the reaction in IL is particularly interesting for water
insoluble products (for example methyl-2-naphthyl
sulfide oxidized with ee=92%). The mixture of IL
containing GOD and Cip can be quantitatively recycled
and reused. Isolation of obtained sulfoxide is easier than
from water, where proteins induce formation of emulsion
with organic solvent. Moreover, the easy recovery of
ionic liquid is environmentally interesting. These obser-
vations indicate that IL are suitable media for biocata-
lytic oxidations.
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Acknowledgements
27. Before washing with water [BMIM]PF6 should be dis-
solved in ethyl acetate to facilitate this process.
We are grateful to Dr Schneider and Novozymes for the
supply of Cip and GOD.