Application of hydrophilic ionic liquids as co-solvents in chloroperoxidase catalyzed oxidations

Article abstract of DOI:10.1016/j.tetlet.2006.05.072

The effect of hydrophilic ionic liquids (ILs) on the activity of chloroperoxidase (CPO) was checked through kinetic and stereochemical studies. The possibility to employ this enzyme in synthesis has been demonstrated investigating the chemo- and stereoselectivity of oxidation of phenyl methylsulfide in several citrate buffer-IL mixtures.

Full text of DOI:10.1016/j.tetlet.2006.05.072

Tetrahedron Letters 47 (2006) 5089–5093
Application of hydrophilic ionic liquids as co-solvents in
chloroperoxidase catalyzed oxidations
*
Cinzia Chiappe, Lisa Neri and Daniela Pieraccini
Dipartimento di Chimica Bioorganica e Biofarmacia, Via Bonanno 33, 56126 Pisa, Italy
Received 31 March 2006; revised 12 May 2006; accepted 15 May 2006
Available online 5 June 2006
Dedicated to the memory of Professor Giancarlo Berti (19 October, 2005)
Abstract—The effect of hydrophilic ionic liquids (ILs) on the activity of chloroperoxidase (CPO) was checked through kinetic
and stereochemical studies. The possibility to employ this enzyme in synthesis has been demonstrated investigating the chemo-
and stereoselectivity of oxidation of phenyl methylsulfide in several citrate buffer–IL mixtures.
Ó 2006 Elsevier Ltd. All rights reserved.
9
The heme enzyme chloroperoxidase (CPO), produced by
the marine fungus Caldariomyces fumago, is a versatile
enzyme which exhibits a broad spectrum of chemical
reactivities, including the reactions typical of peroxi-
dases, the use of halide ions to halogenate a variety of
organic molecules and the catalyses activity in the
alyzed reactions using ionic liquids as co-solvents. Dur-
ing the last years, ionic liquids (ILs) have gained
increased attention as new solvents for performing prac-
1
0,11
tically all types of reactions.
Several papers have
1
2
been published on the use of ILs as reaction media
for biocatalyzed processes evidencing remarkable results
with respect to the yield, enantioselectivity or enzyme
stability.
1
disproportionation of hydrogen peroxide. Due to its
versatility and selectivity CPO is recognized as the most
2
promising enzyme for synthetic applications. Despite
the potentiality, actual synthetic applications of CPO
are hampered by its limited stability due to inactivation
by H O and the low water solubility of many organic
In this letter, we report the kinetic and stereochemical
studies carried out to determine the activity and selectiv-
ity of CPO in the presence of ionic liquids using both
aqueous phosphate (pH 2.7) and citrate buffers (pH
5.0) as co-solvents. The possibility to employ this en-
zyme in synthesis has been demonstrated investigating
the chemo- and stereoselectivity of oxidation of phenyl
methylsulfide in several citrate buffer–IL mixtures.
2
2
substrates of synthetic interest. Attempts to use CPO
in aqueous buffer–organic solvent mixtures or in pure
organic solvents have met with only moderate success,
due to the decreased reaction rate and selectivity in these
3
,4
media. At variance, adsorption on meso- or micro-
5
6
porous solids, covalent bound to silica gel and mi-
7
cro-encapsulation in a microporous silica gel have been
Seven different hydrophilic ILs, 1,3-dimethylimidazo-
shown to be alternative approaches able to ensure suffi-
cient stability to CPO in the presence of hydrogen per-
oxide for carrying out the oxidation reaction with high
activity and/or selectivity. On the other hand, a signifi-
cant stabilization of CPO has been obtained also by
the addition of poly(ethylene glycol)s to the reaction
lium methylsulfate [mmim][MeSO ], 1, 1,3-dimethylimid-
4
azolium dimethylphosphate [mmim][Me PO ], 2, 1-
2
4
ethyl-3-methylimidazolium ethylsulfate [emim][EtSO4],
3, N,N-dimethylmorpholinium methylsulfate [Mor ]-
1
1
[MeSO ], 4, cholinium acetate [N1112OH][OAc], 5,
4
cholinium phosphate [N1112OH][H PO ], 6, and cholin-
2
4
8
mixture. Despite the widespread interest on this topic,
only few papers have been published on peroxidase cat-
ium citrate [N1112OH][Citr], 7, were used as co-solvents
to investigate the influence of ionic liquid structure on
CPO activity and selectivity (Fig. 1). Cholinium based
1
3
ILs, which to the best of our knowledge have been ap-
plied here for the first time in biocatalyzed processes,
have been chosen due to their low cost and extremely
easy preparation procedure.
*
0
Corresponding author. Tel.: +39 050 2219669; fax: +39 050
219660; e-mail: cinziac@farm.unipi.it
2
040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.05.072

5090
C. Chiappe et al. / Tetrahedron Letters 47 (2006) 5089–5093
O
MeSO -
4
N+
R
N
OH
4
: [Mor ][MeSO ]
11 4
N+
X-
+
X-
N
5
: X = CH CO2-
[N1112OH][OAc]
3
1
2
3
: R = Me; X = MeSO4-
[mmim][MeSO4]
6: X= H PO4-
[N11112OH][H PO ]
2
2
4
-
-
7
: X = HOC(CH COOH) COO
[N11112OH][Citr]
: R = Me; X = (CH ) PO [mmim][Me PO ]
2
2
3
2
4
2
4
: R = Et; X = EtSO4-
[emim][EtSO4]
Figure 1.
used for the assay. The chlorinating activity of CPO on 8
was initially measured in a phosphate buffer in the pres-
ence of increasing concentrations of IL. The concentra-
tion of 8, chloride ion and hydrogen peroxide were as
Cl
Cl
Cl
O
OH
O
O
CPO
H O KCl
2
2
4
for the standard assay conditions.
IL/phosphate buffer
8
9
CPO was observed not to chlorinate 8 when the reac-
tions were carried out in the presence of ILs 1, 3, 4
and 5. No absorbance changes were detected for the per-
iod of the assay also adding very low amounts of these
ILs (5%). At variance, CPO activity was observed in
Scheme 1. CPO catalyzed chlorination of 8.
First, CPO activity has been determined by examining
the chlorination of monochlorodimedon, 8 (Scheme 1).
This reaction, initially employed to characterize chloro-
peroxidase, nowadays represents a useful spectrometric
assay to determine enzymatic activity in non-aqueous
the presence of [mmim][Me PO ], [N
][H PO ]
2
4
1112OH
2 4
and [N1112OH][Citr] (2, 6 and 7). The results, expressed
in terms of relative velocity, are reported in Table 1
and depicted in Figure 2. The relative velocity is the ra-
tio of the initial rate in the presence of the ionic solvent,
over the rate in pure aqueous buffer solution. In Figure
2, the data related to the activity of CPO in the presence
4
media.
The ionic liquids used for this test were chosen such that
their cut off point was less than 278 nm, the wavelength
4
of molecular solvents is also reported. Experiments in
Table 1. Relative velocities (V
r
) of CPO chlorination of 8 in the presence of ILs
Run
% (v/v) of co-solvent in potassium phosphate buffer
V
r
pH
1
2
3
4
5
6
7
8
9
5
10
20
30
40
5
10
15
5
[N1112OH][Citr]
[N1112OH][Citr]
[N1112OH][Citr]
[N1112OH][Citr]
[N1112OH][Citr]
0.54
0.27
0.185
0.0409
—
2.86
1.45
—
3.74
[N1112OH][H
[N1112OH][H
[N1112OH][H
[N1112OH][H
2
2
2
2
PO
PO
PO
PO
4
4
4
4
]/[H
]/[H
]/[H
]
3
3
3
PO
PO
PO
4
]
4
]
4
]
2.02
3.53
4.64
5.42
—
—
—
10
11
12
13
14
15
16
17
18
19
20
5
5
5
[N1112OH][OAc]/[HOAc]
[N1112OH][OAc]
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
2
2
2
2
2
2
2
2
2
PO
PO
PO
PO
PO
PO
PO
PO
PO
4
4
4
4
4
4
4
4
4
]
]
]
]
]
]
]
]
]
0.770
0.58
0.455
0.347
0.292
0.898
0.286
0.241
10
15
20
30
30
40
50
90
3.24
a
2.7
b
n.d.
a
Reaction performed in a buffer–ionic liquid mixture (30% v/v), after correcting the pH value to 2.7 using a proper potassium phosphate buffer.
The strong absorbance of the ionic liquids prevented the kinetic study.
b

C. Chiappe et al. / Tetrahedron Letters 47 (2006) 5089–5093
5091
1
0
0
0
0
0
.000
.800
.600
.400
.200
.000
Subsequently, we investigated the CPO catalyzed sulfox-
idation reaction, an oxidative process often performed
under completely different pH conditions (citrate buffer,
pH 5.0).
[
N1112OH][Citrate]
[
mmim][Me PO ]
2
4
AcN
MeOH
DMF
DMSO
CPO from C. fumago is known to be the heme peroxi-
dase of first choice for sulfoxidation reactions owing
2
,15
to its high enantioselectivity.
Sulfoxidation of phenyl
methylsulfide, thioanisole (10), by hydrogen peroxide in
the presence of CPO was therefore investigated both in
pure aqueous citrate buffer solution (0.1 M, pH 5.0)
and in the presence of increasing amounts of ILs
(Scheme 2). Generally, in CPO-catalyzed oxidations
the oxidant is kept as low as possible by a slow addition
to the reaction mixture, to avoid enzyme inactiva-
0
5
10
15
20
25
30
35
40
45
50
%
solvent (v/v)
r
Figure 2. Relative velocities (V ) of CPO chlorination of 8 in the
presence of ILs and molecular solvents.
7
,16
tion.
However, aiming to make CPO a more useful
molecular solvents were carried out under identical con-
ditions to those used in the present investigation.
catalyst for synthetic applications, we have employed
8
the procedure recently reported by Savelli for CPO–
oxidation in water–poly(ethylene glycol)s. H O was
used as oxidant: 1 equiv was added in a unique aliquot
at the beginning of the reaction and 0.5 equiv after
2
2
As shown in Table 1, the enzyme activity varies signifi-
cantly with the nature and concentration of the IL; as
in conventional organic solvents, increasing amounts
of IL progressively lowered the catalytic capability of
the CPO. CPO shows, however, a higher tolerance to-
wards ionic liquids than organic solvents: at least in
the case of [mmim][Me PO ], amounts of IL signifi-
2
h. The reaction was carried out at room temperature
for 4 h. Sulfoxide (11) and sulfone (12) were extracted
by diethyl ether and crude reaction mixtures were ana-
lyzed after addition of anisole as an internal standard
by GC (on a chiral column) to determine the overall
yield, the ratio between sulfoxide/sulfone and the enan-
tiomeric excess of sulfoxide 11.
2
4
cantly higher than 20% v/v can be added before a com-
plete lack of enzyme activity is recorded. In all examined
molecular solvents (dimethyl sulfoxide, dimethyl form-
amide, methanol and acetonitrile) the relative velocity
The results are reported in Table 2. The chiral phenyl
4
decreased below 0.1 in the presence of 20% of organic
methyl sulfoxide obtained always had the R absolute
solvent. Moreover, it is worth noting that in the pres-
ence of small amounts of [N1112OH][H PO ][H PO ]
1
3
configuration, as in pure buffer solution.
2
4
3
4
(
<5%) the enzyme is able to perform the chlorination
As previously observed in the chlorination reaction, the
CPO catalyzed oxidation of phenyl methylsulfide is af-
fected by the nature of the IL. In the presence of
of MCD with a rate superior to that observed in buffer
solution.
[
mmim][MeSO ] [Mor ][MeSO ] and [N1112OH][H2-
4 11 4
Since CPO activity is affected by the hydronium concen-
tration we measured with a glass electrode the pH
change due to the IL addition. Although these measure-
PO ]/[H PO ] (1, 4 and 6), CPO lost completely its activ-
4
3
4
ity and only racemic sulfoxide 11 was isolated. At
variance, enantiomerically pure sulfoxide (R)-11 was ob-
tained performing the incubations in the presence of
1
4
ments reflect both bulk and surface effects, it is evident
that the presence of IL influences the measured pH and
the enzyme activity. The addition of [mmim][Me PO ]
[
mmim][Me PO ], [N1112OH][OAc] and [N1112OH][Citr]
2 4
2
4
(
2, 5 and 7). Using the corresponding aqueous buffer–
(
(
30% v/v) to the potassium phosphate buffer solution
pH 2.7) enhances the pH of the medium to 3.74 deter-
IL mixtures, it is possible to obtain selectively (11:12
ratio may rise the value of 98:2), in high yield (>74%,
run 9) sulfoxide (R)-11 characterized by an enantiomeric
excess >99%. Generally, the higher conversions have
been obtained in 1:1 IL/citrate buffer solutions although
the enzyme maintained part of its activity also in the
presence of higher amounts of IL (70%). The amount
of IL in the presence of which it is possible to perform
sulfide oxidation is therefore significantly higher than
that found for chlorination activity (see above) and than
that recently reported for indene oxidation (20% of
mining a significant decrease in the CPO chlorination
activity (V = 0.292). However, when the same amount
r
of [mmim][Me PO ] was added to a potassium phos-
2
4
phate buffer and the pH was adjusted to 2.7 after IL
addition the V measured was not very far from unity
r
(
V = 0.898). The ability of the IL to affect the medium
r
pH may be therefore considered one of the factors able
to affect the activity of CPO in these non-conventional
media.
O
O O
S
CPO
S
S
CH3
CH3
CH3
+
H O
2
2
IL/citrate buffer
10
11
12
Scheme 2. CPO catalyzed sulfoxidation of 10.

5
092
C. Chiappe et al. / Tetrahedron Letters 47 (2006) 5089–5093
and CPO at room temperature in IL/citrate buffer
Table 2. Oxidation of phenyl methylsulfide 10 with H
2 2
O
b
Run
% (v/v) of co-solvent in citrate
buffer
pH
CPO U
Conv. (%)
11:12
(R)-11 ee (%)
a
1
2
3
4
5
6
7
8
9
0
0
5.0
5.0
3.84
3.84
67.4
0
67.4
0
67.4
67.4
67.4
0
67.4
67.4
67.4
67.4
0
35
8
89:11
67:33
90:10
86:14
95:5
90:10
98:2
70:30
98:2
70:30
100:0
90:10
79:21
90:10
86:14
97
0
>99
0
>99
>99
95
30
30
50
70
30
30
50
70
30
30
30
50
70
[N1112OH][Citr]
[N1112OH][Citr]
[N1112OH][Citr]
[N1112OH][Citr]
38
11
48
17
37
8
76
19
42
25
11
24
10
[mmim][Me
[mmim][Me
[mmim][Me
[mmim][Me
2
PO
2
PO
2
PO
2
PO
4
4
4
4
]
]
]
]
4.93
4.93
0
>99
>99
>99
50
0
38
10
11
12
13
14
15
[N1112OH][OAc]
6.08
4.80
[N1112OH][OAc]/[HOAc]
[N1112OH][OAc]/[HOAc]
[N1112OH][OAc]/[HOAc]
[N1112OH][OAc]/[HOAc]
67.4
67.4
38
a
Citrate buffer 0.1 M, pH 5.0.
b
Determined by GC on a chiral 30 m Chiradex G-TA (ASTEC) column (helium flow 50 kPa, with evaporator and detector set at 200 °C, column
temperature 90 °C per 1 min, 8 °C/min, 170 °C).
[
mmim][MeSO ] in citrate buffer).9a Although more
2. Dembitsky, M. V. Tetrahedron 2003, 59, 4701–4720.
4
3
4
5
. Van Deurzen, M. P. J.; Seelbach, K.; Van Rantwijk, F.;
Kragl, U.; Sheldon, R. A. Biotransform. 1997, 15, 1–16.
. Loughlin, W. A.; Hawkes, D. B. Bioresour. Technol. 2000,
factors are responsible for the activity of enzymes in
ILs, probably in this case the pH of the medium is an
important factor able to affect enzyme activity. Racemic
sulfoxide 11, arising exclusively from the chemical
oxidation, was formed in the presence of ILs able to
shift the pH of the medium at values higher than 6.0
7
1, 167–172.
. Han, Y. J.; Watson, J. T.; Stucky, G. D.; Butler, A. J.
Mol. Catal. B 2002, 17, 1–8; Van de Velde, F.; Bakker, M.;
Van Rantwijk, F.; Rai, G.; Hager, L. P.; Sheldon, R. A.
J. Mol. Catal. B 2001, 11, 765; Aoun, S.; Baboulene, M. A.
J. Mol. Catal. B 1998, 4, 101.
or lower than 2.7 ([N1
112OH
][H PO ]/[H PO ], [Mor ]-
2 4 3 4 11
[
MeSO ] and [mmim][MeSO ]). Even for this oxidation
4 4
process, therefore, the best ILs to use as co-solvents
are [mmim][Me PO ], cholinium citrate and cholinium
acetate.
6. Petri, A.; Gambicorti, T.; Salvadori, P. J. Mol. Catal. B
2004, 27, 103–105.
2
4
7
8
9
. Trevisan, V.; Signoretto, M.; Colonna, S.; Pironti, V.;
Strukul, G. Angew. Chem., Int. Ed. 2004, 43, 4097–4099.
. Spreti, N.; Germani, R.; Incani, A.; Savelli, G. Biotechnol.
Prog. 2004, 20, 96–101.
. (a) Sanfilippo, C.; D’Antona, N.; Nicolosi, G. Biotechnol.
Lett. 2004, 26, 1815–1819; (b) Okrasa, K.; Guib e´ -Jampel,
E.; Therisod, M. Tetrahedron: Asymmetry 2003, 14, 2487–
In conclusion, results reported here are clearly evident
about the potential to use ILs as co-solvents for CPO
catalyzed reactions. As compared to the behavior
observed in conventional organic solvents, CPO in ILs
presents enhanced activity, stability and selectivity.
Moreover, the presence of IL increases substrate solubil-
ity in the reaction medium. However, to take full advan-
tage of these perceived benefits, it is necessary to choose
the ionic liquid accurately. The ability of the IL anion to
modify the medium pH is probably the first parameter
to consider in the selection of the IL to use in CPO cat-
alyzed reactions.
2
490; (c) Machado, M. F.; Saraiva, J. M. Biotechnol. Lett.
2005, 27, 1233–1239.
10. Earle, M. J.; Seddon, K. R. Pure Appl. Chem. 2000, 20,
1391; Welton, T. Chem. Rev. 1999, 99, 2071–2083;
Wasserscheid, P.; Keim, W. Angew. Chem., Int. Ed.
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References and notes
4
37.
1
. Morris, D. R.; Hager, L. P. J. Biol. Chem. 1996, 241,
13. Synthesis of cholinium-based ionic liquids: a choline
hydroxide methanol solution was added dropwise to an
equimolar (for neutral ILs) or a two fold excess (for the
acidic ILs) solution of the organic or inorganic acid. The
mixture was stirred under cooling for 12 h, then methanol
1
763–1768; Zaks, A.; Dodds, D. R. J. Am. Chem. Soc.
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TIBTECH 1999, 17, 163–168; Van Rantwijk, F.; Sheldon,
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was evaporated under reduced pressure. Cholinium citrate
1
[N1112OH][Citr]: H NMR (MeOH-d ) d (ppm): 3.79
6

C. Chiappe et al. / Tetrahedron Letters 47 (2006) 5089–5093
5093
À
13
(
3
(
m, 2H, CH
CH N); 2.64–2.50 (AA’BB’system, 4H). C NMR
MeOH-d ) d (ppm): 177.93; 174.1; 74.15; 68.95; 57.09;
2
OH); 3.26 (m, 2H, CH
2
N); 2.94 (s, 9H,
CH
3
COO ). C NMR (DMSO-d
6
) d (ppm): 173.78;
1
3
3
67.30; 54.94; 53.10; 25,45. ESI-MS (MeOH): positive
ion, 104.12 [N1112OH] ; negative ion, 59 [CH COO] .
+
À
6
3
5
4.73; 44.28. ESI-MS (MeOH): positive ion, 104.12
14. Salis, A.; Pinna, M. C.; Bilani cˇ ova, D.; Monduzzi, M.; Lo
Nostro, P.; Ninham, B. W. J. Phys. Chem. B 2006, 110,
2949–2956.
+
À
[N1112OH] ; negative ion, 191.01 [citrate] . Cholinium
1
phosphate [N1112OH][H
2
4 2
PO ]: H NMR (D O) d (ppm):
3
.76 (m, 2H, CH OH); 3.24 (m, 2H, CH N); 2.91 (s, 9H, 3
15. Fernand e´ z, I.; Khiar, N. Chem. Rev. 2003, 103, 3651–
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16. Colonna, S.; Gaggero, N.; Manfredi, A.; Casella, L.;
Gullotti, M.; Carrea, G.; Pasta, P. Biochemistry 1990, 29,
10465–10468.
2
2
1
3
CH N). C NMR (D O) d (ppm): 68.18; 56.36; 54.64;
4
3 2
1
4.33. Cholinium acetate [N1112OH][OAc]: H NMR
(
DMSO-d ) d (ppm): 4.63 (m, 2H, CH OH); 4.23 (m,
6
2
2
H, CH
2
N); 3.92 (s, 9H, 3 CH
3
N); 2.41 (s, 3H,