Chemoenzymatic Halogenation of Phenols by using the Haloperoxidase from Curvularia inaequalis

Article abstract of DOI:10.1002/cctc.201500862

The vanadium-dependent chloroperoxidase from Curvularia inaequalis is an efficient biocatalyst for the in situ generation of hypohalous acids and subsequent electrophilic oxidation/halogenation reactions. Especially, its superb activity and stability unde

Full text of DOI:10.1002/cctc.201500862

DOI: 10.1002/cctc.201500862
Communications
Chemoenzymatic Halogenation of Phenols by using the
Haloperoxidase from Curvularia inaequalis
[a]
[a]
[b]
[c]
[a]
Elena Fernµndez-Fueyo, Marco van Wingerden, Rokus Renirie, Ron Wever, Yan Ni,
[d]
[a]
Dirk Holtmann, and Frank Hollmann*
The vanadium-dependent chloroperoxidase from Curvularia
inaequalis is an efficient biocatalyst for the in situ generation
of hypohalous acids and subsequent electrophilic oxidation/
halogenation reactions. Especially, its superb activity and stabil-
ity under operational conditions make it an attractive catalyst
for organic synthesis. Herein, the efficient bromination of
thymol was investigated, and turnover numbers of the enzyme
were found to exceed 2000000. The major novelty of the work
is that vanadium chloroperoxidase is more useful as a bromi-
nating enzyme than vanadium bromoperoxidase in terms of
operational stability, besides being far more stable than heme-
containing peroxidases.
dases are of interest here, as their stability towards the oxidant
(H O ) is significantly higher than that of the corresponding
2
2
heme-dependent haloperoxidases, which suggests simpler and
[
3]
more robust reaction schemes.
In the present study, we evaluated the vanadium-dependent
chloroperoxidase from Curvularia inaequalis (CiVCPO) as a cata-
[
3c,4]
lyst for the chemoenzymatic halogenation of phenols.
In
addition to its halogenating activity, the vanadium chloroper-
oxidase also catalyzes the sulfoxidation of sulfides to sulfoxi-
[
4d]
des
and is able to oxidize 2,2’-azino-bis(3-ethylbenzthiazo-
line-6-sulfonic acid) (ABTS), a classical chromogenic heme per-
oxidase substrate, and the industrial sulfonated azo dye Chica-
[
4d]
go Sky Blue 6B.
As a model substrate for our studies we chose thymol
(Scheme 1) because of the interesting antimicrobial activity of
The halogenation of phenols by using elementary hypervalent
halogen species is a well-known and well-established reaction
in organic synthesis. Hypochlorites and hypobromites are fre-
quently used as reagents, but following an in situ generation
strategy to generate the reactive hypohalogenites may be
more advantageous. To attain this goal, vanadium-based cata-
[
5]
its halogenated derivatives.
[
1]
À
À
lysts can be used to catalyze the formation of OBr or OCl
ions from the corresponding halides by using H O as the oxi-
2
2
dant. However, owing to the relatively poor activity of these
catalysts, high molar ratios of substrate to catalyst (S/C) are
necessary to attain full conversion within a reasonable time
frame. Alternatively, haloperoxidases enable significantly lower
catalyst loadings owing to the intrinsically higher activity of
Scheme 1. Simplified reaction scheme for the CiVCPO-mediated halogena-
tion of thymol. CiVCPO catalyzes the formation of hypohalites (XO ), which
then undergo chemical electrophilic substitution at the phenol moiety.
À
[
2]
these catalysts. Particularly, so-called vanadium haloperoxi-
Production of CiVCPO
CiVCPO was obtained from heterologous expression in re-
combinant Escherichia coli by following a previously reported
[
a] Dr. E. Fernµndez-Fueyo, M. van Wingerden, Y. Ni, Dr. F. Hollmann
Department of Biotechnology
Delft University of Technology
[
4c]
procedure. From a 2 L scale fermentation, 30 mg of essen-
tially pure CiVCPO was obtained by using a two-step purifica-
tion procedure. The specific activity (Aspec) [monochlordimedon
Julianalaan 136
2
628BL Delft (The Netherlands)
E-mail: f.hollmann@tudelft.nl
(
MCD) assay] of the enzyme was 22.5Æ0.1 UmgÀ1.
[
b] Dr. R. Renirie
IVAM UvA BV
Plantage Muidergracht 24
Characterization of CiVCPO
1
018TV Amsterdam (The Netherlands)
[
c] Prof. Dr. R. Wever
Van’t Hoff Institute for Molecular Sciences (HIMS), Faculty of Science
University of Amsterdam
In a first set of experiments, we evaluated the basic biochemi-
cal properties of CiVCPO. As shown in Figure 1, the pH range
of CiVCPO is fairly broad with an optimum at approximately
pH 5.
Science Park 904
1
098 XH Amsterdam (The Netherlands)
[
d] Dr. D. Holtmann
Acidic pH values were rather detrimental to the activity of
the enzyme with half-life times of a few hours. Notably, the ap-
parent inactivation is not due to enzyme denaturation but
rather due to dissociation of the catalytically active vanadate
from the active site of the enzyme. Preliminary experiments
DECHEMA Research Institute
Theodor-Heuss-Allee 25
6
0486 Frankfurt am Main (Germany)
Supporting Information (experimental procedures) for this article is avail-
able on the WWW under http://dx.doi.org/10.1002/cctc.201500862.
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Figure 1. pH range of CiVCPO. Activity (^) and stability (&, residual activity
after 24 h incubation) at different pH values. Conditions: 100 mm B&R buffer,
5
2 2
0 mm MCD, 5 mm KBr, 5 mm H O , 10 nm CiVCPO, T=308C; 100% corre-
À1
À1
sponds to a Aspec(CiVCPO) of 118 Umg (TOF=131 s ).
adding additional vanadate indeed suggested that even at low
pH values the full activity can be reconstituted.
Next, we investigated factors influencing the robustness of
the enzyme. In contrast to heme-dependent chloroperoxidases,
CiVCPO exhibited superb stability in the presence of H O2
2
(
Figure 2): upon exposure to even 100 mm H O for more than
2 2
1
1
day, the enzyme activity decreased by only approximately
4%. Compared to the very high sensitivity of heme-depen-
dent peroxidases towards even micromolar concentrations of
H O , but also compared to the vanadium-dependent perox-
2
2
[6]
idase from Ascophyllum nodosum, the stability of CiVCPO is
impressive. Furthermore, the stability of CiVCPO against polar
water miscible and immiscible solvents such as methanol and
ethyl acetate was excellent and showed no significant decrease
even in the presence of 50 vol% after 3 days of incubation. The
unexpected apparent increase in the activity of CiVCPO over
time in the presence of ethyl acetate is most likely due to slow
hydrolysis of ethyl acetate and concomitant acidification of the
incubation medium (see also Figure 1).
Overall, we concluded that CiVCPO is a very robust biocata-
lyst that may potentially be useful for chemical halogenation
reactions.
CiVCPO-mediated thymol halogenation
Encouraged by these promising catalytic and stability proper-
ties, we set off to evaluate the CiVCPO-mediated halogenation
of thymol (Scheme 1). We were pleased to find that under the
Figure 2. Stability of CiVCPO in the presence of H O (top), methanol
2
2
(
middle), and ethyl acetate (bottom). The x axes indicate the concentrations
(
arbitrarily chosen) reaction conditions significant chlorination
and bromination of thymol occurred. Notably, no conversion
of thymol was observed if CiVCPO, H O , or the halide was
of H and the solvents. From left to right (light gray to black), the relative
residual activity determined after 0, 1, 2, 3, and 27 h of incubation for H
and after 0, 19, 45, and 66 h for methanol and ethyl acetate.
2
O
2
2 2
O
2
2
omitted from the reaction mixture. Interestingly, the bromina-
tion reaction was complete within 6 h, whereas the corre-
sponding chlorination reaction (under otherwise identical reac-
tion conditions) gave approximately 50% conversion
(Figure 3). Most likely, this can be attributed to the different ki-
À
netic properties of CiVCPO towards Cl , which necessitates
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Hence, the apparent turnover frequency (TOF) of CiVCPO (de-
termined as an average rate over the entire reaction) increased
À1
from 2.2 to over 55 s .
À
Higher concentrations of Br led to very significant inhibition
of CiVCPO (data not shown), which is why we decided to opt
À
for a continuous provision of both substrates, H O and Br ,
2
2
for some larger-scale reactions. Trials aiming at a slurry-to-
slurry reaction setup (i.e., by using no co-solvents to dissolve
the thymol) were feasible but suffered from severe mass-bal-
ance issues. Therefore, we used ethanol as a co-solvent, which
À1
enabled homogeneous solutions of up to 200 mm (30 gL ) of
thymol under the reaction conditions. A typical time course of
such a reaction is shown in Figure 4.
Within less than 24 h thymol was converted into the corre-
sponding 2,4-dibromothymol. Interestingly, under these reac-
tion conditions hardly any formation of 2-bromothymol was
observed (less than 1 mm), whereas the 4-bromo isomer ap-
peared to be the primary product of the first halogenation
step. Currently, we are lacking a plausible explanation for this
apparent discrepancy. This may possibly be due to the contin-
uously low bromide concentration relative to that for the reac-
tions reported in Table 1. Apparently, the overall product for-
mation rate in this experiment (Figure 4) was limited by the
À
À1
addition rate of H O /Br (8 mmfinal h ). Further investigations
2
2
will be necessary to understand this unexpected selectivity of
the chemical bromination step.
Finally, this setup also allowed for the semipreparative bro-
mination of thymol (1.5 g), which yielded practical amounts of
pure products after column chromatography (94, 690, and
1
38 mg of 2-bromo-, 4-bromo-, and 2,4-dibromothymol, re-
spectively; refer to the Supporting Information for further de-
tails).
Figure 3. Chromatograms of the chlorination (top) and bromination
bottom) reactions. In the case of bromination, the chemical structures were
(
In this study we demonstrated that vanadium-dependent
chloroperoxidase from Curvularia inaequalis (CiVCPO) is indeed
a potentially very useful catalyst for the in situ generation of
hypohalous acids (especially HOBr) for subsequent chemical
oxidation/halogenation reactions. As a model reaction, the
electrophilic halogenation of thymol was chosen.
confirmed with authentic standards. In the case of chlorination, the products
were identified by GC–MS. Reaction conditions: 5 mm thymol in 100 mm
citrate buffer (pH 5), 10 mm KBr or KCl, 18 nm VCPO, and 1 mm H
0 min for 6 h.
2 2
O every
3
either lower pH values or significantly increased chloride con-
À [4f]
centrations compared to Br .
Under semioptimized reaction conditions, full conversion of
À1
Notably, the product distributions of both the bromination
and the chlorination reactions corresponded to the pattern ob-
tained through chemical halo-
thymol (30 gL ) into 2,4-dibromothymol was achieved. Princi-
pally, under kinetic control, selective monobromination is also
genation. Therefore, we assume
[a]
2 2
Table 1. Influence of varying KBr/H O ratios on the chemoenzymatic halogenation reaction.
that the role of CiVCPO is limit-
ed to the formation of freely dif-
À
fusing XO .
In further experiments we fo-
cused on bromination. As
shown in Table 1, the starting
Entry
[H
[mm]
2
O
2
]
0
=[KBr]
0
[2]
[mm]
[3]
[mm]
[4]
[mm]
Product formation
À1
concentration of H O and KBr
rate [mmh
]
2
2
(
using equimolar concentrations
1
2
3
4
5
1
4
8
16
20
0.29Æ0.24
0.69Æ0.028
0.58Æ0.04
1.50Æ0.395
1.96Æ0.09
0.51Æ0.07
3.00Æ0.39
7.34Æ0.42
10.55Æ3.58
15.24Æ0.806
0
0.81
4.63
8.93
14.74
19.8
of H O and KBr) had no signifi-
0.935Æ0.33
1.00Æ0.049
2.68Æ0.43
2.65Æ0.77
2
2
cant effect on the product distri-
bution. The apparent product
formation rate was almost line-
arly dependent on the starting
concentration of both reagents.
[
a] Conditions: 100 mm citrate buffer (pH 5), 20 mm thymol, 100 nm VCPO, T=308C. The reactions were
stopped after 1 h.
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miscible solvents and its facile preparation at scale make it
a very promising catalyst for further applications.
Acknowledgements
This work was financially supported by the European Union
(
KBBE-2013-7-613549, “INDOX”). We thank Remco van Oosten for
excellent technical support. Enzymicals AG (Greifswald, Germany)
is acknowledged for inspiring discussions.
Keywords: biocatalysis
peroxidases · thymol
·
enzymes
·
halogenation
·
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1
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CiVCPO within the first 4 h of the reaction. This underlines the
very high robustness of this enzyme under the strongly oxidiz-
ing reaction conditions. Another indication for this robustness
is the exceptionally high total turnover number of more than
2
000000 (i.e., 0.00005 mol% of catalyst) achieved in these
experiments.
In this respect, CiVCPO clearly excels over other chloroperox-
idases reported such as the heme-dependent chloroperoxidase
[2f]
from Caldariomyces fumago or even the vanadium-depen-
[6]
dent bromoperoxidase from Ascophyllum nodosum. Both en-
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zymes, as a result of their sensitivity to H O , require careful
2
2
control over the in situ concentration of H O . Consequently,
2
2
also for bromination reactions, a vanadium chloroperoxidase
can actually be superior to a vanadium bromoperoxidase. Fur-
thermore, the high robustness of CiVCPO against water (im)-
Received: August 2, 2015
Published online on October 16, 2015
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