Vanadate(v)-dependent bromoperoxidase immobilized on magnetic beads as reusable catalyst for oxidative bromination

Article abstract of DOI:10.1039/c0gc00499e

Vanadate(v)-dependent bromoperoxidase I (Ascophyllum nodosum) was immobilized on magnetic micrometre-sized particles in quantitative yields, with up to 40% retention of initial bromoperoxidase (BPO) activity. The immobilized enzyme was stable with a half-life time of about 160 days. It served as reusable catalyst for bromide oxidation with H₂O₂ in up to 14 consecutive experiments. Reactivity that resulted from enzymatic bromide oxidation was applicable for methyl pyrrole-2-carboxylate conversion into derivatives of naturally occurring compounds (e.g. from Agelas oroides) with product selectivity of up to 75%.

Full text of DOI:10.1039/c0gc00499e

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PAPER
Vanadate(V)-dependent bromoperoxidase immobilized on magnetic beads as
reusable catalyst for oxidative bromination†
a
a
b
b
c
Diana Wischang, Jens Hartung,* Thomas Hahn, Roland Ulber, Tobias Stumpf and
Claudia Fecher-Trostc
Received 30th August 2010, Accepted 27th October 2010
DOI: 10.1039/c0gc00499e
Vanadate(V)-dependent bromoperoxidase I (Ascophyllum nodosum) was immobilized on magnetic
micrometre-sized particles in quantitative yields, with up to 40% retention of initial
bromoperoxidase (BPO) activity. The immobilized enzyme was stable with a half-life time of
about 160 days. It served as reusable catalyst for bromide oxidation with H
2
2
O in up to 14
consecutive experiments. Reactivity that resulted from enzymatic bromide oxidation was
applicable for methyl pyrrole-2-carboxylate conversion into derivatives of naturally occurring
compounds (e.g. from Agelas oroides) with product selectivity of up to 75%.
Introduction
Vanadate(V)-dependent bromoperoxidase I from the brown alga
1
Ascophyllum nodosum [VBrPO(AnI), EC 1.11.1.10] catalyzes the
oxidation of bromide with H
solutions (pH 6.2–6.5). The turnover frequency of this process
exceeds the hitherto most active non-enzymatic alternatives
2
O (Scheme 1) in weakly acidic
2
2
3
by 4–5 orders of magnitudes. The enzyme tolerates elevated
temperatures and a variety of organic co-solvents without
notable loss of its bromoperoxidase (BPO) activity over time
spans typically needed for substrate bromination in organic
4
,5
synthesis.
VBrPOs from other organisms than A. nodosum
2,6,7
were discovered from screenings and purified to homogeneity.
Some bromoperoxidases even were genetically expressed in fungi
or bacteria. None of these enzymes, however, shares similar
favorable characteristics for application in oxidation catalysis
for the pursuit of sustainable hydrocarbon bromination than
VBrPO(AnI).
The debate on the nature of the electrophilic bromination
reagent that is liberated from the enzymatic process has not yet
9–11
been settled (cf. Scheme 1).
The overall reaction, however,
Scheme 1 Stoichiometry for VBrPO(AnI)-catalyzed bromide oxidation
with H (top) and secondary non-enzymatic reactions utilizing
2
O
2
a
Fachbereich Chemie, Organische Chemie, Technische Universit a¨ t
Kaiserslautern, Erwin-Schr o¨ dinger-Straße, D-67663, Kaiserslautern,
Germany. E-mail: hartung@chemie.uni-kl.de; Fax: +49 631 205 3921
electrophilic bromination reagents for arene functionalization (bottom;
a
L = apoenzyme; Ar = aryl or heteroaryl, see below). Proposed
8
b
intermediate in an early phase of the reaction.
Fachbereich Maschinenbau und Verfahrenstechnik, Technische
Universit a¨ t Kaiserslautern, Gottlieb-Daimler-Straße, D-67663,
Kaiserslautern, Germany
Fachbereich Biologie, Proteinfunktion/Proteomics, Technische
Universit a¨ t Kaiserslautern, Paul-Ehrlich-Straße, D-67663,
Kaiserslautern, Germany
has attracted considerable attention. It has been associated with
formation of naturally occurring organobromine compounds,
although none of the proposed pathways have so far been
c
10,12
biosynthetically proven.
In terms of organic synthesis, the
†
Electronic supplementary information (ESI) available: Standard in-
strumentation, BPO-analysis, determination of BPO-activity, synthesis
use of VBrPO(AnI) as catalyst for oxidative brominations in
of methyl 1H-pyrrole-2-carboxylate (3), details for the use of D-Glc/GO
ocean water-like media probably constitutes the most sus-
for in situ H
2
O
2
generation in BPO-catalyzed oxidations, details for the
13–16
tainable version of the on-site bromination of arenes,
as
use of VBrPO(AnI)-preparations 1a and 2b in multiple runs. See DOI:
recently documented by bromofunctionalization of benzenoid,
1
0.1039/c0gc00499e
1
02 | Green Chem., 2011, 13, 102–108
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5
non-benzenoid, and heterocyclic arenes on an analytical scale.
of measured tryptic peptide masses, in comparison with the
known primary structure of the enzyme (PDB 1QI9). Activity
of VBrPO(AnI) preparations ranged between 405 ± 43 and 526 ±
For further exploration of this chemistry and its development
into a widely applicable method, the aspect of catalyst supply,
however, requires particular attention. Since the enzyme is not
yet biotechnologically available, it needs to be extracted from the
genuine source. The process, although well elaborated, is time
and material consuming. BPO immobilization onto a solid
support, in this sense would open perspectives for its repetitive
use and thus a reduction of the amount needed for conducting
larger scale studies. Structural complexity of surfaces, however,
still precludes clear cut predictions on efficiency of catalyst
immobilization, its durability and its activity during turnover
in close proximity to the solid liquid interface.
As part of a project on the chemistry of VBrPO(AnI) in organic
synthesis we chose to immobilize the BPO on magnetic beads. It
was the aim to obtain a robust oxidation catalyst for multiple use
in sustainable hydrocarbon bromination. The most important
findings from this study showed that binding of the enzyme to
epoxide- and amino-functionalized micrometre-sized magnetic
supports was feasible in quantitative yields with up to 40%
retention of the initial BPO activity. This material served for
at least 14 consecutive runs as catalyst for biomimetic methyl
-
1
22
5,23
11 U
T
mg (triiodide assay in phosphate buffer at pH 6.2).
‡
2
Enzyme immobilization
17
Magnetic beads (M) were chosen as support, which were coated
with a matrix of polyvinyl alcohol (PVA) and functionalized by
the supplier with epoxide groups (E), or aminated with an eight
atomic spacer (N) (see Experimental). The magnetite level was
18
19
5
0–60% (w/w), epoxide group loading 200–220 mmol, amino
group loading 650–750 mmol per g of particle. Particles with a
narrower (0.5–1.3 mm for E01 and N11) and a broader (0.7–
4.5 mm for E02 and N12) polydisperse size distribution were
used for both types of functionalized beads. The materials were
sufficiently attracted by a Nd/Fe bar magnet (20 ¥ 10 ¥ 5 mm,
N42 magnetization) to allow swift and quantitative separation
from a suspension mimicking a standard reaction mixture, as
evident from optical density measurements (600 nm) (Fig. 2 and
ESI).
20
pyrrole-2-carboxylate bromination.
Results and interpretation
1
Enzyme purification and characterization
VBrPO(AnI) was separated from a mixture of isoenzymes accord-
ing to a published procedure, starting from lyophyllized A. no-
dosum (Fig. 1). The enzyme was stored in tris-(hydroxymethyl)-
aminomethane(HCl), i.e., Tris(HCl)-buffered stock solutions
◦
21
(
pH 9.0) at 4 C. Bromoperoxidase activity was retained
with a half-life time of approximately 120 days under such
conditions. The identity of the enzyme was verified via MALDI-
TOF analysis of trypsine-digested samples and database analysis
Fig. 2 Optical density measurement (600 nm) for checking magnetic
attraction of applied beads composed of polyvinyl alcohol (PVA)-coated
magnetite (M) particles having epoxide (E) or amino (N) endgroups (for
size distribution of the particles refer to Table 1; Nd/Fe bar magnet).
V
BrPO(AnI) loading onto epoxide-functionalized supports M-
◦
PVA E01 and E02 at 23 C was feasible after adjusting pH to 6.2
with MES buffer and mixing of the components. Preparations
obtained from these manipulations retained 29% (1a) and 17%
(
1
1b) of initial BPO activity as evident from triiodide tests (Table
, entry 1–2). BPO immobilization on amino-functionalized
supports was attainable via in situ-treatment of M-PVA N11 and
24
N12 with glutardialdehyde (GA) in K
pH 8.0) for 1 h at 23 C, which was followed by addition of the
2
HPO buffer (20 mM,
4
◦
V
(
2
BrPO(AnI)-stock solution in aqueous Tris(HCl) buffer (pH 9.0)
Scheme 2). The latter two reactions furnished materials showing
4% (2a) and 37–39% (2b) of the original BPO activity (Table 1,
entries 3–4). The activity values were in a reproducible manner
Fig. 1 Polyacrylamide gel electrophoresis (1 h, 18 mA; non-denaturing
conditions) of crude bromoperoxidase fraction (right) in comparison
to VBrPO(AnI) preparation obtained from hydrophobic interaction and
size exclusion chromatography (left, BPB = bromophenol blue; staining
‡
Notation for enzymatic activity: 1 unit (U) refers to the amount of
enzyme required for turning over 1 mmol of substrate per minute. BPO-
activity determined with the aid of the triiodide assay (T) was abbreviated
as U . Specific BPO-activity referred to the number of units per mg of
enzyme preparation, i.e. U
was performed in a solution prepared from phosphate buffer, H
2
O
2
, aq.
T
-1
KI, and 1,4-phenyldiammonium dichloride).
T
mg .
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Table 1 Characteristics of immobilized VBrPO(AnI)‡
0
a
0
mg-1 b
BPO-loading/mg g-1 bead
loaded
mg-1c
Entry
Support
Particle size/mm
1–2
m
/mg
U
T
U
T
1
2
3
4
5
M-PVA E 01
M-PVA E 02
M-PVA N 11
M-PVA N 12
M-PVA N 12
0.5–1.9
0.7–6.0
0.5–1.9
0.7–6.0
0.7–6.0
1a
1b
2a
2b
2b
0.14
0.14
0.10
0.08
0.10
405 ± 43
405 ± 43
493 ± 6
446 ± 24
493 ± 6
2.82
2.15
2.13
1.60
2.13
118 ± 12
69 ± 7
120 ± 28
168 ± 47
194 ± 39
a
b
c
Amount of VBrPO(AnI) prior to immobilization. Bromoperoxidase (BPO)-activity of VBrPO(AnI) preparation (triiodide test). BPO-activity of
immobilized VBrPO(AnI) (triiodide test).
7
0 days. In solution this activity loss prevailed for the free
enzyme. For immobilized VBrPO(AnI), BPO activity from this
point on remained approximately constant (Fig. 3). Although
triiodide BPO activity tests using suspensions of preparation
1–2 showed stronger scatter than for homogeneous solutions
containing the free enzyme, presumably for the time span re-
quired for quantitative magnetic attraction prior to UV/Vis data
collection, the outlined trends were diagnostic and reproducible.
In view of the activity decay, oxidation experiments summarized
in the following section were conducted with freshly immobilized
materials 1–2.
Scheme 2 Immobilization of VBrPO(AnI) on polyvinyl alcohol (PVA)-
functionalized (E or N) magnetite (M) (GA = glutardialdehyde; solid
supports drawn as circles of an arbitrary radius; for further abbreviations
a
b
see legend of Fig. 2, for results Table 1). MES buffer. Phosphate buffer.
Tris(HCl) buffer.
c
independent from the batch of enzyme used for immobilization
(
section 2.1 and Table 1, entries 4–5). The data also clarified
that VBrPO(AnI) binding was not inhibited by the presence of
an excess of Tris from the buffer.
Yields of VBrPO(AnI) binding to functionalized supports were
essentially quantitative, as judged on the basis of the Bradford
Fig. 3 Time dependence of specific BPO-activity of immobilized
◦
V PO(AnI) stored at 4 C in MES buffer (samples 1a–b) or Tris(HCl)
Br
25,26
assay.
Since crystalline VBrPO(AnI) was not available in
buffer (samples 2a–b; error bars refer to standard deviations from three
sufficient amounts, the protein concentration in solution was
referenced versus bovine serum albumin (BSA). The results
were independently checked via triiodide BPO activity tests
of the VBrPO(AnI) stock solution, the filtrate after separation
of materials 1–2, and MES buffer washings of the latter
preparations.
independent runs).
3
Oxidation catalysis
Conditions suitable for organic compound bromination in
VBrPO(AnI)-catalyzed oxidations were established using methyl
In view of the considerable fraction of nucleophilic end-
1H-pyrrole-2-carboxylate (3) as reporter substrate. The p-excess
8
groups at the periphery of the protein and the inertness of
heteroaromatic compound undergoes substitution of bromine
for hydrogen already with comparatively weakly electrophilic
reagents, such as HOBr or Br , without the necessity of adding
external Lewis acids. It furthermore provides a diagnostic
mixture of mono- and disubstitution products 4 and 5 that
are sufficiently stable to allow quantitative analysis via gas
chromatography and subsequent column chromatographic sep-
aration, for determining the site of aromatic substitution via
active enzyme toward leaching, attachment of VBrPO(AnI) to
applied beads (Scheme 2) was assumed to occur via covalent
bonds. VBrPO(AnI) immobilization to GA-activated amino-
functionalized beads thereby occurred faster than attachment to
epoxide-substituted particles. For practical reasons, however, the
latter preparations offered the advantage of directly providing
immobilized VBrPO(AnI) in MES-buffered solution, i.e. the
medium the oxidation experiments were performed in. For
experiments using preparations 2a and 2b, washing with MES
buffer was necessary to lower pH from 9.0 to 6.2, i.e. the value
required for attaining maximum BPO activity.
2
2
7
2
7,28
NMR spectroscopy.
Since pyrrole-2-carboxylic acid in nature
29
is formed from the amino acid metabolism, the experiments
were considered to be relevant for providing insight into genesis
of naturally occurring, brominated pyrroles.
Long term studies indicated that storage of preparations 1a–
b in MES buffer and materials 2a–b in Tris(HCl) buffer at
3.1 Catalyst activity. From parameter variation it was
◦
4
C progressively decreased in BPO activity during the first
found that BPO activity of 1.3 U
T
was necessary to quantitatively
1
04 | Green Chem., 2011, 13, 102–108
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Table 2 Reaction parameters for effective methyl pyrrole-2-carboxylate bromination‡
final
mg-1 a
b
Entry
n
3
/mmol
Conv. 3/%
Time/h
BPO
U
T
U
T
4/% (4/5)
c
1
2
3
4
5
6
36
36
36
36
200
200
quant.
24
5
3
1
3
V
V
V
V
1a
2b
BrPO(AnI)
1.3
1.3
1.3
1.3
6.9
6.9
117
99
239
231
active
active
94 (93/7)
73 (89/11)
60 (82/18)
26 (81/19)
40 (90/10)
32 (93/8)
d
92
BrPO(AnI)
BrPO(AnI)
BrPO(AnI)
d
77
d
27
d
54
d
75
3
a
0
mg-1 for entries 1–4; for entries 5–6 refer to Table 1; active refers to residual enzyme activity as judged by qualitative
c 0
Initial enzyme activity 526 U
T
b
triiodide tests. Ratio of 4/5-regioisomers of bromopyrrole 4.
1
c
3
= 7.2 mM, H
2
O
2
addition in 3 batches every 30 min; total amount of substrates:
and 1.0 equiv. of NaBr.
d
.0 equiv. of H
2
O
2
and 3.0 equiv. of NaBr.
H
2
O
2
added in a single batch; total amount of substrates: 1.0 equiv. of H O
2 2
convert 36 mM of pyrrole 3 into 99% of bromopyrrole 4 within
4 h in MES buffered solution (Table 2, entry 1). H was added
in portions or in a single batch. Its mode of administration had
no effect at this scale on the outcome of the experiment. pH
of standard reaction mixtures slightly increased from 6.2 at the
beginning to 6.6 after quantitative conversion of substrate 3.
for validating catalytic properties of immobilized VBrPO(AnI).
Comparison of product formation and substrate consumption
clarified that the use of the enzyme in homogeneous solutions
and in immobilized form (i.e. 1a and 2b) afforded almost
equivalent turnover rates. A separate study for maximizing yields
therefore was not performed for validating highest performance
of solid phase bound VBrPO(AnI)-preparations, since those
data were indirectly available from experiments conducted
in homogeneous solution. Additional products for explaining
deviation between turnover of 3 and yield of bromopyrrole 4 in
the analytical scale experiments using immobilized VBrPO(AnI)
1a and 2b (Table 2, entries 5–6) were not detected (GC, NMR).
2
2
O
2
An alternative mode of H
2
2
O administration was pursued by
in situ generation via aerobic oxidation of D-glucose, catalyzed
3
0,31
by D-glucoseoxidase (GO)
1
from Aspergillus niger (1.67 U,
-
1
47 U mg ). In order to effectively combine the two enzymatic
processes, pH of the reaction mixture was set to 5.8. This
value was half way between pH required for maximum activity
of VBrPO(AnI) (6.2) and of the applied GO (5.6). Pyrrole
conversion under such conditions was 97% to furnish 77% of
bromopyrroles 4 and 5 taken together (Scheme 3). Although
interesting by itself, it was refrained from using this method in
the following part of the study since final VBrPO(AnI)-activity
converged toward zero.
VBrPO(AnI)-activity that was conserved in methyl pyrrole-2-
carboxylate bromination in homogeneous solutions was applica-
ble for further experiments after addition of aliquots of substrate
3, H
2
2
O , NaBr, and MES buffer. BPO-activity after the fourth
run, however, fell below a synthetically relevant level (Fig. 4).
Scheme 3 Combination of D-glucoseoxidase (GO)-catalyzed H
2 2
O
generation and VBrPO(AnI)-catalyzed bromide oxidation for sustainable
pyrrole bromination [D-Glc = D-glucose; 6.2 U
T
for VBrPO(AnI); 1.67 U
0
Fig. 4 Efficiency of product formation in VBrPO(AnI)-catalyzed re-
for GO; pH 5.8 (MES); tBuOH/H
2
O; c
3
= 7.2 mM; for stoichiometry
actions in sequential runs [4.96 U
T
corresponding to 0.05 mmol %
of the BPO-catalyzed reaction see Scheme 1].
V
BrPO(AnI); 144 mmol of 3, 1 equiv. of H
2
O
2
and NaBr, H O/tBuOH =
2
7
2
5/25 (v/v); additional product: 10% of methyl 3,4-dibromopyrrole-
-carboxylate (5) in experiment number 1 and <1% in experiment
A survey of the time dependence of bromopyrrole formation
showed that substrate conversion reached a level of approx-
imately 70% after 3 h. Since changes in terms of reactivity
and selectivity originating from the support would be most
evident at this conversion level, a reaction period of 3 h and
thus incomplete substrate turnover was chosen as standard
number 2].
3.2 Repetitive use of immobilized bromoperoxidase. Immo-
bilized samples 1a and 2b, i.e. of the most active materials
from both types of supports were chosen for validating utility
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of immobilized VBrPO(AnI) in oxidation catalysis. The use of
material 2b required an exchange of buffer from Tris(HCl)
originating from the synthesis to MES for conducting oxidative
bromination. MES buffer hereafter served as standard medium
for storing catalyst 2b.
Concluding remarks
Immobilization of VBrPO(AnI) to solid supports provided three
distinctive results relevant for future development of sustainable
oxidative brominations.
(
i) VBrPO(AnI) binding either to epoxide-functionalized or to
Addition of catalyst suspension to aqueous tBuOH contain-
ing reporter substrate 3 was followed by addition of NaBr
glutardialdehyde-activated amino-substituted magnetic beads
was feasible in quantitative yields, with almost 40% of the
original catalyst activity being conserved.
and H
2
2
O . At fixed reaction times of 3 h, catalyst particles
were separated from the solution with a magnet that was held
against the wall of the reaction flask (vide supra). The reaction
medium was decanted off to afford a clear solution that was
worked up for clarifying product identity, yields, and substrate
conversion. Routine analysis showed weak BPO activity in this
solution during the first 3 runs but not beyond this point.
Additional standard checks indicated no detectable BPO activity
in solutions obtained from catalyst washings from runs 1–15.
The next cycle was started by addition of fresh buffer and
substrates in standard concentrations into the flask, which was
followed by removal of the magnet from the exterior of the
reaction vessel. With increasing number of reaction cycles, the
yield of bromopyrrole 4 increased and reached a maximum at
approximately the 5th cycle (Fig. 5). High catalyst performance
prevailed until the 9th cycle. The following runs (10–15), in a
reproducible manner, led to progressive decrease in turnover
and yield. These changes were paralleled by a gradual decrease of
terminal pH of the reaction mixture from 7.8 (1st run) via 7.6 (5th
cycle), 6.8 (9th cycle) to 6.2 (14th cycle), by the time the reaction
was stopped (3 h). The results obtained from oxidations using
catalyst 2b (ESI) were nearly identical in terms of yields and
activity changes upon multiple use, compared to those described
for experiments performed with preparation 1a (Fig. 5).
(
ii) Immobilized VBrPO(AnI) showed similar turnover charac-
teristics for methyl pyrrole-2-carboxylate oxidative bromination
as the free enzyme in homogeneous solutions. The unexpectedly
high performance of immobilized enzyme was explained with
substrate bromination that occurred in bulk solution and not in
proximity to the active site. In this model, diffusion of bromide
to the histidine-bound vanadate(V) co-factor would be rate
determining and not migration of the organic substrate to a
specific binding site.
(iii) Supported bromoperoxidase was applicable for 8–10
consecutive cycles without notable activity loss.
The most significant achievement compared to other
techniques used for instance for immobilization of the
iron-dependent chloroperoxidase from Caldariomyces fumago
31
[
FeClPO(Cf I)] on mesoporous supports was the ease and the
efficiency of magnetic separation of immobilized VBrPO(AnI)
from reaction mixtures. Chemicals originating from a preceding
transformation thus were removable from the catalyst by simply
washing with the buffer needed for the subsequent run. This
aspect has been noted previously for application of immobilized
lipases in biofuel preparation but has not yet been applied to
haloperoxidase chemistry. With this material in hand, applica-
tion of VBrPO(AnI) in oxidative bromination has the potential to
become an interesting alternative in instances where substitution
of molecular bromine is desired for environmental reasons by
32
the reagent combination of H
2
2
O , possibly generated in situ
from aerobic D-Glc oxidation catalyzed by GO, NaBr and an
adequate source of protons.
Experimental
1
General
For instrumentation, comments on laboratory standards, and
details of compound preparations, see the ESI. VBrPO(AnI) was
◦
isolated from A. nodosum collected in April 2004 (France, 48
4
◦
21
3¢ N, 3 58¢ W) according to a published procedure. Magnetic
0
Fig. 5 Conversion of substrate 3 (c = 35 mM; gray columns) and
efficiency of methyl pyrrole-2-carboxylate bromination (black columns)
beads were obtained from chemagen Biopolymer-Technologie
AG, Germany.
upon multiple use of VBrPO(AnI)-preparation 1a (6.9 U
oxidative bromination in aqueous tBuOH (pH 6.2, 23 C) using 1.0
T
) for catalyzing
◦
equiv. each of H
2
O
2
and NaBr (3 h, see text).
2
Immobilization of VBrPO(AnI)
2.1 Immobilization on M-PVA E (epoxy-functionalized sup-
The origin of irreversible loss of catalyst activity remained
unclear. Leaching of active enzyme was excluded on the basis
of the above-mentioned results from routine triiodide tests of
reaction media and catalyst washings. Possible vanadate loss
was checked by soaking the immobilized catalyst in an ortho-
vanadate solution between consecutive oxidation experiments.
This modification, however, had no effect on performance of the
material and activity loss after the 8–10th cycle.
ports). To Tris(HCl) (50 mM, pH 9.0)-buffered VBrPO(AnI)
-
1
solution (0.20 mg mL ) was added MES buffer (1.05 mL,
400 mM, pH 5.5) to adjust pH of the BPO solution to 6.2. M PVA
E01 or E02 (50 mg) was washed with MES-buffer (3 ¥ 1.0 mL;
50 mM, pH 6.2). The 50 mg portion of support was suspended in
-
1
1.0 mL of enzyme solution (protein concentration 0.14 mg mL )
in MES-buffer (109 mM, pH 6.2). The mixture was shaken gently
◦
at 23 C for 48 h. Periodically, samples of the supernatants were
1
06 | Green Chem., 2011, 13, 102–108
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withdrawn and analyzed for protein concentration (Bradford
assay) and enzyme activity (triiodide assay). Solids were washed
with MES-buffer (2 ¥ 1.0 mL; 50 mM, pH 6.2), stored at 4 C in
organic extracts were dried (MgSO
4
). The solvent was removed
◦
under reduced pressure (13 mbar, 40 C) to afford a product
◦
1
mixture which was analyzed by H NMR (CDCl ). Samples
3
MES-buffer (1.0 mL, 50 mM, pH 6.2) and were ready to use as
oxidation catalyst.
of the supernatants from the washing phase as well as the
aqueous layer after extraction were withdrawn and analyzed
for enzyme activity (Triiodide assay). The described procedure
was repeated 15 times with immobilized VBrPO(AnI) 1a from
prior cycle. For analytical data refer to section 4.3.1, for yields
see the ESI, section 8.1. The same procedure was used for the
use of immobilized VBrPO(AnI) 2b. For analytical data refer to
section 4.3.1, for yields see the ESI, section 8.2.
2
.2 Immobilization on M-PVA N (amino-functionalized sup-
ports). M PVA N11 or N12 (50 mg) was washed with distilled
water (3 ¥ 1.0 mL). The supports were activated by adding
1
5
0
.0 mL activation solution {glutardialdehyde solution [4.00 g,
2 2 4
0% (w/v), in H
O, 200 mM] in aqueous KH
PO
(0.27 g,
◦
.02 M)}. The mixture was shaken gently at 23 C for 1 h.
After discarding the supernatant, the activated supports were
washed with distilled water (3 ¥ 1.0 mL) and suspended in
Acknowledgements
-
1
0
.5 mL of enzyme solution (protein concentration 0.20 mg mL )
in Tris(HCl)-buffer (50 mM, pH 9.0). The mixture was shaken
gently at 23 C for 24 h. Periodically, samples of the super-
natants were withdrawn and analyzed for protein concentration
This work was supported by the Deutsche Bundesstiftung
Umwelt (Scholarship for D.W.) and the State of Rheinland-
Pfalz (NanoKat). We also express our gratitude to chemagen for
generous donations of M-PVA E0x and M-PVA N1x particles
used in this study, and to Dr Thomas Sommer (chemagen) for
helpful discussions. This work is part of the Ph.D. thesis of D.W.
◦
(
Bradford assay) and enzyme activity (triiodide assay). Solids
were washed with Tris(HCl)-buffer (2 ¥ 0.5 mL; 50 mM, pH 9.0)
◦
and stored at 4 C in this buffer (0.5 mL, 50 mM, pH 9.0) for
application as oxidation catalyst.
3
Bromination of methyl pyrrole-(1H)-2-carboxylate (3)
Notes and References
3
.1 Reactions in homogeneous solution. A solution of aque-
1 H. Vilter, Phytochemistry, 1984, 23, 1387–1390.
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3
A. Butler and J. V. Walker, Chem. Rev., 1993, 93, 1937–1944.
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2
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three portion (to 333 mL at the beginning, after 0.5 h and 1.0 h)
to a solution of substrate 3 (4.5 mg, 36.0 mmol) in MES-buffer
50 mM, pH 6.2, 3.75 mL) and tBuOH (1.25 mL), NaBr (11.2 mg,
08.0 mmol) and VBrPO(AnI) (7.5 mL, 1.3 U
reaction mixture was stirred at 23 C for 1 d. The aqueous
layer was extracted with Et
extracts were dried (MgSO
reduced pressure (14 mbar, 40 C) to afford a product mixture
which was analyzed by H NMR. pH and bromoperoxidase
activity (triiodide assay) were determined from the aqueous
3
275.
4 E. de Boer, H. Plat and R. Wever, in Biocatalysis in Organic Media,
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(
1
T
, 0.05 mmol%). The
5
6
7
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◦
ˇ
2
O (3 ¥ 5 mL). Combined organic
4
). The solvent was removed under
◦
1
27
layer. Methyl 4-bromo-1H-pyrrole-2-carboxylate (44-Br). Yield:
.4 mg (31.3 mmol, 87%). H NMR (CDCl
3 H, s, –OCH
Schomburg, J. Mol. Biol., 1999, 293, 595–611.
1
6
(
3
, 600 MHz) d 3.87
9
O. Bortolini, M. Carraro, V. Conte and S. Moro, Eur. J. Inorg. Chem.,
3
), 6.88–6.89 (1 H, m, 3-H), 6.94–6.95 (1 H, m,
2003, 42–46.
5
1
-H), 9.39 (1 H, br.s, -NH-). MS (EI) m/z 205 (77), 203 (77),
10 A. Butler and J. N. Carter-Franklin, Nat. Prod. Rep., 2004, 21, 180–
1
88.
73 (100), 171 (100), 146 (26), 144 (26), 119 (18), 117 (18), 64
1
1 F. H. Vaillancourt, E. Yey, D. A. Vosburg, S. Garneau-Tsodikova
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28
(
47). Methyl 5-bromo-1H-pyrrole-2-carboxylate (45-Br). Yield:
1
0
.5 mg (2.5 mmol, 7%). H NMR (CDCl
3
, 600 MHz) d 3.86 (3
12 G. J. Gribble, Environ. Sci. Pollut. Res., 2000, 7, 37–49.
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3 G. Rothenberg and J. H. Clark, Green Chem., 2000, 2, 248–
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H, s, -OCH
9
1
3
), 6.21–6.22 (1 H, m, 4-H), 6.82–6.83 (1 H, m, 3-H),
2
.48 (1 H, br.s, -NH-). MS (EI) m/z 205 (68), 203 (68), 173 (100),
1
0
71 (100), 146 (23), 144 (23), 119 (30), 117 (30), 64 (48). 526 U
T
4
-
1
final
-1
final
mg , 117 U
T
mg , pH 6.6.
15 C. W. Jones, in Applications of Hydrogen Peroxide and Derivatives,
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3
.2 The use of immobilized bromoperoxidase as catalyst.
1
6 For guidelines of sustainable synthesis see: F. M. Kerton in Intro-
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Immobilized VBrPO(AnI) 1a (0.75 mL, 6.9 U , 0.37 mmol%)
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T
17 H. Vilter, Methods Enzymol., 1994, 228, 665–672.
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8 L. S. Wong, F. Khan and J. Micklefield, Chem. Rev., 2009, 109, 4025–
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(
(
(
3) (25.0 mg, 0.20 mmol), NaBr (20.6 mg, 0.20 mmol) and H
1.0 mL, 200 mM, prepared in distilled water) in MES-buffer
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2
O
2
4
1
9 S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst and R.
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◦
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was separated by magnetic decantation and washed with MES-
buffer (2 ¥ 1.0 mL, 50 mM, pH 6.2) for the next cycle. The
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2
O (3 ¥ 5 mL). Combined
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