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

Abstract

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%.

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Introduction

Vanadate(V)-dependent bromoperoxidase I from the brown alga Ascophyllum nodosum [V_BrPO(AnI), EC 1.11.1.10]¹ catalyzes the oxidation of bromide with H₂O₂ (Scheme 1) in weakly acidic solutions (pH 6.2–6.5).² The turnover frequency of this process exceeds the hitherto most active non-enzymatic alternatives by 4–5 orders of magnitudes.³ 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 synthesis.^4,5 V_BrPOs from other organisms than A. nodosum were discovered from screenings and purified to homogeneity.^2,6,7 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 V_BrPO(AnI).

Scheme 1 Stoichiometry for V_BrPO(AnI)-catalyzed bromide oxidation with H₂O₂ (top) and secondary non-enzymatic reactions utilizing electrophilic bromination reagents for arene functionalization (bottom; L = apoenzyme; Ar = aryl or heteroaryl, see below). ^a Proposed intermediate in an early phase of the reaction.⁸

The debate on the nature of the electrophilic bromination reagent that is liberated from the enzymatic process has not yet been settled (cf.Scheme 1).^9–11 The overall reaction, however, 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 biosynthetically proven.^10,12 In terms of organic synthesis, the use of V_BrPO(AnI) as catalyst for oxidative brominations in ocean water-like media probably constitutes the most sustainable version of the on-site bromination of arenes,^13–16 as recently documented by bromofunctionalization of benzenoid, non-benzenoid, and heterocyclic arenes on an analytical scale.⁵ 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.¹⁷BPO immobilization onto a solid support,¹⁸ 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,¹⁹ 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 V_BrPO(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 methylpyrrole-2-carboxylate bromination.²⁰

Results and interpretation

1 Enzyme purification and characterization

V_BrPO(AnI) was separated from a mixture of isoenzymes according to a published procedure, starting from lyophyllized A. nodosum (Fig. 1). The enzyme was stored in tris-(hydroxymethyl)-aminomethane(HCl), i.e., Tris(HCl)-buffered stock solutions (pH 9.0) at 4 °C.²¹Bromoperoxidase 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 of measured tryptic peptide masses, in comparison with the known primary structure of the enzyme (PDB 1QI9). Activity of V_BrPO(AnI) preparations ranged between 405 ± 43 and 526 ± 11 U_T mg⁻¹ (triiodide assay²² in phosphate buffer at pH 6.2).^5,23‡

Fig. 1 Polyacrylamide gel electrophoresis (1 h, 18 mA; non-denaturing conditions) of crude bromoperoxidase fraction (right) in comparison to V_BrPO(AnI) preparation obtained from hydrophobic interaction and size exclusion chromatography (left, BPB = bromophenol blue; staining was performed in a solution prepared from phosphate buffer, H₂O₂, aq. KI, and 1,4-phenyldiammonium dichloride).

2 Enzyme immobilization

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 50–60% (w/w), epoxide group loading 200–220 μmol, amino group loading 650–750 μmol per g of particle. Particles with a narrower (0.5–1.3 μm for E01 and N11) and a broader (0.7–4.5 μm 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).

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% (1b) of initial BPO activity as evident from triiodide tests (Table 1, entry 1–2). BPO immobilization on amino-functionalized supports was attainable viain situ-treatment of M-PVA N11 and N12 with glutardialdehyde (GA)²⁴ in K₂HPO₄ buffer (20 mM, pH 8.0) for 1 h at 23 °C, which was followed by addition of the V_BrPO(AnI)-stock solution in aqueous Tris(HCl) buffer (pH 9.0) (Scheme 2). The latter two reactions furnished materials showing 24% (2a) and 37–39% (2b) of the original BPO activity (Table 1, entries 3–4). The activity values were in a reproducible manner independent from the batch of enzyme used for immobilization (section 2.1 and Table 1, entries 4–5). The data also clarified that V_BrPO(AnI) binding was not inhibited by the presence of an excess of Tris from the buffer.

Table 1 Characteristics of immobilized V_BrPO(AnI)‡

Entry	Support	Particle size/μm	1–2	m ^0 ^a/mg	UT^0 mg^−1 ^b	BPO-loading/mg g^−1 bead	UT^loaded mg^−1^c
a Amount of VBrPO(AnI) prior to immobilization. b Bromoperoxidase (BPO)-activity of VBrPO(AnI) preparation (triiodide test). c BPO-activity of immobilized VBrPO(AnI) (triiodide test).
1	M-PVA E 01	0.5–1.9	1a	0.14	405 ± 43	2.82	118 ± 12
2	M-PVA E 02	0.7–6.0	1b	0.14	405 ± 43	2.15	69 ± 7
3	M-PVA N 11	0.5–1.9	2a	0.10	493 ± 6	2.13	120 ± 28
4	M-PVA N 12	0.7–6.0	2b	0.08	446 ± 24	1.60	168 ± 47
5	M-PVA N 12	0.7–6.0	2b	0.10	493 ± 6	2.13	194 ± 39

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Scheme 2 Immobilization of V_BrPO(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 see legend of Fig. 2, for results Table 1). ^aMES buffer. ^bPhosphate buffer. ^cTris(HCl) buffer.

Yields of V_BrPO(AnI) binding to functionalized supports were essentially quantitative, as judged on the basis of the Bradford assay.^25,26 Since crystalline V_BrPO(AnI) was not available in sufficient amounts, the protein concentration in solution was referenced versus bovine serum albumin (BSA). The results were independently checked viatriiodide BPO activity tests of the V_BrPO(AnI) stock solution, the filtrate after separation of materials 1–2, and MES buffer washings of the latter preparations.

In view of the considerable fraction of nucleophilic endgroups at the periphery of the protein⁸ and the inertness of active enzyme toward leaching, attachment of V_BrPO(AnI) to applied beads (Scheme 2) was assumed to occur via covalent bonds. V_BrPO(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 V_BrPO(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.

Long term studies indicated that storage of preparations 1a–b in MES buffer and materials 2a–b in Tris(HCl) buffer at 4 °C progressively decreased in BPO activity during the first 70 days. In solution this activity loss prevailed for the free enzyme. For immobilized V_BrPO(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 required 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.

Fig. 3 Time dependence of specific BPO-activity of immobilized V_BrPO(AnI) stored at 4 °C in MES buffer (samples 1a–b) or Tris(HCl) buffer (samples 2a–b; error bars refer to standard deviations from three independent runs).

3 Oxidation catalysis

Conditions suitable for organic compound bromination in V_BrPO(AnI)-catalyzed oxidations were established using methyl 1H-pyrrole-2-carboxylate (3) as reporter substrate. The π-excess heteroaromatic compound undergoes substitution of bromine for hydrogen already with comparatively weakly electrophilic reagents, such as HOBr or Br₂, without the necessity of adding external Lewis acids.²⁷ It furthermore provides a diagnostic mixture of mono- and disubstitution products 4 and 5 that are sufficiently stable to allow quantitative analysis viagas chromatography and subsequent column chromatographic separation, for determining the site of aromatic substitutionviaNMR spectroscopy.^27,28 Since pyrrole-2-carboxylic acid in nature is formed from the amino acid metabolism,²⁹ the experiments were considered to be relevant for providing insight into genesis of naturally occurring, brominated pyrroles.

3.1 Catalyst activity. From parameter variation it was found that BPO activity of 1.3 U_T was necessary to quantitatively convert 36 μM of pyrrole 3 into 99% of bromopyrrole 4 within 24 h in MES buffered solution (Table 2, entry 1). H₂O₂ 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. An alternative mode of H₂O₂ administration was pursued by in situ generation via aerobic oxidation of D-glucose, catalyzed by D-glucoseoxidase (GO)^30,31 from Aspergillus niger (1.67 U, 147 U mg⁻¹). 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 V_BrPO(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 V_BrPO(AnI)-activity converged toward zero.

Table 2 Reaction parameters for effective methyl pyrrole-2-carboxylate bromination‡

Entry	n 3 /μmol	Conv. 3/%	Time/h	BPO	UT	UT^final mg^−1 ^a	4/% (4/5)^b
a Initial enzyme activity 526 UT^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 triiodide tests. b Ratio of 4/5-regioisomers of bromopyrrole 4. c c 3 ^0 = 7.2 mM, H2O2 addition in 3 batches every 30 min; total amount of substrates: 1.0 equiv. of H2O2 and 3.0 equiv. of NaBr. d H2O2 added in a single batch; total amount of substrates: 1.0 equiv. of H2O2 and 1.0 equiv. of NaBr.
1	36	quant.^c	24	VBrPO(AnI)	1.3	117	94 (93/7)
2	36	92^d	5	VBrPO(AnI)	1.3	99	73 (89/11)
3	36	77^d	3	VBrPO(AnI)	1.3	239	60 (82/18)
4	36	27^d	1	VBrPO(AnI)	1.3	231	26 (81/19)
5	200	54^d	3	1a	6.9	active	40 (90/10)
6	200	75^d	3	2b	6.9	active	32 (93/8)

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Scheme 3 Combination of D-glucoseoxidase (GO)-catalyzed H₂O₂ generation and V_BrPO(AnI)-catalyzed bromide oxidation for sustainable pyrrole bromination [D-Glc = D-glucose; 6.2 U_T for V_BrPO(AnI); 1.67 U for GO; pH 5.8 (MES); tBuOH/H₂O; c₃⁰ = 7.2 mM; for stoichiometry of the BPO-catalyzed reaction see Scheme 1].

A survey of the time dependence of bromopyrrole formation showed that substrate conversion reached a level of approximately 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 for validating catalytic properties of immobilized V_BrPO(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 V_BrPO(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 V_BrPO(AnI) 1a and 2b (Table 2, entries 5–6) were not detected (GC, NMR).

V_BrPO(AnI)-activity that was conserved in methylpyrrole-2-carboxylate bromination in homogeneous solutions was applicable for further experiments after addition of aliquots of substrate 3, H₂O₂, NaBr, and MES buffer. BPO-activity after the fourth run, however, fell below a synthetically relevant level (Fig. 4).

Fig. 4 Efficiency of product formation in V_BrPO(AnI)-catalyzed reactions in sequential runs [4.96 U_T corresponding to 0.05 mmol % V_BrPO(AnI); 144 μmol of 3, 1 equiv. of H₂O₂ and NaBr, H₂O/tBuOH = 75/25 (v/v); additional product: 10% of methyl 3,4-dibromopyrrole-2-carboxylate (5) in experiment number 1 and <1% in experiment number 2].

3.2 Repetitive use of immobilized bromoperoxidase. Immobilized samples 1a and 2b, i.e. of the most active materials from both types of supports were chosen for validating utility of immobilized V_BrPO(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.

Addition of catalyst suspension to aqueous tBuOH containing reporter substrate 3 was followed by addition of NaBr and H₂O₂. 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).

Fig. 5 Conversion of substrate 3 (c⁰ = 35 mM; gray columns) and efficiency of methylpyrrole-2-carboxylate bromination (black columns) upon multiple use of V_BrPO(AnI)-preparation 1a (6.9 U_T) for catalyzing oxidative bromination in aqueous tBuOH (pH 6.2, 23 °C) using 1.0 equiv. each of H₂O₂ and NaBr (3 h, see text).

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 orthovanadate solution between consecutive oxidation experiments. This modification, however, had no effect on performance of the material and activity loss after the 8–10th cycle.

Concluding remarks

Immobilization of V_BrPO(AnI) to solid supports provided three distinctive results relevant for future development of sustainable oxidative brominations.

(i) V_BrPO(AnI) binding either to epoxide-functionalized or to glutardialdehyde-activated amino-substituted magnetic beads was feasible in quantitative yields, with almost 40% of the original catalyst activity being conserved.

(ii) Immobilized V_BrPO(AnI) showed similar turnover characteristics 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 [Fe_ClPO(CfI)] on mesoporous supports³¹ was the ease and the efficiency of magnetic separation of immobilized V_BrPO(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 preparation³² but has not yet been applied to haloperoxidase chemistry. With this material in hand, application of V_BrPO(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 the reagent combination of H₂O₂, 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. V_BrPO(AnI) was isolated from A. nodosum collected in April 2004 (France, 48° 43′ N, 3° 58′ W) according to a published procedure.²¹ Magnetic beads were obtained from chemagen Biopolymer-Technologie AG, Germany.

2 Immobilization of V_BrPO(AnI)

2.1 Immobilization on M-PVA E (epoxy-functionalized supports). To Tris(HCl) (50 mM, pH 9.0)-buffered V_BrPO(AnI) solution (0.20 mg mL⁻¹) 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 1.0 mL of enzyme solution (protein concentration 0.14 mg mL⁻¹) 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 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 MES-buffer (1.0 mL, 50 mM, pH 6.2) and were ready to use as oxidation catalyst.

2.2 Immobilization on M-PVA N (amino-functionalized supports). M PVA N11 or N12 (50 mg) was washed with distilled water (3 × 1.0 mL). The supports were activated by adding 1.0 mL activation solution {glutardialdehyde solution [4.00 g, 50% (w/v), in H₂O, 200 mM] in aqueous KH₂PO₄ (0.27 g, 0.02 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 0.5 mL of enzyme solution (protein concentration 0.20 mg mL⁻¹) in Tris(HCl)-buffer (50 mM, pH 9.0). The mixture was shaken gently at 23 °C for 24 h. Periodically, samples of the supernatants were withdrawn and analyzed for protein concentration (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)

3.1 Reactions in homogeneous solution. A solution of aqueous H₂O₂ (1.0 mL, 36.0 mM) was added in a dropwise manner in three portion (to 333 μL at the beginning, after 0.5 h and 1.0 h) to a solution of substrate 3 (4.5 mg, 36.0 μmol) in MES-buffer (50 mM, pH 6.2, 3.75 mL) and tBuOH (1.25 mL), NaBr (11.2 mg, 108.0 μmol) and V_BrPO(AnI) (7.5 μL, 1.3 U_T, 0.05 mmol%). The reaction mixture was stirred at 23 °C for 1 d. The aqueous layer was extracted with Et₂O (3 × 5 mL). Combined organic extracts were dried (MgSO₄). The solvent was removed under reduced pressure (14 mbar, 40 °C) to afford a product mixture which was analyzed by ¹H NMR. pH and bromoperoxidase activity (triiodide assay) were determined from the aqueous layer. Methyl 4-bromo-1H-pyrrole-2-carboxylate (4_4-Br).²⁷ Yield: 6.4 mg (31.3 μmol, 87%). ¹H NMR (CDCl₃, 600 MHz) δ 3.87 (3 H, s, –OCH₃), 6.88–6.89 (1 H, m, 3-H), 6.94–6.95 (1 H, m, 5-H), 9.39 (1 H, br.s, -NH-). MS (EI)m/z 205 (77), 203 (77), 173 (100), 171 (100), 146 (26), 144 (26), 119 (18), 117 (18), 64 (47). Methyl 5-bromo-1H-pyrrole-2-carboxylate (4_5-Br).²⁸ Yield: 0.5 mg (2.5 μmol, 7%). ¹H NMR (CDCl₃, 600 MHz) δ 3.86 (3 H, s, -OCH₃), 6.21–6.22 (1 H, m, 4-H), 6.82–6.83 (1 H, m, 3-H), 9.48 (1 H, br.s, -NH-). MS (EI)m/z 205 (68), 203 (68), 173 (100), 171 (100), 146 (23), 144 (23), 119 (30), 117 (30), 64 (48). 526 U_T⁰ mg⁻¹, 117 U_T^final mg⁻¹, pH^final 6.6.

3.2 The use of immobilized bromoperoxidase as catalyst. Immobilized V_BrPO(AnI) 1a (0.75 μL, 6.9 U_T, 0.37 mmol%) was washed with MES-buffer (2 × 1.0 mL, 50 mM, pH 6.2) before added to a solution of methyl 1H-pyrrole-2-carboxylate (3) (25.0 mg, 0.20 mmol), NaBr (20.6 mg, 0.20 mmol) and H₂O₂ (1.0 mL, 200 mM, prepared in distilled water) in MES-buffer (50 mM, pH 6.2, 3.75 mL) and tBuOH (1.25 mL). The reaction mixture was stirred at 23 °C for 3 h. Immobilized V_BrPO(AnI) 1a was separated by magnetic decantation and washed with MES-buffer (2 × 1.0 mL, 50 mM, pH 6.2) for the next cycle. The aqueous layer was extracted with Et₂O (3 × 5 mL). Combined organic extracts were dried (MgSO₄). The solvent was removed under reduced pressure (13 mbar, 40 °C) to afford a product mixture which was analyzed by ¹H NMR (CDCl₃). Samples 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 V_BrPO(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 V_BrPO(AnI) 2b. For analytical data refer to section 4.3.1, for yields see the ESI, section 8.2.

Acknowledgements

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.

Notes and References

1. H. Vilter, Phytochemistry, 1984, 23, 1387–1390.
2. A. Butler and J. V. Walker, Chem. Rev., 1993, 93, 1937–1944.
3. G. E. Meister and A. Butler, Inorg. Chem., 1994, 33, 3269–3275.
4. E. de Boer, H. Plat and R. Wever, in Biocatalysis in Organic Media, ed. C. Laane, J. Tramper and M. D. Lilly, Elsevier, Amsterdam, 1987, pp. 317–322.
5. J. Hartung, Y. Dumont, M. Greb, D. Hach, F. Köhler, H. Schulz, M. Časný, D. Rehder and H. Vilter, Pure Appl. Chem., 2009, 81, 1251–1264.
6. H. Vilter, in Vanadium and its Role in Life, Metal Ions in Biological Systems, ed. H. Sigel and A. Sigel, Dekker, New York, 1995, vol. 31, pp. 325–362.
7. E. E. Coupe, M. G. Smyth, A. Fosberry, R. M. Hall and J. A. Littlechild, Protein Expression Purif., 2007, 52, 265–272.
8. M. Weyand, H. J. Hecht, M. Kiess, M. F. Liaud, H. Vilter and D. Schomburg, J. Mol. Biol., 1999, 293, 595–611.
9. O. Bortolini, M. Carraro, V. Conte and S. Moro, Eur. J. Inorg. Chem., 2003, 42–46.
10. A. Butler and J. N. Carter-Franklin, Nat. Prod. Rep., 2004, 21, 180–188.
11. F. H. Vaillancourt, E. Yey, D. A. Vosburg, S. Garneau-Tsodikova and C. T. Walsh, Chem. Rev., 2006, 106, 3364–3378.
12. G. J. Gribble, Environ. Sci. Pollut. Res., 2000, 7, 37–49.
13. G. Rothenberg and J. H. Clark, Green Chem., 2000, 2, 248–251.
14. A. Podgoršek, M. Zupan and J. Iskra, Angew. Chem., Int. Ed., 2009, 48, 8424–8450.
15. C. W. Jones, in Applications of Hydrogen Peroxide and Derivatives, RSC Clean Technology Monographs, series ed. J. H. Clark, RSC, Cambridge, 1999, pp. 156–162.
16. For guidelines of sustainable synthesis see: F. M. Kerton in Introduction from Chemicals from Biomass, ed. J. Clark and F. Deswarte, Wiley, 2008, ch. 3, pp. 47–76.
17. H. Vilter, Methods Enzymol., 1994, 228, 665–672.
18. L. S. Wong, F. Khan and J. Micklefield, Chem. Rev., 2009, 109, 4025–4053.
19. S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst and R. N. Muller, Chem. Rev., 2008, 108, 2064–2110.
20. S. Forenza, L. Minale and R. Riccio, J. Chem. Soc., Chem. Commun., 1971, 1129–1130.
21. J. Hartung, O. Brücher, D. Hach, H. Schulz, H. Vilter and G. Ruick, Phytochemistry, 2008, 69, 2826–2830.
22. F. Björkstén, Eur. J. Biochem., 1968, 5, 133–142.
23. International Union of Biochemistry (IUB), Report of the Commission on Enzymes, Pergamon Press, Oxford, 1961.
24. S. S. Wong, in Chemistry of Protein Conjugation and Cross-Linking, CRL Press, Boca Raton, 1993, ch. 4, pp. 74–103.
25. M. H. Bradford, Anal. Biochem., 1976, 205, 22–26.
26. N. J. Krüger, Meth. Mol. Biol., 1994, 32, 9–15.
27. C. Schroif-Gregoire, N. Travert, A. Zaparucha and A. Al-Mourabit, Org. Lett., 2006, 8, 2961–2964.
28. B. M. Trost and G. Dong, J. Am. Chem. Soc., 2006, 128, 6054–6055.
29. C. T. Walsh, S. Garneau-Tsodikova and A. R. Howard-Jones, Nat. Prod. Rep., 2006, 23, 517–531.
30. D. D. Keilin and E. F. Hartree, Biochem. J., 1952, 50, 331–341.
31. M. Hartmann and C. Streb, J. Porous Mater., 2006, 13, 347–352.
32. X. Wang, P. Dou, P. Zaho, C. Zhao, Y. Ding and P. Xu, ChemSusChem, 2009, 2, 947–950.

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Footnotes

† Electronic supplementary information (ESI) available: Standard instrumentation, BPO-analysis, determination of BPO-activity, synthesis of methyl 1H-pyrrole-2-carboxylate (3), details for the use of D-Glc/GO for in situ H₂O₂ generation in BPO-catalyzed oxidations, details for the use of V_BrPO(AnI)-preparations 1a and 2b in multiple runs. See DOI: 10.1039/c0gc00499e

‡ Notation for enzymatic activity: 1 unit (U) refers to the amount of enzyme required for turning over 1 μmol of substrate per minute. BPO-activity determined with the aid of the triiodide assay (T) was abbreviated as U_T. Specific BPO-activity referred to the number of units per mg of enzyme preparation, i.e.U_T mg⁻¹.

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