Scheme 1. Key steps for bromide oxidation by hydrogen peroxide in vanadate(V)-dependent bromoperoxidase (V_BrPO)-catalyzed reactions (top; Pn is short for protein), and equilibria associated with hypobromous acid conversion in aqueous solutions of bromide [bottom; K¹ = for example 1.45·10⁸ M^–2 for H₂O at 20 °C (I = 0.1 M, pH 2.6–3.8) or 1.04·10⁸ M^–2 in H₂O at 25 °C (pH 1.5); K² = 16.9 M^–1 in H₂O at 25 °C].

The active role of vanadate as co-factor in the marine bromoperoxidases was discovered 1983 by Vilter, while studying constituents of the brown alga Ascophyllum nodosum (An). Bromoperoxidases supplement the family of iron-heme-dependent chloroperoxidases discovered by Hager seventeen years before. In a more rational terminology, considering the IUPAC-names for bromide and chloride, the enzymes could have been named bromideperoxidase and chlorideperoxidase, since bromo- or chloro-, according the systematic convention, are prefices for the covalently bound halogens.

 Five years after the first report on the bromoperoxidases, Wever and his group confirmed the role of vanadium as new co-factor for oxidative enzymes. In an abstract book, Vilter and the Wever-group announced in 1988 from a joint project the existence of a second vanadate(V)-dependent bromoperoxidase purified from A.nodosum, abbreviated hereafter as V_BrPO(AnII). Preliminary structural and kinetic data of V_BrPO(AnII) were published in the following year by Wever. In 2012, Vilter and a research team including Wischang, Leblanc, Hartung, and co-workers, presented a consistent picture of primary to quaternary structure, and chemical reactivity of the bromoperoxidase II in synthesis of marine natural products.

The first decade of bromoperoxidase science witnessed discovery of further bromoperoxidases by Butler, Harry, Itoh, Jordan, Leblanc, Potin, Vilter, and Wever, showing that the enzymes occur widely in brown algae, such as Macrocystis pyrifera, Fucus distychus, Laminaria digitata, and in in red algae, for example Corallina pilulifera, C. officinalis, C. vancouveriensis.

In the time between 1988–2008, the knowledge about bromoperoxidase biochemistry consistently grew, particularly due to contributions from the Butler- and the Wever-group, and an influential review appearing in 1993. Predominantly the two groups unraveled the mechanism of bromide oxidation by enzyme kinetics, addressed issues of peroxide- and halide-specificity, and expanded the list of chemical transformations to singlet dioxygen generation, thiocyanate oxidation, and sulfoxidation. Claims on organic substrate specificity providing from enantioselective bromocyclization of terpenes derivatives of brominated secondary metabolites, await independent confirmation. By the end of the pioneering era, a mechanistic picture emerged showing that the bromoperoxidase active site primarily serves for catalyzing bromide oxidation by hydrogen peroxide, whereas hydrocarbon bromination predominantly occurs in solution without involving substrate binding to the active site. The chemical nature of the selectivitity determining bromoelectrophile at that time remained unspecified.

Exploring properties of the vanadate-dependent bromoperoxidases brought together biochemists, experts intrested in inorganic vanadium chemistry, and organic chemists working in the field of stereoselective oxidation. Vanadate is a transition metal occurring in ~30 nanomolar concentration in sea water, sharing many chemical characteristics with phosphate. At the activity maximum of most marine bromoperoxidases (pH 6–7), vanadate exists in concentrations below one millimolar as dihydrogenvanadate (H₂VO₄^–), the conjugated base of orthovanadic acid H₃VO₄ (pK_a¹ = 3.5, pK_a² = 7.8, pK_a³ = 12.5). Although vanadate interacts comparatively weakly with histidyl dipeptides, dihydrogenvanadate binds strongly to bromoperoxidase proteins at the t (tele, remote) imidazole-nitrogen of a histidine side chain. The vanadate cofactor and proximate amino acid side chains provide a template for locking water molecules into a supramolecular array. This network of hydrogen-bonded molecules facilitates hydrogen peroxide binding and peroxide activation for bromide oxidation under mild conditions.

The chemical behavior of hydrogen peroxide toward vanadium is different from alkyl hydroperoxides. Rate and affinity for hydrogen peroxide binding to vanadate strongly depend on protonation at vanadate oxygens, as pointed out by Pecoraro and co-workers from density functional theory-calculations and XANES pre-edge spectroscopic measurements on small molecule bromoperoxidase-mimics. From the results the authors concluded that vanadate bound to a nitrogen donor ligand, for example at the V_BrPO(AnI)-active site, needs to be doubly protonated for binding hydrogen peroxide in an exothermic reaction (cf. Scheme 1).

In spite of a growing body of data including Michalis-Menten-parameters, thermal stability, and organic solvent tolerance of V_BrPO(AnI), it took until 2008, until syntheses of organobromines in sea water-like media were addressed. An issue in this chemistry remains supply with bromoperoxidases, still being restricted for V_BrPO(AnI) to isolation from the alga. Clones are available for other bromoperoxidases, but for unknown reasons have not yet been used to cover demand of the enzymes in mechanistic or synthetic organic chemistry.

For a use in synthesis, methods in bromoperoxidase chemistry operating on analytical levels had to be redesigned to effectively turn over higher substrate concentrations, for being competitive to textbook syntheses of organobromines. Applying bromoperoxidases in synthesis furthermore required to develop a predictive mechanistic model based on the chemical nature of the effective bromination reagent from the mixture supposed to be present in solution, such as vanadium-bound hypobromite, hypobromous acid, bromine, and tribromide. Hypobromous acid, bromine, and tribromide, for example, make a difference in organic synthesis, because the reagents show other chemical reactivity and chemoselectivity toward unsaturated hydrocarbons.

With the report on a structural model for the A. nodosum bromoperoxidase II, sufficient data had become available to start correlating structure to reactivity in the chemistry of vanadate-dependent bromoperoxidases. In view of this background, this article summarizes in the first part structure and oxidative chemistry of VBrPO(AnI) and VBrPO(AnII). The first part ends with formation of hypobromous acid from bromide and hydrogen peroxide. Since hypobromous acid is not sufficiently electrophilic for converting in neutral solution the benchmark substrate phenol into bromophenol, the second part addresses the question of electrophilicity of bromine compounds toward p-nucleophilic hydrocarbons systematicly. The major conclusion of this theoretical part is that molecular bromine is the strongest electrophile formed under conditions used for bromoperoxidase-catalyzed oxidation. The physical organic approached to verify that bromine is the effective reagent for bromofunctionalizing unsaturated hydrocarbons in bromoperoxidase-catalyzed oxidation is summarized in the third part. The final part also highlights biomimetic syntheses of brominated pyrrole-2-carboxylates, secondary metabolites from marine sponges of the genera Agelas, Acanthella, or Axinella (Figure 1).

Figure 1. Examples for naturally occurring organobromines relevant for this project.

4. Results:

Figure 2. Vanadate active sites of V_BrPO(AnI) (top; adapted from the X-ray structure) and V_BrPO(AnII) (bottom; molecular modeled from sequence homology).

Scheme 1. Model for interpreting steady-state kinetics of bromide oxidation catalyzed by V_BrPO(AnX) [bi-bi-ping-pong mechanism; X = I, II; PO = V_BrPO(AnX); PO’ and PO’’ = loaded derivatives of PO; see also eqns (1–4); K_m^H2O2 and K_m^Br– are Michaelis-Menten parameters for H₂O₂- and Br^–-binding; K_i^Br– describes competitive enzyme inhibition by bromide; the red dashed line symbolizes a hydrogen bond].

Scheme 2. Equilibria between HOBr, Br₂, and Br₃^– in H₂O [K^Br2 = 1.45·10⁸ M^–2 for H₂O at 20 °C (I = 0.1 M, pH 2.6–3.8) or 1.04·10⁸ M^–2 in H₂O at 25 °C (pH 1.5); K^Br3– = 16.9 M^–1 in H₂O at 25 °C].

Scheme 3. Synthesis of naturally occurring brominated derivatives of O-methyl pyrrole-2-carboxylate (n = 1, 2; X = I, II; 63 µmol% of V_BrPO(AnI) or V_BrPO(AnI), corresponding to 34.6 U_T). ^a 1.1 equiv. of NaBr and H₂O₂. ^b 2.2 equiv. of NaBr and H₂O₂.

4. Concluding remarks:

Mechanistic studies from biochemistry and physical organic chemistry showed that bromination of arenes and heteroarenes in V_BrPO-catalyzed reactions proceeds via molecular bromine as selectivitity determining reagent. Other bromination reagents, such hypobromous acid and tribromide cannot competite with the pronounced electrophilicity of bromine, as expressed by frontier molecular orbital analysis and a linear free energy relationship.

5. Cooperation:

Dr. Catherine Leblanc, CNRS, Station Biologique, Roscoff (Frankreich).

Dr. Hans Vilter, Trier.

6. Leading References:

Formation of Carbon-Bromine Bonds in Bromoperoxidase-Catalyzed Oxidations. D. Wischang, M. Radlow, J. Hartung, DaltonTransactions, 2013, 42, 11926–11940; DOI:10.1039/C3DT51582F.

Molecular Cloning, Structure, and Reactivity of the Bromoperoxidase II from Ascophyllum nodosum. D. Wischang, M. Radlow, H. Schulz, J. Hartung, H. Vilter, L. Viehweger, M. Altmeyer, C. Kegler, J. Herrmann, R. Müller, L. Delage, F. Gaillard, C. Leblanc, Bioorganic Chemistry2012, 44, 25–34; DOI:10.1016/j.bioorg.2012.05.003.

Funding:

Deutsche Bundesstiftung Umwelt, NanoKat.