Vanadate-dependent bromoperoxidases from Ascophyllum nodosum in the synthesis of brominated phenols and pyrroles

Article abstract of DOI:10.1039/c3dt51582f

Bromoperoxidases from the brown alga Ascophyllum nodosum, abbreviated as V_BrPO(AnI) and V_BrPO(AnII), show 41% sequence homology and differ by a factor of two in the percentage of α-helical secondary structures. Protein monomers organize into homodimers for V_BrPO(AnI) and hexamers for V_BrPO(AnII). Bromoperoxidase II binds hydrogen peroxide and bromide by approximately one order of magnitude stronger than V_BrPO(AnI). In oxidation catalysis, bromoperoxidases I and II turn over hydrogen peroxide and bromide similarly fast, yielding in morpholine-4-ethanesulfonic acid (MES)-buffered aqueous tert-butanol (pH 6.2) molecular bromine as reagent for electrophilic hydrocarbon bromination. Alternative compounds, such as tribromide and hypobromous acid are not sufficiently electrophilic for being directly involved in carbon-bromine bond formation. A decrease in electrophilicity from bromine via hypobromous acid to tribromide correlates in a frontier molecular orbital (FMO) analysis with larger energy gaps between the π-type HOMO of, for example, an alkene and the σ*_Br,X-type LUMO of the bromination reagent. By using this approach, the reactivity of substrates and selectivity for carbon-bromine bond formation in reactions mediated by vanadate-dependent bromoperoxidases become predictable, as exemplified by the synthesis of bromopyrroles occurring naturally in marine sponges of the genera Agelas, Acanthella, and Axinella. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3dt51582f

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Vanadate-dependent bromoperoxidases from
Ascophyllum nodosum in the synthesis of brominated
phenols and pyrroles†
Cite this: Dalton Trans., 2013, 42, 11926
Diana Wischang, Madlen Radlow and Jens Hartung*
Bromoperoxidases from the brown alga Ascophyllum nodosum, abbreviated as VBrPO(AnI) and VBrPO-
(AnII), show 41% sequence homology and differ by a factor of two in the percentage of α-helical second-
ary structures. Protein monomers organize into homodimers for VBrPO(AnI) and hexamers for VBrPO-
(AnII). Bromoperoxidase II binds hydrogen peroxide and bromide by approximately one order of magni-
tude stronger than VBrPO(AnI). In oxidation catalysis, bromoperoxidases I and II turn over hydrogen per-
oxide and bromide similarly fast, yielding in morpholine-4-ethanesulfonic acid (MES)-buffered aqueous
tert-butanol (pH 6.2) molecular bromine as reagent for electrophilic hydrocarbon bromination. Alterna-
tive compounds, such as tribromide and hypobromous acid are not sufficiently electrophilic for being
directly involved in carbon–bromine bond formation. A decrease in electrophilicity from bromine via
hypobromous acid to tribromide correlates in a frontier molecular orbital (FMO) analysis with larger
energy gaps between the π-type HOMO of, for example, an alkene and the σ*Br,X-type LUMO of the bro-
mination reagent. By using this approach, the reactivity of substrates and selectivity for carbon–bromine
bond formation in reactions mediated by vanadate-dependent bromoperoxidases become predictable,
as exemplified by the synthesis of bromopyrroles occurring naturally in marine sponges of the genera
Agelas, Acanthella, and Axinella.
Received 14th June 2013,
Accepted 3rd July 2013
DOI: 10.1039/c3dt51582f
www.rsc.org/dalton
1 Introduction
Vanadate-dependent bromoperoxidases (EC 1.11.1.18) catalyze
oxidation of bromide by hydrogen peroxide at ambient temp-
erature and almost neutral pH (Scheme 1).1–4 Since bromide
occurs abundantly in ocean water,5 oxidative transformations
mediated by marine bromoperoxidases were soon after their
discovery associated with (bio)genesis of brominated second-
ary metabolites.6–8 The existence of enzymes able to generate
bromoelectrophiles from sea water brought together biochem-
ists and specialists from bioinorganic chemistry, natural
product chemistry, and sustainable oxidation catalysis to put
the new knowledge into a larger scientific context.9
The role of vanadate as a co-factor in marine bromoperoxidases
was discovered in 1983 by Vilter while studying constituents of the
brown alga Ascophyllum nodosum.14,15 Bromoperoxidases sup-
plement the family of iron-heme-dependent chloroperoxidases
Scheme 1 Key steps for bromide oxidation by hydrogen peroxide in vanadate
(V)-dependent bromoperoxidase (VBrPO)-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)10 or 1.04 × 108 M−2 in H2O at 25 °C (pH
1.5);11 K2 = 16.9 M−1 in H2O at 25 °C12].13
Fachbereich Chemie, Organische Chemie, Technische Universität Kaiserslautern,
Erwin-Schrödinger-Straße, D-67663 Kaiserslautern, Germany.
E-mail: hartung@chemie.uni-kl.de; Fax: +49-631-205-3921
†Electronic supplementary information (ESI) available: Atomic coordinates,
energies, and NBO-analysis of bromoelectrophiles, alkenes, phenol, and O-
methyl pyrrole-2-carboxylate. See DOI: 10.1039/c3dt51582f
11926 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
discovered by Hager seventeen years earlier. In a more rational ter- and proximate amino acid side chains at the active site of bro-
minology, considering the IUPAC-names for bromide and chlor- moperoxidases provide a template for locking water molecules
ide, the enzymes could also have been named bromideperoxidase into a supramolecular array. This network of hydrogen-bonded
and chlorideperoxidase, since bromo- or chloro-, according the sys- molecules facilitates hydrogen peroxide binding to vanadate
tematic convention, are prefixes for covalently bound and peroxide activation for bromide oxidation under mild
halogens.16,17
conditions.36
Five years after the first report on the bromoperoxidases,
The chemical behavior of hydrogen peroxide toward
Wever and his group confirmed the role of vanadium as co- vanadium differs from alkyl hydroperoxide chemistry.37 The
factor for oxidative enzymes.18 In an abstract book, Vilter and rate and affinity for hydrogen peroxide binding to vanadate
the Wever-group announced in 1988 from a joint project the strongly depend on protonation at vanadate oxygens,38 as
existence of a second vanadate(V)-dependent bromoperoxidase pointed out by Pecoraro and co-workers using density func-
in A. nodosum, abbreviated hereafter as VBrPO(AnII).19‡ Prelimi- tional theory calculations39,40 and XANES pre-edge spectro-
nary structural and kinetic data of VBrPO(AnII) were published scopic measurements on bromoperoxidase-mimics.41 From
in the following year by Wever.20 In 2012, Vilter and a research the results, the authors proposed that vanadate at the VBrPO-
team including Wischang, Leblanc, Hartung, and co-workers (AnI)-active site, needs to be doubly protonated for binding
presented a consistent picture of primary to quaternary struc- hydrogen peroxide in an exothermic reaction (cf. Scheme 1).
ture, including chemical reactivity and selectivity of the bromo-
peroxidase II.21
In spite of a continuously growing body of data including
Michalis–Menten-parameters,18 thermal stability,42 and
The first decade of bromoperoxidase science witnessed the organic solvent tolerance43 of VBrPO(AnI), it was not until 2008
discovery of further bromoperoxidases by Butler, Harry, Itoh, that syntheses of organobromines in sea water-like media were
Jordan, Leblanc, Potin, Vilter, and Wever, showing that such explored.44,45 An issue in this chemistry remained the supply
enzymes occur in other brown algae, for example Macrocystis by bromoperoxidases, which is still restricted for VBrPO(AnI) to
pyrifera,22 Fucus distichus,22 Laminaria digitata,23,24 and a wider isolation from the alga.18,46 Clones are available for other
variety of red algae, such as Corallina pilulifera,25 C. officina- bromoperoxidases,47–49 but for unknown reasons have not yet
lis,26 and C. vancouveriensis.27
In the period between 1988 and 2008, the knowledge about tic or synthetic organic chemistry.
been used to cover the demand for the enzymes in mechanis-
bromoperoxidase biochemistry consistently grew, particularly
For use in synthesis, methods in bromoperoxidase chem-
due to contributions from the Butler- and the Wever-group istry referring to analytical levels had to be redesigned to turn
and an influential review1 appearing in 1993. Predomi- over substrates in higher concentrations, for being competitive
nantly the two groups unraveled the mechanism of bromide with conventional organobromine syntheses.50–52 Applying
oxidation by enzyme kinetics,18 addressed issues of peroxide- bromoperoxidases in synthesis furthermore required to
and halide-specificity, and expanded the list of chemical develop a mechanistic model based on the chemical nature of
transformations to singlet dioxygen generation, thiocyanate the relevant bromination reagent from the mixture of
oxidation, and sulfoxidation.28–30 Enantioselective bromocycli- vanadium-bound hypobromite, hypobromous acid, bromine,
zation of terpenes into brominated secondary metabolites31 and tribromide.1,53,54 Particularly hypobromous acid, bromine,
were not yet independently confirmed. By the end of the and tribromide make
a difference concerning chemical
pioneering era, a mechanistic picture emerged describing reactivity and chemoselectivity toward unsaturated hydro-
bromoperoxidases as catalysts for oxidizing bromide by carbons.55–58
hydrogen peroxide without being involved in hydrocarbon
bromination.32
With the structural model for the A. nodosum bromoperoxi-
dase II, sufficient data finally had become available to correlate
Exploring the properties of the vanadate-dependent bromo- structure to reactivity in the chemistry of vanadate-dependent
peroxidases challenged biochemists and particularly experts bromoperoxidases.21 In view of this background, this article
from the vanadium community.9 Vanadate is a transition summarizes in the first part the structure and oxidative chem-
metal sharing many chemical characteristics with phosphate. istry of VBrPO(AnI) and VBrPO(AnII). The first part ends with
At the activity maximum of most marine bromoperoxidases the formation of hypobromous acid from bromide and hydro-
(pH 6–7), vanadate exists at concentrations below one milli- gen peroxide. Since hypobromous acid is not sufficiently elec-
molar as dihydrogenvanadate (H2VO4−), the conjugated base trophilic for converting in neutral solution the benchmark
1
2
3
of orthovanadic acid, H3VO4 (pKa = 3.5, pKa = 7.8, pKa
=
substrate phenol into bromophenol, the second part addresses
12.5). Although vanadate interacts comparatively weakly with the aspect of electrophilicity in bromine chemistry in general.
histidyl dipeptides, dihydrogenvanadate binds to bromoperoxi- The major conclusion of this theoretical part is that molecular
dase proteins specifically at the τ (tele, remote) imidazole- bromine is the strongest electrophile available from bromoper-
nitrogen of a histidine side chain.33–35 The vanadate cofactor oxidase-catalyzed oxidation. The approach to experimentally
verify that bromine also in bromoperoxidase chemistry is the
effective bromination reagent is summarized in the third part.
‡VBrPO is short for vanadate-dependent bromoperoxidase; AnI is short for isoen-
The final part furthermore presents biomimetic syntheses of
brominated pyrrole-2-carboxylates, secondary metabolites
zyme I from Ascophyllum nodosum. For two letter codes and isoenzyme number-
ing of bromoperoxidases isolated from other species, cf. Table 6.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11927

View Article Online
Paper
Dalton Transactions
side chains with PNGase provided no evidence for the carbo-
hydrate nature of obtained fragments.21
The secondary structure of the A. nodosum bromoperoxidase
I in (Tris)/HCl-buffered solution (pH 8.0) shows approximately
evenly distributed fractions of α-helical subunits, β-folded
sheets, turns, and random coils, as calculated from the wave-
length dependence of the molar ellipticity [Θ]m between 184
and 260 nm (Table 2, entry 1). This information differs from
the conformational analysis provided by X-ray diffraction ana-
lysis, showing a fraction of 41% α-helical subunits, 6%
β-sheets, and 51% random coils in the solid state. The ratio of
secondary structural elements of VBrPO(AnII) in the solution
model decreases from 52% for α-helical subunits via random
coils and turns to 5% for β-sheets (Table 2, entry 2).21
Gel filtration experiments provide a molecular mass of
136 kDa for VBrPO(AnI) (Table 3, entry 1), which is in line with
the homodimeric structure found in the solid state by X-ray
diffraction, and in the gas phase by MALDI-ToF-mass spec-
trometry. A molecular mass of 420 kDa (Table 3, entry 2), as
determined from gel filtration, implies hexameric aggregation
of the protein monomer in VBrPO(AnII).21
Fig. 1 Examples for naturally occurring organobromines relevant for this
article (bottom).
from marine sponges of the genera Agelas,59 Acanthella,60 or
Axinella61 (Fig. 1).
2 Structure and reactivity of
bromoperoxidases from Ascophyllum
nodosum
2.2 The vanadate(V)-cofactor
Vanadate is the co-factor responsible for regulating bromoper-
oxidase activity in VBrPO(AnI) and VBrPO(AnII). Apobromoper-
2.1 From primary to quaternary structure
Bromoperoxidases VBrPO(AnI) and VBrPO(AnII) are available oxidases apo-PO(AnI) and apo-PO(AnII), prepared by dialyzing
from A. nodosum in a multistep procedure including freeze- solutions of the active enzymes versus ethylenediaminetetraa-
drying, milling, liquid–liquid partitioning, precipitation, cetate (EDTA), are not able to catalyze bromide oxidation by
dialysis, and chromatography. From different affinities to con- hydrogen peroxide.15,21,64 In solutions of sodium orthovana-
canavalin A-Sepharose, Wever concluded that both bromoper- date, apobromoperoxidase II recovers bromoperoxidase activity
oxidases were glycoproteins, which has not been confirmed by one order of magnitude faster than apobromoperoxidase I
(vide infra).
The three-dimensional structure of VBrPO(AnI) was deter-
(Table 4).21
In the solid state structure obtained for VBrPO(AnI), every
mined using X-ray crystallography (PDP accession code 1QI9) protein monomer binds one vanadate to the tele (τ)-imidazole
from crystals obtained from the native extracted and vanadate- nitrogen of histidine-486. Close contacts exist in this environ-
reconstituted enzyme. The crystals diffracted to a resolution of ment between vanadate oxygens and two guanidinium groups
2 Ångstrøms,34 showing a protein composed of 557 amino from arginine side chains, one hydroxymethyl group from a
acids (Table 1).62 Two protein strands form a twofold-disulfide serine side chain, and the imidazole nucleus from histidine-
bridged homodimer, which is stable under the conditions that 418, which is relevant for acid–base catalysis in bromide oxi-
are used, for example, to record matrix-assisted laser desorption/ dation (Fig. 2, top). Computational studies have shown that
ionization (MALDI)-time-of-flight (ToF) mass spectra.34 The vanadate has to be twofold-protonated in order to bind to the
crystal structure provides no evidence for glycoconjugation of the pyridine-type nitrogen of imidazole.38,39
bromoperoxidase I protein.
The three-dimensional structure of VBrPO(AnII) is not yet
The amino acid sequence of the bromoperoxidase II protein known from crystal structure analysis. In order to model the
was determined from full length cloned cDNA, obtained from structure, particularly at the active site, sequence homology
a tandem mass spectrometry-reverse transcriptase-polymerase was correlated to structural homology using the solid state
chain reaction (RT-PCR)-approach. The clone encodes
a
structure VBrPO(AnI) (Fig. 2, top) as a reference. In the mod-
protein composed of 641 amino acids (Table 1), providing a elled bromoperoxidase II tertiary structure, vanadate was fixed
mature 67.4 kDa-bromoperoxidase II-protein (620 amino at the τ-imidazole nitrogen of the histidine-537 side chain
acids).21 Bromoperoxidase II shares 41% sequence homology (Fig. 2, bottom).65 This positioning leads to close contacts
with VBrPO(AnI) (Table 1), and is 61% homologous to the bro- between the apical vanadate oxygen and the τ-nitrogen of histi-
moperoxidase I from Laminaria digitata. Extracted bromoper- dine-452, which is possibly the acid–base catalyst for bromide
oxidase II protein furnishes in MALDI-ToF-mass spectrometric oxidation (vide infra). Further close contacts in this model exist
analysis weak signals of monomers at m/z = 64.2 to 65.0 kDa, between vanadate oxygens and the hydroxyl group of serine-
and further signals at m/z = 127.7 to 130.0 kDa possibly relat- 450, the ε-ammonium group of lysine-374, and the guanidi-
ing to dimers.63 Attempts to hydrolyse putative carbohydrate nium group of arginine-382 (Fig. 2, bottom). The six cysteine
11928 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

Table 1 Amino acid sequence alignment of vanadate-dependent bromoperoxidases VBrPO(AnX)21,34 (X = I, II) from A. nodosuma
X
Amino acid sequence
a Conserved amino acids are shown in bold. Amino acids associated with the catalytic site in VBrPO(AnI),34 conserved in VBrPO(AnII), in white on red (catalytic histidine), purple (vanadate
binding site), and blue background. Cysteine residues involved in intramolecular disulfide bonds are shown in red, or in bold on yellow background for intramolecular disulfide bonds.

View Article Online
Paper
Dalton Transactions
Table 2 Secondary structures of A. nodosum vanadate(V)-dependent bromo-
peroxidases from CD-spectroscopy21
Entry VBrPO(AnX) α-Helical/% β-Sheet/% Turns/% Random coil/%
1
2
X = I
X = II
26
52
20
5
22
17
32
27
Table 3 Results of gel filtrations of bromoperoxidases from A. nodosum vana-
date(V)-dependent bromoperoxidases
Entry
VBrPO(AnX)
Mcalcd.a/kg mol−1
Mfound/kg mol−1
1
2
X = I
X = II
120
135
136
420
a For homodimer VBrPO(AnX) and the assumed dimer based on the
broken vanadate/apo-BrPO(AnII)-monomer ratio found by vanadium
analysis.
Table 4 Relative rates for vanadate-binding to apobromoperoxidases in solu-
tions of sodium orthovanadate, and vanadete/protein-monomer ratios in recon-
stituted bromoperoxidases21
apo-BrPO(AnX)-
V/proteina
monomer/g mol−1
krel
b
Entry
VBrPO(AnX)
1
2
X = I
X = II
0.9 0.2
0.5 0.1
60 189
67 439
1
23
a From ICP-MS. b In Tris–HCl buffer (pH 9.0, 25 mM, T = 23 °C) and
Na3VO4-solutions [for apo-BrPO(AnI) 0.70 µM (M = 60 189 Da) and for
apo-BrPO(AnII) 0.32 µM (M = 134 878 Da for the entity composed of
mature protein (620 amino acids) binding one vanadate].
Fig. 2 Vanadate active sites of VBrPO(AnI) (top; adapted from the X-ray struc-
ture)34 and VBrPO(AnII) (bottom; molecular modeled from sequence hom-
ology;21 for the color code of amino acid side chain numbers, refer to Table 1).65
kinetics fit into a mechanistic scheme composed of two revers-
ible bimolecular substrate binding steps and a third non-rate
determining dissociative step, reverting the enzyme to the
initial state [bi-bi-ping-pong mechanism; Scheme 2, eqn (1)–
(4)].66–68
In the general mechanism for VBrPO-catalyzed bromide oxi-
dation derived from kinetic investigations and the chemistry
of bromoperoxidase mimics,69 hydrogen peroxide is the first
substrate to bind to the vanadate co-factor, displacing two
hydroxo ligands by one side-on coordinated peroxido ligand in
PO′ (Scheme 2). In the second step of the kinetic scheme, PO′
is protonated and bromide bound, presumably via hydrogen
residues known to form intramolecular disulfide bonds within
the VBrPO(AnI)-monomer are present at similar positions in
the VBrPO(AnII)-protein.21
The only bromoperoxidase-crystal structure available for
comparing the A. nodosum active sites is the vanadate–phos-
phate-exchanged bromoperoxidase-protein from Corallina
officinalis.35 Phosphate in this three-dimensional structure is
hydrogen-bonded to a hydroxyl group of serine-483, glycine-
484, and histidine-485, leading to close contact with the imida-
zole group of histidine-551, the proposed site of vanadate-
binding. Phosphate, however, does not bind to the τ-nitrogen
of histidine to expand its electron shell from eight to ten
valence electrons, as vanadate does. Additional salt bridges
form between negatively charged phosphate oxygens and the
terminal ammonium group of lysine-398, and two guanidi-
nium groups of arginine-406 and -545. This architecture com-
pares to active sites of the A. nodosum bromoperoxidases,
including a cavity named the substrate funnel, connecting the
exterior of the protein to the co-factor.
bonding to the vanadate-bound hydroxo ligand leading to PO″,
Br− 38,40
as expressed by Km
.
Proton transfer, for example, from
histidine-418 in VBrPO(AnI) or histidine-452 in VBrPO(AnII) to
the proximate peroxido oxygen-atom leads to a η2-hydroperox-
ido complex, which is attacked by bromide in a SN2-reaction.
Once the oxygen atom transfer from the peroxido group to
bromide has occurred, hydrolysis follows, providing hypobro-
mous acid and PO, the proposed resting state.
Bromide binding to PO leads to the competitively inhibited
state PO′″, being kinetically characterized by KiBr−, but structu-
rally not yet specified.70 Computational studies on bromoper-
oxidase mimics at the density functional level of theory predict
that bromide binding to coordinatively saturated monoanionic
2.3 Bromide oxidation
The role of the vanadate-active site in bromoperoxidases is to
bind hydrogen peroxide for oxidizing bromide under physio-
logical conditions (Section 3). Results from steady state
11930 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
Specific bromoperoxidase activities of VBrPO(AnI) and of
VBrPO(AnII), as expressed in U0 mg−1 in the MCD-assay,§
compare to the value obtained for the Laminaria saccharina
bromoperoxidase I (Table 6, entries 1, 2 and 9).72 Less active
bromoperoxidases are found in Ceramium rubrum,73 Corallina
pilulifera,25,74 and L. digitata (Table 6, entries 1, 3, and 5),23
whereas far more active bromoperoxidases occur in C. officina-
lis,26 C. vancouveriensis,27 Fucus distichus22 and Macrocystis pyri-
fera22 (Table 6, entries 4, 6, 7, 10). Explaining such differences
would be a step toward finding out whether a kinetic limit for
bromide oxidation in wild-type bromoperoxidases exists, and
whether this limitation can be overcome by a synthetic catalyst.
Since none of the highly active bromoperoxidases so far has
been crystallized and structurally investigated, this field is
wide open for future exploration.34,35
To sum up, bromoperoxidases VBrPO(AnI) and VBrPO(AnII)
differ in primary to quaternary structures, having different
affinities for hydrogen peroxide- and bromide-binding, but
show comparable turnover frequencies for turning over
bromide and hydrogen peroxide. The results from structure-
reactivity analysis led to the question whether products of
bromide oxidation for use in organic synthesis for the two bro-
moperoxidases are similar.
Scheme 2 Model for interpreting steady-state kinetics of bromide oxidation
catalyzed by VBrPO(AnX) [bi-bi-ping-pong mechanism; X = I, II; PO = VBrPO(AnX);
2
H O2
Br−
PO’ and PO’’ = loaded derivatives of PO; see also eqn (1)–(4); Km
and Km
are Michaelis–Menten parameters for H2O2- and Br−-binding; KiBr− describes
competitive enzyme inhibition by bromide; the red dashed line symbolizes a
hydrogen bond].
vanadate is endothermic, whereas bromide binding to a coor-
dinatively unsaturated vanadium(V) peroxido complex is
exothermic.38,40
3 Hypobromous acid, tribromide, and
bromine – the weak, the dormant, and the
strong bromoelectrophile
Important parameters in kinetic experiments are substrate
concentrations, temperature, pH, and ionic strength. A stan-
dard for the ionic strength is 0.8 M, the average value for sea-
water (I ∼ 0.7–0.9 M).71 Many but not all Michaelis–Menten
parameters reported in the literature refer to pH of 5.9,
because bromoperoxidases show maximum activity for
bromide oxidation in the range between 5.5 and 6.51 (Table 7,
entry 1). The standard assay for measuring rates of bromide
oxidation in bromoperoxidase chemistry is the monochlorodi-
medone (MCD)-test (cf. Table 5), showing a time-dependent
decrease of the enol absorption band at 290 nm, as MCD is
converted into bromochlorodimedone (BCD).
3.1 Chemical equilibria in aqueous solution
The general mechanism of VBrPO-catalyzed oxidation states
that hypobromous acid, the primary product of enzymatic
bromide oxidation (Scheme 2),75 is formed by two-electron oxi-
2
dation of bromide via transfer of two 4pz -electrons into the
σ*-orbital of the oxygen–oxygen bond (σ*O,O). The electron
transfer lowers the oxygen–oxygen bond order in the peroxide
to zero, while the bromine–oxygen bond forms.76,77 The rate of
oxidation at neutral pH and ambient temperature is small,
because the electron-accepting σ*O,O-orbital interacts only
weakly with the lone electron pairs at bromide. Reagents
capable of enhancing the electron accepting ability of hydro-
gen peroxide are acyl donors,78 Brønsted-acids,79 or vanadate
(V) in marine bromoperoxidases.13 Substituting a hydrogen
atom in hydrogen peroxide by an electron accepting group con-
tracts orbitals at peroxide oxygens, stabilizing the σ*O,O-orbital.
This change in σ*O,O-orbital energy strengthens the interaction
between the peroxide and bromide, reducing the activation
energy for the two electron oxidation.13
Michaelis–Menten-parameters determined via eqn (5) show
that the affinity for hydrogen peroxide binding to VBrPO(AnII)
is by almost one order of magnitude higher than for VBrPO
(AnI) (Table 5, entry 4). Approximately the same trend is seen
for bromide binding (Table 5, entry 3). The values found for
Br−
inhibitory constant Ki
suggest that bromide at concen-
trations exceeding ∼0.3–0.5 M retards rates of bromoperoxi-
dase-catalyzed oxidations (Table 5, entry 5).
Vmax
The initial product of oxygen atom transfer from activated
peroxides to bromide is coordinated or solvated hypobromite,
reacting in water to hypobromous acid (pKa 8.7). In brines,
ꢀ
ꢁ
Brꢀ
m
½H2O2ꢁ
K
1 þ
½Brꢀꢁ
ꢀ
ꢁ
voxidation
¼
þ ½H2O2ꢁ
ð5Þ
½Brꢀꢁ
1 þ
Brꢀ
K
KmH O
i
2
2
ꢀ
ꢁ
Brꢀ
§One unit (1 U) refers to the amount of enzyme necessary for turning over one
micromole of substrate per minute. The specific activity, on the other hand,
K
m
1 þ
½Brꢀꢁ
refers to the number of units per mg of enzyme, that is, U mg−1
.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11931

View Article Online
Paper
Dalton Transactions
Table 5 Steady-state kinetic parameters for bromoperoxidase catalyzed oxidations (22 0.5 °C)a
Entry
Parameter
VBrPO(AnI)a
VBrPO(AnI)b
VBrPO(AnII)b
VBrPO(AnII)b
1
2
3
4
5
pH
5.9c
107
2.9 0.3
75
5.9c
85
3.7 0.3
55
5.9c
68
0.32 0.11
22.5 3.6
125
7.2d
22
1.22 0.14
7.8 0.8
4242
Vmax/U mg−1 e
KBmr−/mM
7
4
4
1
KHm O /µM
7
6
2
2
KBi r− f/mM
557
255
a Ionic strength adjusted with sodium sulfate to 1.65 M. b Ionic strength adjusted with sodium sulfate to 0.80 M. c MES buffer. d HEPES buffer.
e Determined according to eqn (5), based on the MCD-assay (Table 6). f The estimated precision in KiBr− is 30–60%.
Table 6 Summary of bromoperoxidase activity in the monochlorodimedone (MCD) assay
c
Entry
Organism and isoenzyme
VBrPO(SpX)a
U0MCD mg−1 [pH]
V/proteinb
nagg
1
2
3
4
5
6
7
8
9
10
Ascophyllum nodosum I
Ascophyllum nodosum II
Ceramium rubrum I
VBrPO(AnI)
VBrPO(AnII)
VBrPO(CrI)
VBrPO(CoI)
VBrPO(CpI)
VBrPO(CvI)
VBrPO(FdI)
VBrPO(LdI)
VBrPO(LsI)
VBrPO(MpI)
130–170 [6.5]
90–120 [6.5]
7 [7.4]
550 [6.4]
27 [6.0]
266 [6.0]
1580 [6.5]
42 [6.1]
134 [6.0]
1730 [6.0]
1
0.5
1
1
2
1
1 (0.3)
—
2
2
6
4d
12
12
Corallina officinalis I26
Corallina pilulifera I25,74
Corallina vancouveriensis I27
Fucus distichus I22
e
—
—
2
2
—
e
Laminaria digitata I23
Laminaria saccharina I72
Macrocystis pyrifera I22
e
e
1.5 (0.38)
a Sp = species; X = isoenzyme number. b Ratio of vanadium per protein monomer in vanadate-reconstituted enzymes. Figures in brackets refer to
c
the reported V/protein-ratio of native bromoperoxidases. nagg. = the number of aggregated protein monomers. d Calculated from molecular
masses of 58 kDa for the monomer and 242 kDa for the aggregated bromoperoxidase. e Not reported.
such as sea water, the reaction continues by comproportionat-
ing bromide and the positively polarized bromine substituent
in hypobromous acid to molecular bromine. Bromine sub-
sequently is able to add in an exergonic reaction (ΔG298
=
−7.0 kJ mol−1) one equivalent of bromide to afford tribromide
2
−
Scheme 3 Equilibria between HOBr, Br2, and Br3 in H2O [KBr = 1.45 × 108
(Scheme 3).80–82
M−2 for H2O at 20 °C (I = 0.1 M, pH 2.6–3.8)10 or 1.04 × 108 M−2 in H2O at
In aqueous solution of bromide, such as sea water, hypo-
bromous acid, bromine, and tribromide are in rapid chemical
equilibrium, suggesting that the three compounds co-exist
close to the vanadate active site, in the substrate funnel, and at
the periphery of the bromoperoxidase protein (Scheme 4).
−
25 °C (pH 1.5);11 KBr = 16.9 M−1 in H2O at 25 °C12].
3
The molecular orbital for characterizing electrophilicity of
the bromine compound Br–X is the σ*Br,X-orbital. The energies
for σ*Br,X-orbitals of hypobromous acid, bromine, tribromide,
and other frequently used electrophilic bromination reagents
3.2 A scale for electrophilicity of bromination reagents
In the general mechanism of organobromine synthesis from in organic synthesis are available from density functional
alkenes, arenes, and heteroarenes, the unsaturated hydro- theory calculations, using Becke’s three parameter hybrid func-
carbon is the nucleophile, and the bromination reagent the tional (B3LYP)87,88 in combination with the 6-311++G** basis
electrophile.83,84 The nucleophile/electrophile-concept arises set.89 The method reproduces within satisfactory precision
from chemical kinetics and focuses on differences in acti- geometrical parameters [e.g. dO,Br in HOBr: 1.869 Å calculated
vation energies for explaining reactivity and selectivity.85 Para- versus 1.834 Å from microwave spectroscopy;90 dBr,Br in Br2:
meters for describing the affinity between reactants in 2.332 Å calculated versus 2.286(3) Å from X-ray diffraction at
kinetically controlled reactions are the energies of the highest 250 K;91 dBr,Br in Br3−: 2.639 Å calculated versus 2.557(2) Å
occupied molecular orbital of the donor and the lowest unoc- from X-ray diffraction92], and fundamental frequencies refer-
cupied molecular orbital of the acceptor.86
ring to bromine-79 isotopomers (νO,Br for HOBr: calculated
11932 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
Table 7 Mulliken-charge(s), effective electronegativity (χeff ), and σ*Br,X-bond
energies of bromination reagents Br–X, formed in bromoperoxidase-catalyzed
oxidations, compared to standards in organic synthesis
Mulliken-chargea
χeff
E(σ*X–Br)/eVc
b
Entry
X–Br
1
2
3
4
5
6
H2OBr+
Br2
+0.478
0.000
+0.090
+0.091
3.69
3.22
3.09
2.72
2.88
2.15
−10.2
−2.9
−1.4
−1.1
−0.4
+0.1
HOBr
tBuOBr
NBSd
+0.236
Br3
−0.491 (per)/−0.018 (cen)e
−
Scheme 4 Model for correlating the location and events for organobromine
synthesis in VBrPO(AnX)-catalyzed oxidations.
a Calculated
(B3LYP/6-311++G**//B3LYP/6-311++G**).
b Molecular
electronegativity (χeff) of Br–X according to Sanderson based on
corrected values of Pauling-electronegativities (χO = 3.654, χBr = 3.219,
χN = 3.194, χC = 2.746, χH = 2.592).97 c From the natural bond orbital
608 cm−1, experimental 620 cm−1; νBr,Br for Br2: calculated
311 cm−1 versus 322 cm−1 experimental;93 νBr,Br for Br3−: 53
(NBO)-analysis (B3LYP/6-31G**//B3LYP/6-311++G**). d NBS
=
N-
and 191 cm−1 in dichloromethane versus 83 and 177 cm−1 in bromosuccinimide. e per = peripheral bromine; cen = central bromine.
the gas phase) of bromine compounds.
B3LYP-theory in combination with the 6-311++G**-basis set
furthermore describes oxidation of hydrogen bromide by
hydrogen peroxide, comproportionation of hypobromous acid
(Table 7),96,97 σ*Br,X-energies gradually decrease, leading to a
stronger overlap with the π-type HOMO of an alkene (Fig. 3).
Effective electronegativities reflect changes in the electron-
and hydrogen bromide, and tribromide formation from
bromine and bromide as exergonic processes (Scheme 5).81,82,94
withdrawing capability of atoms, as bonds form and electrons
are delocalized, leading to one χeff-value for a molecule.
The Gibbs-free energy calculated for the gas phase synthesis
Common sense implies that this approach is practical for
of tribromide is smaller than the experimental value in solu-
tion,82 presumably for reasons of solvation effects in the
small molecules (e.g. Table 8, entries 1–3 and 6), but not for
larger ones such as tert-butyl hypobromite or N-bromosuccini-
mide (NBS) (e.g. Table 7, entries 4 and 5), because the accept-
experiment.
According to perturbation theory, the highest occupied
ing ability of a group generally fades at a distance of one to
molecular orbital (HOMO) of an alkene is stabilized by overlap
two bonds.
with the lowest unoccupied orbital (LUMO) of the bromine
compound Br–X (Fig. 3).95 As the accepting power of X
In the electronegativity analysis, bromine is a strong and
hypobromous acid is a weak bromoelectrophile (Table 7,
increases, expressed by effective electronegativities χeff
entries 2 and 3). Tribromide, in this model, is a borderline
case being able to react as electrophile and as nucleophile for
reasons of high σ*Br,X-orbital energy, negative charges at
bromine, and a low χeff-value (Table 7, entry 6).
The reference nucleophile selected for ranking electrophili-
city of Br–X in the frontier molecular orbital (FMO)-approach
is ethene (Scheme 6). Bromonium ion formation from
bromine and ethene or other alkenes proceeds kinetically con-
trolled via early reactant-like transition states.86,98 The energy
ΔE gained by perturbing the alkene π-orbital with the σ*Br,X
-
Scheme 5 Computed (B3LYP/6-311++G**//B3LYP/6-311++G**) free energy of
hypobromous acid-, bromine-, and tribromide formation from hydrogen per-
oxide, hydrogen bromide, and bromide in the gas phase [calculated free ener-
gies for 298.15 K (ΔRG298.15) are shown in bold and zero-point corrected
reaction energies at 0 K (ΔRE0) in italics].
orbital, according to the third term of the Salem–Klopman-
equation, correlates with the reciprocal value of the HOMO–
LUMO-energy gap (Fig. 3).95
Reciprocal values of the energy differences between πC,C
-
and σ*Br,X-orbitals, calculated99 from natural bond orbital
(NBO)-analysis100 at the B3LYP/6-31G**//B3LYP/6-311++G**-
level of theory, correlate for ethene, (E)-but-2-ene, 2-methylbut-
2-ene, 2,3-dimethylbut-2-ene, and prop-1-en-2-ol exponentially
Fig. 3 Diagram displaying stabilizing interactions from frontier molecular
orbital correlation between σ*Br,X- and πC,C-orbitals in the transition state of
alkene bromination by Br–X.
Scheme 6 Benchmark for scaling relative reactivity of bromoelectrophiles from
FMO-theory: bromonium ion formation from ethene and Br–X (cf. Fig. 3 and 4).
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11933

View Article Online
Paper
Dalton Transactions
2
between the 4pz -orbital of bromide and the σ*Br,Br-orbital of
bromine (Fig. 5), which is the familiar mode of interaction
between bromine and a nucleophile (vide supra). Populating
the σ*Br,Br-orbital lowers the MO-bond order in bromine to
0.75, which is slightly overcompensated by the newly formed
bromine–bromine and delocalization of the excess charge
across the three bromines (Fig. 5, bottom).
Protonating at terminal bromine, the most negatively
charged position in tribromide, reduces the donor ability of
bromide, causing the unsymmetric hydrogen tribromide to
fragment into bromine and hydrogen bromide (Scheme 7).
This behavior explains the ability of protic solvents to acceler-
ate tribromide dissociation, liberating bromine for use in
organic synthesis.56
Fig. 4 Correlation of calculated σ*Br,X-energies and reciprocal FMO-energy-
○
◊
●
□
differences (cf. Fig. 3; = ethene, = (E)-but-2-ene, = 2-methylbut-2-ene, =
■
2,3-dimethylbut-2-ene, = prop-1-en-2-ol; NBS = N-bromosuccinimide; 1 a.u. =
27.2144 eV; correlation coefficients R2 for all exponential functions are 0.995
and higher).
with σ*Br,X-energies (Fig. 4). From this information we con-
cluded that FMO-interactions in the gas phase between
alkenes and bromine are stronger than those for hypobromous
acid and for tribromide.
3.3 Tribromide – a borderline case
Among the bromination reagents formed in bromoperoxidase-
catalyzed oxidations, tribromide probably is the one more
difficult to classify in terms of chemical reactivity. Tribromide
is an anion with the terminal bromine atoms being stronger
negatively charged than the central bromine (Fig. 5). Low
effective electronegativity in combination with electron deloca-
lization makes tribromide a soft nucleophile, which is used in
organic synthesis to prepare organobromines via nucleophilic
substitution.101 FMO-correlations (Fig. 4), chemical kinetics,57
and stereoselectivity56 for alkene dibromination in chlorinated
hydrocarbons show that tribromide may also behave as an
electrophile. In protic solvents, this tribromide-specific reactiv-
ity disappears and bromine reactivity shows up, explaining the
use of tribromide salts under such conditions as safe-to-
handle bromine surrogates.56
Scheme 7 Products of tribromide protonation according to density functional
theory calculations (B3LYP/6-311++G**).
Protonating tribromide at the central bromine atom
enhances the accepting ability of the bromine molecule, stabi-
lizing the symmetric hydrogen tribromide isomer. Theory pre-
dicts the molecule to be stable, showing an equilibrium
distance of 1.432 Å and a harmonic stretching frequency of
2581 reciprocal wavenumbers for the hydrogen–bromine bond
in hydrogen tribromide. Both parameters are similar to the
bond length and harmonic stretching frequency of hydrogen
bromide (1.427 Å, 2605 cm−1; B3LYP/6-311++G**-level of
theory). From this information, we concluded that the hydro-
gen–bromine bond dissociation energies in hydrogen tribro-
mide and hydrogen bromide are similar (366.4 kJ mol−1),
although tribromide protonation at the central bromine is
expected to be disfavored for reasons of charge effects.102
To sum up, hypobromous acid, tribromide, and bromine
differ in electronegativity, Mulliken-charges at bromine, and elec-
trophilicity. Hypobromous acid is a weak, tribromide in protic
solvents a dormant, and bromine a strong bromoelectrophile.
The dichotomic behavior of tribromide, according to
theory, originates from changes in electron donating and
accepting abilities of the bromide and the bromine entity, as
an electrophile or a nucleophile approaches. A natural bond
order analysis at the B3LYP/6-31G**//B3LYP/6-311++G**-level
of theory shows that bonding in tribromide occurs via overlap
4 Reactivity and selectivity for carbon–
bromine formation in vanadate-dependent
bromoperoxidase-catalyzed oxidations
4.1 Bromination of phenols
Fig. 5 Bonding in the tribromide anion, described by valence bond theory
(top; numbers refer to calculated Mulliken-charges) and molecular orbital
theory (BO = bond order).
According to reaction theory, hypobromous acid, tribromide,
and bromine show distinctively different chemical behavior
11934 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
toward alkenes. From this information, we expected reactivity preference for tribromophenol formation. The enzymatic reac-
and selectivity for carbon–bromine bond formation in VBrPO tion is the most effective and the only known procedure so far
(AnI)- and VBrPO(AnII)-catalyzed oxidation to be controlled by for selectively preparing bromophenol 2a.
the most electrophilic bromination reagent.
In oxidations catalyzed by VBrPO(AnI), ortho-substituted
The chemical nature of the effective bromination reagent in phenols 1b–f are converted into bromophenols 2b–f and
bromoperoxidase-catalyzed oxidations was experimentally dibrominated derivatives 3b–f (Table 9, entries 2–6). Substrate
uncovered via competition kinetics on phenol bromination, in conversion and yields of bromoarene formation decrease as
combination with a Hammett-type linear free energy relation- the electron accepting ability of substituent R increases
ship [eqn (6–8)]. The energy of the highest occupied π-type (Table 9).
orbital in phenol (1a) (−6.39 eV) is similar to the π-orbital
energy of 2-methylbut-2-ene [−6.42 eV, both values calculated
(B3LYP/6-311++G**//B3LYP/6-311++G**)], allowing us to quali-
Table 8 Products of phenol bromination formed in bromoperoxidase-cata-
lyzed oxidation and non-enzymatic reactions103
tatively adapt the results from the previous chapter to the
chemistry of phenol bromination in bromoperoxidase-cata-
lyzed oxidations.
Phenol (1a) yields in morpholine-4-ethanesulfonic acid
(MES)-buffered aqueous tert-butanol bromophenol 2a as a 9/
91-mixture of ortho/para-regioisomers, besides dibromophenol
3a and tribromophenol 4a, if treated with VBrPO(AnI) and 1.1
equivalents of hydrogen peroxide and sodium bromide. No
phenol bromination occurs in a bromide-free, pH 6.2-buffered
solution of hypobromous acid in aqueous tert-butanol even by
raising the aliquots of the reagent from one (Table 8, entry 2)
to three.103 Molecular bromine preferentially converts phenol
(1a) into tribromophenol 4a. Raising bromine aliquots from
one to three improves the conversion of 1a from 53% (Table 8,
entry 3) to quantitative. Tribromide in this series of exper-
Conv. 2a/%
1a/%a (o/p)b
Entry [Br]
3a/% 4a/% pHc
1
2
3
4
NaBr/H2O2/VBrPO(AnI) 90
69 (9/91)
—
4
—
3
6
—
29
23
6.8
5.7
6.2
6.1
d
d
d
HOBr
Br2
NBu4Br3
6
53
53
17 (18/82)
14 (<2/98) 13
a Continuous addition (syringe pump, 8 h) of a solution of H2O2 (0.825
M, 1.1 equiv.) and NaBr (0.75 mmol, 1.0 equiv.) in aq. MES buffer
iments is equally effective and selective as molecular bromine (500 mM, pH 6.2) {VBrPO(AnI) 55.2 μL, 0.5626 mg mL−1, 558 UT mg−1
,
(Table 8, entry 4).103
17.3 UT, 0.036 mmol%} (entry 1); batch addition of a solution of HOBr
in aq. MES buffer (500 mM) (0.2–0.3 M, 0.75 mmol, 1.0 equiv.) (entry
2); continuous addition (syringe pump, 8 h) of a solution of Br2 (0.75
The results from phenol bromination, in summary, show
that hypobromous acid is not able to convert phenol (1a) in M, 1.0 equiv.) (entry 3); batch addition of TBABr3 (0.75 mmol, 1.0
equiv.) (entry 4); starting material: 0.75 mmol of 1a for all entries. b o
solutions used for conducting bromoperoxidase-catalyzed oxi-
dations into bromoderivatives 2a–4a. Bromine and tribromide
(rtho) and p(ara) relative to OH; unless otherwise stated 1H NMR
yields. c pH-electrode, final pH of the aqueous solution. d Not
provide matching reactivity/selectivity-patterns with a strong detectable.
Table 9 Products of phenol bromination in bromoperoxidase-catalyzed oxidation103
Entry
1–3
R
Conv. 1/%a
2/% (o/p)b
3/%
Mass balance/%c
pHfinal d
Activity UfTinal mg−1 e
1
2
3
4
5
6
a
b
c
d
e
f
H
90 f
86
53
68
43
51
69 (9/91)
4
3
2
10
26
18
89
85
91
98
96
86
6.8
6.5
6.4
6.4
6.3
6.4
133
138
CH3
C(CH3)3
OCH3
Cl
68 (16/84)
42 (36/64)
56 (21/79)
13 (<2/98)
19 (<2/98)
g
—
—
g
129
160
F
a Continuous addition (syringe pump, 8 h) of a solution of H2O2 (0.825 m, 1.1 equiv.) and NaBr (0.75 mmol, 1.0 equiv.) in aq. MES buffer (pH
6.2)–tBuOH = 75/25% (v/v); 0.75 mmol of 1 were used as the starting material. b ortho and para relative to OH; unless otherwise stated 1H NMR
yields. c Sum of recovered 1 and bromides 2–4. d pH electrode. e Initial enzyme activity 558 UT0 mg−1 (entries 1 and 3) and 611 U0T mg−1 (entries 2
and 4–6), with UT0 mg−1 referring to VBrPO(AnI)-activity in the triiodide-assay.75 f Additional product: 6% of 2,4,6-tribromophenol (4a). g No final
bromoperoxidase activity detected.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11935

View Article Online
Paper
Dalton Transactions
4.2 Competition kinetics
electronic effects of R, but not a combination of electronic and
steric effects.105
Competition kinetics conducted under pseudo-first order con-
ditions show that relative rates krel of phenol bromination in a
solution containing sodium bromide, hydrogen peroxide, and
one of the A. nodosum bromoperoxidases, determined accord-
ing to eqn (6) and (7), decrease along the series of ortho-substi-
tuents CH3, C(CH3)3, OCH3, Cl, to F by a factor 32 (Table 10,
entries 1–5; Scheme 8). This trend is similarly seen in oxi-
dations catalyzed by VBrPO(AnII) and in bromine-mediated
reactions (Table 10).
Competition kinetics, unlike synthesis, provides in enzy-
matic reactions, and in reactions starting from molecular
bromine, exclusively products of monobromination, presum-
ably due to a twenty-fold increase of arenol concentration. For
gas chromatographic-analysis, brominated alcohols 2a–f are in
situ quantitatively converted into O-acetyl derivatives, allowing
to insert the ratio of O-acetylbromophenols instead of bromo-
phenols 2a–f into eqn (6).104
½ 2bꢀ fꢁ kR ½ 1bꢀ fꢁ
¼
ð6Þ
½ 2aꢁ
kH ½ 1aꢁ
kR
kH
¼ krel
ð7Þ
ð8Þ
lg krel ¼ ρ ꢂ σm
Enzymatic reactions and phenol bromination by bromine
show in Hammett-type correlations of decadic logarithms of
lg krel versus a standard substituent constant σm, according to
eqn (8), similar ρ-values (Table 11, Fig. 6). The negative sign of
ρ reflects rate enhancing effects of electron-releasing substitu-
ents, classifying the rate determining bromination reagent as
an electrophile. Comparing the absolute value of ρ to refer-
ences from the literature shows that the reactivity and selecti-
vity-determining bromoelectrophile is molecular bromine.106
The estimated reaction parameter for tetrabutylammonium tri-
bromide-mediated phenol bromination (ρ ∼ −3.1) is similar to
controls starting from bromine (Table 11). This observation
implies that tribromide decomposes into bromide and
bromine prior to phenol bromination, as predicted by reaction
theory (Section 3).
All competition kinetic experiments provided ortho- and
para-substituted bromophenols 2a–f. Relative rate constants
summarized in Table 10 exclusively refer to yields of para-bro-
mophenols, which are the major products.21,75 This approach
arises from a fundamental principle of correlation analysis
stating that the substituent parameter σm summarizes
Table 10 Relative rate constants of phenol brominationa in bromoperoxidase-
catalyzed oxidation and for non-enzymatic methods, and σm parameters used
for correlation analysis21,103
Table 11 Reaction parameter
butanol (pH 6.2, 25 °C)21,103
ρ for phenol bromination in aqueous tert-
VBrPO(AnI) VBrPO(AnII) Br2
NBu4Br3
Entry R/1
σm
kRp/kHp
kRp/kHp
kRp/kHp kRp/kHp
Entry
[Br]
ρ
1
2
3
4
5
CH3/1b
−0.11 2.25
1.70
0.71
0.63
0.13
1.35
—
0.97
0.22
0.19
3.15
b
1
2
3
4
V
BrPO(AnI)
−3.1
b
C(CH3)3/1c −0.02 0.98
OCH3/1d
Cl/1e
—
VBrPO(AnII)
Br2
NBu4Br3
−2.5
−1.9
0.05 0.48
0.31 0.10
0.36 0.07
0.99
c
(−3.1)a
—
b
c
F/1f
—
—
a Estimated on the basis of krel values for 1b and 1d in MES-buffered
aqueous acetonitrile (cf. Table 10).
a p(ara) with respect to the hydroxyl group in 1. b Not considered due to
scatter of data in the diagram used for determining krel according to
eqn (6) and (7). c Not considered for correlation analysis for reasons of
significant dibromide formation.
Fig. 6 Correlation of lg krel from phenol bromination by bromine ( ) (corre-
●
■
Scheme 8 Reaction scheme for kinetic experiments in the phenol-competition
system {for data analysis, see eqn (6)–(8); indexing for phenols 1 and bromophe-
nols 2: R = H for a, CH3 for b, C(CH3)3 for c, OCH3 for d, and Cl for e; for [Br] cf.
Table 8}.
lation coefficient R2 = 0.986), tetrabutylammonium tribromide ( ), the combi-
2
○
nation of sodium bromide, hydrogen peroxide and VBrPO(AnI) ( ) (R = 0.995),
and sodium bromide, hydrogen peroxide and VBrPO(AnII) ( ) (R2 = 0.990) versus
□
σm according to eqn (8).
11936 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
5 Biomimetic syntheses
6. Concluding remarks
The information that reactivity in bromoperoxidase-catalyzed oxi- Although structures of vanadate-dependent bromoperoxidases
dation is controlled by molecular bromine allows to develop I and II from the brown alga Ascophyllum nodosum are surpris-
organobromine synthesis based on enzymatic reactions. To eluci- ingly different, the bromine chemistry that follows oxidation
date a taxonomy of functional groups being receptive for electro- catalysis is almost identical. Differences in carbon–bromine
philic bromination under such conditions, we started, for two bond formation relate to the stability of the enzymes under
reasons, from pyrrole-2-carboxylate chemistry.45,75 The first argu- turnover conditions but not to chemical selectivity. Consider-
ment came from theory, because the energies of the highest ing a sequence homology of 41% between VBrPO(AnI) and
occupied π-type orbital in O-methyl-pyrrole-2-carboxylate VBrPO(AnII), we think that the bromoperoxidase chemistry
(−6.45 eV) and in phenol (1a) (−6.39 eV) are similar (both ener- described in this article has potential to serve as representative
gies calculated on the B3LYP/6-311++G**//B3LYP/6-311++G**- example for bromoperoxidases showing similar vanadate
level of theory). The second argument arose from natural active site architecture.
product chemistry,107 showing that brominated pyrrole-2-
carboxylic acid derivatives occur in marine sponges.59–61,108
Mechanistic studies from biochemistry and physical
organic chemistry in the past few years provided a clear
Introducing bromine into the heteroaromatic core of picture of elementary reactions leading from bromide in
O-methyl-pyrrole-2-carboxylate via bromoperoxidase-catalyzed oxi- VBrPO-catalyzed oxidations to brominated aromatic and het-
dations is feasible under conditions similar to those described eroaromatic compounds. For both product classes, molecular
for phenol bromination (Scheme 9, Table 8).103 Oxidations bromine is the effective bromination reagent. Alternative
catalyzed by VBrPO(AnI) provided O-methyl-bromopyrrole-2- reagents, such as hypobromous acid and tribromide, are not
carboxylate as a ∼77/23-mixture of 4/5-regioisomers, or O-methyl- competitive with the pronounced electrophilicity of bromine,
4,5-dibromopyrrole-2-carboxylate as a major product, depending as expressed by frontier molecular orbital analysis and linear
on the chosen substrate ratios (Scheme 9). Triiodide-assays free energy relationship. Bromination of other hydrocarbons
showed residual peroxidase activity in the range of ∼20% after have so far not been investigated with similar precision,
quantitative bromide- and hydrogen peroxide consumption. This leaving open the answer to the question whether the chem-
bromoperoxidase activity can be used for three additional runs by istry of arene bromination transfers to, for example, terpenol
progressively lengthening reaction times for compensating declin- bromination. This question is particularly interesting in view
ing enzymatic activity. Changing the oxidation catalyst from of the multitude of chiral brominated terpenes isolated as
VBrPO(AnI) to VBrPO(AnII) provides a similar product selectivity, enantiomerically pure secondary metabolites from numerous
but with a reduced degree of substrate conversion, leading to a marine organisms.
lower yield of O-methyl-bromopyrrole-2-carboxylate (Scheme 9).103
The final remark in this article relates to a question raised
Pyrrole-2-carboxamide affords 42% bromopyrrole-2-carbox- on a possible reactivity limit for bromide oxidation by hydro-
amide as a 71/29-mixture of 4/5-regioisomers and 9% of 4,5- gen peroxide, catalyzed by protein-bound vanadate. A poss-
dibromopyrrole-2-carboxamide if treated with 1.1 equivalents of ible approach to answer this question is a combination of
sodium bromide and hydrogen peroxide, and catalytic amounts site-directed mutagenesis and molecular modeling, for
of VBrPO(AnI). 4,5-Dibromopyrrole-2-carboxamide is a marine example, by density functional theory, to systematically map
natural product isolated from the sponge Acanthella carteri.21,75
the chemical reactivity of vanadate in an environment com-
posed of protein side chains. From such a study, a robust,
low-priced, highly selective oxidation catalysts could emerge
for organobromine synthesis from sea-water as a bromide
source, as
chemistry.
a contribution to sustainable organobromine
Acknowledgements
This work is part of the Ph.D. theses of D.W. and M.R. For
funding we express our thanks to the Deutsche Bundesstiftung
Umwelt (DBU) for providing a scholarship (for D.W.) and
financial support, the Deutscher Akademischer Austausch-
dienst (travel fund for J.H. for presenting this work at the 8th
International Symposium on the Chemistry, Biological Chem-
istry, and Toxicology of Vanadium, Arlington, U.S.A.), and the
state Rheinland-Pfalz for providing financial support within
the NanoKat-framework.
Scheme 9 Synthesis of naturally occurring brominated derivatives of O-methyl
pyrrole-2-carboxylate (n = 1, 2; X = I, II; 63 µmol% of VBrPO(AnI) or VBrPO(AnI),
corresponding to 34.6 UT). a1.1 equiv. of NaBr and H2O2. b2.2 equiv. of NaBr and
H2O2.21,75
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11937

View Article Online
Paper
Dalton Transactions
24 P. Jordan and H. Vilter, Biochim. Biophys. Acta, 1991, 1073,
98–106.
Notes and references
25 N. Itoh, Y. Izumi and H. Yamada, J. Biol. Chem., 1986, 261,
5194–5200.
26 D. J. Sheffield, T. Harry, A. J. Smith and L. J. Rogers, Phyto-
chemistry, 1992, 32, 21–26.
27 R. R. Everett, J. R. Kanofsky and A. Butler, J. Biol. Chem.,
1990, 265, 4908–4914.
1 A. Butler and J. V. Walker, Chem. Rev., 1993, 93, 1937–
1944.
2 S. L. Neidleman and J. Geigert, in Biohalogenation: Prin-
ciples, Basic Roles and Applications, Ellis Horwood, Chiche-
ster, 1986.
3 A. Butler, Coord. Chem. Rev., 1999, 187, 17–35.
4 M. C. R. Franssen and H. C. van der Plas, Adv. Appl. Micro-
biol., 1992, 37, 41–99.
5 R. Frim and S. D. Ukeles, in Industrial Minerals & Rocks,
ed. J. E. Kogel, N. C. Trived and J. M. Baker, Society for
Mining, Metallurgy and Exploration, Littleton, Colorado,
2006, 7th edn, pp. 285–294.
28 A. Butler and J. N. Carter-Franklin, Nat. Prod. Rep., 2004,
21, 180–188.
29 M. Andersson and S. Allenmark, Tetrahedron, 1998, 54,
15293–15304.
30 H. B. ten Brink, H. L. Holland, H. E. Schoemaker, H. van
Lingen and R. Wever, Tetrahedron: Asymmetry, 1999, 10,
4563–4572.
6 G. W. Gribble, in Naturally Occurring Organohalogen Com-
pounds – A Comprehensive Update, Progress in the Chemistry
of Organic Natural Products, series ed. A. D. Kinghorn, H.
Falk and J. Kobayashi, Springer, Wien, 2010, vol. 91,
pp. 9–348.
7 J.-M. Kornprobst, in Encyclopedia of Marine Natural Pro-
ducts, Wiley-VCH, Weinheim, 2010, vol. 1, pp. 251–422.
8 (a) R. Wever, Nature, 1988, 335, 501; (b) R. M. Moore,
M. Webb, R. Tokarczyk and R. Wever, J. Geophys. Res.,
1996, 101, 20.899–20.908.
9 D. Rehder, Angew. Chem., Int. Ed. Engl., 1991, 30, 148–167.
10 M. Eigen and K. Kustin, J. Am. Chem. Soc., 1962, 84, 1355–
1361.
11 J. M. Pink, Can. J. Chem., 1970, 48, 1169–1171.
12 D. B. Scaife and H. J. V. Tyrrell, J. Chem. Soc., 1958, 80,
386–392.
31 J. N. Carter-Franklin and A. Butler, J. Am. Chem. Soc.,
2004, 126, 15060–15066.
32 F. H. Vaillancourt, E. Yeh, D. A. Vosburg, S. Garneau-
Tsodikova and C. T. Walsh, Chem. Rev., 2006, 106, 3364–3378.
33 A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U. S.
A., 1996, 93, 392–396.
34 M. Weyand, H.-J. Hecht, M. Kieß, M.-F. Liaud, H. Vilter
and D. Schomburg, J. Mol. Biol., 1999, 293, 595–611.
35 M. N. Isupov, A. R. Dalby, A. A. Brindley, Y. Izumi,
T. Tanabe, G. N. Murshudov and J. A. Littlechild, J. Mol.
Biol., 2000, 299, 1035–1049.
36 D. Geibig, R. Wilcken, M. Bangesh and W. Plass, in NIC--
Symposium 2008, John von Neumann Institute for Computing
Jülich, ed. G. Münster, D. Wolf and M. Kremer, NIC Series,
2008, vol. 39, pp. 71–78.
37 R. A. Sheldon, in Aspects of Homogeneous Catalysis, ed.
R. Ugo, Reidel Publishing, Dordrecht, 1981, vol. 4,
pp. 3–69.
13 D. Wischang, O. Brücher and J. Hartung, Coord. Chem.
Rev., 2011, 255, 2204–2217.
14 H. Vilter, K.-W. Glombitza and A. Grawe, Bot. Mar., 1983,
26, 331–340.
38 J. Y. Kravitz and V. Pecoraro, Pure Appl. Chem., 2005, 77,
1595–1605.
15 H. Vilter, Phytochemistry, 1984, 23, 1387–1390.
16 H. L. Holland, in Organic Synthesis with Oxidative Enzymes,
Wiley-VCH, New-York, 1992, ch. 1, pp. 1–40.
17 J. Hartung, Angew. Chem., Int. Ed., 1999, 38, 1209–1211.
18 E. de Boer and R. Wever, J. Biol. Chem., 1988, 263, 12326–
12332.
19 B. E. Krenn, H. Plat, M. G. M. Tromp, H. Vilter and
R. Wever, Abstracts of the 14th International Congress
of Biochemistry, Prague, Czechoslovakia, 1988, vol. 4,
p. 103.
39 G. Zampella, J. Y. Kravitz, C. E. Webster, P. Fantucci,
M. B. Hall, H. A. Carlson, V. L. Pecoraro and L. De Gioia,
Inorg. Chem., 2004, 43, 4127–4136.
40 C. J. Schneider, G. Zampella, C. Greco, V. L. Pecoraro and
L. De Gioia, Eur. J. Inorg. Chem., 2007, 515–523.
41 C. J. Schneider, J. E. Penner-Hahn and V. L. Pecoraro,
J. Am. Chem. Soc., 2008, 130, 2712–2713.
42 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.
20 B. E. Krenn, M. G. M. Tromp and R. Wever, J. Biol. Chem.,
1989, 264, 19287–19292.
21 D. Wischang, M. Radlow, H. Schulz, J. Hartung, H. Vilter,
L. Viehweger, M. Altmeyer, C. Kegler, J. Herrmann,
R. Müller, L. Delage, F. Gaillard and C. Leblanc, Bioorg.
Chem., 2012, 44, 25–34.
22 H. S. Soedjak and A. Butler, Biochim. Biophys. Acta, 1991,
1079, 1–7.
23 C. Colin, C. Leblanc, E. Wagner, L. Delage, E. Leize-
Wagner, A. van Dorsselaer, B. Kloareg and P. Potin, J. Biol.
Chem., 2003, 278, 23545–23552.
43 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. 17–322.
44 A. Podgoršek, M. Zupan and J. Iskra, Angew. Chem., Int.
Ed., 2009, 48, 8424–8450.
45 D. Wischang, J. Hartung, T. Hahn, R. Ulber, T. Stumpf
and C. Fecher-Trost, Green Chem., 2011, 13, 102–108.
46 J. Hartung, O. Brücher, D. Hach, H. Schulz, H. Vilter and
G. Ruick, Phytochemistry, 2008, 69, 2826–2830.
47 For cloning and heterologous expression of a bromoperox-
idase from Corallina officinalis in Escherichia coli, see:
11938 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013

View Article Online
Dalton Transactions
Paper
J. N. Carter, K. E. Beatty, M. T. Simpson and A. Butler,
J. Inorg. Biochem., 2002, 91, 59–69.
66 H. J. Fromm, in Initial Rate Enzyme Kinetics, Springer,
Berlin, New York, 1975.
48 For the expression of bromoperoxidase from C. pilulifera
in Saccharomyces cerevisiae, see: T. Ohshiro, W. Hemrika,
T. Aibara, R. Wever and Y. Izumi, Phytochemistry, 2002, 60,
595–601.
49 For the expression of bromoperoxidase from C. pilulifera
in Escherichia coli, see: M. Shimonishi, S. Kuwamoto,
H. Inoue, R. Wever, T. Ohshiro, Y. Izumi and T. Tanabe,
FEBS Lett., 1998, 428, 105–110.
50 For methods of electrophilic and homolytic organobro-
mine syntheses, see: O. Brücher and J. Hartung, ACS
Catal., 2011, 1, 1448–1454, and references cited therein.
51 For exploratory studies on hydrocarbon bromination in
oxidations catalyzed by acetone-powder from C. vancouver-
iensis, see: M. Shang, R. K. Okuda and D. Worthen, Phyto-
chemistry, 1994, 37, 307–310.
52 For exploratory studies on hydrocarbon bromination in
oxidations catalyzed by the vanadate(V)-dependent bromo-
peroxidase from C. pilulifera, see: N. Itoh, A. K. M.
Q. Hasan, Y. Izumi and H. Yamada, Eur. J. Biochem., 1988,
172, 477–484.
53 A. Butler and M. Sandy, Nature, 2009, 460, 848–854.
54 O. Bortolini, M. Carraro, V. Conte and S. Moro,
Eur. J. Inorg. Chem., 2003, 42–46.
55 H. Keller-Rudek, D. Koschel, P. Merlet, U. Ohms-
Bredemann, J. Wagner and A. Wietelmann, in Gmelin
Handbook of Inorganic and Organometallic Chemistry,
ed. R. Haubold, J. V. Jonanne, H. Keller-Rudek,
D. Koschel, P. Merlet and J. Wagner, Supplement 2,
Springer, Heidelberg, 8th edn, 1992.
56 W. J. Wilson and F. G. Soper, J. Chem. Soc., 1949, 3376–
3379.
57 G. Bellucci, R. Bianchini, R. Ambrosetti and G. Ingrosso,
J. Org. Chem., 1985, 50, 3313–3318.
58 R. S. Brown, H. Slebocka-Tilk, A. J. Bennet, G. Bellucci,
R. Bianchini and R. Ambrosetti, J. Am. Chem. Soc., 1990,
112, 6310–6316.
59 S. Forenza, L. Minale, R. Riccio and E. Fattorusso,
J. Chem. Soc., Chem. Commun., 1971, 1129–1130.
60 I. Mancini, G. Guella, P. Amade, C. Roussakis and
F. Pietra, Tetrahedron Lett., 1997, 38, 6271–6274.
61 N. S. Reddy, P. Ramesh, T. P. Rao and Y. Venkateswarlu,
Ind. J. Chem B., 1999, 38, 1145–1147.
62 E. de Boer, H. Platt, M. G. M. Tromp, R. Wever,
M. C. Franssen, H. C. van der Plas, E. M. Meijer and
H. E. Schoemaker, Biotechnol. Bioeng., 1987, 30, 607–610.
63 C. Leblanc, D. Wischang and J. Hartung, unpublished
results.
67 C.-R. Yang, B. E. Shapiro, E. D. Mjolsness and
G. W. Hatfield, Bioinformatics, 2005, 21, 774–780.
68 W. W. Cleland, Biochim. Biophys. Acta, 1963, 67, 104–137.
69 D. Rehder, in Bioinorganic Vanadium Chemistry, Wiley,
Chichester, 2008, pp. 105–128.
70 H. Dau, J. Dittmer, M. Epple, J. Hanss, E. Kiss, D. Rehder,
C. Schulzke and H. Vilter, FEBS Lett., 1999, 457, 237–240.
71 M. Whitefield, Mar. Chem., 1973, 1, 251–266.
72 E. de Boer, M. G. M. Tromp, H. Plat, G. E. Krenn and
R. Wever, Biochim. Biophys. Acta, 1986, 872, 104–115.
73 B. E. Krenn, H. Plat and R. Wever, Biochim. Biophys. Acta,
1987, 912, 287–291.
74 N. Itoh, H. Sasaki, N. Ohsawa, M. S. Shibata and J. Miura,
Phytochemistry, 1996, 42, 277–281.
75 D. Wischang and J. Hartung, Tetrahedron, 2011, 67, 4048–
4054.
76 (a) J. H. Espenson, O. Pestovsky, P. Huston and S. Staudt,
J.
Am.
Chem.
Soc.,
1994,
116,
2869–2877;
(b) M. S. Reynolds, S. J. Morandi, J. W. Raebiger,
S. P. Melican and S. P. E. Smith, Inorg. Chem., 1994, 33,
4977–4984.
77 E. A. Shilov, J. Am. Chem. Soc., 1938, 60, 490–491.
78 F. Beer, G. Düsing and H. Pistor, in Wasserstoffperoxid und
seine Derivate – Chemie und Anwendung (translates into:
Hydrogen peroxide and its derivatives – chemistry and appli-
cation), ed. W. Weigert, Hüthig, Heidelberg, 1998,
pp. 23–35.
79 C. W. Jones, in Application of Hydrogen Peroxide and
Derivatives, RSC Clean Technology Monographs, Cam-
bridge, 1999, pp. 156–162.
80 G. Jones and S. Baeckström, J. Am. Chem. Soc., 1934, 56,
1517–1523.
81 H. A. Young, J. Am. Chem. Soc., 1950, 72, 3310–3312.
82 C. M. Kelley and H. V. Tartar, J. Am. Chem. Soc., 1956, 78,
5752–5756.
83 (a) G. Bellucci, R. Bianchini and S. Vecchiani, J. Org.
Chem., 1986, 51, 4224–4232; (b) J. Berthelot, C. Guette,
P.-L. Desbène, J.-J. Basselier, P. Chaquin and D. Masure,
Can. J. Chem., 1989, 67, 2061–2066.
84 (a) S. Doonan, in The Chemistry of Functional Groups – The
Chemistry of the Carbon-Halogen Bond, ed. S. Patai, Wiley,
Chichester, 1973, pp. 865–915; (b) Y. Sasson, in The Chem-
istry of Functional Groups – Supplement D2, The Chemistry
of Halides, Pseudo-Halides, and Azides, ed. S. Patai and
Z. Z. Rappoport, Wiley, Chichester, 1995, pp. 535–628.
85 G. W. Klumpp, in Reaktivität in der Organischen Chemie,
Thieme, Stuttgart, 1977, vol. 1, pp. 139–231.
64 H. Vilter, Metal ions in biological systems, in Vanadium
and its Role in Life, ed. H. Sigel and A. Sigel, Dekker,
New York, 1995, vol. 31, pp. 325–362.
65 By mistake the proposed vanadate(V)-binding site in ref.
21 was abbreviated as His-517. The correct position of this
amino acid according to the numbering outlined in
Table 1 is His-537.
86 N. T. Anh, in Frontier Orbitals, Wiley, Chichester, 2007.
87 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.
88 C. Lee, W. Yang and R. G. Parr, Phys. Rev., 1988, B37, 785–
789.
89 For the assessment of the B3LYP/6-311++G**-theory in
bromonium ion formation, see: V. I. Teberedikis and
M. P. Sigalas, Tetrahedron, 2003, 59, 4749–4756.
This journal is © The Royal Society of Chemistry 2013
Dalton Trans., 2013, 42, 11926–11940 | 11939

View Article Online
Paper
Dalton Transactions
90 Y. Koga, H. Takeo, S. Kondo, M. Sugie, C. Matsumura,
G. A. McRae and E. A. Cohen, J. Mol. Spectrosc., 1989, 138,
467–481.
91 B. M. Powell, K. M. Heal and B. H. Torrie, Mol. Phys.,
1984, 34, 929–939.
92 S. L. Lawton, E. R. McAfee, J. E. Benson and
R. A. Jacobson, Inorg. Chem., 1973, 12, 2939–2944.
93 C. F. Shaw III, J. Chem. Educ., 1981, 58, 343–348.
J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul,
S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin,
D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng,
A. Nanayakkara, M. Challacombe, P. M. W. Gill,
B. Johnson, W. Chen, M. W. Wong, C. Gonzalez and
J. A. Pople, GAUSSIAN 03 (Revision E.01), Gaussian, Inc.,
Wallingford, CT, 2004.
94 O. Maass and P. G. Hiebert, J. Am. Chem. Soc., 1924, 46, 100 E. D. Glendening, J. K. Badenhoop, A. E. Reed,
290–308.
J. E. Carpenter, J. A. Bohmann, C. M. Morales and
F. Weinhold, NBO 5.9, Theoretical Chemistry Institute,
University of Wisconsin, Madison, WI, 2009, http://www.
chem.wisc.edu/~nbo5
95 I. Fleming, in Molecular Orbitals and Organic Chemical
Reactions, Wiley-VCH, Weinheim, 3rd edn, 2012,
pp. 106–109.
96 S. G. Bratsch, J. Chem. Educ., 1985, 62, 101–103.
97 R. T. Sanderson, J. Chem. Educ., 1988, 65, 112–118.
101 G. A. Olah, B. G. B. Gupta, R. Malhotra and S. C. Narang,
J. Org. Chem., 1980, 45, 1638–1639.
98 J. E. Dubois and G. Mouvier, Tetrahedron Lett., 1963, 20, 102 K. P. Huber and G. Herzberg, in Molecular Spectra and
1325–1331.
Molecular Structure Constants of Diatomic Molecules, Van
99 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
Nostrand, New York, 1979.
M. A. Robb, J. R. Cheeseman, J. A. Montgomery Jr., 103 D. Wischang and J. Hartung, Tetrahedron, 2012, 68, 9456–
T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, 9463.
S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, 104 By mistake, substrates and reactants were interchanged in
G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji,
M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,
the version of eqn (6) provided in ref. 21. The correct form
of this equation is given in this article.
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, 105 C. Hansch, A. Leo and R. W. Taft, Chem. Rev., 1991, 91,
M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, 165–195.
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, 106 O. S. Tee and N. R. Iyengar, J. Am. Chem. Soc., 1985, 107,
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, 455–459.
C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, 107 C. T. Walsh, S. Garneau-Tsodikova and A. R. Howard-
G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, Jones, Nat. Prod. Rep., 2006, 23, 517–531.
S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, 108 H. Hoffmann and T. Lindel, Synthesis, 2003, 1753–
D. K. Malick, A. D. Rabuck, K. Raghavachari,
1783.
11940 | Dalton Trans., 2013, 42, 11926–11940
This journal is © The Royal Society of Chemistry 2013