Bromination of phenols in bromoperoxidase-catalyzed oxidations

Article abstract of DOI:10.1016/j.tet.2012.08.081

Phenol and ortho-substituted derivatives furnish products of selective para-bromination, if treated with sodium bromide, hydrogen peroxide, and the vanadate(V)-dependent bromoperoxidase I from the brown alga Ascophyllum nodosum. Relative rates of bromination in morpholine-4-ethane sulfonic acid (MES)-buffered aqueous tert-butanol (pH 6.2) increase by a factor 32, as the ortho-substituent in a phenol changes from F via Cl, OCH₃, C(CH ₃)₃, and H to CH₃. The polar effect in phenol bromination by the enzymatic method, according to a Hammett-correlation (ρ=-3), compares to reactivity of molecular bromine under identical conditions (ρ=-2). Hypobromous acid is not able to electrophilically substitute bromine for hydrogen at pH 6.2 in aqueous tert-butanol. The tribromide anion behaves in MES-buffered aqueous tert-butanol as electrophile (ρ～-3), showing a similar polar effect in phenol bromination as molecular bromine.

Full text of DOI:10.1016/j.tet.2012.08.081

Tetrahedron 68 (2012) 9456e9463
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Bromination of phenols in bromoperoxidase-catalyzed oxidations
*
Diana Wischang, Jens Hartung
€
€
Fachbereich Chemie, Organische Chemie, Technische Universitat Kaiserslautern, Erwin-Schrodinger-Straße, D-67663 Kaiserslautern, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 April 2012
Received in revised form 24 August 2012
Accepted 27 August 2012
Available online 1 September 2012
Phenol and ortho-substituted derivatives furnish products of selective para-bromination, if treated with
sodium bromide, hydrogen peroxide, and the vanadate(V)-dependent bromoperoxidase I from the
brown alga Ascophyllum nodosum. Relative rates of bromination in morpholine-4-ethane sulfonic acid
(MES)-buffered aqueous tert-butanol (pH 6.2) increase by a factor 32, as the ortho-substituent in a phenol
changes from F via Cl, OCH3, C(CH3)3, and H to CH3. The polar effect in phenol bromination by the en-
zymatic method, according to a Hammett-correlation (
mine under identical conditions (
¼ꢀ2). Hypobromous acid is not able to electrophilically substitute
bromine for hydrogen at pH 6.2 in aqueous tert-butanol. The tribromide anion behaves in MES-buffered
r
¼ꢀ3), compares to reactivity of molecular bro-
Keywords:
r
Ascophyllum nodosum
Aromatic substitution
Bromine
Bromoperoxidase
Hammett-correlation
Hypobromous acid
Linear free energy-relationship
Phenol
aqueous tert-butanol as electrophile (
molecular bromine.
r
wꢀ3), showing a similar polar effect in phenol bromination as
Ó 2012 Elsevier Ltd. All rights reserved.
Tribromide
1. Introduction
progenitors, proposed on the basis of purely structural arguments,
are carbonecarbon double bonds of donor-substituted alkenes or
Bromide, dissolved in ocean water (cBr
ꢀw1 mM) or deposited in
arenes, and the aromatic core of p
-excess heteroarenes.9,10
minerals, is the major resource for production of molecular bro-
mine, the most important reagent for synthesis of organo-
bromines.1e3 In an oxidative environment, such as the marine
boundary layer or in hydrogen peroxide-producing compartments
of living cells, bromide is rapidly oxidized into hypobromous acid,
molecular bromine, and tribromide, to mention the major prod-
ucts.4 All products of peroxidative bromide oxidation are able to
convert hydrocarbons into organobromines, for example, as part of
natural product synthesis, although with different functional group
selectivity.5
Naturally occurring organobromines show considerable struc-
tural diversity regarding site of bromosubstitution and carbon
skeleton the halogen atom is attached to.6 Simple bromoalkanes,
such as bromomethane or bromopentanes, are similarly found in the
environment as more complex metabolites, for example, bromo-
substituted fatty acids, phenylpropanes, or amino acids. Probably
the most complex structures arise from highly substituted, chiral,
cyclic terpenes, and acetogenins.7,8 Functional groups that appear to
be particularly receptive for bromination in putative biogenic
Biosynthetic pathways for organobromine formation are largely
unexplored.11 From biochemical experiments it is known, that
hydrogen peroxide oxidizes bromide at pH 6e7, if catalyzed by
vanadate(V)-dependent bromoperoxidases (VBrPOs).12e14 Accord-
ing to the general mechanism of VBrPO-catalyzed bromide oxida-
tion (Scheme 1),12,14 hydrogen peroxide binds first to vanadate(V),
in a reversible proton-assisted step. The peroxido-loaded cofactor is
the active form of the enzyme and able react with bromide in
a second reversible reaction, to furnish a structurally uncharac-
terized but kinetically relevant intermediate. Oxygen atom transfer
from the peroxido complex to bromide follows, possibly resulting
in a short-lived intermediate,15 which rapidly hydrolyzes, to re-
generate by the end of the catalytic cycle the resting state of the
enzyme. Hydrolysis of the assumed intermediate furthermore
provides one molecule of hypobromous acid, which is considered
to be the primary product of enzymatic bromide oxidation (Scheme
1, top).10,16 In an aqueous solution of bromide, such as ocean water,
hypobromous acid, rapidly equilibrates to provide a mixture of
molecular bromine, tribromide (Scheme 1, bottom), and possibly
further products.3,12,17
The chemical behavior of hypobromous acid, bromine, and
tribromide toward functional groups relevant for explaining
organobromine formation in nature is distinctively different.
* Corresponding author. Tel.: þ49 631 205 2431; fax: þ49 631 205 3921; e-mail
address: hartung@chemie.uni-kl.de (J. Hartung).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.08.081

D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
9457
2. Results and discussion
2.1. Concept
• bromoperoxidase-catalyzed oxidation – primary product formation
O
L
O
O
V
Br– / H2O
HO
2 H2O
The strategy used in this study to reference reactivity and se-
lectivity in bromoperoxidase-catalyzed oxidation combines
a product study for determining individual reactivity of ortho-
substituted phenols in enzymatic reactions with competition ki-
netics for determining polar substituent effects on rates in phenol
bromination. The results from bromoperoxidase-catalyzed oxida-
tions subsequently were compared to controls, using hypobromous
acid, molecular bromine, and tetrabutylammonium tribromide as
bromination reagents (Scheme 2).
O
O–
H+ / H2O2
L
HOBr
V
OH
HO
VBrPO(AnI)
OH
OH
• thermodynamic equilibration – secondary product formation
k rel
R
R
K1
+ [ Br ]
+ [ H ]
–
HOBr + Br + H +
Br2 + H2O
tBuOH / H2O
pH 6.2
K 2
Br
–
–
Br2 + Br
Br3
1
2
Scheme 1. Proposed mechanism for bromide oxidation catalyzed by the vanadate(V)-
dependent bromoperoxidase I (VBrPO) from Ascophyllum nodosum (An) (top), and
equilibria associated with secondary product formation in bromide-containing brines
(bottom) [K1¼for example, 1.45ꢁ108 Mꢀ2 for H2O at 20 ꢂC (I¼0.1 M, pH 2.6e3.8)26 or
1.04ꢁ108 Mꢀ2 in H2O at 25 ꢂC (pH 1.5);27 K2¼16.9 Mꢀ1 in H2O at 25 ꢂC28].3
[ Br ] = Br2, HOBr, NBu4Br3,
NaBr / H2O2 / VBrPO(AnI)
1, 2
R
a
b
c
d
e
f
H
Hypobromous acid bromohydroxylates alkenes to afford 2-
CH3
C(CH3)3
OCH3
Cl
bromoalcohols (bromohydrins). Unless activated by
a strong
Brønsted-acid, hypobromous acid is comparatively inert toward
arenes and not able to electrophilically displace bromine for hy-
drogen. Tribromide, a weakly bound adduct between bromine and
bromide, shows chemical reactivity at the borderline between
nucleophilic and electrophilic,18e20 and reacts with alkenes in polar
aprotic solvents with different selectivity for vicinal dibromination
than molecular bromine.21e23 In protic solvents, such as water24 or
acetic acid,25 tribromide seems to predominantly serve as safe-to
handle substitute for bromine, liberating the halogen at a steady
rate for converting, for example, phenol, 2-naphthol, aniline, and
other strongly activated arenes into bromoderivatives with
bromine-like selectivity.
In bromoperoxidase chemistry, the chemical nature of the
reagent that controls reactivity and selectivity for carbonebromine
bond formation, so far has not been determined. Endogeneous
substrates that bind to the active site in vanadate(V)-dependent
bromoperoxidases are bromide and hydrogen peroxide. A binding
site for an organic substrate close to the vanadate co-factor has
not yet been identified. This observation correlates with the lack
in hydrocarbon specificity and regio- or stereoselectivity for
carbonebromine bond formation in bromoperoxidase-catalyzed
oxidations.3,10
In a project dealing with biomimetic synthesis of brominated
natural products, we encountered the problem to predict selectivity
for preparing the correct starting material via bromoperoxidase-
catalyzed oxidation. We therefore specified the chemical nature of
the bromoelectrophile that is directly involved in carbonebromine
bond formation in enzymatic reactions. The major results from
a combined synthetic and competition kinetic study on phenol
bromination show that bromination in oxidations catalyzed by the
vanadate(V)-dependent bromoperoxidase I from the brown alga
Ascophyllum nodosum [VBrPO(AnI)] shows considerable parallels to
the chemistry of molecular bromine in water. This information
allowed us to derive a reaction model for explaining a striking dif-
ference between phenol- and anisol bromination.
F
Scheme 2. Summary of bromination reagents and indexing of phenols used in this
study ([H]¼e.g., Hþ).
The approach in chemistry to quantify polar substituent effects
on chemical reactivity is correlation analysis.29,30 In the present
study, we used the phenol competition system to determine rela-
tive rates of bromination in bromoperoxidase-catalyzed oxidations,
and in non-enzymatic references (Scheme 2). Correlation of decadic
logarithms of relative rate constants lg krel¼lg (kR/kH) with sub-
stituent constants31 sm, according to equation 1, provides the re-
action parameter
r. Sign and magnitude of r characterize
responsivity of polar substituent effects for accelerating or retard-
ing the rate determining step, and helps to characterize the
chemical nature of the reagent that is directly involved in the re-
activity determining step.
kR
lgkrel ¼ lg
¼
r
$
sm
(1)
kH
2.2. Bromoperoxidase isolation and preparation of bromina-
tion reagents
The bromoperoxidase used in this study is the isoenzyme I from
the brown alga A. nodosum [VBrPO(AnI), EC 1.11.1.18, PDB-code
1QI9].32,33 The enzyme was isolated from specimen collected in
Roscoff, France, following an established freezeedrying, milling,
and liquideliquid partitioning process.34 The crude bromoperox-
idase fraction, which precipitated upon addition of acetone from
the final extract of the extraction process, was dialyzed against
sodium metavanadate for restoring bromoperoxidase activity of the

9458
D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
Table 1
protein. From the mixture of active bromoperoxidases, VBrPO(AnI)
was separated by hydrophobic interaction chromatography and
subsequent size exclusion chromatography, providing samples of
specific activity between 558ꢃ11 U0T mgꢀ1 to 611ꢃ6 UT0 mgꢀ1, based
on the triiodide assay35,36 (pH 6.2; phosphate buffer).
For synthetic and kinetic experiments on phenol bromination
with the VBrPO(AnI), we used a solvent/buffer-combination com-
posed of 25 volume percent of tert-butanol for dissolving the or-
ganic components, 75 volume percent of water as major reaction
medium, and morpholine-4-ethane sulfonic acid (MES) to maintain
pH of the solutions close to 6.2. In reference reactions for phenol
bromination starting from hypobromous acid, molecular bromine,
and tetrabutylammonium tribromide, we tried as close as possible
to adhere to these conditions.
The applied solution of hypobromous acid was prepared
from bromine and silver nitrate in aqueous MES-buffer, containing
25vol% of tert-butanol. From the solubility product of silver
bromide in water [4.948$10ꢀ3 (mol/L),2 25 ꢂC],37 we estimated
a residual bromide concentration in this solution of 7ꢁ10ꢀ7 M.
Since bromine in the required concentration of 0.75 M did not
quantitatively dissolve in the solvent of interest, we added the
halogen for synthesis and for competition kinetics as solution in
tert-butanol via syringe pump to solutions containing a phenol 1 in
aqueous MES-buffered tert-butanol.
Tribromide salts having tetrabutylammonium or cesium as
countercation, are sparingly soluble in tert-butanol and aqueous
solutions of this alcohol. We therefore added tetrabutylammonium
tribromide as single batch to MES-buffered solutions of a phenol 1
in aqueous tert-butanol for bromophenol syntheses, and as ho-
mogeneous solution in acetonitrile via syringe pump for competi-
tion kinetic experiments.
Products of phenol bromination formed in bromoperoxidase-catalyzed oxidation
and non-enzymatic reactions
OH
OH
OH
OH
[Br]
Br
Br
Br
+
+
Br
tBuOH / H2O
pH 6.2 / 23 °C
Br
Br
4a
1a
2a
3a
Entry [Br]
[Br]eq. Conv. 1a/%a 2a/% (o/p)b 3a/% 4a/% pHc
1
2
3
4
5
6
7
8
9
NaBr/H2O2/VBrPO(AnI)
1
1
2
3
1
2
3
1
2
3
90
69 (9/91)
dd
dd
dd
4
6
6.8
HOBr
HOBr
HOBr
Br2
Br2
Br2
6
dd dd 5.7
dd dd 3.1
dd dd 1.0
dd
6
53
80
quant.
53
17 (18/82)
33 (18/82)
13(<2/98)
3
7
8
29
38
77
23
59
6.2
5.9
5.3
6.1
6.0
5.8
NBu4Br3
NBu4Br3
NBu4Br3
14 (<2/98) 13
10 (<2/98) 14
83
quant.
10
dd
dd 99
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 (500 mM, pH 6.2)
{VBrPO(AnI) [55.2 mL, 0.5626 mg/mL, 558 UT/mg, 17.3 UT, 0.036 mmol%} (entry 1);
batch addition of a solution of HOBr in aq MES buffer (500 mM) (0.2e0.3 M,
0.75e2.25 mmol, 1.0e3.0 equiv, pH 1) (entries 2e4); continuous addition (syringe
pump, 8 h) of a solution of Br2 (0.75e2.25 M, 1.0e3.0 equiv) (entries 5e7); batch
addition of TBABr3 (0.75e2.25 mmol, 1.0e3.0 equiv) (entries 8e10); 0.75 mmol of
1a were used as starting material for all entries; the final reaction medium was aq
MES buffer (pH 6.2)/tBuOH¼75/25% (v/v) for all entries.
b
ortho and para relative to OH; unless otherwise stated 1H NMR yields.
pH-electrode (for pH 3.1e6.8) or pH-indicator paper (for pH<3), pH-value after
c
reaction of the aqueous layer.
d
Not detectable.
2.3. Phenol bromination in aqueous tert-butanol
2.3.1. Bromophenol synthesis. For phenol bromination via enzy-
matic oxidation, we used 17.3 units (UT) of VBrPO(AnI), referenced
versus the triiodide assay, and the solvent/buffer-system outlined
in the previous section. To minimize bromoperoxidase-activity loss,
we used sodium bromide and the organic substrate in an equimolar
ratio and added not more than 1.1 equiv of hydrogen peroxide as
0.825 M solution in aqueous tert-butanol (pH 6.2) with a syringe
pump to the solution containing the enzyme, the phenol 1, and
sodium bromide in the same solvent.14 Under such conditions, 90%
of phenol (1a) (0.75 mmol) were converted into bromophenols
2ae4a with a total yield of 79% (Table 1, entry 1). By the end of the
reaction, the original pH of the mixture had increased to 6.8. A
noteworthy feature of the enzymatic reaction is the selectivity for
providing monobromophenol 2a as major product (vide infra).
If 1 equiv of hypobromous acid is added to a solution of phenol
(1a) in MES-buffered (500 mM) aqueous tert-butanol, the pH of the
solution immediately changes from 6.2 to 5.7. This solution, how-
ever, does not afford within a standard reaction time of 72 h any of
the bromophenols 2ae4a (Table 1, entry 2). By successively in-
creasing the amount of hypobromous acid from one via two to three
aliquots, the pH gradually declines from 5.7 to 1, still without any
sign of bromophenol formation (Table 1, entries 2ꢀ4). As wereduced
the buffer capacity to 50 mM, the pH of the reaction mixture de-
creased to 1, directly after adding the first equivalent of hypobro-
mous acid. From this solution we isolated 21% of bromophenol (2a)
with an ortho/para-selectivity of 24:76. The yield of bromophenol 2a
further increased as we doubled (47%) and tripled (57%) the amount
of hypobromous acid, without finding experimental evidence for
dibromophenol- and tribromophenol formation (Supplementary
data). From the results we concluded that hypobromous acid un-
der mildly acidic conditions, used for conducting bromoperoxidase-
catalyzed oxidations, is not able to brominate phenol (1a).
Molecular bromine rapidly converts phenol (1a) in solutions of
aqueous tert-butanol (pH 6.2) into bromophenols 2ae4a, with the
substrate conversion and product formation improving as the
amount of bromine increases from one to three aliquots (Table 1,
entries 5e7). The main product from the synthesis is 2,4,6-
tribromophenol (4a), which correlates with information from
a standard laboratory procedure.38 The yields of bromophenols
remained slightly below the values obtained from bromoperoxidase-
catalyzed oxidations.
Tetrabutylammonium tribromide, if added as neat solid to
solution of phenol (1a) in aqueous tert-butanol (pH 6.2), furnishes
a slurry that turns into a homogeneous clear solution by the end
of the reaction. From this solution, we isolated tribromophenol
4a in yields that gradually increases as the amount of tribromide
is raised from one (23%) via two (59%) to three aliquots (99%;
Table 1, entries 8e10). Singly and doubly brominated products
2a and 3a appeared as minor components in experiments
performed with one and two equiv of the bromination reagent.
In
a control on the solvent effect in tetrabutylammonium
tribromide-mediated phenol bromination, ratios of mono- versus
di- and tribromide, and the yield of products 2ae4a remained
unchanged, as we replaced tert-butanol by acetonitrile, or ad-
ministered the bromination reagent either as neat material
batchwise or as homogeneous solution in acetonitrile with a sy-
ringe pump. From this information we concluded that acetonitrile
and tert-butanol have
a similar solvent effect in tetrabuty-
lammonium tribromide-mediated phenol bromination and are
able to replace one another without changing selectivity.
To sum up, molecular bromine, tetrabutylammonium tri-
bromide, and the combination of sodium bromide, hydrogen per-
oxide, and VBrPO(AnI) are able to brominate phenol (1a) in aqueous
tert-butanol (pH 6.2). The enzymatic oxidation selectively furnishes

D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
9459
monobromophenol 2a, whereas bromine- and tribromide-
2.4. Competition kinetics and correlation analysis
mediated reactions give tribromide 4a as major product.
With the knowledge about the performance of VBrPO(AnI) in
synthetic scale oxidative brominations, we turned our attention to
competition kinetics for determining relative reactivity of phenols
under such conditions. The underlying reaction model thereby
considers, for reasons given above, no organic substrate binding to
the vanadate active site. Phenol bromination in this model occurs at
some distance from the active site, and it was the aim of the kinetic
part of the study to clarify whether the reactivity determining
bromoelectrophile compares to a reagent familiar from the chem-
istry of bromine in water.
In the general experimental set-up for competition kinetics
under pseudo-first order conditions, bromoperoxidase-catalyzed
oxidations furnished exclusively products of monobromination.
The fractions of ortho-substituted products and the yields of bro-
mophenols 2aef obtained from the diluted solutions used for
competition kinetics [62ꢃ4% for 2aþ2b, 81ꢃ17% for 2aþ2c, 34ꢃ3%
for 2aþ2d, 92ꢃ4% for 2aþ2e, and 78ꢃ11% for 2aþ2f] in most in-
stances were higher than those obtained from the more concen-
trated solutions applied for synthesis (Supplementary data).
Competition kinetics for phenol bromination by tetrabutylammo-
nium tribromide surprisingly provided substantial amounts of
dibrominated halophenols 3eef. All attempts to circumvent this
problem by lowering phenol concentrations, or using phenols in
larger excess, failed to solve this problem. We therefore excluded
substrates 1e and 1f from correlation analysis for tribromide-
mediated reactions.
2.3.2. Bromination of ortho-substituted phenols in bromoperoxidase-
catalyzed oxidation. Prior to conducting competition kinetic ex-
periments, we verified that ortho-substituted phenols 1bef provide
in bromoperoxidase-catalyzed oxidations comparable yields of
bromophenols 2bef. The substrates chosen for relative rate anal-
ysis, and thus the synthetic part of the study, bear a methyl-, tert-
butyl-, methoxy-, chloro-, and a fluoro- substituent in ortho-posi-
tion to the phenolic hydroxyl group. The hydroxyl group is a strong
para-directing group in electrophilic aromatic substitution, which
we hypothesized also to operate as underlying mechanism in en-
zymatic reactions. Decisive selectivity for aromatic substitution
para to the hydroxyl group, being meta to R, is a prerequisite for
obtaining meaningful information from Hammett-correlations. For
the synthetic study, we restricted ourselves to bromoperoxidase-
catalyzed oxidations, since bromine and tetrabutylammonium tri-
bromide have been successfully used before for this purpose.39e43
For attaining maximum yields of bromophenols 2bef, and
minimizing bromoperoxidase activity loss, we added 1.1 equiv of
hydrogen peroxide as a 0.825 M, MES-buffered solution of tert-
butanol via syringe pump to a reactant solution composed of one
equivalent of sodium bromide, 17.3 UT of VBrPO(AnI), and an ortho-
substituted phenol (compounds 1bef; 0.75 mmol) in aqueous tert-
butanol (pH 6.2, MES-buffer). From likewise prepared reaction
mixtures, we isolated bromophenols 2bef as major compounds
(Table 2). The yields of target products 2bef depended on the na-
ture of the ortho-substituent in 1bef, and gradually decreased
along the series of ortho-substituents R¼CH3 (68%), via OCH3 (56%),
C(CH3)3 (42%), F (19%) to Cl (13%). In the latter two instances, we
isolated 18e26% of dibromides 3e and 3f (Table 2, entries 5e6),
leading to a satisfactory mass balance for sodium bromide utiliza-
tion between 46% (for 1c) and 95% (1a; cf. Table 2).
Pre-kinetic evaluations furthermore clarified that bromophe-
nols 2aef had to be derivatized for obtaining reproducible yields
from gas chromatographic analysis. We found that the combination
of acetic anhydride and catalytic amounts of zinc perchlorate-
hexahydrate44 quantitatively copies ratios of 2a versus 2bef into
ratios of O-acetyl derivatives 5a versus 5bef (Scheme 3,
Supplementary data), which solved the analytical problem.
Table 2
Products of phenol bromination in bromoperoxidase-catalyzed oxidation
VBrPO(AnI)
OH
OH
OH
OAc
R
R
R
Br
H2O2 / NaBr
1a
Ac2O
+
2a
Br
tBuOH / H2O
H
k
Zn(ClO4)2
Br
25 °C
pH 6.2
pH 6.2 / 23 °C
Br
3
5a (quant.)
OAc
2
1
[ Br ]
tBuOH
H2O
final d
Entry 1e3 R
Conv. 1/%a 2/% (o/p)b 3/% Mass
pH
Activity
balance/%c
UT mgꢀ1e
final
R
Ac2O
1b–f
1
2
3
4
5
6
a
b
c
d
e
f
H
90f
86
69 (9/91)
68 (16/84)
42 (36/64)
4
3
2
89
85
91
6.8
6.5
6.4
6.4
6.3
6.4
133
138
dg
2b–f
Br
R
CH3
C(CH3)3 53
OCH3
Cl
F
Zn(ClO4)2
k
5b–f (quant.)
68
43
51
56 (21/79) 10 98
13 (<2/98) 26 96
19 (<2/98) 18 86
dg
129
160
a
Continuous addition (syringe pump, 8 h) of a solution of H2O2 (0.825 M,
R
H
1.1 equiv) and NaBr (0.75 mmol, 1.0 equiv) in aq MES buffer (pH 6.2)/tBuOH¼75/25%
(v/v); 0.75 mmol of 1 were used as starting material.
[ 5b–f ]
[ 1b–f ]
[ 1a ]
k
k
(eq. 2)
=
b
ortho and para relative to OH; unless otherwise stated 1H NMR yields.
Sum of recovered 1 and bromides 2e4.
[ 5a ]
c
d
Scheme 3. Reaction scheme for kinetic experiments in the phenol-competition system
{for data analysis, see equations 1e2; [Br]¼Br2, NBu4Br3, or NaBr/H2O2/VBrPO(AnI)}.
pH electrode.
e
Initial enzyme activity 558 UT0 mgꢀ1 (entries 1 and 3) and 611 U0T mgꢀ1 (entries 2
and 4e6).
f
Additional product: 6% of 2,4,6-tribromophenol (4a).
No final bromoperoxidase activity detected.
g
From competition experiments performed at three different
ratios of phenol 1a versus one of the reporter substrates 1bef at
fixed bromide-, hydrogen peroxide-, and bromoperoxidase con-
centration we derived from linear correlations according to equa-
tion 2 relative rate constants krel¼kR/kH (Supplementary data).
Correlations considering exclusively products of para-bromination
hereafter are abbreviated as krpel ¼ kpR=kpH, and those taking
By the end of the enzymatic oxidation, the pH had slightly in-
creased to 6.4e6.8. Final bromoperoxidase activity had declined to
18e27% of the initial value, except of experiments on guaiacol- and
tert-butyl phenol bromination, which consistently led to quantita-
tive VBrPO(AnI)-activity loss (Table 2, entries 3 and 4).

9460
D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
products of ortho- and para-bromination into account as
whereas others, such as the original values proposed by Ham-
mett,29 and alternative values put forward by Taft, resulted in
a scatter of data.30,31
rel
k
¼ kRop=kHop. The correlation coefficients (R2) of the fits for cal-
op
culating krel-values were satisfactory (R2 for krpel¼0.995 for 1b,
0.909 for 1c, 0.963 for 1d, 0.855 for 1e, and 0.999 for 1f).
The
r-value for describing para-substitution of 1 (rp¼ꢀ3.1) in
The kinetic data show that the rate of phenol bromination in
VBrPO(AnI)-catalyzed oxidations is slightly more negative than the
value referring to ortho- and para-bromination (rop¼ꢀ2.3; Table 4,
entry 1). Since ortho-substitution adds a steric component to
chemical reactivity that is not covered by sm, and kroepl summarizes
effects from two elementary reactions, which not necessarily have
to respond similarly to polar effects, we restricted ourselves for the
general mechanistic discussion to compare of rp-values rather than
the rop-values.
rel
bromoperoxidase-catalyzed oxidation, as expressed in krpel and k
,
op
gradually increases, along the sequence of ortho-substituents in 1
from R¼CH3 via C(CH3)3, OCH3, and Cl to F (Table 3). Relative rates
for the most reactive phenol (1b, R¼CH3) and the least reactive
substrate (1f, R¼F) differed by a factor 32 for krpel and 16 for korepl. The
difference between the two values arises from a change in ortho/
para-selectivity from 26:74 for phenols 1bed [R¼CH3, C(CH3)3, and
OCH3] to 47:53 for 1eef (R¼Cl, F; Supplementary data).
Table 4
Reaction parameter
Table 3
r
for phenol bromination in aqueous tert-butanol (pH 6.2, 25 ꢂC)
Relative rate constants of phenol brominationa in bromoperoxidase-catalyzed oxi-
dation and non-enzymatic methods, and sm parameters31 used for correlation
analysis
Entry
[Br]
rp
rop
1
2
3
NaBr/H2O2/VBrPO(AnI)
Br2
NBu4Br3
ꢀ3.1
ꢀ2.3
Entry R/1
sm
VBrPO(AnI)
Br2
NBu4Br3
ꢀ1.9
ꢀ1.9
(ꢀ3.1)a
(ꢀ3.0)a
kpR=kpH kRop=koHp kRp=kHp koRp=kHop kpR=kpH koRp=koHp
Estimated on the basis of krel values for 1b and 1d in MES-buffered aqueous
a
1
2
3
4
5
CH3/1b
ꢀ0.11 2.25
1.89
0.67
0.43
0.15
0.12
1.35
db
1.17
db
3.15
db
2.96
db
acetonitrile (see Table 3).
C(CH3)3/1c ꢀ0.02 0.98
OCH3/1d
Cl/1e
0.05 0.48
0.31 0.10
0.36 0.07
0.97
0.22
0.19
1.18
0.21
0.18
0.99
dc
dc
1.37
dc
dc
F/1f
The reaction parameters for phenol bromination in aqueous
tert-butanol (pH 6.2) by molecular bromine
VBrPO(AnI)-catalyzed oxidations,
a
o(rtho) and p(ara) with respect to the hydroxyl group in 1; op refers to values
(
rp¼ꢀ1.9), via
considering products of ortho- and para-substitution.
(
rp¼ꢀ3.1), and the approxi-
b
Not considered due to a poor quality in correlation analysis.
Not considered for correlation analysis due to dibromide formation.
mated value for tetrabutylammonium tribromide-mediated bro-
minations (Table 4) are within a reasonable confidence level for
correlation analysis similar. Since electron releasing substituents
enhance relative rates of phenol bromination, the sign of rp is
negative and shows that the attacking bromination reagent is an
electrophile.
According to the data summarized in the previous paragraphs,
reactivity and selectivity of phenol bromination in bromoperoxidase-
catalyzed oxidation is best described by the chemistry of molecular
bromine in water.45,46 Alternative reagents, such as hypobromous
acid or the tribromide-anion, are expected to co-exist with
bromine in typical bromoperoxidase reaction mixtures, but are not
expected to be directly involved in the carbonebromine bond
forming step.
c
The reactivity of phenols 1bef toward molecular bromine in
aqueous tert-butanol (pH 6.2) follows the same trend as described
for VBrPO(AnI)-catalyzed oxidations. The fastest (1b, R¼CH3) and
the slowest reaction (1f, R¼F) differed by a factor 7.1 for krpel and 6.5
for kroepl. The ortho/para-ratio in all phenol brominations by bromine
under pseudo-first order conditions was close to 25:75.
The data available for classifying the polar effect in phenol
bromination by tetrabutylammonium tribromide show that ortho-
cresol 1b reacts by a factor 3.2 for para-bromination and 2.2 for
ortho- and para-bromination faster than phenol (1a), whereas ref-
erence 1a is approximately similar reactive as guaiacol 1d. The or-
tho/para-ratio in the competition experiment is about 10:90 for 2b,
and 30:70 for 2d.
2.4.1. Mechanistic implications. The noteworthy propensity of
molecular bromine for electrophilically displacing hydrogen in
phenols under physiological conditions arises from a mechanism
for aromatic substitution, which proceeds via cyclohexadienone
intermediates (e.g., 6) instead of cyclohexadienyl cations (e.g.,
From correlation of decadic logarithms of relative rate constants
k
r
rel and substituent constants sm, we obtained reaction parameters
for the three bromination methods (Table 3). The sm-parameter
selected for correlation analysis refers to fluorine-19 NMR-chemical
shift-changes caused by substituent R in meta-substituted fluo-
robenzenes,31 and is almost independent from solvent effects. This
substituent parameter provided excellent correlations (Fig. 1),
7; Scheme 4).45,47,48 4-Bromocyclohexadienones
6
or the
6-bromoisomers (not shown in Scheme 4), according to
this mechanism, are formed in the reaction between a phenolate
1e and molecular bromine. Although the phenolate fraction in
aqueous solution at pH 6.3, the average value in bromoperoxidase-
catalyzed oxidations, is very small (2ꢁ10ꢀ4 for 1aꢀ/1a; for other
1ꢀ/1-ratios,49e54 see the Supplementary data), this mechanism is
the kinetically favored pathway for phenol bromination. The rate
constant, for example, for the reaction between 1aꢀ and bromine
(k¼2.4ꢁ1010 Me1 se1; pH 7, 25 ꢂC) is close to the diffusion limit,56
whereas bromophenol formation from 1a and bromine is by five
orders of magnitude slower (4.1ꢁ105 Me1
s
e1; pH 7, 25 ꢂC). The
alternative mechanism furthermore helps to explain the magni-
tude of experimentally determined r-values for phenol bromina-
tion in water, being less negative for reactions proceeding via
uncharged cyclohexadienone intermediates [ꢀ3.1 (Table 4, entry
1), ꢀ2.955 or ꢀ5.2 under slightly different conditions56] than for
Fig. 1. Correlation of log krel from phenol bromination by bromine (B) (correlation
coefficient R2¼0.958), tetrabutylammonium tribromide (6), and the combination of
sodium bromide, hydrogen peroxide, and VBrPO(AnI) (C) (R2¼0.995) versus sm ac-
cording to equation 1.
electrophilic
halogenations
via
cyclohexadienyl
cations
(ꢀ8>
r
>ꢀ12).57,58

D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
9461
chemistry of molecular bromine in water. This information closes
the gap between the mechanistic details for bromide oxidation
derived from steady-state kinetics (Scheme 1), and reactivity/
selectivity-guidelines required to apply the enyzme in synthesis.
VBrPO(AnI) is readily available from renewable resources, retains
full bromoperoxidase activity if stored in Tris/HCl-buffer, is com-
paratively thermostable, and tolerates significant concentrations of
organic co-solvents, and substrates. On the basis of the findings
summarized in this article, we think that synthesis of organo-
bromines via bromoperoxidase-catalyzed oxidation using hydro-
gen peroxide as terminal oxidant and ocean water as bromide
source is an attractive perspective, worth-while to pursue for fur-
ther developing sustainable synthesis.
Alternative bromine compounds for mechanistically explaining
phenol bromination, such as hypobromous acid and tribromide, are
for thermochemical reasons expected to co-exist with bromine in
typical bromoperoxidase reaction mixtures, but seem not to be
directly involved in carbon-bromine bond formation. Hypobro-
mous acid, for example, does not react with phenols at pH 6.2 in
aqueous morpholine-4-ethane sulfonic acid-buffered solution. The
tribromide ion dissociates in protic solvents providing reactivity
and selectivity for arene bromination that is largely governed by
molecular bromine.24,25
cyclohexadienone route
cyclohexadienyl cation route
–
OH
+
O
OH
R
+
R
R
– H
H
Br2
+ MBr
+
+
M
H
H
Br
1–
1
7
– Br–
R
Br2
O
OH
+
OH
R
R
– HBr
+ MBr
+
M
H
Br
Br
Br
2
6
7
Scheme 4. Mechanistic pathways for phenol bromination in aqueous solution45,56
[R¼for example, CH3, C(CH3)3, H, OCH3, Cl, F; Mþ¼Lewis- or a Brønsted-acid].
As the mechanistic picture for organic substrate bromination in
oxidations catalyzed by VBrPO(AnI) becomes clearer, the role of the
bromoperoxidase protein on selectivity deserves closer attention.
Once the primary product hypobromous acid diffuses from the
active site (cf. Scheme 1) and secondary products, such as bromine
and tribromide have been formed, hydrocarbon bromination still
can take place at the outer rim of the bromoperoxidase protein. The
fact that phenol (1a) furnishes tribromophenol 4a from the reaction
between molecular bromine in aqueous solution, but mono-
bromophenol 2a in the bromoperoxidase-catalyzed reaction points
to such a selectivity effect. Competition kinetic data show, that this
selectivity effect is not relevant for describing chemical reactivity,
as expressed in relative rate constants and the Hammett-parameter
To test the hypothesis that phenol bromination in
bromoperoxidase-catalyzed oxidation follows the cyclo-
hexadienone route, we investigated the chemistry of anisole
bromination in enzymatic reactions (Scheme 5). Bromination of
the phenol ether has to follow the cyclohexadienyl cation route
and should therefore occur with a slower rate than phenol
bromination.
VBrPO(AnI)
H2O2 / NaBr
OMe
OMe
tBuOH / H2O
pH 6.2 / 23 °C
20% conv.
r
. A selectivity effect originating from the bromoperoxidase protein
or possibly from an external co-factor is an attractive feature for
attaining new means of stereocontrol in this chemistry and there-
fore is being addressed at the moment in our laboratory.
Br
9 (18%)
8
Scheme 5. Anisole bromination in bromoperoxidase-catalyzed oxidation.
4. Experimental
4.1. General remarks
Under standard conditions of bromoperoxidase-catalyzed oxi-
dations used in this study, anisole bromination consistently
stopped at about 22% conversion to furnish 18% of para-bromoa-
nisole 9. Attempts to raise the yield of bromoether 9 by increasing
the amount of enzyme to 34.6 UT, and adding further sodium
bromide- and hydrogen peroxide aliquots, until the reaction mix-
ture contained a twofold excess of the reagents, failed to improve
rate and efficiency of anisole turnover. A competition kinetic ex-
periment performed with a 100/10-anisole/phenol-ratio refer-
enced versus one equivalent of sodium bromide and hydrogen
peroxide, provided 14% of bromoanisole 9 (ortho:para¼43:57) and
86% of bromophenol 2a (ortho:para¼24:76). From these data we
estimated that anisole bromination occurs by a factor of 60 slower
than phenol bromination, which is in line with a mechanistic in-
terpretation outlined above.
Standard instrumentation and general remarks have been dis-
closed previously (see also ESI). All solvents and reagents were
purified following recommended standard procedures.59
VBrPO(AnI) was isolated from A. nodosum collected in April 2009
(France, 48ꢂ 430 N, 3ꢂ 580 W) as described previously.34
4.2. Phenol bromination
4.2.1. Phenol bromination with HOBrdgeneral procedure A. A so-
lution of HOBr60,61 (0.2e0.3 M, 0.75e2.25 mmol, 1.0e3.0 equiv,
pHfinal 1 for all reactions, 3.0e9.0 mL) in aq MES buffer (500 mM)
was added to a solution of substrate 1a (0.75 mmol) in MES-buffer
(500 mM, pHfinal 6.2, 2e8 mL) and BuOH (3.3 mL). The reaction
t
mixture was stirred at 23 ꢂC for 3 d. The aqueous layer was
extracted with Et2O (3ꢁ20 mL). Combined organic extracts were
dried (MgSO4). The solvent was removed under reduced pressure
(40 mbar, 40 ꢂC) to afford a product mixture, which was analyzed
by 1H NMR and GC, using pentachlorobenzene (1H NMR) as an
internal standard in comparison to spectroscopic data from au-
thentic references. The pH-value of the aqueous layer was de-
3. Concluding remarks
The chemistry of phenol bromination that follows bromide ox-
idation by hydrogen peroxide in reactions catalyzed by the
vanadate(V)-dependent bromoperoxidase I from the brown alga A.
nodosum [VBrPO(AnI)] shows considerable parallels to the
termined at the end of reaction. For spectroscopic data of 2a4-Br
,

9462
D. Wischang, J. Hartung / Tetrahedron 68 (2012) 9456e9463
2a2-Br, 3a2,4-Br , and 4a2,4,6-Br refer to Section 4.3.2 of the experi-
(22), 93 (9). 2,4,6-Tribromophenol (4a2,4,6-Br ).62 dH (600 MHz;
2
3
3
mental part. For yields refer to Table 1.
CDCl3) 5.88 (1H, s, eOH), 7.59 (2H, s, 3-H, and 5-H). m/z (EI, 70 eV)
334 (39), 332 (94), 330 (100), 328 (38), 172 (14), 170 (9). 558
U0T mgꢀ1, 133 UfiTnal mgꢀ1, pHfinal 6.80.
4.2.2. Phenol bromination with Br2dgeneral procedure B. A solution
of Br2 (120e360 mg, 0.75e2.25 mmol) in tBuOH (1.0 mL) was added
with a syringe pump (8 h, 2.08 mL/min) to a solution of substrate 1a
4.4. Competition kinetics
(0.75 mmol) in MES-buffer (500 mM, pHfinal 6.2,11.0 mL) and tBuOH
(2.3 mL). The reaction mixture was stirred at 23 ꢂC for 3 d. The
aqueous layer was extracted with Et2O (3ꢁ20 mL). Combined or-
ganic extracts were washed with aq Na2S2O3 solution (0.1 M,
25 mL) and dried (MgSO4). The solvent was removed under reduced
pressure (40 mbar, 40 ꢂC) to afford a product mixture, which was
analyzed by 1H NMR and GC, using pentachlorobenzene (1H NMR)
as an internal standard in comparison to spectroscopic data from
authentic references. The pH-value of the aqueous layer was de-
4.4.1. Competition kinetics for VBrPO(AnI)dgeneral procedure E. In
three separate reactions phenol (1a) and 2-substituted phenol
derivative 1bꢀ1f in the proportions 1:1, 1:2, and 2:1 (200
1.0 equiv or 400 mol, 2.0 equiv) were added to a stock solution A
[3.75 mL, corresponding to 0.1 equiv NaBr (20 mol)], consisting of
mmol,
m
m
MES-buffer (50 mM, pH 6.2, 225 mL), and NaBr (124 mg,
1.20 mmol). MES-buffer (50 mM, pH 6.2, 3.75 mL), tBuOH (2.5 mL)
and VBrPO(AnI) [14.5
mL (0.4505 mg/mL; 611 UT/mg), 4.0 UT,
termined at the end of reaction. For spectroscopic data of 2a4-Br
,
0.27 mmol%] were added. After the addition of H2O2 (2 mL, 10 mM,
0.1 equiv) in a dropwise manner within 2 min, the reaction mixture
was stirred at 25 ꢂC for 24 h, acidified with 2 M HCl (pH 1) and
extracted with Et2O (2ꢁ15 mL, 1ꢁ10 mL). Combined organic ex-
tracts were dried (MgSO4). The volatiles were removed under re-
duced pressure (13 mbar, 40 ꢂC). To the crude product mixture
a stock solution B (0.3 mL), consisting of Zn(ClO4)2ꢁ6H2O (30.0 mg,
2a2-Br, 3a2,4-Br , and 4a2,4,6-Br refer to section 4.3.2. of the experi-
2
3
mental part. For yields refer to Table 1.
4.2.3. Phenol bromination with NBu4Br3dgeneral procedure C. NBu4Br3
(362e723 mg, 0.75e2.25 mmol) was added to a solution of substrate
1a (0.75 mmol) in MES-buffer (500 mM, pHfinal 6.2,11.0 mL) and tBuOH
(3.3 mL). The suspension was stirred at 23 ꢂC for 3 d. The aqueous layer
was extracted with Et2O (3ꢁ20 mL). Combined organic extracts were
dried (MgSO4). The solvent was removed under reduced pressure
(40 mbar, 40 ꢂC) to afford a product mixture, which was analyzed by
1H NMR and GC, using pentachlorobenzene (1H NMR) as an internal
standard in comparison to spectral data from authentic references. The
pH-value of the aqueous layer was determined at the end of reaction.
80.0 mmol) in Ac2O (551 mg, 5.40 mmol) and Et2O (0.9 mL), was
added. The homogenous reaction mixture was stirred at 25 ꢂC for
18 h. The purified product mixture by adsorption chromatography
(SiO2, EtOAc) was analyzed by GC, using pentachlorobenzene (1c)
or n-decane (1a, 1b, 1de1f) as an internal standard in comparison
to spectroscopic data from authentic references.
For spectroscopic data of 2a4-Br, 2a2-Br, 3a2,4-Br , and 4a2,4,6-Br refer to
Acknowledgements
2
Section 4.3.2 of the experimental part. For yields refer to Tab3le 1.
This work was supported by the Deutsche Bundesstiftung
Umwelt (grant 20008/982; scholarship for D.W.) and NanoKat. The
study is part of the Ph.D. thesis of D.W. We express our gratitude to
Swen Ehnert for technical assistance, and Dr. Hans Vilter and Dipl.-
4.3. VBrPO(AnI)-catalyzed reactions
4.3.1. General procedure D. A solution of H2O2 (1.0 mL, 0.825 M)
€
and NaBr (77.2 mg, 0.75 mmol) in MES-buffer (500 mM, pH 6.2)
Chem. Oliver Brucher for helpful discussions.
was added with a syringe pump (8 h, 2.08
substrate 1, or 8 (0.75 mmol) and VBrPO(AnI) [55.2
mg/mL; 558 UT/mg), 17.3 UT, 0.036 mmol% for 1a, 1c, and 8; 62.8
m
L/min) to a solution of
mL (0.5626
Supplementary data
mL
(0.4505 mg/mL; 611 UT/mg), 17.3 UT, 0.031 mmol% for 1b and 1def]
in MES-buffer (500 mM, pH 6.2, 10.0 mL) and tBuOH (3.3 mL). The
reaction mixture was stirred at 23 ꢂC for 3 d. The aqueous layer was
extracted with Et2O (4ꢁ10 mL). Combined organic extracts were
dried (MgSO4). The solvent was removed under reduced pressure
(14 mbar, 40 ꢂC) to afford a product mixture, which was analyzed by
1H NMR or GC, using pentachlorobenzene (1H NMR, 1aed and), 1-
bromodecane (1H NMR, for 1e), or n-decane (GC, for 8) as an in-
ternal standard in comparison to spectroscopic data from authentic
Instrumentation, reagent specification, details about competi-
tion kinetics, experimental procedures, spectral, and analytical data
of compounds. Supplementary data related to this article can be
found at http://dx.doi.org/10.1016/j.tet.2012.08.081.
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