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atom regioselectively on an array of aromatic substrates.[12] The
proposed mechanism involves the in situ production of hypo-
halous acid, which binds to an active-site lysine residue; this
residue is then thought to guide the regioselective attack of
the substrate.[13] In contrast, heme-dependent enzymes release
free hypohalous acid, which reacts with a substrate in solution
and thus lacks good regiocontrol.[10] Early reports of vanadium-
dependent enzymes suggested that they are also unselective
due to the escape of hypohalous acid[10] but, more recently,
a number of reports have now emerged indicating that good
regiocontrol is possible.[14]
Results and Discussion
Development of halogenation assay
The basis for the assay is the distinct UV/Vis spectrum of qui-
none–aniline adduct that is formed by the stoichiometric Mi-
chael addition of the amino component to the ortho-benzoqui-
none, formed by the oxidation of catechol by HRP.[20] Previous-
ly, this adduct formation had been used to quantify the con-
centration of peroxidases in solution, but by using an excess of
HRP and H2O2 it is instead possible to use the adduct pro-
duced by the reaction for quantification of the amino compo-
nent. If the reaction mixture contains multiple amino compo-
nents, each resulting adduct can be differentiated by their re-
spective lmax peaks in the overall UV/Vis spectra. In the case of
arylamines and halogenated arylamines it was found that the
lmax values were typically in the range of ꢀ520 and ꢀ425 nm,
respectively (Figure 2).
In all cases, any efforts aimed at discovering new halogenas-
es[10] or genetically reengineering existing enzymes[15] require
the capacity to detect and quantify halogenation activity. One
possible approach to this end is the application of assays for
the detection of free hypohalous acid in solution,[16,17] but this
is an indirect method of detecting enzyme turnover and would
not be applicable to halogenating enzymes that bind hypoha-
lous acid in their active site, such as the tryptophan halogenas-
es. As a result, most analyses of these substrates has been con-
ducted with serial and inherently low-throughput methods
such as HPLC, LC–MS and NMR.[11,18] Therefore, the further de-
velopment of halogenating enzymes would greatly benefit
from a high-throughput screen, which would aid in the screen-
ing of enzyme libraries.
Previously, it has been shown that the oxidation of catechols
by horseradish peroxidase (HRP) to their corresponding 1,2-
benzoquinone (ortho-quinone) can be combined with a subse-
quent Michael addition of an aniline to the quinone in a “one-
pot” reaction.[19] Because the resulting quinone–aniline adduct
was highly coloured, this reaction sequence has been applied
in an assay for peroxidase activity.[20] We have observed that
different anilines, even those closely related in structure, result
in the formation of adducts that have significantly different
UV/Vis spectra. In comparing halogenated anilines to their un-
halogenated parent compounds, these spectral differences
were sufficiently large to be visually distinguishable.
Figure 2. UV/Vis spectra of adduct formed from 4-methyl-catechol (4-MC)
with either 2-aminobenzoic acid (0.5 mm, X=H) or 2-amino-6-chlorobenzoic
acid (0.5 mm, X=Cl) in K2HPO4 (50 mm) at pH 7.4.
Based on these observations, we now report the develop-
ment of this reaction sequence into a colorimetric assay for
the detection and quantification of halogenated arylamines.
The assay described is generally applicable to a wide range of
arylamines, rapid and high-throughput. Furthermore, since the
oxidation of the catechol is also enzymatically driven, it can be
performed sequentially after an enzymatic halogenation reac-
tion in a “one-pot” procedure (Scheme 1).
A number of experimental considerations were taken into
account to simplify the analysis and enable a one-pot proce-
dure. Firstly, 4-methylcatechol (4-MC) was employed as the cat-
echol component, because it was known to be a good sub-
strate for HRP. The resulting ortho-quinone gives only a single-
conjugate product, because the 4-methyl substituent blocks
further addition at this position.[21] To prevent any oxidation of
the arylamine by HRP at low pH,[22] all assays were carried out
in pH 7.4 buffered conditions. To
confirm that only the desired
product was formed under these
conditions, test reactions were
carried out with 4-MC and either
2-aminobenzoic acid or 2-amino-
6-chlorobenzoic acid; HPLC and
MS analysis of the reaction mix-
tures confirmed single-product
Scheme 1. General scheme illustrating the HRP-catalysed oxidation of catechols to their corresponding ortho-qui-
none (upper pathway) and the formation of the arylamine–catechol adduct. This reaction can be coupled to a bio-
catalytic halogenation reaction (lower pathway).
formation and that the reaction
was complete within 5 min.
Chem. Eur. J. 2014, 20, 16759 – 16763
16760 ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim