title: A high-throughput assay for arylamine halogenation based on a peroxidase-mediated quinone-amine coupling with applications in the screening of enzymatic halogenations.
abstract: Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more "green" halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a "one-pot" workflow.
authors.affiliation: Manchester Institute of Biotechnology and School of Chemistry, University of Manchester, 131 Princess Street, Manchester M1 7DN (UK).
authors.forename: Joseph
authors.initials: J
authors.lastname: Hosford
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Shepherd
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
authors.forename: Lu Shin
authors.initials: LS
authors.lastname: Wong
mesh_terms: D000588:Amines / Q000737:chemistry* / Q000378:metabolism; D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D054879:Halogenation; D057166:High-Throughput Screening Assays; D006735:Horseradish Peroxidase / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000588:Amines; D016227:Benzoquinones; C004532:quinone; D006735:Horseradish Peroxidase
keywords: Michael addition; enzyme catalysis; halogenation; high-throughput screening