title: Characterisation and quantitation of a selenol intermediate in the reaction of ebselen with thiols.
abstract: The reaction of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) with thiols was investigated with particular attention to the formation of an ebselen selenol intermediate. The selenol intermediate could be trapped in a mixture of ebselen and thiols with 1-chloro-2,4-dinitrobenzene and the resulting product displayed unique spectral characteristics. The reaction of authentic, synthesised ebselen selenol with 1-chloro-2,4-dinitrobenzene (CDNB) was shown to give rise to the same compound (2,4-dinitrophenyl (N-phenyl-2-carboxamido phenyl) selenide as characterized by light spectroscopy, NMR, IR and elemental analysis. The determination of the absorbtion coefficient at 400 nm (E = 7.5 mM-1 cm-1) and the initial rate constant of the reaction (1.4 +/- 0.3 mM-1 min-1) allows for the convenient quantification of ebselen selenol concentrations by initial rate measurements after addition of CDNB. The choice of 400 nm to monitor the reaction excludes the interference of other intermediates in the reaction of ebselen with thiols as well as the reaction of the thiols with CDNB. When the assay is applied to typical incubation conditions used for investigating the glutathione peroxidase-like activity of ebselen it was shown that as much as 10-20% of ebselen is in the selenol form. If a stronger reductant (dithiothreitol) is used 60% is in the selenol form. These data could also be confirmed by the direct determination of ebselen selenol by UV spectroscopy, due to its peak absorption at 370 nm (E = 2 mM-1 cm-1). In conclusion, this investigation demonstrates, for the first time, the identity and quantity of ebselen selenol in the reaction of ebselen with thiols and also describes a convenient assay for its quantification. These observations allow further possibilities for investigation of the molecular species responsible for the antioxidant and peroxidase activities of ebselen.
authors.affiliation: Department of Toxicology, Karolinska Institute, Stockholm, Sweden.
authors.forename: I A
authors.initials: IA
authors.lastname: Cotgreave
authors.forename: R
authors.initials: R
authors.lastname: Morgenstern
authors.forename: L
authors.initials: L
authors.lastname: Engman
authors.forename: J
authors.initials: J
authors.lastname: Ahokas
mesh_terms: D000813:Anilides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D001393:Azoles / Q000378:metabolism*; D001549:Benzamides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D054833:Isoindoles; D009682:Magnetic Resonance Spectroscopy; D016566:Organoselenium Compounds / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D000813:Anilides; D001393:Azoles; D001549:Benzamides; D004137:Dinitrochlorobenzene; D054833:Isoindoles; D016566:Organoselenium Compounds; D013438:Sulfhydryl Compounds; C075952:2-selenylbenzanilide; C042986:ebselen; D005979:Glutathione Peroxidase; D005978:Glutathione