title: Chemical reactivity and microbicidal action of bethoxazin.
abstract: Bethoxazin is a new broad spectrum industrial microbicide with applications in material and coating preservation. However, little is known of its reactivity profile and mechanism of action. In this study, we examined the reactivity of bethoxazin toward biologically important nucleophilic groups using UV-vis spectroscopy and LC-MS/MS techniques and found the molecule to be highly electrophilic. Bethoxazin reacted with molecules containing free sulfhydryl groups such as GSH and human serum albumin to form covalent adducts that were detectable by MS, but did not react with amino, carboxylic, phenolic, amino oxo, alcoholic, and phosphate functional groups. Bethoxazin potently inhibited the catalytic activity of yeast DNA topoisomerase II and the growth of yeast BY4742 cells at low micromolar concentrations. However, the reduced form of bethoxazin and GSH-treated bethoxazin were both inactive in these assays. The experimentally determined relative reactivity of bethoxazin and its reduced form analog correlated with their biological activities as well as their quantum-mechanically calculated electrophilicity properties. Taken together, the results suggest that bethoxazin may exert its microbicidal action by reacting with sensitive endogenous sulfhydryl biomolecules of microbial cells. Consistent with this view, the inhibitory activity of bethoxazin on topoisomerase II may be due to its ability to react with critical free cysteine sulfhydryl groups on the enzyme. Our studies have provided for the first time a better understanding of the reactivity of bethoxazin, as well as some insights into the mechanism by which the compound exerts its microbicidal action.
authors.affiliation: Faculty of Pharmacy, University of Manitoba, Winnipeg, Canada R3E 0T5. lean_chee@umanitoba.ca
authors.forename: Gaik-Lean
authors.initials: GL
authors.lastname: Chee
authors.forename: Bharat
authors.initials: B
authors.lastname: Bhattarai
authors.forename: R
authors.initials: R
authors.lastname: Daniel Gietz
authors.forename: Samaa
authors.initials: S
authors.lastname: Alrushaid
authors.forename: John L
authors.initials: JL
authors.lastname: Nitiss
authors.forename: Brian B
authors.initials: BB
authors.lastname: Hasinoff
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry / Q000494:pharmacology; D003497:Cyclic N-Oxides / Q000737:chemistry* / Q000494:pharmacology*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011422:Propionates / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D013843:Thiazines / Q000737:chemistry* / Q000494:pharmacology*; D059003:Topoisomerase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D015003:Yeasts / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D003497:Cyclic N-Oxides; D011422:Propionates; D012709:Serum Albumin; D013843:Thiazines; D059003:Topoisomerase Inhibitors; C017526:3-methylthiopropionate; C572565:bethoxazin