Bioactivation of S-(2,2-Dihalo-1,1-difluoroethyl)-l- cysteines and S-(Trihalovinyl)-l-cysteines by Cysteine S-Conjugate β-Lyase:  Indications for Formation of both Thionoacylating Species and Thiiranes as Reactive Intermediates

Abstract

The covalent binding of reactive intermediates, formed by beta-elimination of cysteine S-conjugates of halogenated alkenes, to nucleophiles was studied using 19F-NMR and GC-MS analysis. beta-Elimination reactions were performed using rat renal cytosol and a beta-lyase model system, consisting of pyridoxal and copper(II) ion. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFE-Cys) was mainly converted to products derived from difluorothionoacetyl fluoride, namely, difluorothionoacetic acid, difluoroacetic acid, and N-difluorothionoacetylated TFE-Cys. In the presence of o-phenylenediamine (OPD), as a bifunctional nucleophilic trapping agent, the major product formed was 2-(difluoromethyl)benzimidazole. This product results from initial reaction of difluorothionoacetyl fluoride with one of the amino groups of OPD, followed by a condensation reaction between the thionoacyl group and the adjacent amino group of OPD. In incubations with S-(2-chloro-1,1,2-trifluorofluoroethyl)-L-cysteine (CTFE-Cys) and S-(2,2-dichloro-1,1-difluorofluoroethyl)-L-cysteine (DCDFE-Cys), formation of thionoacylated cysteine S-conjugates was also observed by GC-MS analysis, indicating formation of the corresponding thionoacyl fluorides. However, according to 19F-NMR analysis, chlorofluorothionoacyl fluoride-derived products accounted for only 10% of the CTFE-Cys converted. In the presence of OPD, next to the corresponding 2-(dihalomethyl)benzimidazoles, 2-mercaptoquinoxaline was identified as the main product in incubations with CTFE-Cys. When chlorofluorothionoacylating species were generated from the unsaturated S-(2-chloro-1,2-difluorovinyl)-L-cysteine (CDFV-Cys), 2-(chlorofluoromethyl)benzimidazole and 2-mercaptoquinoxaline were also found as OPD adducts. However, with CDFV-Cys the ratio of 2-(chlorofluoromethyl) benzimidazole to 2-mercaptoquinoxaline was 12-fold higher than in the case of CTFE-Cys. These results suggest an important second mechanism of formation of 2-mercaptoquinoxaline with CTFE-Cys. The formation of 2-mercaptoquinoxaline could also be explained by reaction of OPD with 2,3,3-trifluorothiirane as a second reactive intermediate for CTFE-Cys. Comparable results were obtained when comparing OPD adducts from DCDFE-Cys and TCV-Cys. Both DCDFE-Cys and TCV-Cys form dichlorothionoacylating species. However, DCDFE-Cys forms 21-fold more 2-mercaptoquinoxaline than TCV-Cys, which may be explained by its capacity to form 3-chloro-2,2-difluorothiirane next to dichlorothionoacyl fluoride. In order to explain the apparent differences in the preference of thiols to form different reactive intermediates, free enthalpies of formation (delta 1G) of thiolate anions and their possible rearrangement products, thionoacyl fluorides and thiiranes, derived from TFE-Cys, CTFE-Cys, and DCDFE-Cys, were calculated by ab initio calculations. For TFE-thiolate, formation of difluorothionoacetyl fluoride is energetically favored over formation of the thiirane. In contrast, the thiirane pathway is favored over the thionoacyl fluoride pathway for CTFE- and DCDFE...

Theoretical Evaluation of Two Plausible Routes for Bioactivation of S-(1,1-Difluoro-2,2-dihaloethyl)-l-cysteine Conjugates: Thiirane vs Thionoacyl Fluoride Pathway

“…The results indicated that the thionoacyl fluoride pathway was thermodynamically favored for 2,2-difluoro-DFET, while the thiirane pathway was favored for the two chlorinated 2,2-dihaloDFETs. The results of the Commandeur et al (31) study…”

Visible‐light Photochemistry and Phototoxicity of Thiarubrines

“…The photosulfides may also exert toxic effects because episulfides have been reported to be highly reactive (24,25) and early studies show that episulfides possess antituberculous (26) and insecticidal (27) properties. As well, episulfide intermediates have been implicated in the nephrotoxicity of halogenated alkenes (28,29). A third, possibly toxic event in the thiambrine-thiophene pathway is the extrusion of the episulfide sulfur from photosulfides, giving a reactive form of sulfur.…”

Chemical Toxicology of Reactive Intermediates Formed by the Glutathione-Dependent Bioactivation of Halogen-Containing Compounds

“…Commandeur et al (108) determined the free enthalpies of formation (∆ f G) for the tetrahaloethanethiolates formed from S-(1,1,2,2-tetrahaloethyl)-L-cysteines. With 1,1,2,2-tetrafluoroethanethiolate, the formation of 2,2-difluorothioacetyl fluoride was energetically favored over 2,2,3-trihalothiirane formation.…”