BIOACTIVATION OF S-(2,2-DIHALO-1,1-DIFLUOROETHYL)-L-CYSTEINES AND S-(TRIHALOVINYL)-L-CYSTEINES BY CYSTEINE S-CONJUGATE BETA-LYASE - INDICATIONS FOR FORMATION OF BOTH THIONOACYLATING SPECIES AND THIIRANES AS REACTIVE INTERMEDIATES

The covalent binding of reactive intermediates, formed by beta-elimina tion of cysteine S-conjugates of halogenated alkenes, to nucleophiles was studied using F-19-NMR and CC-MS analysis. beta-Elimination reacti ons were performed using rat renal cytosol and a beta-lyase model syst em, consisting of pyridoxal and copper(II) ion. S-(1,1,2,2-Tetrafluoro ethyl)-L-cysteine (TFE-Cys) was mainly converted to products derived f rom difluorothionoacetyl fluoride, namely, difluorothionoacetic acid, difluoroacetic acid, and N-difluorothionoacetylated TFE-Cys. In the pr esence of o-phenylenediamine (OPD), as a bifunctional nucleophilic tra pping agent, the major product formed was 2-(difluoromethyl)benzimidaz ole. This product results from initial reaction of difluorothionoacety l fluoride with one of the amino groups of OPD, followed by a condensa tion reaction between the thionoacyl group and the adjacent amino grou p of OPD. In incubations with S-(2-chloro-1,1,2-trifluoroethyl)-L-cyst eine (CTFE-Cys) and -(2,2-dichloro-1,1-difluorofluoroethyl)-L-cysteine (DCDFE-Cys), formation of thionoacylated cysteine S-conjugates was al so observed by GC-MS analysis, indicating formation of the correspondi ng thionoacyl fluorides. However, according to F-19-NMR analysis, chlo rofluorothionoacyl fluoride-derived products accounted for only 10% of the CTFE-Cys converted. In the presence of OPD, next to the correspon ding 2-(dihalomethyl)benzimidazoles, 2-mercaptoquinoxaline was identif ied as the main product in incubations with CTFE-Cys. When chlorofluor othionoacylating species were generated from the unsaturated S-(2-chlo ro-1,2-difluorovinyl)-L-cysteine (CDFV-Cys), 2-(chlorofluoromethyl)ben zimidazole and 2-mercaptoquinoxaline were also found as OPD adducts. H owever, 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-mercaptoqui noxaline could also be explained by reaction of OPD with 2,3,3-trifluo rothiirane as a second reactive intermediate for CTFE-Cys. Comparable results were obtained when comparing OPD adducts from DCDFE-Cys and TC V-Cys. Both DCDFE-Cys and TCV-Cys form dichlorothionoacylating species . However, DCDFE-Cys forms al-fold more 2-mercaptoquinoxaline than TCV -Cys, which may be explained by its capacity to form 3-chloro-2,2-difl uorothiirane 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(f)G) of t hiolate anions and their possible rearrangement products, thionoacyl f luorides and thiiranes, derived from TFE-Cys, CTFE-Cys, and DCDFE-Cys, were calculated by ab initio calculations. For TFE-thiolate, formatio n of difluorothionoacetyl fluoride is energetically favored over forma tion of the thiirane. In contrast, the thiirane pathway is favored ove r the thionoacyl fluoride pathway for CTFE- and DCDFE-thiolates. The r esults of these quantum chemical calculations appear to be consistent with the experimental data.