CYSTEINE CONJUGATE BETA-LYASE-CATALYZED BIOACTIVATION OF BROMINE-CONTAINING CYSTEINE S-CONJUGATES - STOICHIOMETRY AND FORMATION OF 2,2-DIFLUORO-3-HALOTHIIRANES

1,1-Dichloroalkene-derived S-(1-chloroalkenyl)-L-cysteine conjugates, but not 1,1-difluoroalkene-derived S-(2,2-dihalo-1,1-difluoroethyl)-L- cysteine conjugates, are mutagenic in the Ames test. Recent studies ha ve showed, however, that bromine-containing, 1,1-difluoroalkene-derive d S-(2-bromo-2-halo-1,1-difluoroethyl)-L-cysteine conjugates are mutag enic [Finkelstein, M. B., et al. (1994) Chem. Res. Toxicol. 7, 157-163 ] and that alpha-thiolactones are formed as reactive intermediates and glyoxylate as a terminal product [Finkelstein, M. B., et al. (1995) J . Am. Chem. Sec. 117, 9590-9591]. The present studies were undertaken to examine the stoichiometry of cysteine conjugate beta-lyase-catalyze d product formation from a panel of bromine-containing and bromine-lac king cysteine S-conjugates and to search for additional metabolites. T he cysteine S-conjugates were incubated with rat renal homogenates, an d pyruvate:product (glyoxylate, bromide, fluoride, dihaloacetate, trih aloethene) ratios were measured. Pyruvate: glyoxylate ratios for S-(2- bromo-1,1,2-trifluoroethyl)-L-cysteine S-(2-bromo-2-chloro-1,1-difluor oethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L-cysteine r anged from 1:0.13 to 1:0.16. With S-(2-bromo-2-chloro-1,1-difluoroethy l)-L-cysteine and S-(2-bromo-1, 1,2-trifluoroethyl)-L-cysteine, pyruva te:bromide ratios were 1:1, but with the dibrominated conjugate S-(2,2 -dibromo-1,1-difluoroethyl)-L-cysteine, the pyruvate:bromide ratio was 1:1.2. All bromine-containing cysteine S-conjugates gave less than co mplete conversion to fluoride. A search for additional metabolites led to the consideration of 2,2-difluoro-3-halothiiranes as putative inte rmediates. 2,2-Difluoro-3-halothiiranes may arise by internal displace ment of bromide and cyclization of 2-bromo-2-halo-1,1-difluoroethaneth iolates, which are beta-elimination products of cysteine S-conjugates. Such halogenated thiiranes may eliminate sulfur to give 1,1-difluoro- 2-haloethenes. GC/MS analysis showed that trifluoroethene, 2-chloro-1, 1-difluoroethene, and 2-bromo-1,1-difluoroethene were terminal product s of S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine, S-(2-bromo-2-chloro- 1,1-difluoroethyl)-L-cysteine, and S-(2,2-dibromo-1,1-difluoroethyl)-L -cysteine, respectively. The bromine-lacking conjugate S-(2-chloro-1,1 ,2-trifluoroethyl)-L-cysteine did not yield glyoxylate or trifluoroeth ene as products, but the formation of chlorofluoroacetate was confirme d. The pyruvate:chlorofluoroacetate ratio was 1:0.38, indicating that other products are formed. This is the first report of the stoichiomet ry of the beta-lyase-catalyzed biotransformation of haloalkene-derived cysteine S-conjugates and of the formation of 2,2-difluoro-3-halothii ranes as reactive intermediates in the biotransformation of bromine-co ntaining cysteine S-conjugates.