Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine

Abstract

19F NMR spectroscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N alpha-acetyl-N epsilon-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by 19F and 13C NMR spectroscopy and mass spectrometry. N alpha-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation was greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However N alpha-acetyllysine, at concentrations of greater than 100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Both stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated nephrotoxicity.

Novel Mechanism of Surface Catalysis of Protein Adduct Formation

“…In this case, adducts were observed with the N-terminal glycyl amino group. Analogous observations regarding the formation of thionoacyl adducts of albumin with metabolically generated thionoacyl fluoride intermediates led to a similar conclusion and to a proposed catalytic effect of histidine or tyrosine side chains, which was studied by the addition of imidazole and phenol to the system (2).…”

“…As noted above, Hayden et al (2) found that imidazole and phenol catalyzed adduct formation with metabolically generated thionoacyl fluorides. Khalifah and Sutherland (15) found that imidazole stimulated alkylation of alcohol dehydrogenase at low concentrations but was inhibitory at high concentrations.…”

“…Aspartyl and glutamyl carboxyl groups have also been found in the vicinity of highly glycated lysyl residues (6,13). Similarly, tyrosyl side chains could be transiently acetylated and become involved in analogous transacetylation reactions (2). It seems likely that all of the catalytic effects observed at the active sites of enzymes may play a role in non-enzymatic protein modification, although the effects in general are expected to be considerably smaller because of the lack of defined surface residue stereochemical constraints (29).…”

“…Ethanol was purchased from Amersham Pharmacia Biotech. [ Synthesis of [1][2][3][4][5][6][7][8][9][10][11][12][13] C]Acetyl Salicylic Acid-[1-13 C]Acetyl salicylic acid was synthesized following an approach similar to that described by Gerig et al (16) by the reaction of [1-13 C]acetyl chloride with salicylic acid. Salicylic acid (1.5 g) was dissolved in 90 ml of methylene chloride containing 1.8 ml of pyridine.…”

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

“…The thioacyl halides formed from S-(1,1,2,2-tetrafluoroethyl)-L-cysteine covalently modify rat renal tubular proteins (42): Incubation of [ 35 S]S-(1,1,2,2-tetrafluoroethyl)-L-cysteine with renal tubular cells and analysis by 19 F NMR spectroscopy demonstrated the formation of N -(difluorothionoacetyl)lysine adducts (Scheme 1). Similarly, in rats given S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine or S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, covalently modified renal proteins were identified by 19 F NMR spectroscopy as N -(chlorofluorothionoacetyl)lysine or N -(difluorothionoacetyl)lysine adducts (43); renal proteins incubated with 2-chloro-1,1,2-trifluoroethyl 2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiolate, also showed the formation of N -(chlorofluorothionoacetyl)lysine adducts.…”