Synthesis of Adducts with Amino Acids as Potential Dosimeters for the Biomonitoring of Humans Exposed to Toluenediisocyanate

Abstract

Toluenediisocyanates (TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. Protein adducts of TDI might be involved in the etiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. The functional groups of cysteine, tyrosine, serine, lysine, tryptophan, histidine, and N-terminal amino acids are potential reaction sites for isocyanates. Especially the N-terminal amino acids, valine, and aspartic acid of hemoglobin and albumin, respectively, are reactive toward electrophilic xenobiotics. To develop methods for the quantitation of protein adducts of 2,4- and 2,6-TDI, we reacted 3-nitro-4-methylphenyl isocyanate (1a) with single amino acids and reduced the nitro group using catalytic hydrogenation or ammonium formate with palladium on carbon yielding N-[(3-amino-4-methylphenyl)carbamoyl]valine (2a), N-[(3-amino-4-methylphenyl)carbamoyl]aspartic acid (8a), N(alpha)-acetyl-N(epsilon)-[(3-amino-4-methylphenyl)carbamoyl]lysine (12a), and N(alpha)-acetyl-O-[(3-amino-4-methylphenyl)carbamoyl]serine (15a). The same reactions were performed with 5-nitro-2-methylphenyl isocyanate (1b) and 3-nitro-2-methylphenyl isocyanate (1c). The valine adducts were boiled in acid to obtain the corresponding hydantoins: 3-(3-amino-4-methylphenyl)-5-isopropylimidazoline-2,4-dione (5a), 3-(5-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5b), and 3-(3-amino-2-methylphenyl)-5-isopropylimidazoline-2,4-dione (5c). A method for the detection of N-terminal adducts with valine in biological samples was developed. The tripeptide adduct N-[(3-amino-4-methylphenyl)carbamoyl]valyl-glycyl-glycine (19a) was hydrolyzed with acid in the presence of globin and the internal standard N-[(3-amino-4-methylphenyl-d(6))carbamoyl]valyl-glycyl-glycine (19d). The released hydantoins were determined by LC/MS/MS and after derivatization with pentafluoropropionic anhydride by GC/MS. The determination limit was 0.16 pmol/sample. The same N-terminal adduct with valine was found in globin of a TDI-worker and in two women with polyurethane covered breast implants.