REACTIONS OF 4-METHYLPHENYL ISOCYANATE WITH AMINO-ACIDS

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lu ng sensitization and asthma. Protein adducts of isocyanates might be i nvolved in the aetiology of sensitization reactions. blood protein add ucts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4-methylphenyl isocyanate ( 4MPI) with the tripeptide valyl-glycyl-glycine and with single amino a cids yielding N-(4-methylphenyl-carbamoyl)-L -valyl-glycyl-glycine (4M PI-Val-Gly-Gly), N-(4-methylphenyl-carbamoyl)-L-valine (4MPI-Val), N-( 4-methylphenyl-carbamoyl)-L-aspartic-acid (4MPI-Asp), pha-acetyl-S-(4- methylphenyl-carbamoyl)-L-cysteine (4MPI-AcCys), acetyl-N-(4-methylphe nyl-carbamoyl)-epsilon-lysine (4MPI-AcCys), alpha-acetyl-O-(4-methylph enyl-carbamoyl)-tyrosine (4MPI-AcTyr) and lpha-acety-O-(4-methylphenyl -carbamoyl)-D,L-serine (4MPI-AcSer). The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4-methylaniline (4MA). The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 degrees C with 6 M HCI in and/or 0.1 M NaOH at room te mperature, following methods applied for the analyses of biological sa mples of arylisocyanate-exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 degrees C. The hydrolysates were analysed using HPLC with UV-detectio n and quantified against the internal standard, 4-fluoroaniline or 4-c hloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; a fter 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI-Val and 4MPI-Asp yielded the respective hydant oins 3-(4-methylphenyl)-5-isopropyl-1,3-imidazolin and 1-(4-methylphen yl)-2,5-dioxoperhydro-4-imidazolyl) acetic acid. For future studies, w e propose to hydrolyse biological samples with 0.3 NI NaOH at 100 degr ees C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also resu lt from arylamine exposure. Therefore, we propose to analyse the N-ter minal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.