2,4-toluenediisocyanate and hexamethylene-diisocyanate adducts with blood proteins: assessment of reactivity of amino acid residues in vitro

Diisocyanates, reactive compounds used in plastics industry and potent occupational allergens, readily bind to proteins both in vitro and in vivo, however, the pattern of adducts with individual amino acids has not been investigated systematically. In this study, potential of the proteinogenic amino acid residues for carbamoylation with 2,4-toluenediisocyanate (2,4-TDI) and hexamethylenediisocyanate (HDI) was evaluated. The diisocyanates were incubated in an in vitro system (buffer pH 7.4/dioxane 50:50) with: (a) a series of Nalpha-benzyloxycarbonyl amino acids (Z-amino acids) and N-acetylcysteine (Ac-Cys), model compounds for non-N-terminal amino acids of the protein chain; (b) dipeptides Val-Phe and Asp-Phe, model compounds for N-termini of globin and albumin, respectively. Reactivity of the compounds tested, evaluated from their depletion during incubation with the diisocyanates (measured by HPLC), was in the order: Ac-Cys = Asp-Phe > Val-Phe = Nalpha-Z-Lys > Nalpha-Z-His for 2,4-TDI, and Ac-Cys > Asp-Phe > Val-Phe = Nalpha-Z-Lys > Nalpha-Z-His > N-Z-Tyr for HDI, however, the adducts with Ac-Cys were unstable. Reactions of other amino acid residues (e.g. Ser, Thr, Met, Trp, Arg, Asn, Gln) with 2,4-TDI and HDI were not observed. Thus, N-terminal amino acids and Lys residues are likely to produce most abundant adducts with diisocyanates in proteins. Further, three amino compounds with increasing pKa values (Val-Phe, Val and Nalpha-Z-Lys) were incubated with 2,4-TDI and N-acetyl-S-4-(2-amino)tolylcarbamoyl]cysteine, a 2,4-TDI-derived thiocarbamate with carbamoylating activity, in media with 10% and no dioxane, respectively. Here, reactivity of the amino compounds was decreasing in the order: Val-Phe > Val > Nalpha-Z-Lys, which reflects the mechanism of the amine-isocyanate reaction. The experiments also demonstrate the effect of a solvent (organic phase content) on the yield of the carbamoylation reactions.