Nuclear Magnetic Resonance Studies on Covalent Modification of Amino Acids Thiol and Amino Residues by Monofunctional Aryl ¹³C-Isocyanates, Models of Skin and Respiratory Sensitizers: Transformation of Thiocarbamates into Urea Adducts

Abstract

Exposure to aryl isocyanates, intermediates in the manufacture of polyurethanes, provokes lung sensitization and asthma but also occupational allergic contact dermatitis, sensitization occurring from a single accidental exposure. The initial step in the sensitization process is believed to be the covalent binding of the -N triple bond C triple bond O group with nucleophilic residues on proteins. While a wide knowledge exists on the reactivity of skin sensitizers toward amino acids, little is known about respiratory sensitizers such as aryl isocyanates. (13)C-Labeled monofunctional aryl isocyanates were synthesized, and their reactivities toward nucleophilic amino acids, GSH, and a model peptide were studied by (13)C and [(1)H-(13)C] NMR spectroscopy. An acetonitrile/buffer solution was used as a solvent to avoid the hampering of the follow up of the reactivity by the isocyanate hydrolysis competing reaction. The compounds reacted with thiol groups, through the formation of thiocarbamate bonds and with amino groups to form urea derivatives. The reactivity was confirmed with GSH, containing both free amino and thiol groups, and with a model peptide, particularly in the case of the reaction with lysine. The use of (13)C NMR to follow the aryl isocyanates reversible conjugation with thiol groups is also reported. Particularly, it is shown that thiocarbamate adducts can be converted into adducts of the urea kind by reaction with amino groups. These results confirmed the hypothesis by which thiol-containing peptides/proteins may act as carriers of isocyanates for possible reaction at a later time and/or place with other nucleophiles and confirmed the role of lysine as a good competing nucleophilic amino acid. The reactivity of aryl isocyanates with thiol and amino groups needs thus to be considered in their assigned sensitization processes.

Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure

“…PBS), formation of antigenic MDI-albumin occurred at GSH concentrations within the range (100 μM - 1 mM) found in the lower airway fluid [33, 34]. The pH was also an important factor in GSH-mediated transcarbamoylation of albumin, consistent with classic descriptions of thiol-cyanate linkages [40], and the previously described pH-sensitivity of S-linked MDI-cysteine, TDI-GSH, and other monoisocyanate-GSH conjugates [11, 41, 51]. The effect of pH, as well as NaCl, on GSH carbamoylation of human albumin is intriguing with respect to the differences in composition of exposure sites vs. internal tissues, e.g.…”

“…In the absence of NaCl the “dose-response curve” was shifted to the right. When GSH was prepared in unbuffered solution (water), limited MDI reactivity/transcarbamoylation was observed, which may be due to GSH's innately low pH, and the acid-stability of thiol-cyanate reaction products [41, 51]. Consistent with this hypothesis, higher pH levels during the carbamoylation reaction (co-incubation of albumin with GSH-MDI), resulted in MDI-albumin products with relatively increased antigenicity (Fig 5C).…”

“…Evidence supporting a “shuttling” capacity of GSH has been derived largely from in vitro studies of toluene diisocyanate (TDI) and aryl mono-isocyanates, demonstrating that GSH conjugates can carbamoylate other peptides/proteins, including albumin [11, 38, 41, 42]. In contrast, GSH mediated transcarbamoylation of MDI, which possesses unique physical properties compared with other mono- and di-isocyanates, remains relatively under-studied.…”

“…shuttle) in the conjugation of albumin by MDI, and identify specific reaction conditions that further modulate this process. The experimental strategy builds upon limited published data on GSH reactivity with MDI, and other aryl mono-isocyanates, which largely used NMR to characterize thiol-MDI conjugation and disassociation [41, 45, 46]. The present investigation utilizes a combination of LC-MS/MS and electrophoretic analyses to further characterize GSH-MDI reaction products and their unique modification of human albumin, under physiological exposure conditions (neutral pH, normal saline, temperature, aqueous solvent).…”

Toluene Diisocyanate Reactivity with Glutathione Across a Vapor/Liquid Interface and Subsequent Transcarbamoylation of Human Albumin

“…This hypothesis is supported by in vitro studies demonstrating that GSH can form conjugates with TDI, capable of transcarbamoylating a model peptide (10). Similar findings have been demonstrated in studies with other mono-isocyanates, most notably, 2-chloroethyl-isocyanate, released by the cancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea, methylisocyanate, the cause of the catastrophic industrial disaster in Bhopal India in 1984, and phenyl isocyanate, a sensitizer in animal studies (23–26). Thus, substantial evidence for reversible formation of thiocarbamate linkages between isocyanates and GSH, suggest this process might contribute to TDI’s toxicity and/or allergenicity.…”