Kinetics and Mechanistic Studies of the Hydrolysis of Diisocyanate-Derived Bis-thiocarbamates of Cysteine Methyl Ester

Abstract

Diisocyanates (dNCOs) are the most commonly reported cause of chemically induced occupational asthma, but the ultimate antigenic form is unknown. Reactions of the three most common monomeric dNCOs, hexamethylene dNCO (HDI), methylene diphenylisocyanate (MDI), and toluene dNCO (TDI), with cysteine methyl ester (CME) gave the corresponding bis-dithiocarbamates (HDI-CME, TDI-CME, and MDI-CME). The dissociation kinetics of these bis-thiocarbamates, in aqueous conditions, was followed spectrophotometrically under varying pH and temperature conditions. Reaction of the adducts with methylamine or human serum albumin (HSA) produced diurea, monourea, and diamine products, and this was consistent with the base-catalyzed elimination reaction (E1cB) pathway being the dominant, but not exclusive, dissociation mechanism. The hydrolysis of the adducts was first-order with respect to OH(-) concentration and overall second-order (HDI-CME, k = 3.36 x 10(2) M(-)(1) min(-)(1); TDI-CME, k = 2.49 x 10(4) M(-)(1) min(-)(1); and MDI-CME, k = 5.78 x 10(4) M(-)(1) min(-)(1) at pH 7.4) with deviation from second-order when the dNCO had an aromatic functional group. Arrhenius plots gave activation energies (HDI-CME, E(a) = 70.6 kJ/mol; TDI-CME, E(a) = 46.1 kJ/mol; and MDI-CME, E(a) = 44.5 kJ/mol) that were consistent with the following order of stability: HDI-CME > TDI-CME > MDI-CME. Therefore, the stability of different dNCO-derived thiocarbamates in aqueous environments can vary greatly. Thiocarbamate dissociation rates and type of products formed may potentially influence antigenicity and subsequent hypersensitivity/toxic reactions following dNCO exposures.

Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate

“…This may be related to differences in exposure route, the stability of metabolic intermediates, or the differences in the chemical nature of the protein-HDA bonds between aromatic and aliphatic amines, which could influence the yields of various protein adducts (albumin vs. Hb). The bis-dithiocarbamate intermediate of HDI, which can be easily transported into RBCs, is more stable compared to toluene diisocyanate or MDI (Chipinda et al 2006). Therefore, the stability of this metabolic intermediate would allow greater opportunity for cellular uptake and subsequent Hb adduct formation following further transformation.…”

“…Individual differences in acetylator genotype (fast vs. slow) that drive these dominant enzymatic-directed pathways have been linked with asthma risk (Wikman et al 2002). The non-enzymatic formation of protein adducts may occur via hydrolysis of bis-dithiocarbamate intermediates resulting from the reaction of NCO with thiols, such as cysteine (Chipinda et al 2006). In a human case study, HDI protein adducts were detected in lung tissue following exposure to HDI, demonstrating that blood proteins are suitable markers for exposure and target-dose estimation (Redlich et al 1997).…”

“…The mechanism by which reactive diisocyanates are transported across the epithelial layer of the respiratory tract, into the blood, and through the erythrocyte membrane to react with Hb is unknown, but may involve the formation of mono- and bis-thiocarbamate intermediates (Chipinda et al 2006). Such products formed by the reaction of diisocyanate with cysteine would allow transport across cellular membranes.…”

“…Such products formed by the reaction of diisocyanate with cysteine would allow transport across cellular membranes. It has been postulated that the bis-thiocarbamate product could be carried from the lung to a distal site, whereupon hydrolysis would occur, releasing a free diisocyanate that could react with another nucleophile (Chipinda et al 2006). Because the rates of hydrolysis among the different diisocyanate intermediates vary widely, the biological availability of reactive intermediates may also vary.…”

Structural elucidation of isocyanate-peptide adducts using tandem mass spectrometry

“…A comparison of the relative reactivity of the peptides with isocyanates is difficult to make as the pH of non-buffered aqueous solutions will vary with the chemical nature of the peptide dissolved. Although current investigations into the effect of pH on isocyanate-peptide binding are underway in our laboratory, previous studies have dem- onstrated that buffers, such as phosphate and carbonate, act as competing nucleophiles in this system [23].…”