Selected Chemistry of Biologically-Active Thiols : N-acetylpenicillamine and 2-mercaptobenzothiazole in Nitrosothiol Formation and Role in allergic contact dermatitis Respectively

Two biologically-active thiols, N-acetylpenicillamine (NAP) and 2-mercaptobenzothiazole (MBT), were studied in this thesis. NAP is known to combine with nitric oxide (NO) to produce S-nitroso-N-acetylpenicillamine (SNAP) and MBT is a known allergen. The formation, reaction dynamics, and detailed kinetics and mechanism of the reaction between nitrous acid (HNO2), prepared in situ, and NAP to produce SNAP were studied. The reaction is first order in nitrite, NAP and acid in pH conditions at or slightly higher than the pKa of HNO2. Both HPLC and quadrupole time-of-flight mass spectrometry techniques confirmed the formation of SNAP and the absence of any other products. Cu(I) ions were found to be effective SNAP-decomposition catalysts. The formation of SNAP occurs through two distinct pathways. One involves the direct reaction of NAP and HNO2 to form SNAP and eliminate water, and the second pathway involved the initial formation of the nitrosyl cation, NO+, which then nitrosates the thiol. The bimolecular rate constant for the reaction of NAP and HNO2 was derived as 2.69 M-1 s-1, while that of direct nitrosation by the nitrosyl cation was 3.00 x 104 M-1 s -1. A simple reaction network made up of four reactions was found to be sufficient in simulating the formation kinetics and acid-induced decomposition of SNAP. The chemical mechanism leading to MBT\u27s allergenicity is unknown. It was hypothesized that the thiol group is critical to MBT\u27s covalent binding/haptenation to nucleophilic protein residues. Hypochlorous acid oxidized MBT to the disulfide, 2, 2\u27-dithiobis(benzothiazole) (MBTS), within the glove matrix. Cysteine reduced MBTS to MBT with subsequent formation of the mixed disulfide 2-amino-3-(benzothiazol-2-yl disulfanyl)-propionic acid. Simultaneous reduction of MBTS and disulfide formation with Cys34 on bovine serum albumin was observed, suggesting a potential route of protein haptenation through covalent bonding between cysteinyl residues on proteins and the MBT/MBTS thiol moiety. Guinea pigs were sensitized to MBT using a modified guinea pig maximization assay (GPMT) and cross-reactivity towards MBTS and the free thiol-lacking or blocked compounds benzothiazole (BT), 2-hydroxybenzothiazole (HBT) and 2-(methylthio)benzothiazole (MTBT) assessed. MBT and MBTS, but not BT, HBT or MTBT elicited allergic contact dermatitis (ACD) in MBT-sensitized animals

Characterization and Comparative Analysis of 2,4-Toluene Diisocyanate and 1,6-Hexamethylene Diisocyanate Haptenated Human Serum Albumin and Hemoglobin

Diisocyanates (dNCOs) are lowmolecularweight chemical sensitizers that reactwith autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCOspecific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may bemodified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb–TDI antigenicity was approximately 30% that of HSA–TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity