Covalent Adduction of Nitrogen Mustards to Model Protein Nucleophiles

Abstract

Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine.

Cross-Linking of Thioredoxin Reductase by the Sulfur Mustard Analogue Mechlorethamine (Methylbis(2-chloroethyl)amine) in Human Lung Epithelial Cells and Rat Lung: Selective Inhibition of Disulfide Reduction but Not Redox Cycling

“…23, 29, 42, 43 Our BIAM experiments showed that treatment of oxidized TrxR with HN2 blocks iodoacetamide binding at pH 8.5 in a concentration-dependent manner, confirming that noncatalytic cysteine residues are also targets for HN2 modification. This is supported by our LC-MS/MS analysis that showed that HN2 covalently binds to other cysteine residues in TrxR including cysteine 177, 189, 382, and 383.…”

“…The selection of non-thiol-containing amino acids was based on earlier studies showing that these amino acids were alkylated by HN2 and potential nucleophilic residues for modification by electrophiles. 29, 30 Modifications on selenium-containing peptides were searched manually by distinctive isotope patterns and confirmed by the critical b and y ions in the MS/MS. The MS/MS data from ETD fragmentation were also searched against the rat IPI database using the ZCore algorithm, with similar modification settings as the SEQUEST search.…”

Mass spectral studies of silyl derivatives of partially hydrolyzed products of nitrogen mustards: Important markers of nitrogen mustard exposure

“…present in biological systems. 9 Moreover, the protonated salts of NMs which are not scheduled in the Convention are highly stable and can easily be synthesized. Hence, NMs can easily be stockpiled, transported and finally can be deployed by simple deprotonation of their salts, indicating that NMs could readily be used as a weapon in terrorist activities.…”

Keratinous Materials as Novel Absorbent Systems for Toxic Pollutants

“…A study is reported on using model peptides containing cysteine, lysine and histidine residues to predict the interaction mechanisms of the nitrogen mustards (mechloroethamine and tris-2-chlorethylamine) to amino acid residues of proteins by means of liquid chromatographytandem mass spectrometry. It is hypothesized that mustard agents react with all model peptides via initial formation of a reactive aziridinium intermediate, followed by covalent adduction to amino acid residues, and form stable adducts 91 . Keratins in human skin can interact with some model organophosphates such as paraoxon, parathion, diazinon and diisopropylfluorophosphate, and form covalent adducts with some amino acids such as tyrosine and serine via nucleophilic addition 92 .…”