Synthesis and Characterization of Styrene Oxide Adducts with Cysteine, Histidine, and Lysine in Human Globin

• Published in: Chemical research in toxicology 2007-10, Vol.20 (10), p.1442-1452
• Main Authors: Jágr, Michal, Mráz, Jaroslav, Linhart, Igor, Stránský, Vladimír, Pospíšil, Martin
• Format: Article
• Language: English
• Subjects: Biomarkers; Chromatography, High Pressure Liquid; Cysteine - chemistry; Environmental Monitoring; Environmental Pollutants - chemistry; Environmental Pollutants - metabolism; Epoxy Compounds - chemical synthesis; Epoxy Compounds - chemistry; Epoxy Compounds - metabolism; Gas Chromatography-Mass Spectrometry; Globins - chemical synthesis; Globins - chemistry; Globins - metabolism; Histidine - chemistry; Humans; Lysine - chemistry; Magnetic Resonance Spectroscopy; Stereoisomerism
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx700057t

Styrene 7,8-oxide (SO), a reactive metabolic intermediate of the industrial chemical styrene, binds covalently at nucleophilic amino acid residues of blood proteins in vivo and in vitro. In this study, SO adducts with cysteine, lysine, and histidine were synthesized, characterized, and then used as authentic standards to assign and quantitate the SO adducts in globin incubated with SO. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine were prepared by direct alkylation of cysteine with (R)-SO or (S)-SO. To prepare the SO adducts with lysine and histidine, Nα-Boc-protected amino acids were alkylated with (R)-SO or (S)-SO followed by deprotection of the Boc group to obtain Nε-(2-hydroxy-1-phenylethyl)lysine and Nε-(2-hydroxy-2-phenylethyl)lysine as well as Nπ-(2-hydroxy-1-phenylethyl)histidine, Nπ-(2-hydroxy-2-phenylethyl)histidine, Nτ-(2-hydroxy-1-phenylethyl)histidine, and Nτ-(2-hydroxy-2-phenylethyl)histidine. The individual regioisomers were isolated from their mixtures by semipreparative HPLC, and their structure was assigned using NMR techniques. The SO-modified globin, isolated from human hemoglobin incubated in vitro with racemic SO at a molar ratio SO/globin of 100:1 or 10:1, was digested with pronase and subjected to LC/MS and GC/MS analysis. All known regioisomers of the SO adducts were detected, with S-(2-hydroxy-1-phenylethyl)cysteine, Nε-(2-hydroxy-1-phenylethyl)lysine, and Nτ-(2-hydroxy-2-phenylethyl)histidine being the most abundant in the modified globin. Deuterated analogues of the SO adducts were employed as internal standards. The SO–amino acid adducts described here appear to be suitable biomarkers for long-term exposures to styrene or SO.