title: Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminal valine of mouse and rat hemoglobin.
abstract: Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primary reactive metabolite, butadiene monoxide (BMO). However, mechanistic details concerning the relative reactivity, regioselectivity, and stereospecificity of BMO with the N-terminal valine of hemoglobin are lacking. In the studies presented here, L-valinamide was used as a model for the N-terminal valine of hemoglobin to compare the nucleophilic reactivity, regioselectivity, and stereoselectivity of the reaction both in aqueous solution and within a protein microenvironment. Four products produced by the reaction of L-valinamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and characterized by (1)H NMR and GC/MS. These four reaction products were used as analytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results show that the adducts formed by reaction at C-2 were formed at a ratio of approximately 2:1 compared to the adducts formed by reaction at C-1. The stereoisomers of each respective regioisomer were produced with similar rates of formation. The reaction of BMO with the N-terminal valine of hemoglobin was also studied in vitro using intact erythrocytes from Sprague-Dawley rats and B6C3F1 mice. After cleavage of the N-modified valine by the N-alkyl Edman degradation procedure using pentafluorophenylisothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitation of the four expected products by silylation of the PFPTH-valine-BMO derivatives. The hemoglobin results contrast with the valinamide results in that the reaction of BMO with the N-terminal valine residue in both rat and mouse hemoglobin produced mostly C-1 adducts. The rates obtained with rat hemoglobin were much slower than the rates obtained with mouse hemoglobin or with valinamide. These results, and the finding that the reaction with rat hemoglobin produced a higher ratio of C1:C2 adducts in comparison with the reaction with mouse hemoglobin, indicate the importance of measuring all four adducts when comparing the relative rates of adduct formation both with model compounds and among different species.
authors.affiliation: Department of Comparative Biosciences and Center for Environmental Toxicology, University of Wisconsin, Madison, Wisconsin 53706-1102, USA.
authors.forename: T S
authors.initials: TS
authors.lastname: Moll
authors.forename: A A
authors.initials: AA
authors.lastname: Elfarra
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange; D004852:Epoxy Compounds / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D006454:Hemoglobins / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008815:Mice, Inbred Strains; D009153:Mutagens / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013237:Stereoisomerism; D014633:Valine / Q000737:chemistry*
chemical_list: D004852:Epoxy Compounds; D006454:Hemoglobins; D009153:Mutagens; C024370:3,4-epoxy-1-butene; D014633:Valine