Characterization of the reactivity, regioselectivity, and stereoselectivity of the reactions of butadiene monoxide with valinamide and the N-terminalvaline of mouse and rat hemoglobin

Occupational exposure to 1,3-butadiene (BD) has been monitored by measuring the level of hemoglobin N-terminal valine adduct formation with the primar y reactive metabolite, butadiene monoxide (BMO). However, mechanistic detai ls concerning the relative reactivity, regioselectivity, and stereospecific ity of BMO with the N-terminal valine of hemoglobin are lacking. In the stu dies presented here, L-valinamide was used as a model for the N-terminal va line 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-va linamide with racemic BMO (two pairs of diastereomers produced by reactions at C-1 and C-2 of the epoxide moiety) were synthesized, purified, and char acterized by H-1 NMR and GC/MS. These four reaction products were used as a nalytical standards for kinetic studies of the reaction of valinamide with BMO at physiological pH (7.4) and temperature (37 degrees C). The results s how that the adducts formed by reaction at C-2 were formed at a ratio of ap proximately 2:1 compared to the adducts formed by reaction at C-1. The ster eoisomers of each respective regioisomer were produced with similar rates o f formation. The reaction of BMO with the N-terminal valine of hemoglobin w as 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 Ed man degradation procedure using pentafluorophenyl-isothiocyanate (PFPITC), a novel procedure was developed that allowed GC/MS detection and quantitati on of the four expected products by silylation of the PFPTH-valine-BMO deri vatives. The hemoglobin results contrast with the valinamide results in tha t the reaction of BMO with the N-terminal valine residue in both rat and mo use hemoglobin produced mostly C-1 adducts. The rates obtained with rat hem oglobin were much slower than the rates obtained with mouse hemoglobin or w ith valinamide. These results, and the finding that the reaction with rat h emoglobin produced a higher ratio of C1:C2 adducts in comparison with the r eaction with mouse hemoglobin, indicate the importance of measuring all fou r adducts when comparing the relative rates of adduct formation both with m odel compounds and among different species.