Moll T S, Elfarra A A
Department of Comparative Biosciences and Center for Environmental Toxicology, University of Wisconsin, Madison, Wisconsin 53706-1102, USA.
Chem Res Toxicol. 1999 Aug;12(8):679-89. doi: 10.1021/tx990043x.
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.
职业性接触1,3 - 丁二烯（BD）是通过测量血红蛋白N - 末端缬氨酸与主要反应性代谢产物丁二烯 monoxide（BMO）形成加合物的水平来进行监测的。然而，关于BMO与血红蛋白N - 末端缬氨酸的相对反应性、区域选择性和立体特异性的机理细节尚不清楚。在本文所呈现的研究中，L - 缬氨酰胺被用作血红蛋白N - 末端缬氨酸的模型，以比较在水溶液和蛋白质微环境中反应的亲核反应性、区域选择性和立体选择性。通过L - 缬氨酰胺与外消旋BMO反应生成的四种产物（由环氧化物部分的C - 1和C - 2处反应产生的两对非对映异构体）被合成、纯化，并通过¹H NMR和GC/MS进行表征。这四种反应产物被用作在生理pH（7.4）和温度（37℃）下缬氨酰胺与BMO反应动力学研究的分析标准品。结果表明，与在C - 1处反应形成的加合物相比，在C - 2处反应形成的加合物的生成比例约为2:1。每个相应区域异构体的立体异构体以相似的生成速率产生。还使用来自Sprague - Dawley大鼠和B6C3F1小鼠的完整红细胞在体外研究了BMO与血红蛋白N - 末端缬氨酸的反应。在使用五氟苯基异硫氰酸酯（PFPITC）通过N - 烷基埃德曼降解程序裂解N - 修饰的缬氨酸后，开发了一种新方法，通过对PFPTH - 缬氨酸 - BMO衍生物进行硅烷化，实现了GC/MS对四种预期产物的检测和定量。血红蛋白的结果与缬氨酰胺的结果形成对比，因为BMO与大鼠和小鼠血红蛋白中的N - 末端缬氨酸残基反应主要生成C - 1加合物。用大鼠血红蛋白获得的速率比用小鼠血红蛋白或缬氨酰胺获得的速率慢得多。这些结果，以及与小鼠血红蛋白反应相比，与大鼠血红蛋白反应产生更高比例的C1:C2加合物这一发现，表明在比较模型化合物之间以及不同物种之间加合物形成的相对速率时，测量所有四种加合物的重要性。
