Rink Stacia M, Warner Don L, Klapars Artis, Vedejs Edwin
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Biochemistry. 2005 Oct 25;44(42):13981-6. doi: 10.1021/bi050426w.
The aziridinomitosene derivative (1S,2S)-6-desmethyl(methylaziridino)mitosene (4) was shown to alkylate plasmid DNA at pH 7.4 in the absence of a reducing agent [Vedejs, E., Naidu, B. N., Klapars, A., Warner, D. L., Li, V. -s., Na, Y., and Kohn, H. (2003) J. Am. Chem. Soc. 125, 15796-15806], an activity not found in the parent mitomycins. We sought to evaluate aziridinomitosene 4 for the presence of DNA interstrand cross-linking activity using nonreductive reaction conditions. Radiolabeled DNA treated with 4 was analyzed by denaturing polyacrylamide gel electrophoresis (DPAGE), a technique that readily separates the less mobile cross-linked ds DNA from the more mobile ss DNA products. Nonreduced 4 produced an interstrand cross-link (ICL) in duplex DNA containing 5'-d(CG) sites, and the yield of ICL was comparable to that obtained from reduced MC under similar conditions. A ds DNA having the central tetranucleotide 5'-d(ACGT) provided the greatest ICL yield from both nonreduced 4 and reduced MC. Substitution of 5'-d(CG) with the inverted sequence 5'-d(GC) completely abolished interstrand cross-linking by 4, revealing 5'-d(CG) as its specific site of ICL formation. Replacement of dG at 5'-d(CG) with 2'-deoxyinosine (dI), which lacks the exocyclic C2 amino group present in dG, also prevented DNA ICL formation by 4, revealing an essential role for the dG C2 amino group in the interstrand cross-linking reaction between 4 and duplex DNA. This report directly demonstrates the presence of bifunctional alkylating activity in a nonreduced aziridinomitosene and clearly shows that unreduced 4 alkylates residues in the minor groove of ds DNA, cross-linking with the same 5'-d(CG) sequence specificity displayed by reduced MC.
氮丙啶丝裂霉素衍生物（1S，2S）-6-去甲基（甲基氮丙啶基）丝裂霉素（4）在pH 7.4且无还原剂的条件下可使质粒DNA烷基化[维德伊斯，E.，奈杜，B. N.，克拉帕尔斯，A.，华纳，D. L.，李，V. -s.，纳，Y.，以及科恩，H.（2003年）《美国化学会志》125，15796 - 15806]，这是其母体丝裂霉素所没有的活性。我们试图在非还原反应条件下评估氮丙啶丝裂霉素4是否具有DNA链间交联活性。用4处理过的放射性标记DNA通过变性聚丙烯酰胺凝胶电泳（DPAGE）进行分析，该技术能轻易地将迁移性较差的交联双链DNA与迁移性较好的单链DNA产物分开。未还原的4在含有5'-d（CG）位点的双链DNA中产生了链间交联（ICL），且ICL的产率与在类似条件下还原型丝裂霉素C（MC）得到的产率相当。具有中央四核苷酸5'-d（ACGT）的双链DNA从未还原的4和还原型MC中都获得了最高的ICL产率。用反向序列5'-d（GC）取代5'-d（CG）完全消除了4引起的链间交联，表明5'-d（CG）是其ICL形成的特定位点。用缺乏dG中存在的环外C2氨基的2'-脱氧肌苷（dI）取代5'-d（CG）中的dG，也阻止了4引起的DNA ICL形成，揭示了dG的C2氨基在4与双链DNA之间的链间交联反应中的重要作用。本报告直接证明了未还原的氮丙啶丝裂霉素中存在双功能烷基化活性，并清楚地表明未还原的4使双链DNA小沟中的残基烷基化，与还原型MC显示出相同的5'-d（CG）序列特异性交联。
