Ding Hui, Majumdar Ananya, Tolman Joel R, Greenberg Marc M
Department of Chemistry and Biomolecular NMR Center, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
J Am Chem Soc. 2008 Dec 31;130(52):17981-7. doi: 10.1021/ja807845n.
Recently, a phenylselenyl-modified thymidine (2) was shown to produce DNA interstrand cross-links (ICLs) via two mechanisms. Photolysis of 2 generates 5-(2'-deoxyuridinyl)methyl radical (1), the reactive intermediate that results from formal hydrogen atom abstraction from the thymine methyl group. This reactive intermediate reacts with the opposing dA and is the first example of a DNA radical that produces ICLs. Kinetic competition studies support the proposal that the rate-limiting step in ICL formation from 1 involves rotation about the glycosidic bond and that the rate constant for this process is influenced by the flanking sequence. Cross-links also form with the opposing dA when 2 is treated with mild oxidants that result in the formation of an intermediate methide-like species (4). Kinetic experiments reveal that 4 reacts with azide, a model nucleophile, via an S(N)2' pathway. Previous experiments suggested that the same product is produced via 1 or 4 but that the initially formed cross-link rearranges during the enzyme digestion and isolation procedures. In situ product analysis by NMR using synthetic, doubly labeled duplex DNA containing (13)C-2 and (15)N(1)-dA provides definitive evidence that the kinetic ICL products formed via the radical and oxidative pathways are the same and correspond to that arising from formal alkylation of N(1)-dA. Furthermore, analysis of the thermodynamic product formed upon rearrangement indicates that the primary product isomerizes via an associative mechanism in DNA.
最近，一种苯基硒基修饰的胸腺嘧啶核苷（2）被证明可通过两种机制产生DNA链间交联（ICL）。2的光解产生5-（2'-脱氧尿苷基）甲基自由基（1），这是一种反应性中间体，由胸腺嘧啶甲基的形式氢原子抽象产生。这种反应性中间体与相对的dA反应，是产生ICL的DNA自由基的第一个例子。动力学竞争研究支持了这样的提议，即从1形成ICL的限速步骤涉及围绕糖苷键的旋转，并且该过程的速率常数受侧翼序列的影响。当2用导致形成中间体类似甲基化物的物种（4）的温和氧化剂处理时，也会与相对的dA形成交联。动力学实验表明，4通过S(N)2'途径与亲核试剂模型叠氮化物反应。先前的实验表明，通过1或4产生相同的产物，但最初形成的交联在酶消化和分离过程中会重新排列。使用含有(13)C-2和(15)N(1)-dA的合成双标记双链DNA通过NMR进行原位产物分析提供了确凿的证据，即通过自由基和氧化途径形成的动力学ICL产物是相同的，并且对应于由N(1)-dA的形式烷基化产生的产物。此外，对重排后形成的热力学产物的分析表明，主要产物在DNA中通过缔合机制异构化。
