John Jay College of Criminal Justice , 524 West 59th Street , New York , New York 10019 , United States.
Departamento de Química Orgánica, Facultade de Química , Universidade de Santiago de Compostela , 15782 Santiago de Compostela , Spain.
Chem Res Toxicol. 2018 Aug 20;31(8):762-771. doi: 10.1021/acs.chemrestox.8b00102. Epub 2018 Aug 2.
Mitomycin C (MC) is an anticancer agent that alkylates DNA to form monoadducts and interstrand cross-links. Decarbamoylmitomycin C (DMC) is an analogue of MC lacking the carbamate on C10. The major DNA adducts isolated from treatment of culture cells with MC and DMC are N-deoxyguanosine (dG) adducts and adopt an opposite stereochemical configuration at the dG-mitosene bond. To elucidate the molecular mechanisms of DMC-DNA alkylation, we have reacted short oligonucleotides, calf thymus, and M. luteus DNA with DMC using biomimetic conditions. These experiments revealed that DMC is able to form two stereoisomeric deoxyadenosine (dA) adducts with DNA under bifuntional reduction conditions and at low temperature. The dA-DMC adducts formed were detected and quantified by HPLC analysis after enzymatic digestion of the alkylated DNA substrates. Results revealed the following rules for DMC dA alkylation: (i) DMC dA adducts are formed at a 48- to 4-fold lower frequency than dG adducts, (ii) the 5'-phosphodiester linkage of the dA adducts is resistant to snake venom diesterase, (iii) end-chain dA residues are more reactive than internal ones in duplex DNA, and (iv) nucleophilic addition by dA occurs on both faces of DMC and the ratio of stereoisomeric dA adducts formed is dependent on the end bases located at the 3' or 5' position. A key finding was to discover that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by DMC: at 0 °C, both dA and dG alkylation occur, whereas at 37 °C, DMC preferentially alkylates dG residues.
丝裂霉素 C（MC）是一种抗癌药物，可使 DNA 烷化形成单加合物和链间交联。去甲酰丝裂霉素 C（DMC）是 MC 的类似物，缺少 C10 上的氨基甲酰基。从用 MC 和 DMC 处理培养细胞中分离出的主要 DNA 加合物是 N-脱氧鸟嘌呤（dG）加合物，并在 dG-丝裂霉素键处具有相反的立体化学构型。为了阐明 DMC-DNA 烷化的分子机制，我们使用仿生条件使短寡核苷酸、小牛胸腺和 M. luteus DNA 与 DMC 反应。这些实验表明，在双功能还原条件下和低温下，DMC 能够形成两种立体异构脱氧腺苷（dA）与 DNA 的加合物。通过对烷基化 DNA 底物进行酶消化，通过 HPLC 分析检测和定量检测形成的 dA-DMC 加合物。结果揭示了 DMC dA 烷化的以下规则：（i）DMC dA 加合物的形成频率比 dG 加合物低 48-4 倍，（ii）dA 加合物的 5'-磷酸二酯键对蛇毒二酯酶具有抗性，（iii）双链 DNA 中末端链的 dA 残基比内部的 dA 残基更具反应性，以及（iv）dA 的亲核加成发生在 DMC 的两面，形成的立体异构 dA 加合物的比例取决于位于 3'或 5'位置的末端碱基。一个关键的发现是发现温度在 DMC 对双链 DNA 的区域选择性烷化中起着决定性的作用：在 0°C 时，dA 和 dG 烷化都会发生，而在 37°C 时，DMC 优先烷基化 dG 残基。
