Synthetic Approaches to Carcinogen-Linked Oligonucleotides

许多烷基化试剂与DNA反应，得到主要的N7-脱氧鸟苷(DG)加合物。主要作用于N7-DG的药物的遗传毒性和细胞毒性通常归因于其他不太丰富的DNA损伤。PPG应用的主要假设是阳离子N7-DG加合物在细胞内转化为N5取代的甲酰胺嘧啶(Fapy-DG)损伤，后者对烷化剂的遗传毒性和细胞毒性有显著影响。该计划项目交互地使用有机合成、生物分析化学、结构生物学、酶学和分子生物学来阐明来自内源和外源亲电体和化疗药物的Fapy-DG加合物改变DNA复制和修复的分子细节。项目1将利用生物分析质谱学、化学合成和酶学来解决这一假设。努力将重点放在甲酰胺嘧啶(Fapy-DG)，如替莫唑胺(MeFapy-DG)、环氧氯丙烷(OxEt-Fapy-DG)、硫代替巴(AE-Fapy-DG)和氮化物(NM-Fapy-DG)等甲基化试剂造成的损伤，包括链间交联链(FapyG-NM-FapyG和FapyG-NMG)。替莫唑胺、硫代替巴和氮芥是临床上常用的化疗药物，我们推测相应的N5取代的Fapy-DG损伤在它们的作用机制中发挥了重要作用。 项目1的具体目标将确定哺乳动物细胞中存在N5取代的Fapy-DG损伤(特定目标1)，以位置特异的方式化学合成包含所需Fapy-DG损伤的寡核苷酸(特定目标2)，并确定Fapy-DG损伤在体外的错误编码潜力(特定目标3)。我们将Fapy-DG损伤定点整合到寡核苷酸中的能力是 是项目1、2和3研究计划的核心。项目1将与DNA合成资源核心密切合作。

Many alkylating agents react with DNA to afford predominanfiy N7-adducts of deoxyguanosine (dG). The genotoxicity and cytotoxicity of agents that react primarily at N7-dG is often attributed to other less abundant DNA lesions. The overarching hypothesis of this PPG application is the cationic N7-dG adducts are converted into N5-substituted formamidopyrimidine (Fapy-dG) lesions in cells, and the latter lesions contribute significantly to the genotoxicity and cytotoxicity of alkylating agents. This Program Project interactively uses organic synthesis, bioanalytical chemistry, structural biology, enzymology, and molecular biology to elucidate the molecular details by which Fapy-dG adducts derived from endogenous and exogenous electrophiles and chemotherapeutic agents alter DNA replication and repair. Project 1 will utilize bioanalytical mass spectrometry, chemical synthesis, and enzymology to address this hypothesis. Efforts will focus on formamidopyrimidine (Fapy-dG) lesions derived from methylating agents such as temozolomide (MeFapy-dG), chlorooxirane (OxEt-Fapy-dG), thioTEPA (AE-Fapy-dG) and nitrogen mustards (NM-Fapy-dG), including interstrand cross-links (FapyG-NM-FapyG, and FapyG-NMG). Temozolomide, thioTEPA, and nitrogen mustards are clinically used chemotherapeutic agents and we hypothesize that the corresponding N5-substituted Fapy-dG lesion plays an important role in their mechanism of action. The Specific Aims of Project 1 will establish the presence of N5-substituted Fapy-dG lesions in mammalian cells (Specific Aim 1), chemically synthesized oligonucleotides containing the desired Fapy-dG lesion in a site-specifically manner (Specific Aim 2), and determine the miscoding potential of the Fapy-dG lesion in vitro (Specific Aim 3). Our ability to site-specifically incorporate the Fapy-dG lesions into oligonucleotide is central to the research plans of Projects 1, 2 and 3. Project 1 will work closely with the DNA Synthesis Resource Core.