Synthetic Approaches to Carcinogen-Linked Oligonucleotides

许多烷化剂与DNA反应，主要提供脱氧鸟苷（dG）的N7-加合物。主要在N7-dG反应的药物的遗传毒性和细胞毒性通常归因于其他丰度较低的DNA损伤。该PPG应用的首要假设是阳离子N7-dG加合物在细胞中转化为N5-取代的甲酰胺基嘧啶（Fapy-dG）病变，后者病变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性和外源性亲电试剂和化疗剂的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，化学合成和酶学来解决这一假设。工作重点将放在甲酰胺基嘧啶（Fapy-dG）损伤，这些损伤来自甲基化剂，如替莫唑胺（MeFapy-dG）、氯环氧乙烷（OxEt-Fapy-dG）、硫代TEPA（AE-Fapy-dG）和氮芥（NM-Fapy-dG），包括链间交联（FapyG-NM-FapyG和FapyG-NMG）。替莫唑胺、硫代替派和氮芥是临床上使用的化疗药物，我们假设相应的N5-取代的Fapy-dG损伤在其作用机制中起重要作用。 项目1的特定目的将确定哺乳动物细胞中存在N5取代的Fapy-dG病变（特定目的1），以位点特异性方式化学合成含有所需Fapy-dG病变的寡核苷酸（特定目的2），并确定Fapy-dG病变在体外的错误编码潜力（特定目的3）。我们将Fapy-dG损伤位点特异性掺入寡核苷酸的能力是 项目1、2和3的研究计划的核心。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.