Regio/stereochem defined, diol epoxide adducted ras

描述：本提案是一项请求， 多环芳烃化学致癌领域研究者计划 芳烃（PAH）。多环芳烃，由于无处不在的环境 污染物，对人类构成癌症威胁。这个阶级的许多成员经历了 代谢活化为四种反应性二醇环氧化物。在DNA存在的情况下， 这些代谢物大部分与嘌呤残基结合形成共价损伤。 第一步是形成共价的二烯烃环氧-DNA加合物 在生物级联反应中最终导致突变， 肿瘤发生虽然烃类代谢产物的结构特征 与高致癌活性相关的化合物， 最近的研究集中在回答DNA加合物的问题上 结构和生物学随后的DNA结合这些代谢物。的 任何烃的四种代谢产生的异构二醇环氧化物，只有一种 异构体显示出高致瘤活性。然而，所有四种二醇环氧化物 烷基化DNA，产生总共16个加合物。因此一个核心问题 对化学致癌作用的全面理解在于确定 异构体DNA加合物影响局部DNA结构和 修复.为了解决这个问题，有必要制定一个 一套用于物理、生化和生物实验的探针。迄今 任何PAH的所有16种加合物都不能用于全面的比较， 问题研究PI提出了合理的，高度非对映选择性合成所有 两种结构不同的多环芳烃苯并[a]芘和 苯并[c]菲，在统一的，生物学上重要的DNA序列内 contexts. PAH结构的差异提供了一个测试的一般性， 合成，而序列背景的一致性提供了比较 以及结构和生物学效应的概括。本研究的 PI选择了人ras序列（A为N-ras原癌基因密码子60 - 62）， G蛋白在第61位密码子和c-Ki-ras原癌基因第11 - 13位密码子的修饰 密码子12处的修饰），两者都富含嘌呤。本研究将 因此提供了16 N-ras和16 c-Ki-ras寡聚体，其具有两种立体异构体 两种PAH二醇环氧化物。就其本身而言，这是一个不平凡的，以前 完成任务。然后PI将进行热评估 PAH修饰的双链体的变性、UV和CD性质与正常 靶和在病变对面具有错配碱基的靶。后者是 这是重要的，因为据信与加合物相对的错误掺入可能 对诱变事件至关重要由于结构信息可以从 根据核磁共振研究，合成的目的是生产足够的材料， 为此PI还将与其他团体和主题合作， 所涉及的是（a）NMR分析，（B）可能的X射线结构，（c）突变 分析，（d）DNA修复，和（e）荧光研究。

DESCRIPTION: This proposal is a request for support for a beginning investigator's program in the area of chemical carcinogenesis by polycyclic aromatic hydrocarbons (PAHs). PAHs, by virtue of being ubiquitous environmental pollutant, pose a cancer threat to humans. Many members of this class undergo metabolic activation to four reactive diol epoxides. In the presence of DNA, these metabolites bind largely to purine residues forming covalent lesions. Formation of covalent diolepoxide-DNA adducts is implicated as the first step in the biological cascade that ultimately leads to mutagenesis and tumorigenesis. Although structural features o f the hydrocarbon metabolites associated with high carcinogenic activity have been known for quite some time, recent studies have become focused on answering questions on DNA adduct structure and biology subsequent to DNA binding by these metabolites. Of the four metabolically produced isomeric diol epoxides of any hydrocarbon, only one isomer exhibits high tumorigenic activity. However, all four diol epoxides alkylate DNA producing a total of 16 adducts. Thus a question that is central to the overall understanding of chemical carcinogenesis lies in determining how isomeric DNA adducts affect local DNA structure and enzymatic replication of repair. In order to address this question, it then becomes necessary to have a set of probes for physical, biochemical and biological experimentation. To date all 16 adducts of any PAH are not available for comprehensive, comparative studies. The PI proposes rational, highly diastereoselective syntheses of all 16 epoxide adducts of two structurally different PAHs benzo[a]pyrene and benzo[c]phenanthrene, within uniform, biologically important DNA sequence contexts. The differences in PAH structure provide a test for the generality of the syntheses, while uniformity of sequence context provides for comparisons and generalizations of structural and biological effects. For this study, the PI has chosen the human ras sequences (N-ras protooncogene codons 60-62 for A modification at codon 61 and c-Ki-ras protooncogene codons 11-13 for G modification at codon 12), both of which are purine rich. This study will therefore provide 16 N-ras and 16c-Ki-ras oligomers with two stereoisomers of two PAH diol epoxides. In and of itself, this is a nontrivial, previously accomplished task. The PI will then undertake evaluation of thermal denaturation, UV and CD properties of the PAH modified duplexes with normal targets and targets having mismatched bases opposite the lesion. The latter is important because it is believed that misincorporation opposite an adduct could be critical to the mutagenic event. Since structural information can be derived from NMR studies, the syntheses are designed to produce sufficient materials for this. The PI will also collaborate with other groups and the topics to be addressed are (a) NMR analyses, (b) possibly x-ray structures, (c) mutation analyses, (d) DNA repair, and (e) fluorescence studies.