BIOSYNTHESIS OF ARA-A, 2'-DEOXYCOFORMYCIN

ARA-A、2-脱氧福霉素的生物合成

• 批准号: 3133259
• 负责人: DAVID C. BAKER
• 金额: $ 12.62万
• 依托单位: UNIVERSITY OF ALABAMA IN TUSCALOOSA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3133259
• 关键词: Streptomyces; adenosine deaminase; antiviral agents; diazepine; drug design /synthesis /production; drug screening /evaluation; genital herpes; hypoxanthines; nucleoside analog

The proposed research involves the biosynthesis of the four nucleoside analogs, 2'-deoxycoformycin (NSC-218321, 2'-dCF), 2'-chlorodeoxycoformycin (2'-C1dCF), coformycin and 9-Beta-D-arabinofuranosyladenine (NSC-404241, ara-A) which are elaborated by Streptomyces antibioticus. The research goals of this application are: (i) to study the biosynthesis of 2'-dCF; (ii) to determine the mechanism by which adenosine is converted to ara-A by the partially purified adenosine-2'-epimerase isolated from S. antibioticus, and (iii) to perform molecular cloning of the genomic DNA for adenosine-2'epimerase. Information will be gained with respect to the biosynthetic interrelationship of these antiviral/antitumor nucleoside analogs. Mutants which have additional growth requirements as compared to the original S. antibioticus strain will be selected. Once the mutants are obtained, it will be possible to elucidate the biosynthetic interrelationship of the the nucleoside antibiotics with respect to requirements for amino acids, carbohydrates or nucleosides. Experiments are described to determine how the "extra" carbon from C-1 of D-ribose is inserted between N-1 and C-6 of the purine ring to form the 1,3-diazepine ring, how the reduction at C-2' of the D-ribosyl moiety of the 2'-dCF occurs and how the chlorine is inserted at C-2' to form 2'-C1dCF. 1H, 2H, 13C NMR spectroscopy, as well as MS techniques, will be used to determine product composition and labeling. The application of molecular cloning to the Streptomyces will make it possible to isolate and analyze the DNA and to study the regulation of adenosine-2'-epimerase. The introduction of the epimerase DNA into S. lividans followed by expression of this enzyme will provide us with sufficient epimerase to complete enzyme kinetic studies in the conversion of adenosine to ara-A.

拟议的研究涉及四种核苷的生物合成 类似物，2'-脱氧考福霉素（NSC-218321，2'-dCF），2'-氯脱氧考福霉素 (2'-C1dCF)、辅福霉素和 9-Beta-D-阿拉伯呋喃糖腺嘌呤 (NSC-404241， ara-A)，由抗生素链霉菌 (Streptomyces Antibioticus) 精制而成。 研究 本申请的目标是： (i) 研究 2'-dCF 的生物合成； (ii) 确定腺苷转化为ara-A的机制 部分纯化的腺苷-2'-差向异构酶从 S. 抗生素，以及（iii）对基因组DNA进行分子克隆 腺苷-2'差向异构酶。 将获得有关以下方面的信息 这些抗病毒/抗肿瘤核苷的生物合成相互关系 类似物。 与其他突变体相比，具有额外生长要求的突变体 将选择原始的抗生素链球菌菌株。 一旦突变体 获得后，将有可能阐明生物合成 核苷类抗生素之间的相互关系 对氨基酸、碳水化合物或核苷的需求。 实验 描述了如何确定 D-核糖 C-1 中的“额外”碳 插入嘌呤环的N-1和C-6之间形成1,3-二氮杂卓 环，如何在 2'-dCF 的 D-核糖基部分的 C-2' 处还原 发生以及氯如何插入到 C-2' 处形成 2'-C1dCF。 1小时，2小时， 13C NMR 光谱以及 MS 技术将用于确定 产品成分和标签。 分子克隆技术的应用 链霉菌将使分离和分析 DNA 成为可能 研究腺苷-2'-差向异构酶的调节。 的介绍 将差向异构酶 DNA 导入变青链球菌，然后表达该酶 为我们提供足够的差向异构酶来完成酶动力学研究 腺苷转化为ara-A。