BIOSYNTHESIS OF ARA-A, 2'-DEOXYCOFORMYCIN

ARA-A、2-脱氧福霉素的生物合成

• 批准号: 3133260
• 负责人: DAVID C. BAKER
• 金额: $ 14.68万
• 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 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3133260
• 关键词: Streptomyces; adenosine deaminase; antileukemic agent; antineoplastics; 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.

这项研究涉及四种核苷的生物合成， 类似物，2 '-脱氧coformycin（NSC-218321，2'-dCF），2 '-氯脱氧coformycin （2 ′-C1 dCF）、coformycin和9-β-D-阿拉伯呋喃糖基腺嘌呤（NSC-404241， ara-A），其由链霉菌（Streptomyceslasticus）加工。 研究 本申请的目的是：（i）研究2 ′-dCF的生物合成; (ii)为了确定腺苷被转化为ara-A的机制， 从S. （iii）进行基因组DNA的分子克隆， 腺苷-2 '差向异构酶。 将获得关于 这些抗病毒/抗肿瘤核苷的生物合成相互关系 类似物 与具有额外生长要求的突变体相比， 原始的S.将选择嗜热链球菌菌株。 一旦变种人 获得，将有可能阐明生物合成 核苷类抗生素与 需要氨基酸、碳水化合物或核苷。 实验 来确定来自D-核糖的C-1的“额外”碳是如何 插入嘌呤环的N-1和C-6之间，形成1，3-二氮杂卓 环，如何还原在C-2'的D-核糖基部分的2'-dCF 以及氯如何插入C-2'形成2'-C1 dCF。 1H，2H， 13 C NMR光谱以及MS技术将用于确定 产品组成和标签。 分子克隆技术在 链霉菌可以分离和分析DNA， 研究腺苷-2 '-差向异构酶的调控。 的出台 差向异构酶DNA转化为S.在表达这种酶之后， 为我们提供足够的差向异构酶，以完成酶动力学研究， 腺苷转化为ara-A。