BIOSYNTHESIS OF ARA-A, 2'-DEOXYCOFORMYCIN

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)、考福霉素和9-β-D-阿拉伯糖基腺嘌呤(NSC-404241， Ara-A)，由抗菌链霉菌阐述。这项研究 本应用的目的是：(I)研究2‘-DCF的生物合成； (2)确定腺苷转化为Ara-A的机制 分离得到部分纯化的腺苷-2‘-差向异构酶。 以及(Iii)进行基因组DNA的分子克隆，以便 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 类比。对生长有额外要求的突变株 最初的抗生素菌株将被选中。一旦变种人 一旦获得，就有可能阐明生物合成 核苷类抗生素与抗癌药物的相互关系 对氨基酸、碳水化合物或核苷的要求。实验 用来确定D-核糖C-1中的“额外”碳是如何 插入在嘌呤环的N-1和C-6之间以形成1，3-二氮杂 环，2‘-DCF的D-核糖部分如何在C-2’还原 以及氯如何插入C-2‘形成2’-C1dCF。1H、2H、 将使用13C核磁共振波谱以及MS技术来确定 产品组成和标签。分子克隆技术在生物医学研究中的应用 链霉菌将使分离和分析DNA和 研究腺苷-2‘-差向异构酶的调节作用。引入了 变铅青链霉菌DNA异构体酶的表达 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。

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.