BIOSYNTHESIS OF ARA-A, 2'-DEOXYCOFORMYCIN

拟议的研究涉及四核苷的生物合成

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.