Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

Synthesis of (3'-5'),(2'-5')-linked di- and tri-adenylyl methylphosphonate analogs.

Stereoselectivity was found during the coupling reaction, to form 2',5'- and 3',5'-linked di- and triadenylyl methylphosphonate. The configuration of phosphorus was determined by 1HNMR NOE.     

Interaction of 3'-fluoro-3'-deoxyanalogs of a trimer of (2'-5')oligoadenylic acid with (2'-5')A3-antibodies: stereochemical aspect

Mouse antibodies to (2'-5')oligoadenylates were obtained by the immunization of animals with the (2'-5')oligoadenylic acid trimer conjugated with bovine serum albumin through a 2',3'-levulinic acid residue. Using radioimmunoassay, the reactivity of mouse polyclonal antibodies to the (2'-5')oligoadenylic acid trimer was studied for the trimer analogues containing 9-(3-deoxy-3-fluro-beta-D- xylofuranosyl)adenine and 3'-deoxy-3'-fluoro-adenosine in various positions of the chain. It was found that (a) the three-dimensional structure of short oligonucleotides is an important factor in the antibody recognition; (b) antibodies are more sensitive to modifications of the 5'-terminal and central ribose fragments of the (2'-5')oligoadenylic acid trimer; (c) the 3'-hydroxyl group plays a secondary role in the formation of the antigen determinant.     

Lipid derivatives of (2'-5')oligo A

The synthesis of adenylyl-(2'-5')-adenylyl-(2'-5')-2', 3'-O-(1-methoxyhexadecylidene)-adenosine (III) and its 5'-phosphorylated analogue (V) is described. Phosphorylation was achieved by (2-cyanoethyl)-phosphodichloridite agent followed by iodine oxidation.     

The human 2'-5'oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2'-5' instead of 3'-5' phosphodiester bond formation

The demonstration by Kerr and colleagues that double-stranded (ds) RNA inhibits drastically protein synthesis in cell-free systems prepared from interferon-treated cells, suggested the existence of an interferon-induced enzyme, which is dependent on dsRNA. Consequently, two distinct dsRNA-dependent enzymes were discovered: a serine/threonine protein kinase that nowadays is referred to as PKR and a 2'-5'oligoadenylate synthetase (2'-5'OAS) that polymerizes ATP to 2'-5'-linked oligomers of adenosine with the general formula pppA(2'p5'A)(n), n>or=1. The product is pppG2'p5'G when GTP is used as a substrate. Three distinct forms of 2'-5'OAS exist in human cells, small, medium, and large, which contain one, two, and three OAS units, respectively, and are encoded by distinct genes clustered on the 2'-5'OAS locus on human chromosome 12. OASL is an OAS like IFN-induced protein encoded by a gene located about 8 Mb telomeric from the 2'-5'OAS locus. OASL is composed of one OAS unit fused at its C-terminus with two ubiquitin-like repeats. The human OASL is devoid of the typical 2'-5'OAS catalytic activity. In addition to these structural differences between the various OAS proteins, the three forms of 2'-5'OAS are characterized by different subcellular locations and enzymatic parameters. These findings illustrate the apparent structural and functional complexity of the human 2'-5'OAS family, and suggest that these proteins may have distinct roles in the cell.     

Synthesis and template-directed polymerization of adenylyl(3'-5')adenosine cyclic 2', 3'-phosphate.

Adenylyl(3'-5')adenosine cyclic 2',3'-phosphate (A-A greater than p) was synthesized and its polymerization was attempted under various conditions inthe presence of poly(uridylic acid) and1,3-propanediamine. Reaction at -20 degrees C for 16 days gave polymerized products (up to the 8-mer) in 15% yield and was proved to be dependent on the template. Reaction at 0 degrees C for 16 days gave more extensive (up to the 10-mer) and more efficient (35%) polymerization. The newly formed phosphodiester linkage was exclusively 2'-5'. These results are discussed in comparison with the monomer-condensation reaction.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Stability of (2'-5')oligoriboadenylates in various sera

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.     

Inhibition of ribosomal peptidyltransferase with cytidyl-3' leads to 5'-[2'(3')-O-L-phenylalanyl]-L-adenosine.

The title compound 1e, obtained by chemical synthesis, is an inhibitor of E. coli ribosomal peptidyltransferase. A 50% inhibition of peptidyltransferase-catalyzed N-Ac-Phe-puromycin formation at puromycin concentration 1 × 10^?4 M with 70 S ribosome-poly U-N-Ac[¹⁴C-Phe-tRNA complex occurred at 5 × 10^?4 M of 1e. In contrast, the parent compound 2′(3′)-O-L-phenylalanine-L-adenosine (1b) is a much weaker inhibitor causing only 5% inhibition at 1 × 10^?3 M. Alkaline hydrolysis of compound 1e to cytidylyl-3′→5′-L-adenosine (1c) results in a greatly diminished inhibition which, however, exceeds that of 1b by a factor of two. The inhibition of peptidyltransferase with 1e can be reversed by puromycin. The latter effect levels off at 40% inhibition.     

The 2'-5' adenylate (2-5A) system in erythropoiesis

The trimer and tetramer core forms of 2'-5' Adenylate (2-5A) were tested in vitro for their effect on mouse liver CFU-E. Both forms of 2-5A core partially inhibited the CFU-E response to erythropoietin when given as a single dose at time 0 hours. Approximately 25% fewer colonies were seen after 2-days growth following exposure of the CFU-E to 0.1 mM 2-5A core. A 0.01 mM concentration of exogenous 2-5A core was not inhibitory. Endogenous 2-5A synthetase activity was assayed in spleen cell lysates from mice made anemic by several injections of phenylhydrazine. This method of treatment stimulated erythropoiesis, and this was directly correlated with an increase in the rate of enzyme activity measured by lysate conversion of 14C-ATP to 2-5A (our in press data suggests that the same may occur with hypoxic stimulation). This suggested to us that endogenous 2-5A synthetase and its 2-5A, a known inhibitor of DNA synthesis and protein synthesis, may help to regulate some of the late events in erythropoiesis.     

Studies towards the large scale chemical synthesis of the precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3',4',5',5'-2H4 and -2',3',4',5',5'-2H5 from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-alpha,beta-D-allofuranose-3,4-d2 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 16 (precursor of ribonucleosides-3',4',5',5'-2H4) or to 1-O-methyl-3,5-di-O-benzyl-alpha,beta-D-ribofuranose-3,4,5,5'-d4 18 (precursor of ribonucleosides-3',4',5',5'-2H4).