Nucleic Acids Research

Specific synthesis of some oligoadenylates including A2'p5'A2'p5'Ap (2'), the 2'-phosphorylated oligoribonucleotide core of the recently discovered protein synthesis inhibitor pppA2'p5'A2'p5'A is described using a novel solid-phase method. The CD spectra of A2'p5'Ap(2'), A2'p5'A2'p5'Ap (2') and A2'p5'A2'p5'A (derived by treatment of the phosphorylated synthetic trimer with E.coli alkaline phosphatase) are presented. Comparison of the latter spectrum with that of A2'p5'A2'p5'A obtained similarly from a biologically derived sample of pppA2'p5'A2'p5'A provides further evidence that this molecule is in fact the first naturally-occurring 2'-5'-linked oligoribonucleotide. INTRODUCTI ON The unusual trinucleoside pentaphosphate, pppA2'p5'A2'p5'A inter alia is synthesised from ATP by a synthetase enzyme activated in interferontreated cells and rabbit reticulate lysates by double-stranded RNA . Cellfree and intact cell protein synthesis are inhibited by subnanomolar concentrations of the material and the dephosphorylated core is only 2 slightly less inhibitory in vivo . The activity appears to be mediated by a nuclease which degrades mRNA and there is evidence that a 2'-5' oligo3 adenylate synthetase activity is widely distributed in mammalian cells However, these potentially important anti-metabolites are quickly deactivated, particularly in cell-free systems 2. This rapid degradation may be accomplished by some kind of 3'-exonuclease, since the compounds are resistant to known ribonucleases. In this context it might be expected that phosphorylation at the terminal 2'-position would afford a degree of protection to these oligomers and perhaps increase the duration of their biological action. We now describe the chemical synthesis of A2'p5'A2'p5'Ap (2') by a novel solid phase procedure analogous to that already described for oligodeoxyribonucleotides ' . Synthesis of this compound and of the C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England 2569 Nucleic Acids Research more typical dinucleotide, A3p5lAp(31), provides a useful starting point for the development of a general solid phase method for oligoribonucleotides. It should be emphasised that the experiments reported here represent an initial feasibility study of the application of the polyamide solid-phase method to the oligoribonucleotide series; optimisation of the synthetic strategy and of reaction conditions has not yet been established. In addition we report the CD spectral properties of chemically synthesised 2'-51 linked oligoriboadenylates which provide further evidence that the proposed structure of biologically derived pppA2'p5'A2'p5'A is correct. RESULTS AND DISCUSSION Oliqoribonucleotide Synthesis Only twice has solid-phase oligoribonucleotide synthesis been attempted . The recent success of oligodeoxyribonucleotide synthesis on a polar poly(N,N-dimethylacrylamide) resin4' prompted us to investigate its use for oligoribonucleotide synthesis. We chose in these initial experiments to retain the main elements which had proved of value in the deoxy series viz: 1) a reversible linkage between polymer and growing oligonucleotide chain, stable during chain assembly but specifically cleaved at the end of the synthesis, 2) nucleotide addition via a phosphodiester approach, and 3) oligonucleotide isolation by high performance liquid chromatography (hplc). However chain extension from 5' to 31 by use of nucleoside-5'phosphates was impractical. We preferred to use the mure readily accessible protected monomers containing 2' or 3' phosphates, and to extend chains in the reverse direction. The required 5'-O-monomethoxytrityl-N, 2'-Odibenzoyl adenosine 3'-phosphate and 5'-O-monomethoxytrityl-N, 3'-0dibenzoyl adenosine 2'-phosphate were prepared by standard techniques Resin was functionalised by reaction of amino groups on the resin with the activated ester, C6C150CO. (CH2)2C6H5S(CH2)20H, as previously 4595 described'. (MeOTr)bzA(Bz)p(2') could now be coupled to the support (Scheme 1). Acidolysis of terminal 5'-monomethoxytrityl groups was effective using anhydrous acetic acid/methanol (4:1). Cleavage of oligonucleotides from the support required specific oxidation of the linkage to the corresponding sulphone followed byeA-elimination. It was expected that the elimination conditions developed for use with oligodeoxyribonucleotides (O.22M sodium hydroxide in aqueous dioxan, room temperature, 2 x 5min.) would