Synthesis, Characterisation and Biological Significance of (2′‐5′)Oiigoadenylate Derivatives of NAD+, ADP‐Ribose and Adenosine(5′)Tetraphosph0(5′)Adenosine

The (2′‐5′)oligoadenylate synthetase [ppp(A2′p)n,A synthetase] from interferon‐treated HeLa cells has been used to synthesize NAD+2′p5′A and NAD′2′p5′A2′p5′A. High‐performance liquid chromatography conditions have been developed for the separation of these products and the structures of the purified nucleotides confirmed by proton nuclear magnetic resonance spectroscopy. Using A5′p45′A and ADP‐ribose as primers the corresponding series of (2′‐5′)oligoadenylate products have been similarly synthesised and purified. All of the analogues whose structure includes pp(A2′p)2A were found to displace ppp(A2′p)3A[32P]pCp in the radiobinding assay for ppp(A2′p)nA (n = 2 to ≥ 4) [M. Knight et al. (1980) Nature (Lond.) 288, 189–192] at the same concentration (nanomolar) as does ppp(A2′p),A. They also have the same stability as ppp(A2′p)nA under protein‐synthesis conditions in Ehrlich ascites tumour cell extracts. A5′p,5′(A2′p)4A and A5′p45′(A2′p)3A inhibit protein synthesis in Ehrlich ascites tumour extracts as effectively as ppp(A2′p)2A. A5′p45′(A2′p)3A activates the ppp(A2′p),A‐dependent nuclease in reticulocyte lysates at the same concentration as ppp(A2′p)3A. The biological activity of these molecules may reflect their conversion to ppp(A2′p)nA. NAD+ is a preferred primer for the ppp(A2′p)nA synthetase in HeLa extracts. Despite this, using thc radiobinding assay in combination with high‐performance liquid chromatographic analysis, no evidence was found for the natural occurrence of NAD+2′p5′A2′p5′A or the corresponding A5′p45′A or ADP‐ribose derivatives in trichloroacetic acid/ether extracts froin interferon‐treated, encephalomyocarditis‐virus‐infected mouse L cells. In contrast ppp(A2′p)2A and ppp(A2′p)3A were detected at levels corresponding to approximately 100 nM in these cells. It appears therefore that none of the derivatives of NAD+ A5′p45′A and ADP‐ribose having two or more additional AMP residues in 2′‐5′ linkage, occurs naturally in these cells at concentrations ≥ 5 nM under conditions in which the ppp(A2′p)nA synthetase is actively synthesising ppp(A2′p)nA.