Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2' .fwdarw. 5')adenylyl(2' .fwdarw. 5')adenosine

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Abstract
The oligonucleotides A5''pp5''A2''p5''A2''p5''A and A5''ppp5''A2''p5''A2''p5''A were prepared by reaction of AMP or ADP, respectively, with the 5''-(phosphoimidazolidate) of A2''p5''A2''p5''A. A5''pppp5''A2''(p5''A)n (n = 1-3) were synthesized by reaction of p5''A2''(p5''A)n (n = 1-3) with adenosine 5''-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P NMR. The products A5''pppp5''A2''p5''A and A5''pppp5''A2''p5''A2''p5''A were found to be identical with 2 of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5''pppp5''A2''p5''A2''p5''A2''p5''A and A5''pppp5''A2''p5''A2''p5''A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5''ppp5''A2''p5''A2''p5''A and A2''pppp5''A2''p5''A were .apprx. 100 times less active than 2-5A, and A5''pp5''A2''p5''A2''p5''A was without translational inhibitory activity. When affinity for the 2-5A dependent endonuclease was determined (by displacement of 2-5A [32P]pCp from endonuclease), all of the analogs, and 2-5A itself, had similar affinities for the endonuclease except for A5''pppp5''A2''p5''A, which was bound .apprx. 100 times less effectively. Under conditions of the radiobindng assay, A5''pppp5''A2''p5''A2''p5''A was degraded (t1/2 [half-life] = 2 h) to ATP, ADP, AMP, ppp5''A2''p5''A2''p5''A and p5''A2''p5''A2''p5''A. The same products were obtained when degradation was carried out under protein synthesis conditions at 30.degree. C except that the half-life of A5''pppp5''A2''p5''A2''p5''A was reduced to 3 min. The other unsymmetrical di- and triphosphates A5''pp5''A2''p5''A2''p5''A and A5''ppp5''A2''p5''A2''p5''A were degraded much more slowly than the tetraphosphate, and they did not give rise to 2-5A as a degradation product. When the degradation of A5''pppp5''A2''p5''A2''p5''A was examined in incubation mixtures containing human serum or [human lymphoblastoid] Nalmalwa cell extract, it was found that the tetraphosphate was quite stable to the action of human serum but was readily degraded to 2-5A and p5''A2''p5''A2''p5''A by extracts of the human lymphoblastoid cells. Thus, capping (with adenosine) of the .beta.- or .gamma.-phosphates of the established translational inhibitors pp5''A2''p5''A2''p5''A or ppp5''A2''p5''A2''p5''A led to a loss of ability to activate the 2-5A-dependent endonuclease even though such oligomers still were bound to the endonuclease and 2-5A itself. Capping with an adenosine tetraphosphate moiety gave a 2-5A derivative that was stable in the external milieu of the cell but that was rapidly cleaved by the enzyme(s) of the cytosol to give 2-5A itself.

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