Respective role of each of the purine N7 nitrogens of 5'-O-triphosphoadenylyl(2' .fwdarw. 5')adenylyl(2' .fwdarw. 5')adenosine in binding to and activation of the RNase L of mouse cells

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Abstract
Through a combination of chemical and enzymatic approaches a series of sequence-specific tubercidin-substituted ppp5''A2''p(5''A2''p)n5''A (n = 1 to about 10; 2-5A) analogues were generated. In addition to the previously developed methodology of Imai and Torrence [Imai, J., and Torrence, P.F. (1985) J. Org. Chem. 50, 1418-1420], a new approach to synthesis of 2'',5''-linked oligonucleotides utilized adenosine in 3'',5'' linkage as a precursor to the targeted 5''-terminus of the desired product. For instance, A3''p5''A could be condensed under conditions of lead ion catalysis with tubercidin 5''-phosphate to give A3''p5''A2''p5''(c7A). Treatment with the 3'',5''-specific nuclease P1 led to p5''A2''p5''(c7A). The combined use of the above procedures led to the synthesis of p5''(c7A)2''p5''A2''p5''A, p5''A2''p5(c7A)2''p5''A, p5''A2''p5''A2''p5''(c7A), and p5''A2p5(c7A)2''p5(c7A), which were converted to their corresponding 5''-triphosphates by the usual methods. Evaluation of these analogues for their ability to bind to and activate the 2-5A-dependent endonuclease (RNase L) of mouse L cells showed that there were small changes (.ltoreq. 10-fold) in the ability of the four tubercidin analogues to bind to RNase L. However, whenever the first and/or third adenosine nucleotide units were replaced by tubercidin, a dramatic decrease in ability to activate RNase L occurred. Only the second (from the 5''-terminus) adenosine residue could be replaced by tubercidin without any effect on RNase L activation ability.
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