Phosphorothioate analogs of 2',5'-oligoadenylate. Enzymic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

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Abstract
The chiral and achiral phosphorothioate analogues of 2'',5''-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5''-O-(2-thiotriphosphate) [(Sp)-ATP.beta.S and (Rp)-ATP.beta.S, respectively] and adenosine 5''-O-(3-thiotriphosphate) (ATP.gamma.S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2'',5''-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP.gamma.S and (Sp)-ATP.beta.S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP.beta.S was more than 50-fold less efficient a substrate. The .beta.- and .gamma.-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2'',5''-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Kariko, K, Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., and Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Kariko, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., and Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A. The .gamma.-phosphorothioates displaced the p3A4[32P]pCp probe from RNase L with equivalent affinity to p3A3 (IC50 5 .times. 10-9 M), while the (Sp)-.beta.-phosphorothiates had had a slightly increased binding affinity (IC50 1 .times. 10-6 M). This same dramatic difference was observed in the activation process of RNase L as determined in the core-cellouse and rRNA cleavage assays; i.e., the (Sp)-.beta.-,.gamma., and (Rp)-.beta.-phosphorothioates activated RNase L to hydrolyze poly(U)[32P]pCp 50% at 5 .times. 10-10, 1 .times. 10-9, and 3 .times. 10-7 M, respectively. The proposed structures of the 2''5''-phosphorothioate trimer analogues synthesized from (Sp)-ATP.beta.S, (RP)-ATP.beta.S, and ATP.gamma.S are 5''-O-[(Sp)-2-P-thiotriphosphoryl]adenylyl-(2''-5'')adenylyl(2''-5'')adenosine, 5''-O-[(Rp)-2-P-thiotriphosphoryl]adenyly(2''-5'')adenyly(2''-5'')adenosine, and 5''-O-[3-thiotriphosphoryl]adenylyl(2''-5'')adenylyl(2''-5'')adenosine,respectively.

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