BIOORGANIC CHEMISTRY 19,283-299 (1991) Uridine Analogs of 2’,5’-Oligoadenylates: On the Biological Role of the Middle Base of 2-5A Trimerl YUKIO KITADE,~ DAVID K. ALSTER,~ AYSUN PABUCCUOGLU,~ AND PAUL F. TORRENCE* Section on Biomedical Chemistry, Laboratory of Analytical Chemistry, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892 Received November 13, 1990 In order to further delineate the role of the second nucleotide residue of 2-5A in its interaction with the 2-SA-dependent endonuclease, RNase L, a series of uridine-substituted sequence-specific analogs were synthesized and evaluated for their ability to bind to and activate the nuclease. Substitution of only the S-terminal adenosine by uridine caused up to a lOO-fold loss in binding and activation of RNase L. Replacement of the middle adenosine residue of 2-SA trimer by uridine also resulted in some loss of binding and activation ability. When the 2’-terminal adenosine was replaced by uridine, a dramatic decrease in activation ability was observed. The results reinforced earlier conclusions that elements of the adenine base of the 5’-nucleotide are involved in binding to the 2-SA-dependent endonuclease, whereas activation is dependent upon structural determinants in the adenine moiety of the third adenosine nucleotide residue of 2-5A. These results also implicated some as yet unde- fined structural or conformational feature associated with the second nucleotide unit of 2-5A that may be involved in binding to or activation of RNase L. 8 1~1 Academic PUSS, IK. INTRODUCTION Among the most notable of the diverse biological activities of interferon are the antiviral effects which are mediated through several mechanisms including the 2- 5A6 system (I, 2). 2-5A represents a unique oligonucleotide series of the form ’ A part of this work was presented at the Joint Meeting of the American Society of Biological Chemists and the American Chemical Society, June 8-12, 1986 Washington, D.C., and ak~ at the 107th Congress of the Pharmaceutical Society of Japan, April, 1987, Kyoto, Japan. 2 Present address: Medicinal Chemistry Department, Gifu Pharmaceutical University, 5-6-l Mita- hora-higashi, Gifu 502 Japan. 3 Present address: Section on Endocrinology and Metabolism, Tulane Medical School, 1430 Tulane Ave., New Orleans, LA 70112. 4 Present address: Aegean University, Faculty of Pharmacy, Bomova-Izmir, Turkey sTo whom correspondence should be addressed. 6 Abbreviations used: 2-SA, ppp5’A2’p5’A2’pSfA; DMF, dimethylformamide; HPLC, high perfor- mance liquid chromatography; Hepes, N-2-hydroxyethylpiperazine-N’-Zethanesulfonic acid. In refer- ence to the 2-5A trimer, adei , adeZ, and ade, refer to the adenines of the 5’-terminal nucleotide unit, the middle nucleotide, and the 2’-terminal nucleotide, respectively; similarly, rib,, ribZ, and rib3 refer to the similarly located ribose moieties. The 2-SA-dependent endonuclease is also referred to as RNase L. The derivative in which the 2’-terminal ribose unit has been oxidized, reacted with n-hexylamine, 283 0045-2068/91 $3.00 Copyright Q 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.