Probing the activation site of ribonuclease L with new N6-substituted 2',5'-adenylate trimers.

Abstract 2-5A trimer [5′-monophosphoryladenylyl(2′-5′)adenylyl(2′-5′)adenosine] activates RNase L. While the 5′-terminal and 2′-terminal adenosine N 6 -amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5′-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5′- O -dimethoxytrityl-3′- O -( tert -butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine 2′-(2-cyanoethyl N , N -diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N 6 -substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5′A2′p5′A2′p5′A. Thus, the spatial domain about this N 6 -amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.