*Modified (2*′*,5*′*)Oligonucleotides: The Influence of Structural and Steriochemical Factors… DOI: http://dx.doi.org/10.5772/intechopen.108630*

to activate endonuclease. As a result of these studies, structural requirements emerged that are important for RNase L activation (reviewed in [4, 13]). Adenine bases were modified in several positions [14, 15], or even substituted by other base moieties [15, 16]. Substitution of H-8 in adenine with a Br atom [17], a methyl group [18], or a hydroxyl group [19] seems to change mainly the base-to-ribose orientation, which may have implications for endonuclease binding capacity. Despite the assumption of minor role of the central adenosine residue [14], its substitution by uridine reduces binding and activation significantly [16]. These studies have generally shown that the presence of adenosine in all positions of the 2-5A chain is crucial for the mediator activity and minimal modifications of heterocyclic bases might be acceptable.

Substitution of the adenosine moiety with 1-deazaadenosine (c1 A) or 3-deazaadenosine (c3 A) at different positions of the 2-5A chain first allowed to establish the role played by each of the adenine heterocycle nitrogen atoms in RNase L binding and activation [20–22].

The 2-5A analogs where adenosine was sequentially substituted by inosine show that (A1**)** NH2–C6–N1 moiety of the adenosine residue is critical for binding to RNase L. The same 2′-terminal (A3) adenosine residue moiety is required for the enzyme activation [14]. However, during the examination of the structure/biological activity dependence, a substitution of adenosine by inosine shall be considered as it causes two simultaneous alterations of the chemical structure including the transformation of adenine primary exocyclic 6-amino group into a keto function and the transformation of adenine N1 nitrogen atom into a NH part of hypoxanthine (**Figure 2**). Thus, previous data did not allow us to draw a definite conclusion as to what caused the loss of binding and/or activating ability of the above analogs substituted for inosine—the loss of exocyclic adenosine NH2-C6 group or transition of the tertiary adenine N1 nitrogen atom to the secondary atom in hypoxanthine? The use of 2-5A 1-deazaadenosine analogs resolved the above ambiguity since the substitution of adenosine by 1-deazaadenosine removes solely the adenine N1 nitrogen atom turning it into a CH moiety yet leaving the exocyclic primary amino function unchanged (**Figure 2**) [20, 21].

The pA(c1 A)A deaza analog containing c1 A in the trimer middle chain link was highly active which is consistent with data on the inosine analog pA(**I**)A activity [14]. Substitution of (A1) moiety with both c1 A, p(c1 A)A2, and inosine, p(I)A2 reduces the ability to activate RNase L by about a factor of 33. So the loss of activity is due to the absence of tertiary N1 nitrogen atom and not the absence of the exocyclic amino group. These results are consistent with data provided in [23] which showed that 2-5A

**Figure 2.**

*Structural formulas of adenosine (A), 1-dezazaadenosine (c1 A), and inosine (I).* analog containing 1-(β-D-ribofuranosyl)-1H-1,2,4-triazolo-3-carboxamide (ribavirin) in (A1) moiety activates recombinant RNase L from human CEM cell extracts more effectively compared to the parent trimer. The carboxamide group of ribavirin is probably better in mimicking NH2–C6–N1 moiety 5′-terminal adenosine compared to c1 A. It should also be noted that N1 of (A1**)** nitrogen atom of adenosine moiety can be pivotal in binding (2′-5′) oligomers with RNase L. Deazaadenosine analog pA2( 1 A) showed a 20-fold increase in activating ability compared to pA2(**I**) and as much as a 5-fold decrease compared to the parent tetramer pA4. Obviously, the NH2-C6 exocyclic amino group of 2-5A (A3) adenosine moiety is responsible for the conformational "switch" that induces activation of RNase L.

It was shown that substitution of 5′-terminal or 2′-terminal adenosine for c3 A produced respective analogs including p5′(c3 A)2′p5′A2′p5′A and p5′A2′p5′A2′p5′(c3 A) which were not inferior to the parent tetramer in activating RNase L (EC50 ≤ 1 nM) [22]. In contrast, p5′A2′p5′(c3 A)2′p5′A showed a reduced ability to activate RNase L (EC50 ≤ 10 nM). These data are consistent with substantial stereochemical discrepancies between A2′p5′(c3 A)2′p5′A and the parent core (2′,5′) trimer whereas a specific recognition of N3 atom of mid-adenosine (A2) is unlikely. The extensive conformational analysis of c3 A-substituted core trimers compared to the initial parent core trimer showed close stereochemical resemblance between the parent core trimer and (c3 A)2′p5′A2′p5′A and A2′p5′A2′p5′(c3 A) analogs which is a strong evidence of syn orientation of the base with respect to the glycoside bond. Conversely, A2′p5′(c3 A)2′p5′A analog deviated rather significantly from the spatial arrangement of the parent core trimmer. The extensive conformational analysis of the c3 A-substituted core trimers versus the parent natural core trimer displayed close stereochemical similarity between the natural core trimer and (c3 A)2′p5′A2′p5′A and A2′p5′A2′p5′(c3 A) analogs, thereby strong evidences for the syn base orientation about the glycosyl bond of the c3 A residue of the latter were found. On the contrary, an analog A2′p5′(c3 A)2′p5′A displayed rather essential deviations from the spatial arrangement of the parent natural core trimer.

Synthesis data of 2-5A analogs containing 6-(benzylamino)purine riboside (AdoBn), a nucleoside with cytokinin activity are of interest. The second type of modification of the heterocyclic base was a replacement for virazole (ribavirin), a synthetic nucleoside that exhibits a wide range of antiviral activity against a wide variety of viruses. Compounds with high antiviral activity were found among the obtained oligomers. Studies of biological properties exhibited by these 2-5A analogs [23, 24] showed that these compounds had HIV-1 reverse transcriptase inhibitory and recombinant human ribonuclease L activity [25]. Specifically, trimers containing AdoBn at any positions of the oligonucleotide chain have been shown to impede the HIV-1-induced formation of syncytium by 1500 times (vs. three times for 2′,5′A3 parent trimer). It is also in evidence that all virazole-containing trimers at a concentration of 300 μmol have been shown to inhibit HIV-1 reverse transcriptase by 99.5–99.7% (33% for parent trimer). The ability of AdoBn-containing compounds to inhibit this enzyme is based on the position of the modified nucleoside unit in the oligomer chain and is greatest for the trimer containing the above moiety in the 5′-terminal position. The ability of the studied 2′,5′-oligonucleotides to activate recombinant human ribonuclease L defined as the percentage of poly(U)-3′-[32P]pCp hydrolysis in the presence of these compounds also depends on the structure of these oligoadenylates. Thus, the trimer containing a virazole moiety in the 5′-terminal position of the chain inhibited ribonuclease L by 87.7%

(50% for the parent trimer). AdoBn-containing compounds as the 5′-terminal and central link—by 37.4% and 34.8%, respectively, whereas the heterobase modified 2′-terminal unit of oligoadenylates resulted in a complete loss of ability to activate ribonuclease L [25].
