**3.1 The role of structural and stereochemical factors in binding and activation of RNase L**

The synthesis of parent 2-5A analogs modified in the carbohydrate moiety and the study of their physicochemical and biological properties helped to establish the role of structural and stereochemical factors of this unique class of cell biomolecules.

The substitution of ribose 3′-OH groups of ppp(A2′p5′)2A with hydrogen proved that 3′-OH groups of (A2) moiety are essential for biological activity (**Figure 3**). However, this group is perhaps required for degradation by 2′-5′-phosphodiesterase since (A2′p5)2A with (A2) substituted by xyloadenosine was resistant to 2′-5′-phosphodiesterase activity [26]. For 2′-5′-phosphodiesterase, 3′-OH groups of the second residue and free 2′- and 3′-OH groups of the 2′-terminal residue [27–30] are required.

The results of examinations to assess relationship between the structure and spatial arrangement (stereochemistry), on the one hand, and the biological properties of 2-5A analogs, on the other hand, clearly showed that the known properties can be enhanced or new properties can be added by changing the structure and/or stereochemistry of their molecules. For example, the use of fluorodeoxyadenylates, parent oligomer analogs, in which adenosine moieties are consecutively substituted by a conformationally rigid fluoronucleoside molecule for enzyme reaction tests allowed to differentiate the role of structural and stereochemical factors and demonstrate the effect of molecule stereochemistry on their biological properties [31].

Substitution of the 3′-hydroxyl group of adenosine furanose ring with a fluorine atom in two different configurations, *ribo* (AF) and *xylo* (AF ), contributes to the overriding population of different conformations of the pentofuranose moiety due to stereochemical differences in the gauche effects of the fluorine atom and other electronegative substituents of furanose rings. It was demonstrated that the conformational features of individual fluoronucleosides АF and AF , which are mainly in the

#### **Figure 3.**

*Stereochemical structures of 3*′*-fluoro-ribo-adenosine (AF), 3*′*-fluoro-xylo-adenosine (AF ), and the structure of 5*′*-phosphates of the 2*′*,5*′*-oligoadenylic acid trimer.*

N- or S-conformation, respectively, are also preserved in the respective fluoronucleoside moiety of the trimer. These datademonstrated that the fluorine atom present in the carbohydrate part of the molecule is a key factor of the conformation of these molecules [31].

Stereochemical features of 2-5A *xylo*- and *ribo*-fluorodeoxy analogs make these compounds unique stereochemical analogs of the parent oligomer, which enables to discriminate the role of structural and stereochemical factors in biological processes. Indeed, on the one hand, a pair of AF and A<sup>F</sup> substituted analogs in a certain position of the chain is structurally related to 3′-deoxyadenosine (cordycepin) analogs, while on the other hand, the sugar rings AF and A<sup>F</sup> are in different conformations, S and N, respectively. These stereochemical differences between AF and A<sup>F</sup> are preserved when included in the (2′-5′) oligomer molecule instead of adenosine, and determine largely its stereochemistry. The *syn*↔*anti* orientation change of the heterocyclic base of AF and A<sup>F</sup> fluoronucleosides with prevalent syn- or anti-conformers, respectively, results in a variety of stacking interactions of heterocyclic bases and thereby changed oligonucleotide chain conformation as a whole. The introduction *of xylo-*fluoronucleoside (A<sup>F</sup> ) into the central (A2) 2-5A moiety produced an oligomer more resistant to (2′,5′)-phosphodiesterase compared to the parent trimer. At the same time, the presence of 3′-deoxy-, 3′-fluoro*xylo*(A<sup>F</sup> ), or 3′-fluoro-*ribo*(AF) adenosine in the (A3) moiety of oligoadenylate produced different degrees of hydrolysis despite their structural resemblance suggesting the role of the spatial arrangement of the molecule in the recognition thereof by phosphodiesterase active site [32].

The study of the ability of 2-5A fluorodeoxyanalogs to bind and activate RNase L showed that the analog whose fluorine atom in the *xylo-*configuration in the midmoiety is nine times more active than the parent mediator, 2-5A and about 280 times more effective than isomeric *ribo*-trimer [32–34]. The results suggest that *anti*↔*syn* stereochemical differences between pppAA(AF) and pppAA(AF ), on the one hand, and related 8-bromo- and -methyl-analogs, on the other hand, cause differences in the degree of RNase L activation. Data presented in [32–34] suggest the *syn*-orientation is a major contribution to this process. The pppA(A<sup>F</sup> )AF poor ability to activate RNase L supports this assumption.

It is noteworthy that 2-5A fluorine-substituted analogs are important for the analysis of stereochemical patterns of RNase L activation. The *syn*-orientation of the base both at 5′- terminal of the oligomer and in the central site and the prevalent *N*-conformation of pentofuranose residues are probably required to form a productive complex between the enzyme, mediator, and substrate. In addition, the *syn*-orientation of the base in (A3) moiety of the oligomer is positive for RNase L activation. These reasonably substantiated assumptions are quite paradoxical and a question suggests itself: could this unusual *syn*-orientation of bases being in the transient state during RNase L activation be the second - after the unusual (2′-5′) phosphodiester bond in the oligomer—unique property of this mediator?

The *syn-*orientation of the heterocyclic base in (A1) or (A2) units of the oligomer chain, together with the predominant *N*-conformation of pentofuranose residues, is obviously required to form a productive complex between the enzyme, mediator, and substrate. The s*yn*-orientation of the base in (A3) moiety of the oligomer is also positive for RNase L activation. The trimer stereochemical features have been established to be pivotal in shaping RNase L binding and activation rather than the presence of C3′-OH group in 2-5A (A2) moiety.
