5‘-Modified G-Quadruplex Forming Oligonucleotides Endowed with Anti-HIV Activity:  Synthesis and Biophysical Properties

Oligodeoxyribonucleotides of sequence d(^5‘TGGGAG^3‘) carrying bulky aromatic groups at the 5‘ end were found to exhibit potent anti-HIV activity [Hotoda, H., et al. (1998) J. Med. Chem. 41, 3655−3663 and references therein]. Structure−activity relationship investigations indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5‘-end, were both essential for their antiviral activity. In this work, we synthesized some representative examples of the anti-HIV active Hotoda's 6-mers and analyzed the resulting G-quadruplexes by CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case of the sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drug optimization, we developed an alternative protocol to synthesize the 5‘-DBB-thymidine phosphoramidite building block in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes of the 5‘-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of the molecular modeling studies. The aromatic groups at the 5‘ position of d(^5‘TGGGAG^3‘) dramatically enhance both the equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigated quadruplexes was found to correlate with the reported IC₅₀ values, thus furnishing quantitative evidence for the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biological activity.