Synthesis and properties of DNAzymes carrying conformationally constrained nucleosides

L. ROBALDO; F IZZO; M. DELLAFIORE; C PROIETTI; P. ELIZALDE; J MONTSERRAT; A. IRIBARREN

Melbourne

Congreso; 19th International Conference on Organic Chemistry; 2012

Oligonucleotides can perform other functions in addition to the traditional transmission and storage of genetic information. In particular, DNAzymes are oligonucleotides that catalyze the sequence specific hydrolysis of RNA. The 10–23 DNAzyme consists of a 15-nucleotides catalytic core and two recognition arms that bind to the target mRNA (Fig.1).   For biological purposes, oligonucleotides must be chemically modified in order to increase their stability against degradation by nucleases.   In this work we show the synthesis and the properties of modified DNAzymes carrying in their catalytic core 2’-C-methyl-2’-deoxynucleosides. These analogs show differential preferred sugar conformations depending on the absolute configuration at the 2’-C (Fig. 2).[1] Therefore, (2’R)-2’-deoxy-2’-C-methyluridine, (2’S)-2’-deoxy-2’-C-methyluridine, (2’R)-2’-deoxy-2’-C-methylcytidine and (2’S)-2’-deoxy-2’-C-methylcitidine were synthesized and introduced in one, two and three positions of the 10–23 DNAzyme catalytic core. In the case of DNAzyme Dz-4S7R8R, 72% of the activity was maintained at 1 mM Mg2+. When conformationally locked nucleosides, like LNA-C or anhydrouridine, were introduced at the same positions, no catalytic activity was observed.[2],[3]     Preliminary in silico studies showed that these results could be partially explained by the conformational flexibility showed by 2’-C-methylnucleosides when incorporated into loop environments. In relation to DNAzyme stability, some triple modified molecules were three times more resistant against endonuclease degradation and have four to five times higher activity/resistance ratios than nonmodified sequences.   One DNAzyme carrying three 2’-C-methylpyrimidines was tested against Stat 3, a promoter of human breast cancer, showing a complete abrogation of progestin-induced proliferation [1] Cicero, D. O.; Gallo, M.; Neuner, P. J.; Iribarren, A. M. Tetrahedron 2001, 57, 7613. [2] Robaldo, L.; Montserrat, J. M.; Iribarren, A. M. Bioorg. Med. Chem. Lett. 2010, 20, 4367. [3] Robaldo,L.; Izzo, F.; Dellafiore,M.; Proietti,C.; Elizalde,P.V.; Montserrat,J.M.; Iribarren,A.M.