Double sugar and phosphate backbone-constrained nucleotides: synthesis, structure, stability, and their incorporation into oligodeoxynucleotides.

Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S(p))- or (R(p))-D(2)-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio calculations showed that in (S(p))- and (R(p))-D(2)-CNA the following occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) = 54 degrees ; (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)], epsilon (cis), zeta [anticlinal(+)] for (S(p))-D(2)-CNA, and delta [gauche(+)], epsilon (cis), zeta [anticlinal(-)] for (R(p))-D(2)-CNA. It has been found that F(-) ion can catalyze the isomerization of pure (S(p))-D(2)-CNA or (R(p))-D(2)-CNA to give an equilibrium mixture (K = 1.94). It turned out that at equilibrium concentration the (S(p))-D(2)-CNA isomer is preferred over the (R(p))-D(2)-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered dioxaphosphorinane ring in D(2)-CNA was found to be dependent on the internucleotidic phosphate stereochemistry. Thus, both (S(p))- and (R(p))-D(2)-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximately 70-90%, carba-LNA-3',5'-phosphodiester (22) approximately 10%, and carba-LNA-6',3'-phosphodiester (23) <10%. In contrast, the (S(p))-D(2)-CNA was about 2 times more stable than (R(p))-D(2)-CNA under hydrazine hydrate/pyridine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S(p))-D(2)-CNA-incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S(p))-D(2)-CNA-modified AONs by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S(p))-D(2)-CNA modification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes the AON/DNA and AON/RNA duplex by about -6 to -9 degrees C/modification. Treatment of (S(p))-D(2)-CNA-modified AON with concentrated aqueous ammonia gave carba-LNA-6',5'-phosphodiester modified AON ( approximately 80%) plus a small amount of carba-LNA-3',5'-phosphodiester-modified AON ( approximately 20%). It is noteworthy that Carba-LNA-3',5'-phosphodiester modification stabilized the AON/RNA duplex by +4 degrees C/modification (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6', 5'-phosphodiester modification destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 degrees C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6',5'-phosphodiester modified AONs are much more weakly stacked than carba-LNA-3',5'-phosphodiester-modified AONs.

Fine tuning of electrostatics around the internucleotidic phosphate through incorporation of modified 2',4'-carbocyclic-LNAs and -ENAs leads to significant modulation of antisense properties.

In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the internucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic part and their integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH(3)) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resistance without loss of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreased cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or C8') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.