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

Abstract

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.

New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies

“…During our effort to functionalize the carbocyclic moiety of the 5-membered carba-LNA 13 through new synthetic routes 27 , we attempted to remove the 6′-hydroxyl group of 6′-hydroxy-6′-methyl-carba-LNA 27 , prepared from 6′-hydroxy-carba-LNA 27,28 Unexpectedly, a unique stereospecifically rearranged compound 1 (Scheme 1) was obtained 28 during the radical deoxygenation of 6′-methyloxalyl-6′-methyl-carba-LNA in addition to a diastereomeric mixture of C6′-reduced products. 27,28 It turned out that this radical rearrangement only takes place when the tertiary radical is formed from the 5-membered conformationallyconstrained 6′-methyloxalyl-6′-methyl-carba-LNA 27 .…”

Synthesis and Structure of New Methylene-Bridged Hexopyranosyl Nucleoside (BHNA)

“…with Bu 3 SnH in presence of a catalytic amount of AIBN in anhydrous toluene, under an identical condition as used for compound 3 from 2, gave hithertofore an uncharacterized 34 radical rearranged product, protected BHNA 5, in 27% yield along with the known C6′ reduced products 6 (18%) and 7 (15%). 34 The protected BHNA 5 was debenzylated using 20% Pd(OH) 2 /C and ammonium formate under reflux in methanol to give fully deprotected BHNA 8 quantitatively.…”

“…34 The protected BHNA 5 was debenzylated using 20% Pd(OH) 2 /C and ammonium formate under reflux in methanol to give fully deprotected BHNA 8 quantitatively. to give C4′ radical, which upon quenching by H • could give hexopyranosyl nucleoside (Path "B" in Figure 4).…”

“…34 Crich et al have shown that the C4' radical of 2',3'-dideoxythymidine is known to rearrange to C1' radical by scission of the C1'-O4' bond to give the acyclic product, but this reaction does not take place with 2'-deoxy-3'-methoxythymidine. 35 They suggested that different stereoelectronic effect in these two radical intermediates leads to their different fates.…”

“…Recently, our group has reported synthesis of conformationally-constrained carba-LNA and carba-ENA nucleosides through radical cyclization reactions. 20,33,34 It is known that carbon radical in the sugar moiety of nucleoside can induce C-O bond scission under anaerobic conditions: For example, C4′ radical leads to scission of C1′-O4′ bond 35 followed by rearrangement to C1′ radical ( Figure 2A). C1′ may lead to scission of C4′-O4′ bond and subsequent rearrangement to C4′ radical ( Figure 2B).…”