Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.
Oligoribonucleotides containing formacetal internucleoside linkages have been prepared and studied by UV melting experiments. In RNA duplexes, the formacetal substitution is stabilizing (Deltat(m)=0 to +0.9 degrees C per modification) at physiological salt concentrations (0.1 M) but destabilizing (Deltat(m)=-0.4 to -0.8 degrees C per modification) at high salt concentrations (1 M); this suggests that reduction of electrostatic repulsion contributes substantially to the stabilization. The presence of 2'-O-Me substituents increases the stabilities of the duplexes (Deltat(m)=+0.5 to +1.1 degrees C per modification). The positive effects of formacetals and 2'-O-Me groups were independent and additive. (1)H NMR studies on monomeric model compounds containing 3'-(ethyl phosphate) or 3'-O-ethoxymethyl groups showed that the formacetal and 2'-O-Me substitutions shift the conformational equilibria of the ribose residues towards the North conformers by 5 to 12 %. Although the preference for the North conformers qualitatively correlates with increased duplex stabilities, changes in thermodynamic parameters (DeltaH degrees and TDeltaS degrees ) for formation of oligonucleotide duplexes and differences in dependence on concentrations of sodium acetate, sodium chloride and sodium perchlorate suggest that solvation effects are also important for the duplex stabilities. Overall the formacetal linkages fit well in A-type RNA duplexes, making them potentially interesting modifications for RNA-based gene-control strategies (e.g., antisense and RNA interference).
Controlled enzymatic synthesis is an alluring alternative to solid-phase synthesis and polymerase-mediated incorporation of nucleotides for the crafting of chemically modified, therapeutic oligonucleotides. While this approach has met some success for the elaboration of long, unmodified DNA sequences, very little research efforts have been dedicated to xeno nucleic acids (XNAs). Here, we have evaluated the possibility of using various 3'-O-blocking groups for controlled synthesis of locked nucleic acids (LNA). LNA nucleosides were equipped with protecting groups used in synthetic organic chemistry and were evaluated for their stability. The most promising candidates, benzoyl-and pivaloyl-protected nucleosides, were converted to the corresponding nucleotides. The resulting modified nucleotides were shown to be accepted by various polymerases. While single incorporation events were observed in high yields, strong esterase activity of polymerases represents a lasting hurdle that needs to be overcome. Overall, this article represents an additional step towards the controlled enzymatic synthesis of LNA-containing oligonucleotides and could be extended to other sugar or nucleobase modified nucleotides.
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