Synthesis and Properties of RNA Analogues Having Amides as Interuridine Linkages at Selected Positions

Abstract

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.

Amides as Excellent Mimics of Phosphate Linkages in RNA

“…Backbone-modified RNA may find broad application in the fundamental biology and biomedicine of noncoding RNAs, providing that the modifications mimic the structure of the phosphodiester linkage and do not alter the conformation of RNA. In particular, the potential of RNA interference to become a new therapeutic strategy has revitalized interest in chemical modifications that may optimize the pharmacological properties of short interfering RNAs (siRNAs).[1] We are interested in hydrophobic nonionic mimics of the phosphate backbone, such as formacetals [2] and amides, [3] that may confer high nuclease resistance to siRNAs along with reduced charge and increased hydrophobicity. Earlier studies showed that 3'-CH 2 -CO-NH-5' internucleoside amide linkages (abbreviated here as AM1) were well-tolerated in the DNA strand of an A-type DNA-RNA heteroduplex.…”

“…The tert-butyldimethylsilyl (TBS) groups in the known 3'-allyluridine 1 [3] were replaced with 5'-O-methoxytrityl (MMT) and 2'-O-acetyl protecting groups suitable for solid-phase RNA synthesis. Two-step oxidative degradation of the alkene gave the carboxylic acid part 6 of the r(U AM1 A) dimer.…”

Designing an effective approach for obtaining methylenecarboxylate analogues of adenophostin A. Preliminary results

“…Olefin 11 was dissolved in dioxane-H 2 O, and catalytic amounts of OsO 4 were added with stoichiometric NMO acting as the reoxidizing agent. 14 The crude reaction product was then treated with NaIO 4 to afford aldehyde 12 in 74% yield. Subsequent oxidation with sodium chlorite in a buffered t-BuOH-H 2 O mixture in the presence of a halogen scavenger generated the corresponding carboxylic acid.…”

Toward Amide-Modified RNA: Synthesis of 3‘-Aminomethyl-5‘-carboxy-3‘,5‘-dideoxy Nucleosides

“…22,23 Among the few exceptions are amides (3′-CH 2 -CONH-5′ and 3′-CH 2 NHCO-5′), [24][25][26] 27,28 formacetal (3′-OCH 2 O-5′), [29][30][31][32] and thioformacetal (3′-SCH 2 O-5′) 31,32 backbones. Recently, we showed that the two isomeric amide linkages (Figure 1, 1 and 2) were excellent mimics of the phosphodiester backbone in RNA duplexes.…”