Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5′-C-Methyl Pyrimidine-Modified Oligonucleotides

Abstract

Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.

The chemical evolution of oligonucleotide therapies of clinical utility

“…This simple combination of backbone and sugar modification provides additional resistance to exonucleases—the primary effectors of RNA degradation—and an order of magnitude increase in oligonucleotide accumulation in vivo . Methylation of the 5′ carbon to give ( S )-5′- C -methyl-RNA 93 has also been used to enhance 3′-exonuclease resistance.…”

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

“…Compared to the successful ribose 2′-position modifications mentioned above, modifications at the C4′ and C5′ positions of ribose have been explored in less detail. Recently we examined a series of chirally pure ( R )- and ( S )-5′- C -methyl pyrimidine modifications and found that they affect thermodynamic stability negatively and in a manner that depends on the configuration at C5′ ( 15 ). In regard to protection against nucleases, the ( S )-epimers with various 2′-substituents were more effective than the corresponding ( R )-epimers.…”

Effect of uridine protecting groups on the diastereoselectivity of uridine-derived aldehyde 5’-alkynylation

“…Their widespread applications span from therapeutic agents, such as antibacterial [ 3 – 5 ], antiviral [ 6 ] or antitumor [ 7 – 8 ] drugs, to epigenetic modulators [ 9 ] or chemical tools [ 10 – 11 ]. Furthermore, they represent central monomers for oligonucleos(t)ide synthesis [ 12 – 17 ]. In the past decades, chemical modifications of these crucial building blocks have been extensively studied, both on the nucleobase itself and on the sugar moiety [ 6 ].…”

“…It can also be created directly on the nucleoside, either by functionalization of an alkene at C-5’ [ 27 – 32 ] or by the diastereoselective addition of a nucleophile on a carbonyl group. The latter can involve either the reduction of a ketone at C-5’ [ 12 , 33 – 35 ], or the addition on an aldehyde of various nucleophiles such as enolates [ 15 , 36 – 38 ], allylborane [ 39 ], dialkyl phosphites [ 40 ], TMSCN [ 41 ] or Grignard reagents [ 12 , 17 , 35 , 42 – 47 ]. Aside from the use of chiral ligands promoting an excellent facial discrimination of the aldehyde [ 34 ], the addition of Grignard reagents usually proceed with moderate diastereoselectivity and yield ( Table 1 ) [ 12 , 35 , 43 – 46 48 ].…”