Five- and six-membered conformationally locked 2',4'-carbocyclic ribo-thymidines: synthesis, structure, and biochemical studies.
Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC), TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 degrees C and 1.5 degrees C/modification for carbocyclic-LNA-T and carbocyclic-ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-modified AONs (fully degraded <9 h) and aza-ENA ( approximately 85% stable in 48 h). Clearly, remarkably enhanced lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired pharmacokinetic properties because of their unique stability and consequently a net reduction of the required dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as potential antisense therapeutic agents.
Srivastava
Puneet
P
Department of Bioorganic Chemistry, Biomedical Center, Uppsala University, Uppsala, Sweden.
Barman
Jharna
J
Pathmasiri
Wimal
W
Plashkevych
Oleksandr
O
Wenska
Małgorzata
M
Chattopadhyaya
Jyoti
J
D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D044967:Serum / Q000737:chemistry; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry*
D002241:Carbohydrates; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D009943:Organophosphorus Compounds; C477371:locked nucleic acid; C434331:phosphoramidite; D012260:Ribonucleases; D013936:Thymidine