The Five- and Six-Membered Conformationally- Locked 2',4'-Carbocyclic ribo-Thymidine: 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 5-membered and 6membered carbocyclic analogs of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields. Their structures have been confirmed unambiguously by the long range H-C NMR correlation (HMBC), TOCSY, COSY and nOe experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently incorporated in to the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified AON/RNA heteroduplexes by 3.5-5 °C and 1.5 °C /modification for carbocyclic-LNAT, and carbocyclic-ENA-T respectively. Whereas the relative RNase H cleavage rates with carbocyclicLNA-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 the carbocyclic-ENA into AONs leads to Journal of the American Chemical Society (Accepted for publication on 26-Apr-2007, Manus # ja071106y). 2 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 (≈85 % stable in 48 h). Clearly, remarkably enhanced life-time 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 agent.